WO2022077276A1 - Application of combination of nicotinamide mononucleotide and lactobacillus fermentum in preparation of formulation for relieving skin photoaging - Google Patents
Application of combination of nicotinamide mononucleotide and lactobacillus fermentum in preparation of formulation for relieving skin photoaging Download PDFInfo
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- WO2022077276A1 WO2022077276A1 PCT/CN2020/120924 CN2020120924W WO2022077276A1 WO 2022077276 A1 WO2022077276 A1 WO 2022077276A1 CN 2020120924 W CN2020120924 W CN 2020120924W WO 2022077276 A1 WO2022077276 A1 WO 2022077276A1
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Definitions
- the invention belongs to the field of biomedicine, and in particular relates to the application of nicotinamide mononucleotide and Lactobacillus fermentum in combination in preparing a preparation for relieving skin photoaging.
- the skin is one of the largest and most complex organs in the human body, accounting for about 15% of body weight, and it is also the first line of defense against various environmental aggressions.
- Skin aging is an important part of body aging, which is not only detrimental to beauty, but also closely related to the occurrence of many skin diseases such as seborrheic keratosis, solar keratosis, basal cell carcinoma, and squamous cell carcinoma.
- Photoaging refers to the premature aging of the skin caused by repeated light exposure, and its clinical manifestations, histopathological and biochemical changes are different from the natural aging of the skin.
- Photoaging leads to a decrease in the number of mature type I collagen and elastic fibers in the dermis of the skin, and its clinical features are mainly manifested as skin roughness, loss of elasticity, deepening and thickening of wrinkles, leather-like appearance, and hyperpigmentation in exposed areas such as the face, neck, and forearm and telangiectasia.
- NMSCs non-melanoma skin cancers
- BCCs basal cell carcinomas
- SCCs squamous cell carcinomas
- Skin anti-aging has become a research hotspot of many scholars and clinicians in today's society, and it is also one of the hot areas that many beauty seekers pay attention to. In view of this, it is particularly important to establish a feasible photoaging model for in-depth study of its occurrence and development mechanism and screening of anti-photoaging drugs. Studies have shown that the occurrence and development of UV radiation-mediated skin photoaging involves multiple pathways, including apoptosis, proliferation, autophagy, DNA repair, checkpoint signal transduction, cell transduction, and inflammation.
- UVA 315nm-400nm
- UVB 280nm-315nm
- UVC 200nm-280nm
- UVB 280-315nm
- UVB UVB radiation only accounts for 1-2% of the sun's ultraviolet rays, it is considered to be the main environmental carcinogen that causes skin cancer and is related to the occurrence and development of tumors.
- the animal model is the most widely used photoaging model at home and abroad, and UVB is often used for modeling, because the skin tissue changes caused by UVB are very similar to human photoaging skin.
- Nicotinamide ribonucleotide is synthesized from nicotinamide (Nic) and 5'-phosphate pyrophosphate by nicotinamide phosphotransferase (NAMPT) and is a key intermediate of NAD + .
- NAMPT nicotinamide phosphotransferase
- NMN has been shown to enhance NAD + biosynthesis and improve various pathologies in mouse disease models, such as myocardial and cerebral ischemia, neurodegenerative diseases such as Alzheimer's disease, and diabetes.
- NMN nicotinamide mononucleotide
- Lactobacillus fermentum is a gram-positive bacterium and is widely distributed in nature. They have important application value in important fields closely related to human life, such as industry, agriculture, animal husbandry, food and medicine.
- the health benefits of Lactobacillus fermentum include regulating the immune system, regulating the balance of intestinal flora, lowering serum cholesterol and reducing the risk of tumors.
- some Lactobacillus fermentum were found to have other important biological functions, such as anti-aging and antioxidant activities, attracting more and more researchers' attention.
- Some studies have found that oral administration of live bacteria of Lactococcus lactis subsp. cremoris H61 or heat-killed bacteria can improve the skin condition of Japanese women.
- the invention explores the relevant reagents for relieving skin photoaging, adopts UVB-induced skin damage mouse model, and then gives mice nicotinamide mononucleotide combined with L. fermentum bacterial suspension for gavage treatment, and finally through the oxidation of serum and skin index, inflammatory index, mRNA expression level and protein expression of related genes in skin and liver to evaluate the improvement effect of NMN combined with L. fermentum on UVB-induced skin damage in mice, so as to find new ways to prevent skin aging or develop NMN combined
- the new probiotics of Lactobacillus fermentum provide theoretical reference and available raw materials.
- the purpose of the present invention is to provide a new use of nicotinamide mononucleotide (NMN) combined with L. fermentum.
- NPN nicotinamide mononucleotide
- the first aspect of the present invention provides the application of nicotinamide mononucleotide in combination with Lactobacillus fermentum L. fermentum in the preparation of preparations for relieving skin aging.
- the application of nicotinamide mononucleotide in combination with Lactobacillus fermentum in the preparation of preparations for relieving skin photoaging is provided, and the preparation forms include medicines, health products and skin care products.
- Lactobacillus fermentum Lactobacillus fermentum form is different: when the preparation is a drug, health product or food additive, the Lactobacillus fermentum Lactobacilus fermentum is in the form of intermittently sterilized Lactobacillus fermentum cells or lysate; when the preparation is a skin care product When produced, Lactobacillus fermentum Lactobacilus fermentum is in the form of metabolites, cytoplasmic fragments, cell wall components or polysaccharide complexes of Lactobacillus fermentum.
- the intermittently sterilized Lactobacillus fermentum cells are in the form of freeze-dried powder
- the concentration ratio between the nicotinamide mononucleotide and the intermittently sterilized Lactobacillus fermentum cells is 1:1
- the nicotinamide mononucleotide The concentration was 30 mg/mL
- the concentration of Lactobacillus fermentum cells was 10 9 CFU/mL.
- the photoaging skin is skin aging caused by ultraviolet radiation
- the skin aging caused by ultraviolet radiation is skin photoaging induced by medium-wave ultraviolet radiation.
- the preparation for relieving skin photoaging is a preparation for alleviating oxidative stress damage induced by ultraviolet radiation and reducing inflammatory damage in the body.
- the preparation is an agent for activating the AMPK signaling pathway, thereby inhibiting the activation of the NF ⁇ B-p65 signaling pathway, and further reducing the damage of the body caused by oxidative stress and inflammatory responses.
- the active ingredients of the preparation for relieving skin photoaging in the present invention only include nicotinamide mononucleotide and Lactobacilus fermentum.
- the medicines for relieving skin photoaging are gels, ointments, emulsions, aerosols, injections, mixtures, oral ampoules, tablets or capsules;
- the health care products for relieving skin photoaging are capsules, tablets, oral Liquid or solid beverages;
- skin care products for relieving skin photoaging are liquids, creams, moisturizing powders or masks.
- the present invention conducts experiments through the UVB-induced mouse skin injury model, and it is verified through experiments that NMN combined with L. fermentum activates the AMPK signaling pathway, thereby inhibiting the activation of the NF- ⁇ B signaling pathway and reducing the damage of inflammatory mediators to mice.
- activated AMPK can reduce the oxidative damage of the skin and improve the overall antioxidant capacity of the body by regulating the levels of related oxidative stress indicators in the serum, liver and skin of mice. Therefore, the present invention has certain reference significance for the prevention and treatment of skin damage caused by UVB, and provides a theoretical basis and an available strain source for the development of the preparation of NMN combined with Lactobacillus fermentum.
- Figure 1 is a comparison chart of the liver organ indexes of mice in different experimental groups. The data are presented as mean ⁇ standard deviation ( ⁇ SD), where * means p ⁇ 0.05, ** means p ⁇ 0.01, and *** means p ⁇ 0.001;
- Figure 2 is the observation of the pathological morphology of the liver in different experimental groups
- Figure 3 is the observation of skin pathological morphology in different experimental groups, (A) skin H&E staining; (B) skin Masson staining; (C) skin TB staining;
- Figure 3 shows the mRNA expression levels of SOD1, SOD2, CAT and GSH in skin and liver tissues, data are presented as mean ⁇ standard deviation ( ⁇ SD), where * means p ⁇ 0.05, ** means p ⁇ 0.01, *** means p ⁇ 0.001;
- Figure 4 shows the mRNA expression levels of AMPK, NF- ⁇ Bp65, I ⁇ B- ⁇ , SOD1 and CAT in skin and liver tissues, data are presented as mean ⁇ standard deviation
- * means p ⁇ 0.05, ** means p ⁇ 0.01, *** means p ⁇ 0.001;
- Figure 5 shows the mRNA expression levels of PGC-1 ⁇ , APPL1, mTOR, FOXO1, TNF- ⁇ , IL-6, IL-10 and GSH in skin and liver tissues, data are presented as mean ⁇ standard deviation
- * means p ⁇ 0.05, ** means p ⁇ 0.01, and *** means p ⁇ 0.001.
- NMN was provided by Yinfu Pharmaceutical Technology Co., Ltd. (Shanghai, China). The purity of NMN was determined by high performance liquid chromatography, and the purity was >98.5%.
- Lactobacillus fermentum was isolated and purified from natural fermented yak yogurt in Xinjiang.
- mice Forty 7-week-old female Kunming mice were purchased from the Experimental Animal Center of Chongqing Medical University [Chongqing, China, SCXK(YU) 2018-0003]. Mice were housed under constant temperature and humidity conditions (temperature 25 ⁇ 2°C, relative humidity 50 ⁇ 5%), 12h light/dark cycle. Mice were allowed free access to standard rat chow and drinking water.
- mice Forty mice were randomly divided into 4 groups with 10 mice in each group, namely normal group, model group, VC group, and NMN+L.fermentum group (NMN+L).
- the UVB modeling method is as follows: the whole experimental period is 4 weeks. From the 3rd week, the skin damage model of the mice in the other groups except the normal group is established by using ultraviolet radiation equipment, irradiating for 2h every day. Before UV irradiation, an electric shaver was used to shave about 2 cm 2 of hair on the back of the mice.
- mice were administered free food and water; the mice in the VC group were given VC solution by gavage at a dose of 300 mg/kg.bw every day; Mice were orally gavaged with NMN, and at the same time, 1.0 ⁇ 10 9 CFU/mL bacterial suspension was orally administered to mice every day. All experiments were approved by the Chongqing Functional Food Collaborative Innovation Center.
- Example 2 NMN+L.fermentum can improve liver organ index and tissue pathological morphology in mice after UV irradiation
- mice After four weeks of culture, the mice were sacrificed by devertebral method, the mice were dissected to remove the liver and the hairless skin of the back of the mice, and the organ index of the liver was calculated. The results are shown in Figure 1.
- liver and skin tissues were cut and fixed in 4% paraformaldehyde solution, and the remaining liver and skin tissues were stored in a -80°C refrigerator for later use.
- the liver was stained with H&E and the skin was stained with H&E, Masson's trichrome and toluidine blue. Finally, an upright microscope was used to observe the pathological morphology of the tissue, and the results are shown in Figure 2 and Figure 3:
- the mouse organ index can directly reflect the structural changes and functions of organs, and is of great significance for evaluating the functional properties of test samples.
- the liver organ index of the mice in the model group was significantly reduced, indicating that UV irradiation not only directly accelerates skin aging, but also indirectly causes liver aging. oxidative stress or induced inflammatory response.
- the liver organ index of the mice was significantly improved, indicating that NMN combined with L. fermentum can maintain the normal weight of the mouse liver and delay the aging of the liver.
- the liver structure of the mice in the normal group was complete, and the hepatocytes were neatly arranged around the central vein in a satellite emission pattern.
- the nuclei were large and round, and there was no infiltration of inflammatory cells.
- the liver cells in the VC group were relatively disordered, with partial necrosis and infiltration of inflammatory cells around the central vein, and the overall structural integrity of the liver was worse than that in the normal group.
- the phenomenon of cell infiltration; compared with the model group and the VC group, the shape of the liver cells in the NMN+L group was significantly more complete, and there was almost no cell necrosis and inflammatory cell infiltration, and the overall structure of the liver was similar to the normal group.
- H&E staining is mainly used to observe the overall structure of the skin
- Masson staining is mainly used to observe collagen fibers in the skin
- TB staining is mainly used to observe mast cells in the skin.
- the thickness of the skin dermis of the mice in the NMN+L group was significantly increased, and the collagen fiber bundles were not broken, shrunken and glued, and the overall structure was similar to that of the normal group.
- Collagen fibers were blue-purple after Masson staining. It can be seen from Figure 3(B) that the normal group has a large number of collagen fibers, and the distribution is uniform and orderly. The number of collagen fibers in the model group was significantly less than that in the normal group, and the phenomenon of breakage and shrinkage appeared. The number of collagen fibers in the dermis of the skin of the mice in the VC group was less than that in the normal group, but higher than that in the model group. Compared with the model group and the VC group, the number of collagen fibers in the dermis of the mice in the NMN+L group increased more, the arrangement was more orderly, and there was almost no shrinkage or breakage.
- the skin dermis of the UVB group mice became thinner, the number of collagen fibers decreased, the number of mast cells increased, and at the same time, the liver tissue also showed a certain degree of pathological changes, indicating that the UVB-induced skin damage model was successful.
- the pathological morphology of the skin was greatly improved, and the liver of the mice also remained in a normal shape without obvious damage.
- Example 3 NMN+L.fermentum can improve oxidative stress injury in UVB mice
- Oxidative stress is one of the important factors of UVB-induced skin aging. Under normal circumstances, the generation and scavenging of oxygen free radicals are in a state of balance. When stimulated by the outside world, the body will generate a large amount of oxygen free radicals due to local hypoxia, resulting in cell apoptosis or damage.
- Superoxide dismutase (SOD) is an enzyme containing metal cofactors, which can remove (disproportionate) the superoxide anion produced by the body and play an important role in the process of anti-aging and oxidation. With the progress of lipid peroxidation in the body, a large amount of SOD can be consumed, which in turn induces serious damage to the body.
- Catalase is an enzyme scavenger, also known as catalase, which is a conjugated enzyme with iron porphyrin as a prosthetic group. It can promote the decomposition of H 2 O 2 into molecular oxygen and water, so as to protect cells from the poison of H 2 O 2 , and is one of the key enzymes in the biological defense system.
- MDA is the final product of lipid oxidation. It can affect mitochondrial respiratory chain complexes and key enzyme activities in mitochondria in vitro. Its production can also aggravate membrane damage. Therefore, the determination of malondialdehyde content can reflect lipid peroxidation in the body. degree, which indirectly reflects the degree of cell damage.
- Glutathione as an important antioxidant in the body, can scavenge free radicals in the human body; because GSH itself is susceptible to oxidation by certain substances, it can protect the sulfhydryl groups in many proteins and enzymes in the body It is not oxidized by harmful substances, so as to ensure the normal functioning of molecular physiological functions such as proteins and enzymes.
- Advanced glycation end products AGEs are the end products of non-enzymatic glycation reactions, and their production and accumulation in serum and tissues increase with age. The protein modification of AGEs in the human body is closely related to the occurrence of senile diseases, so it can be used as an important indicator to test the aging process.
- the skin When continuously exposed to UV light, the skin will produce a strong oxidative stress response, releasing a large amount of reactive oxygen species (ROS), and ROS as an upstream signal will further initiate the NF- ⁇ B-mediated inflammatory pathway, thereby making the skin appear dry , itching, erythema and edema and other inflammatory symptoms.
- ROS reactive oxygen species
- the nuclear transcription factor NF- ⁇ B and its inhibitory protein I ⁇ B are combined together and exist in the cell in a resting manner.
- I ⁇ B- ⁇ is a repressor protein of NF- ⁇ B, which can mask the nuclear localization signal of NF- ⁇ B, so that NF- ⁇ B exists in the cell in the form of an inactive complex. Studies have shown that almost all NF- ⁇ B inducers rapidly activate NF- ⁇ B due to the degradation of I ⁇ B- ⁇ , so the activation of NF- ⁇ B can be prevented by preventing the phosphorylation of I ⁇ B- ⁇ .
- TNF- ⁇ is a cytokine with a wide range of biological activities, which is mainly secreted by activated monocyte-macrophages and T cells, which can lead to the aggregation of inflammatory cells and stimulate the production of inflammatory cytokines by inflammatory cells, eventually leading to Inflammation.
- IL-6 is a pleiotropic pro-inflammatory cytokine with biological activity, and its role is similar to IL-1 ⁇ . It can also activate the NF- ⁇ B signaling pathway to a certain extent, thereby causing chronic inflammation in the skin.
- IL-6 can increase the permeability of the skin, allowing inflammatory factors to infiltrate the inflammatory site and initiate inflammation.
- IL-10 is an important negative regulatory cytokine, which has a blocking effect on multiple links in the development of immune inflammatory response, has a wide range of immunosuppressive activities, and can inhibit the production of activated monocytes-macrophages
- Other cytokines including IL1, IL6, granulocyte colony stimulating factor, TNF, platelet activating factor, etc. have strong immunosuppressive functions.
- T-SOD, CAT, MDA and AGEs in mouse serum were determined according to the instructions of conventional biochemical kits, and the levels of TNF- ⁇ , IL-6 and IL-10 in mouse serum were determined according to the instructions of ELISA kits. The results are shown in the table. 1 shown.
- T-SOD, CAT, Na + K + -ATP and NAD + in mouse serum were determined according to the instructions of conventional biochemical kits, and the levels of TNF- ⁇ and IL-10 in serum and skin tissue were determined according to the instructions of ELISA kits, respectively , and the results are shown in Table 2.
- NMN combined with L. fermentum can increase the enzyme activities of T-SOD and CAT in serum and skin tissue of mice, increase the mRNA expression of SOD and CAT in liver and skin, and reduce the content of AGEs and MDA in serum.
- NMN combined with L. fermentum can greatly increase the levels of T-SOD and CAT in serum, and the enzyme activity of T-SOD is even higher than that of the normal group.
- the above results indicate that NMN combined with Lactobacillus fermentum can resist the oxidative stress of the skin caused by UVB irradiation in mice by increasing the activity of antioxidant enzymes, and can also improve the overall antioxidant level of the body from the levels of serum and liver.
- NMN combined with L. fermentum can down-regulate the expression of pro-inflammatory cytokines TNF- ⁇ and IL-6 and up-regulate the expression of anti-inflammatory cytokine IL-10 from the serum level, skin tissue level and mRNA level, respectively, thereby reducing the The degree of inflammatory damage caused by these inflammatory mediators to skin-injured mice.
- Example 4 NMN+L.fermentum can activate AMPK signaling pathway
- AMP-activated protein kinase is a serine/threonine protein kinase, which is a heterotrimer formed by catalytic subunits ⁇ and ⁇ and regulatory subunit ⁇ . AMPK is mainly involved in the regulation of glucose, lipid and energy metabolism in the body. Studies have shown that AMPK activation can inhibit inflammation and oxidative stress.
- AMPK can also act on nuclear transcription factor NF- ⁇ B and other inflammatory factors to regulate inflammation through various pathways, becoming a potential therapeutic target for various metabolic diseases such as diabetes and obesity and various inflammatory diseases.
- NF- ⁇ B nuclear transcription factor
- AMPK can effectively regulate the expression, migration and adhesion of inflammatory cytokines, thereby affecting the inflammatory response.
- inhibition of AMPK activity can significantly enhance the expression of inflammatory factors such as TNF- ⁇ , IL-1 ⁇ , and IL-6, thereby aggravating inflammatory damage.
- high expression of AMPK promotes the activity of factors such as SIRT1, FOXO, and PGC1 ⁇ , which in turn inhibits NF- ⁇ B activity. After cellular aging, NF- ⁇ B signaling is enhanced due to decreased AMPK activity.
- Adiponectin receptor-binding protein belongs to the upstream gene of AMPK. It is a functional protein composed of 709 amino acids and located on chromosome 3. It can mediate a variety of cell signal transduction and play a role in a variety of cellular responses. It can regulate the inflammatory response of cells, anti-oxidation and arteriosclerosis.
- Target of rapamycin mTOR is a downstream molecule in the AMPK signaling pathway, which mainly controls cell growth by regulating protein translation, and is involved in regulating cell immunity and apoptosis.
- Activated AMPK can inhibit the activity of mTOR, inhibit the synthesis and translation of proteins, and then play a negative role in regulating tumor growth.
- Activation of the mTOR pathway is thought to be closely related to the pathogenesis of cutaneous melanoma.
- Forkhead transcription factor O1 (FOXO1) is a member of the forkhead box family, which is mainly involved in the regulation of various physiological and biochemical processes such as cellular oxidative stress, cycle arrest, autophagy, and metabolism.
- peroxisome proliferator gamma coactivator alpha can participate in mitochondrial biosynthesis by regulating adaptive heat production, glucose and lipid metabolism, and blood sugar balance, and improve mitochondrial respiration and oxidation. ability to regulate fatty acid oxidation.
- PGC-1 ⁇ can regulate the body's adaptive heat production, glucose and lipid metabolism and blood sugar balance, participate in mitochondrial biosynthesis, and improve mitochondrial respiration and oxidation capacity.
- NMN combined with L. fermentum is used to display the related factors in the AMPK signaling pathway and the level changes of the related factors through this signal.
- the experimental method was as follows: liver and skin tissues were homogenized and total RNA was extracted with Trizol reagent, and then the RNA was reverse transcribed into cDNA using a cDNA kit. Next, 1 ⁇ L of cDNA, 10 ⁇ L of TaqMan TM Multiplex Master Mix, 2 ⁇ L of 10 ⁇ M primer and 7 ⁇ L of ddH 2 O were mixed evenly, and amplification detection was performed on a real-time fluorescence quantitative PCR instrument.
- the amplification conditions were: deformation at 95°C for 15s, annealing at 55°C for 30s, and extension at 72°C for 35s, for a total of 40 cycles. Finally, the relative expression of each target gene was calculated by 2 - ⁇ CT , in which ⁇ -actin was used as an internal reference gene.
- the primer sequences used are shown in Table 3.
- Figure 4 shows the mRNA expression of AMPK, NF- ⁇ B p65, I ⁇ B- ⁇ , SOD1 and CAT in skin and liver tissues.
- UVB irradiation increased the mRNA expression levels of NF- ⁇ Bp65 in the skin and liver of mice, and decreased the expressions of I ⁇ B- ⁇ , AMPK, SOD and CAT, among which the model group had the most obvious changes. .
- the mRNA expressions of I ⁇ B- ⁇ , AMPK, SOD and CAT in the skin and liver of the VC group and the NMN+L group were increased to varying degrees, while the expression of NF- ⁇ Bp65 was decreased, among which NMN+
- the mRNA expression levels of the above indicators in the L group were close to those in the normal group.
- AMPK mRNA and protein in the liver and skin of mice in the model group was significantly lower than that in the normal group, indicating that UVB irradiation caused energy metabolism disorders in mice, and increased oxidative stress and inflammatory responses.
- NMN combined with L. fermentum gavage treatment can significantly increase the mRNA expression of AMPK in liver and skin tissues, indicating that NMN combined with L. fermentum can effectively promote cellular energy synthesis and reduce the damage caused by oxidative stress and inflammatory reactions.
- Figure 5 shows the mRNA expression levels of PGC-1 ⁇ , APPL1, mTOR, FOXO1, TNF- ⁇ , IL-6, IL-10 and GSH in skin and liver tissues.
- the mRNA expressions of PGC-1 ⁇ , APPL1, FOXO1, IL-10 and GSH in the skin and liver of the normal group mice were the highest, and the expressions of mTOR, TNF- ⁇ and IL-6 were the lowest, while the model Compared with the normal group, the expression levels of the above indexes in the skin and liver of the mice in the group showed a completely opposite trend, and there were significant differences between the two.
- VC and NMN combined with L.
- UVB irradiation can reduce the mRNA expression of APPL1, FOXO1 and PGC-1 ⁇ and increase the mRNA expression of mTOR in mouse skin and liver.
- the mRNA expressions of APPL1, LKB1, SIRT1, FOXO1 and PGC-1 ⁇ in the skin and liver of mice were significantly increased, while the mRNA expression level of mTOR decreased, indicating that NMN combined with L. fermentum can effectively By activating the AMPK signaling pathway, it can improve UVB-induced oxidative stress and inflammation in mice.
- NMN combined with L. fermentum has a relatively obvious improvement effect on UVB-induced skin damage in mice.
- the possible mechanism may be combined with NMN and L. fermentum by activating the AMPK signaling pathway, thereby inhibiting the activation of the NF- ⁇ B signaling pathway, and reducing the damage of inflammatory mediators to mice.
- activated AMPK can reduce the oxidative damage of the skin and improve the overall antioxidant capacity of the body by regulating the levels of related oxidative stress indicators in the serum, liver and skin of mice.
- This study has important reference significance for the prevention and treatment of UVB-induced skin damage, and provides a theoretical basis and available strain sources for the development of NMN combined with Lactobacillus fermentum health food.
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Abstract
Provided is an application of a combination of nicotinamide mononucleotide (NMN) and Lactobacillus fermentum (L. fermentum) in the preparation of a formulation for relieving skin photoaging. Experiments verify that NMN and L. fermentum are combined to activate the AMPK signaling pathway, so as to inhibit the activation of the NF-κB signaling pathway, thus reducing the damage of an inflammatory medium to a mouse. In addition, the activated AMPK can reduce the oxidative damage of skin and improve the overall antioxidant capacity of a body by adjusting levels of associated oxidative stress indicators in the serum, liver, and skin of a mouse. Therefore, the present invention has certain reference significance for the prevention and treatment of UVB induced skin damage, and provides a theoretical basis and available strain sources for the development of NMN and L. fermentum combined formulation.
Description
本发明属于生物医药领域，具体涉及烟酰胺单核苷酸与发酵乳杆菌Lactobacilus fermentum联合在制备缓解皮肤光老化制剂中的应用。The invention belongs to the field of biomedicine, and in particular relates to the application of nicotinamide mononucleotide and Lactobacillus fermentum in combination in preparing a preparation for relieving skin photoaging.
皮肤是人体最大也是最复杂的器官之一，约占体重的15％，它也是抵御各种环境侵害的第一道防线。皮肤衰老是机体衰老的一个重要组成部分，不仅有损于美容，且与许多皮肤病如脂溢性角化、日光性角化、基底细胞癌、鳞状细胞癌的发生密切相关。光老化指皮肤由于反复光暴露引起的提前老化，其临床表现、组织病理及生物化学变化均与皮肤自然老化有所不同。光老化导致皮肤真皮中成熟I型胶原及弹力纤维数量的减少，其临床特征主要表现为曝光部位如面部、颈部、前臂皮肤粗糙、弹性丧失、皱纹增深增粗、皮革样外观、色素沉着及毛细血管扩张等。研究表明大约65％的黑色素瘤病例和90％的非黑色素瘤皮肤癌(NMSC)，包括基底细胞癌(BCC)和鳞状细胞癌(SCC)，都与皮肤光老化有关。导致皮肤光老化的外界因素较多，例如紫外线(UV)、红外线、化学烟雾、粉尘和雾霾等，其中以UV辐射最为显著。The skin is one of the largest and most complex organs in the human body, accounting for about 15% of body weight, and it is also the first line of defense against various environmental aggressions. Skin aging is an important part of body aging, which is not only detrimental to beauty, but also closely related to the occurrence of many skin diseases such as seborrheic keratosis, solar keratosis, basal cell carcinoma, and squamous cell carcinoma. Photoaging refers to the premature aging of the skin caused by repeated light exposure, and its clinical manifestations, histopathological and biochemical changes are different from the natural aging of the skin. Photoaging leads to a decrease in the number of mature type I collagen and elastic fibers in the dermis of the skin, and its clinical features are mainly manifested as skin roughness, loss of elasticity, deepening and thickening of wrinkles, leather-like appearance, and hyperpigmentation in exposed areas such as the face, neck, and forearm and telangiectasia. Studies have shown that approximately 65% of melanoma cases and 90% of non-melanoma skin cancers (NMSCs), including basal cell carcinomas (BCCs) and squamous cell carcinomas (SCCs), are associated with skin photoaging. There are many external factors that cause skin photoaging, such as ultraviolet (UV), infrared, chemical smog, dust and haze, among which UV radiation is the most significant.
皮肤抗衰老已经成为当今社会众多学者、临床医师的研究热点，也是众多求美者关注的热门领域之一。鉴于此，建立切实可行的光老化模型对于深入研究其发生发展机制及筛选抗光老化的药物显得尤为重要。研究表明紫外线辐射介导的皮肤光老化的发生和发展涉及到多种途径，包括细胞凋亡、增殖、自噬、DNA修复、检查点信号转导、细胞转导和炎症等。紫外辐射按波长长短通常分为长波紫外线UVA(315nm-400nm)、中波紫外线UVB(280nm-315nm)、短波紫外线UVC(200nm-280nm)。虽然UVB(280-315nm))的辐射只占太阳紫外线的1-2％，但它被认为是导致皮肤癌的主要环境致癌物，与肿瘤的发生和发展都有关系。有研究发现生活在强日照地区的慢性免疫抑制患者发生皮肤红肿的几率更高。在接受持续免疫抑制治疗的器官移植受者中，皮肤癌的发病率也很高。目前动物模型是迄今为止国内外应用最为广泛的光老化模型，该模型也常采用UVB进行造模，因为UVB引起的皮肤组织改变与人光老化的皮肤极其相似。Skin anti-aging has become a research hotspot of many scholars and clinicians in today's society, and it is also one of the hot areas that many beauty seekers pay attention to. In view of this, it is particularly important to establish a feasible photoaging model for in-depth study of its occurrence and development mechanism and screening of anti-photoaging drugs. Studies have shown that the occurrence and development of UV radiation-mediated skin photoaging involves multiple pathways, including apoptosis, proliferation, autophagy, DNA repair, checkpoint signal transduction, cell transduction, and inflammation. Ultraviolet radiation is usually divided into long-wave ultraviolet UVA (315nm-400nm), medium-wave ultraviolet UVB (280nm-315nm), and short-wave ultraviolet UVC (200nm-280nm) according to the length of the wavelength. Although UVB (280-315nm) radiation only accounts for 1-2% of the sun's ultraviolet rays, it is considered to be the main environmental carcinogen that causes skin cancer and is related to the occurrence and development of tumors. Studies have found that chronically immunosuppressed patients living in areas with strong sunlight have a higher chance of developing skin redness. Skin cancer rates are also high among organ transplant recipients receiving continuous immunosuppressive therapy. At present, the animal model is the most widely used photoaging model at home and abroad, and UVB is often used for modeling, because the skin tissue changes caused by UVB are very similar to human photoaging skin.
烟酰胺单核苷酸(nicotinamide ribonucleotide，NMN)由烟酰胺(nicotinamide,Nic) 和5’-磷酸基焦磷酸通过烟酰胺磷酸基转移酶(NAMPT)合成，是NAD
+的关键中间体。NMN已被证明可以增强NAD
+的生物合成，改善小鼠疾病模型的各种病理，如心肌和脑缺血、阿尔茨海默病等神经退行性疾病和糖尿病。NMN大部分药理作用是通过促进NAD
+合成进行的，因为直接给药高剂量的NAD
+会出现失眠、疲劳、焦虑等副作用，并且NAD
+对质膜的穿透能力也比NMN差。在小鼠模型中最新发现的抗衰老、延长寿命的特性，使NMN更有吸引力。研究表明补充烟酰胺单核苷酸(NMN)可以改善小鼠随着年龄增长的代谢和应激反应，所以NMN被认为是一种很有前途的治疗与年龄相关的生理功能障碍和疾病的方法。
Nicotinamide ribonucleotide (NMN) is synthesized from nicotinamide (Nic) and 5'-phosphate pyrophosphate by nicotinamide phosphotransferase (NAMPT) and is a key intermediate of NAD + . NMN has been shown to enhance NAD + biosynthesis and improve various pathologies in mouse disease models, such as myocardial and cerebral ischemia, neurodegenerative diseases such as Alzheimer's disease, and diabetes. Most of the pharmacological effects of NMN are carried out by promoting the synthesis of NAD + , because direct administration of high doses of NAD + will cause side effects such as insomnia, fatigue, anxiety, and the ability of NAD + to penetrate the plasma membrane is also worse than that of NMN. Newly discovered antiaging, lifespan-extending properties in mouse models make NMN even more attractive. Studies have shown that nicotinamide mononucleotide (NMN) supplementation can improve metabolic and stress responses in mice with age, so NMN is considered a promising treatment for age-related physiological dysfunctions and diseases .
发酵乳杆菌属于革兰氏阳性菌，在自然界中分布极其广泛，它们在工业、农牧业、食品和医药等与人类生活密切相关的重要领域都具有很重要的应用价值。发酵乳杆菌对人体健康的益处包括调节免疫***、调节肠道菌群平衡、降低血清胆固醇和降低肿瘤风险等。近年来，一些发酵乳杆菌被发现具有其他重要的生物学功能，如抗衰老和抗氧化活性，吸引了越来越多研究者的关注。有研究发现，口服Lactococcus lactis subsp.cremoris H61的活菌或者加热处死的菌都可以改善日本妇女的皮肤状态，小鼠实验还证明此菌株能够减少皮肤溃疡的发病率，减少骨质疏松和减少脱发等。另有研究采用细胞抗氧化活性(CAA)法测定了Lactobacillus plantarum MA2不同生长期分离物的抗氧化能力，结果表明该菌株具有较高的抗氧化潜力。近年来，随着发酵乳杆菌对人体多种生理功效的不断发掘，寻找新的发酵乳杆菌以及研究新型发酵乳杆菌对人体的健康功能也越来越受到食品、医学等领域的关注。Lactobacillus fermentum is a gram-positive bacterium and is widely distributed in nature. They have important application value in important fields closely related to human life, such as industry, agriculture, animal husbandry, food and medicine. The health benefits of Lactobacillus fermentum include regulating the immune system, regulating the balance of intestinal flora, lowering serum cholesterol and reducing the risk of tumors. In recent years, some Lactobacillus fermentum were found to have other important biological functions, such as anti-aging and antioxidant activities, attracting more and more researchers' attention. Some studies have found that oral administration of live bacteria of Lactococcus lactis subsp. cremoris H61 or heat-killed bacteria can improve the skin condition of Japanese women. Mouse experiments have also proved that this strain can reduce the incidence of skin ulcers, reduce osteoporosis and reduce hair loss. Wait. Another study used the cellular antioxidant activity (CAA) method to determine the antioxidant capacity of Lactobacillus plantarum MA2 isolates at different growth stages, and the results showed that the strain has high antioxidant potential. In recent years, with the continuous exploration of various physiological effects of Lactobacillus fermentum on the human body, the search for new Lactobacillus fermentum and the research on the health function of new Lactobacillus fermentum on the human body have also attracted more and more attention in the fields of food and medicine.
发明内容SUMMARY OF THE INVENTION
本发明对缓解皮肤光老化的相关试剂进行探索，采用UVB诱导皮肤损伤小鼠模型，然后给予小鼠烟酰胺单核苷酸联合L.fermentum菌悬液灌胃处理，最后通过血清和皮肤的氧化指标、炎症指标以及皮肤和肝脏中相关基因的mRNA表达水平和蛋白表达来评价NMN联合L.fermentum对UVB致小鼠皮肤损伤的改善作用，如此为寻找新的预防皮肤衰老的方法或者开发NMN联合发酵乳杆菌的新型微生态制剂等提供理论参考和可用原料。The invention explores the relevant reagents for relieving skin photoaging, adopts UVB-induced skin damage mouse model, and then gives mice nicotinamide mononucleotide combined with L. fermentum bacterial suspension for gavage treatment, and finally through the oxidation of serum and skin index, inflammatory index, mRNA expression level and protein expression of related genes in skin and liver to evaluate the improvement effect of NMN combined with L. fermentum on UVB-induced skin damage in mice, so as to find new ways to prevent skin aging or develop NMN combined The new probiotics of Lactobacillus fermentum provide theoretical reference and available raw materials.
本发明的目的在于提供一种烟酰胺单核苷酸(NMN)与发酵乳杆菌L.fermentum联合的新用途。The purpose of the present invention is to provide a new use of nicotinamide mononucleotide (NMN) combined with L. fermentum.
本发明第一方面，提供了烟酰胺单核苷酸与发酵乳杆菌L.fermentum联合在制备缓解 皮肤老化制剂中的应用。特别的，提供了烟酰胺单核苷酸与发酵乳杆菌Lactobacilus fermentum联合在制备缓解皮肤光老化制剂中的应用，该制剂形式包括药物、保健品以及护肤品。The first aspect of the present invention provides the application of nicotinamide mononucleotide in combination with Lactobacillus fermentum L. fermentum in the preparation of preparations for relieving skin aging. In particular, the application of nicotinamide mononucleotide in combination with Lactobacillus fermentum in the preparation of preparations for relieving skin photoaging is provided, and the preparation forms include medicines, health products and skin care products.
制剂形式不同，发酵乳杆菌Lactobacilus fermentum形式不同：当制剂为药物、保健品或食品添加剂时，发酵乳杆菌Lactobacilus fermentum的形式为间歇灭菌的发酵乳杆菌菌体或溶胞产物；当制剂为护肤品时，发酵乳杆菌Lactobacilus fermentum的形式为发酵乳杆菌Lactobacilus fermentum的代谢产物、细胞质片段、细胞壁组分或多糖复合体。Different preparation forms, Lactobacillus fermentum Lactobacillus fermentum form is different: when the preparation is a drug, health product or food additive, the Lactobacillus fermentum Lactobacilus fermentum is in the form of intermittently sterilized Lactobacillus fermentum cells or lysate; when the preparation is a skin care product When produced, Lactobacillus fermentum Lactobacilus fermentum is in the form of metabolites, cytoplasmic fragments, cell wall components or polysaccharide complexes of Lactobacillus fermentum.
其中，间歇灭菌的发酵乳杆菌菌体为冷冻干燥的粉末形式，烟酰胺单核苷酸与间歇灭菌的发酵乳杆菌菌体之间的浓度比为1:1，烟酰胺单核苷酸浓度为30mg/mL，发酵乳杆菌菌体的浓度为10
9CFU/mL。
Wherein, the intermittently sterilized Lactobacillus fermentum cells are in the form of freeze-dried powder, the concentration ratio between the nicotinamide mononucleotide and the intermittently sterilized Lactobacillus fermentum cells is 1:1, and the nicotinamide mononucleotide The concentration was 30 mg/mL, and the concentration of Lactobacillus fermentum cells was 10 9 CFU/mL.
优选的，光老化皮肤为紫外线辐射引起的皮肤老化，该紫外线辐射引起的皮肤老化为中波紫外线辐射诱导的皮肤光老化。Preferably, the photoaging skin is skin aging caused by ultraviolet radiation, and the skin aging caused by ultraviolet radiation is skin photoaging induced by medium-wave ultraviolet radiation.
优选的，缓解皮肤光老化制剂为缓解因紫外线照射诱发的氧化应激损伤并降低机体炎症损伤的制剂。该制剂为激活AMPK信号通路的试剂，进而抑制NFκB-p65信号通路活化，进一步减少机体因氧化应激反应和炎症反应产生的损伤。Preferably, the preparation for relieving skin photoaging is a preparation for alleviating oxidative stress damage induced by ultraviolet radiation and reducing inflammatory damage in the body. The preparation is an agent for activating the AMPK signaling pathway, thereby inhibiting the activation of the NFκB-p65 signaling pathway, and further reducing the damage of the body caused by oxidative stress and inflammatory responses.
本发明中的缓解皮肤光老化制剂的活性成分仅包括烟酰胺单核苷酸和发酵乳杆菌Lactobacilus fermentum。The active ingredients of the preparation for relieving skin photoaging in the present invention only include nicotinamide mononucleotide and Lactobacilus fermentum.
优选的，缓解皮肤光老化的药物为胶剂、膏剂、乳剂、气雾剂、注射剂、合剂、口服安瓿剂、片剂或胶囊剂；缓解皮肤光老化的保健品为胶囊剂、片剂、口服液或固体饮料；缓解皮肤光老化的护肤品为液状、面霜、湿粉或面膜。Preferably, the medicines for relieving skin photoaging are gels, ointments, emulsions, aerosols, injections, mixtures, oral ampoules, tablets or capsules; the health care products for relieving skin photoaging are capsules, tablets, oral Liquid or solid beverages; skin care products for relieving skin photoaging are liquids, creams, moisturizing powders or masks.
发明的作用与效果The role and effect of the invention
本发明通过UVB诱导的小鼠皮肤损伤模型进行实验，通过实验验证，NMN联合L.fermentum通过激活AMPK信号通路，进而抑制NF-κB信号通路的活化，减少炎症介质对小鼠的损伤。此外，激活的AMPK能够通过调节小鼠血清、肝脏和皮肤中相关氧化应激指标的水平，减少皮肤的氧化损伤和提高机体的整体抗氧化能力。因此，本发明对UVB致皮肤损伤的预防和治疗具有一定参考意义，为开发NMN联合发酵乳杆菌的制剂提供了理论依据和可用菌种来源。The present invention conducts experiments through the UVB-induced mouse skin injury model, and it is verified through experiments that NMN combined with L. fermentum activates the AMPK signaling pathway, thereby inhibiting the activation of the NF-κB signaling pathway and reducing the damage of inflammatory mediators to mice. In addition, activated AMPK can reduce the oxidative damage of the skin and improve the overall antioxidant capacity of the body by regulating the levels of related oxidative stress indicators in the serum, liver and skin of mice. Therefore, the present invention has certain reference significance for the prevention and treatment of skin damage caused by UVB, and provides a theoretical basis and an available strain source for the development of the preparation of NMN combined with Lactobacillus fermentum.
图1为不同实验组小鼠肝脏脏器指数对比图，数据呈现为平均值±标准偏差(±SD)，其中*表示p<0.05，**表示p<0.01，***表示p<0.001；Figure 1 is a comparison chart of the liver organ indexes of mice in different experimental groups. The data are presented as mean ± standard deviation (±SD), where * means p<0.05, ** means p<0.01, and *** means p<0.001;
图2为不同实验组肝脏病理学形态观察；Figure 2 is the observation of the pathological morphology of the liver in different experimental groups;
图3为不同实验组皮肤病理学形态观察，(A)皮肤H&E染色；(B)皮肤Masson染色；(C)皮肤TB染色；Figure 3 is the observation of skin pathological morphology in different experimental groups, (A) skin H&E staining; (B) skin Masson staining; (C) skin TB staining;
图3为皮肤和肝脏组织中SOD1、SOD2、CAT和GSH的mRNA表达水平，数据呈现为平均值±标准偏差(±SD)，其中*表示p<0.05，**表示p<0.01，***表示p<0.001；Figure 3 shows the mRNA expression levels of SOD1, SOD2, CAT and GSH in skin and liver tissues, data are presented as mean ± standard deviation (± SD), where * means p<0.05, ** means p<0.01, *** means p<0.001;
图4为皮肤和肝脏组织中AMPK、NF-κBp65、IκB-α、SOD1和CAT的mRNA表达水平，数据呈现为平均值±标准偏差
其中*表示p<0.05，**表示p<0.01，***表示p<0.001；
Figure 4 shows the mRNA expression levels of AMPK, NF-κBp65, IκB-α, SOD1 and CAT in skin and liver tissues, data are presented as mean ± standard deviation Where * means p<0.05, ** means p<0.01, *** means p<0.001;
图5为皮肤和肝脏组织中PGC-1α、APPL1、mTOR、FOXO1、TNF-α、IL-6、IL-10和GSH的mRNA表达水平，数据呈现为平均值±标准偏差
其中*表示p<0.05，**表示p<0.01，***表示p<0.001。
Figure 5 shows the mRNA expression levels of PGC-1α, APPL1, mTOR, FOXO1, TNF-α, IL-6, IL-10 and GSH in skin and liver tissues, data are presented as mean ± standard deviation Where * means p<0.05, ** means p<0.01, and *** means p<0.001.
菌种保藏信息：发酵乳杆菌Lactobacilus fermentum保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏地址为北京市朝阳区北辰西路1号院3号，保藏日期为2019年07月15号，保藏编号为CGMCCNo.18222。Culture preservation information: Lactobacillus fermentum is deposited in the General Microbiology Center of China Microbial Culture Collection Management Committee, and the preservation address is No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing. The preservation date is July 15, 2019. The number is CGMCCNo.18222.
下面结合实施例和附图对本发明进行详细描述。但下列实施例不应看作对本发明范围的限制。The present invention will be described in detail below with reference to the embodiments and accompanying drawings. However, the following examples should not be construed as limiting the scope of the present invention.
实施例1：实验材料和方法Example 1: Experimental Materials and Methods
(1)NMN来源(1) NMN source
NMN由音芙医药科技有限公司(上海，中国)提供，通过高效液相色谱仪测定NMN的纯度，纯度>98.5％。NMN was provided by Yinfu Pharmaceutical Technology Co., Ltd. (Shanghai, China). The purity of NMN was determined by high performance liquid chromatography, and the purity was >98.5%.
(2)实验菌株(2) Experimental strains
发酵乳杆菌Lactobacilus fermentum分离纯化自新疆自然发酵牦牛酸乳。Lactobacillus fermentum was isolated and purified from natural fermented yak yogurt in Xinjiang.
(3)实验动物(3) Experimental animals
40只7周龄雌性昆明小鼠购自重庆医科大学实验动物中心[重庆，中国，SCXK(YU)2018-0003]。小鼠饲养在恒定温度与湿度的条件下(温度25±2℃，相对湿度50±5％)，12h 亮/暗循环。允许小鼠自由摄入标准鼠粮和饮用水。Forty 7-week-old female Kunming mice were purchased from the Experimental Animal Center of Chongqing Medical University [Chongqing, China, SCXK(YU) 2018-0003]. Mice were housed under constant temperature and humidity conditions (temperature 25±2°C, relative humidity 50±5%), 12h light/dark cycle. Mice were allowed free access to standard rat chow and drinking water.
(4)UVB诱导皮肤氧化损伤(4) UVB-induced skin oxidative damage
40只小鼠随机分为4组，每组10只，分别为正常组、模型组、VC组、NMN+L.fermentum组(NMN+L)。UVB造模方法如下：整个实验周期为4周，从第3周起，采用紫外辐射设备对除正常组外的其余组小鼠进行皮肤损伤模型的建立，每天照射2h。其中在进行UV照射前，需采用电动剃须刀剃除小鼠背部约2cm
2的毛发。每组小鼠的具体处理方式如下：正常组和模型组整个自由饮食和饮水；每天按照300mg/kg.bw的剂量给VC组小鼠灌胃VC溶液；每天按照300mg/kg.bw的剂量给小鼠经口灌胃NMN，同时每天给小鼠经口灌胃1.0×10
9CFU/mL的菌悬液。所有实验均由重庆市功能性食品协同创新中心批准。
Forty mice were randomly divided into 4 groups with 10 mice in each group, namely normal group, model group, VC group, and NMN+L.fermentum group (NMN+L). The UVB modeling method is as follows: the whole experimental period is 4 weeks. From the 3rd week, the skin damage model of the mice in the other groups except the normal group is established by using ultraviolet radiation equipment, irradiating for 2h every day. Before UV irradiation, an electric shaver was used to shave about 2 cm 2 of hair on the back of the mice. The specific treatment methods of each group of mice were as follows: the normal group and the model group were given free food and water; the mice in the VC group were given VC solution by gavage at a dose of 300 mg/kg.bw every day; Mice were orally gavaged with NMN, and at the same time, 1.0×10 9 CFU/mL bacterial suspension was orally administered to mice every day. All experiments were approved by the Chongqing Functional Food Collaborative Innovation Center.
实施例2：NMN+L.fermentum能够改善小鼠紫外照射后的肝脏脏器指数以及组织的病理学形态Example 2: NMN+L.fermentum can improve liver organ index and tissue pathological morphology in mice after UV irradiation
培养四周后，用脱脊椎法处死小鼠，解剖小鼠取出小鼠的肝脏和背部无毛皮肤，并计算肝脏的脏器指数，结果如图1所示。After four weeks of culture, the mice were sacrificed by devertebral method, the mice were dissected to remove the liver and the hairless skin of the back of the mice, and the organ index of the liver was calculated. The results are shown in Figure 1.
剪取黄豆大小的肝脏和皮肤组织固定在4％的多聚甲醛溶液中，剩余的肝脏和皮肤组织保存在-80℃冰箱备用。肝脏进行H&E染色，皮肤进行H&E、Masson’s trichrome和toluidine blue染色。最后采用正置显微镜观察组织的病理学形态，结果如图2和图3所示：Soybean-sized liver and skin tissues were cut and fixed in 4% paraformaldehyde solution, and the remaining liver and skin tissues were stored in a -80°C refrigerator for later use. The liver was stained with H&E and the skin was stained with H&E, Masson's trichrome and toluidine blue. Finally, an upright microscope was used to observe the pathological morphology of the tissue, and the results are shown in Figure 2 and Figure 3:
2.1肝脏脏器指数2.1 Liver organ index
小鼠脏器指数能够直接反映器官的结构变化和功能，对于评价受试样品的功能特性具有重要意义。在本研究中，经UVB照射后，模型组小鼠的肝脏脏器指数出现明显降低，说明紫外线照射不仅会直接加速皮肤的衰老，还会间接引起肝脏的衰老，其中的原因可能与紫外线诱发机体氧化应激反应或者诱发炎症反应相关。但是通过给予小鼠NMN联合L.fermentum灌胃处理后，小鼠肝脏脏器指数得到明显提高，说明NMN联合L.fermentum能够维持小鼠肝脏的正常重量，延缓肝脏的衰老。The mouse organ index can directly reflect the structural changes and functions of organs, and is of great significance for evaluating the functional properties of test samples. In this study, after UVB irradiation, the liver organ index of the mice in the model group was significantly reduced, indicating that UV irradiation not only directly accelerates skin aging, but also indirectly causes liver aging. oxidative stress or induced inflammatory response. However, after intragastric administration of NMN combined with L. fermentum, the liver organ index of the mice was significantly improved, indicating that NMN combined with L. fermentum can maintain the normal weight of the mouse liver and delay the aging of the liver.
由图1可知，正常组小鼠肝脏脏器指数显著高于模型组。与模型组比较，VC组和NMN+L组的肝脏脏器指数均有不同程度的升高，其中NMN+L组升高更明显，与正常组之间无明显差异。It can be seen from Figure 1 that the liver organ index of mice in the normal group was significantly higher than that in the model group. Compared with the model group, the liver organ indexes of the VC group and the NMN+L group were increased to different degrees, and the NMN+L group increased more obviously, and there was no significant difference between the normal group and the VC group.
2.2肝脏病理学形态2.2 Pathological morphology of liver
如图2所示，正常组小鼠肝脏结构完整，肝细胞以卫星发射状整齐有序地排列在中央 静脉周围，细胞核大而圆，不存在炎症细胞浸润的现象；模型组小鼠肝脏细胞排列比较紊乱，中央静脉周围的肝细胞出现部分坏死，并存在炎症细胞浸润的现象，肝脏整体结构完整性差于正常组；VC组小鼠肝脏细胞结构较模型组好，但仍旧存在部分细胞坏死和炎症细胞细胞浸润的现象；与模型组和VC组相比，NMN+L组小鼠肝细胞形态明显更完整，几乎不存在细胞坏死和炎症细胞浸润的情况，肝脏整体结构与正常组相近。As shown in Figure 2, the liver structure of the mice in the normal group was complete, and the hepatocytes were neatly arranged around the central vein in a satellite emission pattern. The nuclei were large and round, and there was no infiltration of inflammatory cells. The liver cells in the VC group were relatively disordered, with partial necrosis and infiltration of inflammatory cells around the central vein, and the overall structural integrity of the liver was worse than that in the normal group. The phenomenon of cell infiltration; compared with the model group and the VC group, the shape of the liver cells in the NMN+L group was significantly more complete, and there was almost no cell necrosis and inflammatory cell infiltration, and the overall structure of the liver was similar to the normal group.
2.3皮肤病理学形态2.3 Skin pathological morphology
H&E染色主要用来观察皮肤的整体结构，Masson染色主要用来观察皮肤中的胶原纤维，TB染色则主要用于观察皮肤中的肥大细胞。H&E staining is mainly used to observe the overall structure of the skin, Masson staining is mainly used to observe collagen fibers in the skin, and TB staining is mainly used to observe mast cells in the skin.
由图3(A)可知，正常组小鼠皮肤结构完整，表皮层较薄，无过度角质化的角质层；真皮层较厚，胶原束形态结构完整，排列有序，分布均匀。模型组小鼠真皮层厚度明显变薄，胶原纤维束的数量显著减少，皮下组织排列紊乱，分界不明显；此外，可见附属器周围有炎症细胞浸润。与模型组比较，VC组小鼠皮肤真皮层厚度有所增加，但是胶原纤维分散疏松。与模型组和VC组相比，NMN+L组小鼠皮肤真皮层厚度明显增加，胶原纤维束不存在断裂、皱缩和胶黏的情况，整体结构与正常组接近。It can be seen from Figure 3(A) that the skin structure of the mice in the normal group is complete, the epidermis layer is thin, and there is no excessive keratinized stratum corneum; The thickness of the dermis of the mice in the model group was significantly thinner, the number of collagen fiber bundles was significantly reduced, the subcutaneous tissue was disorderly arranged, and the demarcation was not obvious; in addition, inflammatory cell infiltration around the adnexa was seen. Compared with the model group, the thickness of the dermis of the mice in the VC group increased, but the collagen fibers were loosely dispersed. Compared with the model group and the VC group, the thickness of the skin dermis of the mice in the NMN+L group was significantly increased, and the collagen fiber bundles were not broken, shrunken and glued, and the overall structure was similar to that of the normal group.
胶原纤维经Masson染色后呈现蓝紫色。由图3(B)可知，正常组胶原纤维数量多，分布均匀有序。模型组胶原纤维的数量明显少于正常组，且出现了断裂和皱缩的现象。VC组小鼠皮肤真皮层中的胶原纤维数量比正常组少，但比模型组高。与模型组和VC组相比，NMN+L组小鼠皮肤真皮层中的胶原纤维数量增加更多，排列更为整齐有序，几乎不存在皱缩、断裂等现象。Collagen fibers were blue-purple after Masson staining. It can be seen from Figure 3(B) that the normal group has a large number of collagen fibers, and the distribution is uniform and orderly. The number of collagen fibers in the model group was significantly less than that in the normal group, and the phenomenon of breakage and shrinkage appeared. The number of collagen fibers in the dermis of the skin of the mice in the VC group was less than that in the normal group, but higher than that in the model group. Compared with the model group and the VC group, the number of collagen fibers in the dermis of the mice in the NMN+L group increased more, the arrangement was more orderly, and there was almost no shrinkage or breakage.
由TB染色(图3(C))结果可知，与正常组比较，模型组小鼠真皮层中的肥大细胞数量显著升高，说明UVB照射会诱发皮肤肥大细胞的产生，从而导致皮肤炎症。与模型组相比，VC组小鼠皮肤中的肥大细胞有所减少，但数量仍旧明显多于正常组。给予小鼠NMN和发酵乳杆菌处理之后，NMN组和NMN+L组小鼠皮肤中的肥大细胞数量得到明显减少，结果接近于正常组。The results of TB staining (Fig. 3(C)) showed that compared with the normal group, the number of mast cells in the dermis of the mice in the model group was significantly increased, indicating that UVB irradiation could induce the production of skin mast cells, leading to skin inflammation. Compared with the model group, the mast cells in the skin of the mice in the VC group decreased, but the number was still significantly higher than that in the normal group. After the mice were treated with NMN and Lactobacillus fermentum, the number of mast cells in the skin of the mice in the NMN group and the NMN+L group was significantly reduced, and the results were close to the normal group.
本发明中，UVB组小鼠皮肤真皮层变薄，胶原纤维数量减少，肥大细胞数量增多，同时肝脏组织也出现了一定程度的病理改变，说明UVB诱导的皮肤损伤模型成功。给予皮肤损伤小鼠NMN联合L.fermentum处理之后，皮肤病理学形态得到了极大改善，小鼠的肝脏也保持在正常的形态，未出现明显损伤。In the present invention, the skin dermis of the UVB group mice became thinner, the number of collagen fibers decreased, the number of mast cells increased, and at the same time, the liver tissue also showed a certain degree of pathological changes, indicating that the UVB-induced skin damage model was successful. After the skin injury mice were treated with NMN combined with L. fermentum, the pathological morphology of the skin was greatly improved, and the liver of the mice also remained in a normal shape without obvious damage.
实施例3：NMN+L.fermentum能够改善UVB小鼠氧化应激损伤Example 3: NMN+L.fermentum can improve oxidative stress injury in UVB mice
氧化应激反应是UVB诱发皮肤衰老的重要因素之一。机体正常情况下，氧自由基的生成和清除处于平衡状态，当受到外界刺激时，机体会因为局部缺氧导致氧自由基大量生成，从而导致细胞凋亡或者损伤。超氧化物歧化酶(SOD)是一种含金属辅助因子的酶，它可清除(歧化)机体产生的超氧阴离子，在抗衰老、氧化过程中发挥着重要作用。随着机体脂质过氧化反应的进行，可导致SOD的大量消耗，进而诱发严重的机体损伤。过氧化氢酶(CAT)是一种酶类清除剂，又称为触酶，是以铁卟啉为辅基的结合酶。它可促使H
2O
2分解为分子氧和水，从而使细胞免于遭受H
2O
2的毒害，是生物防御体系的关键酶之一。MDA是脂质氧化的终产物，它可在体外影响线粒体呼吸链复合物及线粒体内关键酶活性，它的产生还能加剧膜的损伤，因而测定丙二醛的含量可反映机体脂质过氧化的程度，从而间接反应细胞损伤程度。谷胱甘肽(GSH)作为体内一种重要的抗氧化剂，能够清除掉人体内的自由基；由于GSH本身易受某些物质氧化，所以它在体内能够保护许多蛋白质和酶等分子中的巯基不被有害物质氧化，从而保证蛋白质和酶等分子生理功能的正常发挥。晚期糖基化终末产物(AGEs)是非酶糖基化反应的终产物，它在血清、组织中的生成和积聚会随着年龄的增长而增多。人体内AGEs的蛋白修饰与老年性疾病的发生密切相关，因此它可被作为测试老化进程的重要指标。
Oxidative stress is one of the important factors of UVB-induced skin aging. Under normal circumstances, the generation and scavenging of oxygen free radicals are in a state of balance. When stimulated by the outside world, the body will generate a large amount of oxygen free radicals due to local hypoxia, resulting in cell apoptosis or damage. Superoxide dismutase (SOD) is an enzyme containing metal cofactors, which can remove (disproportionate) the superoxide anion produced by the body and play an important role in the process of anti-aging and oxidation. With the progress of lipid peroxidation in the body, a large amount of SOD can be consumed, which in turn induces serious damage to the body. Catalase (CAT) is an enzyme scavenger, also known as catalase, which is a conjugated enzyme with iron porphyrin as a prosthetic group. It can promote the decomposition of H 2 O 2 into molecular oxygen and water, so as to protect cells from the poison of H 2 O 2 , and is one of the key enzymes in the biological defense system. MDA is the final product of lipid oxidation. It can affect mitochondrial respiratory chain complexes and key enzyme activities in mitochondria in vitro. Its production can also aggravate membrane damage. Therefore, the determination of malondialdehyde content can reflect lipid peroxidation in the body. degree, which indirectly reflects the degree of cell damage. Glutathione (GSH), as an important antioxidant in the body, can scavenge free radicals in the human body; because GSH itself is susceptible to oxidation by certain substances, it can protect the sulfhydryl groups in many proteins and enzymes in the body It is not oxidized by harmful substances, so as to ensure the normal functioning of molecular physiological functions such as proteins and enzymes. Advanced glycation end products (AGEs) are the end products of non-enzymatic glycation reactions, and their production and accumulation in serum and tissues increase with age. The protein modification of AGEs in the human body is closely related to the occurrence of senile diseases, so it can be used as an important indicator to test the aging process.
当持续暴露在紫外线下时，皮肤会产生强烈的氧化应激反应，释放大量的活性氧(ROS)，而ROS作为上游信号会进一步启动NF-κB介导的炎症通路，从而使皮肤表现出干燥、瘙痒、红斑和水肿等炎症症状。机体正常情况下，核转录因子NF-κB和其抑制蛋白IκB结合在一起，并以静息的方式存在细胞内。一旦NF-κB被激活，它就会由细胞液转移至细胞核，从而进一步增加促炎性细胞因子TNF-α、IL-6、IL-12、COX-2、iNOS等的释放，诱发机体炎症损伤。IκB-α是NF-κB的阻遏蛋白，它可以掩蔽NF-κB的核定位信号，从而使NF-κB以非活性复合物形式存在于细胞内。研究表明几乎所有的NF-κB诱导物都会因为IκB-α的降解而迅速激活NF-κB，所以可以通过阻止IκB-α的磷酸化来防止NF-κB的活化。在本研究中，经UVB照射后的小鼠皮肤和肝脏组织中NFκB-p65的mRNA表达显著升高，IκB-α的表达明显降低，说明紫外线照射确实能够激活NF-κB。而NMN联合L.fermentum能够通过上调皮肤和肝脏组织中IκB-α的mRNA表达，从而较好地抑制NFκB-p65信号通路的活化。When continuously exposed to UV light, the skin will produce a strong oxidative stress response, releasing a large amount of reactive oxygen species (ROS), and ROS as an upstream signal will further initiate the NF-κB-mediated inflammatory pathway, thereby making the skin appear dry , itching, erythema and edema and other inflammatory symptoms. Under normal circumstances, the nuclear transcription factor NF-κB and its inhibitory protein IκB are combined together and exist in the cell in a resting manner. Once NF-κB is activated, it will be transferred from the cytosol to the nucleus, thereby further increasing the release of pro-inflammatory cytokines TNF-α, IL-6, IL-12, COX-2, iNOS, etc., inducing inflammatory damage in the body . IκB-α is a repressor protein of NF-κB, which can mask the nuclear localization signal of NF-κB, so that NF-κB exists in the cell in the form of an inactive complex. Studies have shown that almost all NF-κB inducers rapidly activate NF-κB due to the degradation of IκB-α, so the activation of NF-κB can be prevented by preventing the phosphorylation of IκB-α. In this study, the mRNA expression of NFκB-p65 in the skin and liver tissues of UVB-irradiated mice was significantly increased, and the expression of IκB-α was significantly decreased, indicating that UVB irradiation can indeed activate NF-κB. NMN combined with L. fermentum can inhibit the activation of NFκB-p65 signaling pathway by up-regulating the mRNA expression of IκB-α in skin and liver tissue.
为探究UVB照射诱发的皮肤炎症反应程度，本发明测定了与NF-κB活化相关的TNF-α 和IL-6的水平。TNF-α是一种具有广泛生物活性的细胞因子，其主要由活化的单核一巨噬细胞和T细胞分泌，可导致炎性细胞聚集，并刺激炎性细胞产生炎性细胞因子，最终导致炎症反应。IL-6是一种具备生物活性的多向性促炎细胞因子，其作用与IL-1β类似，它同样可在一定程度上激活NF-κB信号通路，从而引起皮肤发生慢性炎症反应。除此之外，IL-6的过度表达可增加皮肤的通透性，使炎症因子浸润到炎症部位，启动炎症发生。IL-10是一种重要的负向调节细胞因子，对免疫炎性反应发展过程中的多个环节均有阻断作用，具有广泛的免疫抑制活性，可抑制活化的单核-巨噬细胞产生其他细胞因子(包括ILl、IL6、粒细胞集落刺激因子、TNF、血小板活化因子等)，有很强的免疫抑制功能。In order to explore the degree of skin inflammation induced by UVB irradiation, the present invention measured the levels of TNF-α and IL-6 related to the activation of NF-κB. TNF-α is a cytokine with a wide range of biological activities, which is mainly secreted by activated monocyte-macrophages and T cells, which can lead to the aggregation of inflammatory cells and stimulate the production of inflammatory cytokines by inflammatory cells, eventually leading to Inflammation. IL-6 is a pleiotropic pro-inflammatory cytokine with biological activity, and its role is similar to IL-1β. It can also activate the NF-κB signaling pathway to a certain extent, thereby causing chronic inflammation in the skin. In addition, the overexpression of IL-6 can increase the permeability of the skin, allowing inflammatory factors to infiltrate the inflammatory site and initiate inflammation. IL-10 is an important negative regulatory cytokine, which has a blocking effect on multiple links in the development of immune inflammatory response, has a wide range of immunosuppressive activities, and can inhibit the production of activated monocytes-macrophages Other cytokines (including IL1, IL6, granulocyte colony stimulating factor, TNF, platelet activating factor, etc.) have strong immunosuppressive functions.
3.1小鼠血清中氧化应激指标和炎症指标水平3.1 The levels of oxidative stress indicators and inflammatory indicators in mouse serum
根据常规生化试剂盒说明测定小鼠血清中T-SOD、CAT、MDA和AGEs的水平，根据ELISA试剂盒说明测定小鼠血清中TNF-α、IL-6和IL-10的水平，结果如表1所示。The levels of T-SOD, CAT, MDA and AGEs in mouse serum were determined according to the instructions of conventional biochemical kits, and the levels of TNF-α, IL-6 and IL-10 in mouse serum were determined according to the instructions of ELISA kits. The results are shown in the table. 1 shown.
由表1可知，模型组小鼠血清中T-SOD、CAT和IL-10的水平显著低于模型组，而MDA、AGES、TNF-α和IL-6的水平显著高于正常组(p＜0.05)。与模型组相比，VC组和NMN+L组小鼠血清中T-SOD、CAT和IL-10的水平均有所提高，MDA、AGES、TNF-α和IL-6的水平有所降低。值得注意的是，NMN+L组小鼠血清中的T-SOD、CAT、IL-10、MDA、AGES、TNF-α和IL-6的水平均更接近于正常组，其中T-SOD的酶活力更是显著高于正常组(p＜0.05)。It can be seen from Table 1 that the serum levels of T-SOD, CAT and IL-10 in the model group were significantly lower than those in the model group, while the levels of MDA, AGES, TNF-α and IL-6 were significantly higher than those in the normal group (p< 0.05). Compared with the model group, the serum levels of T-SOD, CAT and IL-10 in the VC group and NMN+L group were increased, and the levels of MDA, AGES, TNF-α and IL-6 were decreased. It is worth noting that the levels of T-SOD, CAT, IL-10, MDA, AGES, TNF-α and IL-6 in the serum of the mice in the NMN+L group were closer to those in the normal group, in which the enzymes of T-SOD The vitality was significantly higher than that of the normal group (p<0.05).
表1 小鼠血清中相关氧化应激指标和炎症指标的水平Table 1 Levels of related oxidative stress indexes and inflammatory indexes in mouse serum
注：表中数据呈现为平均值±标准偏差
同一列中的不同字母(a-c)表示组别之间存在显著差异(p<0.05)。
Note: The data in the table are presented as mean ± standard deviation Different letters (ac) in the same column indicate significant differences between groups (p<0.05).
3.2小鼠皮肤中相关氧化应激指标和炎症指标的水平3.2 Levels of related oxidative stress markers and inflammatory markers in mouse skin
根据常规生化试剂盒说明测定小鼠血清中T-SOD、CAT、Na
+K
+-ATP和NAD
+的水平，根据ELISA试剂盒说明书分别测定血清和皮肤组织中TNF-α和IL-10的水平，结果如表2所示。
The levels of T-SOD, CAT, Na + K + -ATP and NAD + in mouse serum were determined according to the instructions of conventional biochemical kits, and the levels of TNF-α and IL-10 in serum and skin tissue were determined according to the instructions of ELISA kits, respectively , and the results are shown in Table 2.
由表2可知，正常组小鼠皮肤组织中T-SOD、CAT、IL-10、Na
+K
+-ATP和NAD
+水平分别为26.68±6.52U/mgprot、23.07±3.41U/mgprot、632.98±82.99pg/mL、0.86±0.15U/mgprot和16.98±0.15nmol/min/mgprot，均显著高于模型组，而TNF-α水平为102.18±15.55ng/L，显著高于正常组(p＜0.05)。与模型组相比，VC组和NMN+L组小鼠血清中以上指标的水平都得到不同程度的改善，其中给予NMN和发酵乳杆菌联合处理后的小鼠血清中的以上指标的水平与正常组之间不存在显著差异。
It can be seen from Table 2 that the levels of T-SOD, CAT, IL-10, Na + K + -ATP and NAD + in the skin tissue of the normal mice were 26.68±6.52U/mgprot, 23.07±3.41U/mgprot, 632.98± 82.99pg/mL, 0.86±0.15U/mgprot and 16.98±0.15nmol/min/mgprot, all significantly higher than the model group, while the TNF-α level was 102.18±15.55ng/L, significantly higher than the normal group (p<0.05 ). Compared with the model group, the levels of the above indexes in the serum of the mice in the VC group and the NMN+L group were improved to varying degrees. There were no significant differences between groups.
表2 小鼠皮肤组织中相关氧化应激指标与炎症指标的水平Table 2 Levels of related oxidative stress indexes and inflammatory indexes in mouse skin tissue
注：表中数据呈现为平均值±标准偏差
同一列中的不同字母(a-d)表示组别之间存在显著差异(p<0.05)。
Note: The data in the table are presented as mean ± standard deviation Different letters (ad) in the same column indicate significant differences between groups (p<0.05).
由上述结果可知，NMN联合L.fermentum能够提高小鼠血清和皮肤组织中的T-SOD、CAT酶活力，提高SOD和CAT在肝脏和皮肤中的mRNA表达，降低血清中AGEs和MDA的含量。更加值得注意的是，NMN联合L.fermentum能极大的提高血清中T-SOD、CAT水平，其中T-SOD的酶活力甚至高于正常组。以上结果说明NMN联合发酵乳杆菌能够通过提高抗氧化酶活力来抵抗小鼠因UVB照射引起的皮肤的氧化应激反应，还能从血清、肝脏等层面提高机体的整体抗氧化水平。It can be seen from the above results that NMN combined with L. fermentum can increase the enzyme activities of T-SOD and CAT in serum and skin tissue of mice, increase the mRNA expression of SOD and CAT in liver and skin, and reduce the content of AGEs and MDA in serum. What is more noteworthy is that NMN combined with L. fermentum can greatly increase the levels of T-SOD and CAT in serum, and the enzyme activity of T-SOD is even higher than that of the normal group. The above results indicate that NMN combined with Lactobacillus fermentum can resist the oxidative stress of the skin caused by UVB irradiation in mice by increasing the activity of antioxidant enzymes, and can also improve the overall antioxidant level of the body from the levels of serum and liver.
此外，NMN联合L.fermentum可以分别从血清水平、皮肤组织水平和mRNA水平下调促炎细胞因子TNF-α、IL-6的表达，上调抑炎细胞因子IL-10的表达，从而很好地减少了这些炎症介质对皮肤损伤小鼠的炎症损伤程度。In addition, NMN combined with L. fermentum can down-regulate the expression of pro-inflammatory cytokines TNF-α and IL-6 and up-regulate the expression of anti-inflammatory cytokine IL-10 from the serum level, skin tissue level and mRNA level, respectively, thereby reducing the The degree of inflammatory damage caused by these inflammatory mediators to skin-injured mice.
实施例4：NMN+L.fermentum能够激活AMPK信号通路Example 4: NMN+L.fermentum can activate AMPK signaling pathway
腺苷酸活化蛋白激酶(AMP-activated protein kinase，AMPK)是一种丝氨酸/苏氨酸蛋白激酶，是由催化亚基α和β及调节亚基γ形成的异源三聚体。AMPK在体内主要参与调节糖、脂及能量代谢，有研究表明AMPK活化可以抑制炎症反应及氧化应激反应。AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase, which is a heterotrimer formed by catalytic subunits α and β and regulatory subunit γ. AMPK is mainly involved in the regulation of glucose, lipid and energy metabolism in the body. Studies have shown that AMPK activation can inhibit inflammation and oxidative stress.
AMPK除了维持细胞能量稳态外，也可通过多种途径作用于核转录因子NF-κB及其他炎症因子发挥炎症调节作用，成为糖尿病、肥胖等多种代谢疾病及多种炎症疾病潜在治疗靶点。有研究显示AMPK能够有效调节炎性细胞因子表达、迁移与黏附，从而影响炎症反应。实验证明抑制AMPK活性则可明显增强炎症因子TNF-α、IL-1β、IL-6等炎症因子表达，从而加重炎症损伤。此外，在年轻细胞中，高表达量的AMPK可促进SIRT1、FOXO和PGC1α等因子活性，进而抑制NF-κB活性。在细胞老化后，NF-κB信号传导则会因为AMPK活性降低而增强。In addition to maintaining cellular energy homeostasis, AMPK can also act on nuclear transcription factor NF-κB and other inflammatory factors to regulate inflammation through various pathways, becoming a potential therapeutic target for various metabolic diseases such as diabetes and obesity and various inflammatory diseases. . Studies have shown that AMPK can effectively regulate the expression, migration and adhesion of inflammatory cytokines, thereby affecting the inflammatory response. Experiments have shown that inhibition of AMPK activity can significantly enhance the expression of inflammatory factors such as TNF-α, IL-1β, and IL-6, thereby aggravating inflammatory damage. In addition, in young cells, high expression of AMPK promotes the activity of factors such as SIRT1, FOXO, and PGC1α, which in turn inhibits NF-κB activity. After cellular aging, NF-κB signaling is enhanced due to decreased AMPK activity.
此外，AMPK信号通路被激活以后，会引发该通路上其他相关基因的改变。脂联素受体结合蛋白(APPL1)属于AMPK的上游基因，是由709个氨基酸构成并定位于3号染色体的功能性蛋白，它能够介导多种细胞信号传导，在多种细胞反应中发挥作用，可调节细胞的炎症反应、抗氧化及动脉硬化等。雷帕霉素靶蛋白(mTOR)是位于AMPK信号通路的下游分子，其主要通过调节蛋白质翻译来控制细胞生长，参与调节细胞的免疫及凋亡过程。活化的AMPK可抑制mTOR的活性，抑制蛋白质的合成翻译，进而起到负性调控肿瘤生长的作用。mTOR通路的激活被认为与皮肤黑色素瘤的发生机制密切相关。Forkhead转录因子O1(FOXO1)是叉头框家族成员之一，主要参与调节细胞氧化应激、周期阻滞、自噬、代谢等多种生理生化过程，与参与细胞死亡和氧化应激反应相关的细胞代谢的各种基因相关。过氧化物酶体增殖受体γ辅助激活因子α(PGC-1α)作为AMPK的调控因子可通过调节机体适应性产热、糖脂代谢、血糖平衡，参与线粒体生物合成，提高线粒体的呼吸与氧化能力，调节脂肪酸氧化。PGC-1α能够调节机体适应性产热、糖脂代谢和血糖平衡，参与线粒体生物合成，提高线粒体的呼吸与氧化能力。In addition, when the AMPK signaling pathway is activated, it will trigger changes in other related genes in the pathway. Adiponectin receptor-binding protein (APPL1) belongs to the upstream gene of AMPK. It is a functional protein composed of 709 amino acids and located on chromosome 3. It can mediate a variety of cell signal transduction and play a role in a variety of cellular responses. It can regulate the inflammatory response of cells, anti-oxidation and arteriosclerosis. Target of rapamycin (mTOR) is a downstream molecule in the AMPK signaling pathway, which mainly controls cell growth by regulating protein translation, and is involved in regulating cell immunity and apoptosis. Activated AMPK can inhibit the activity of mTOR, inhibit the synthesis and translation of proteins, and then play a negative role in regulating tumor growth. Activation of the mTOR pathway is thought to be closely related to the pathogenesis of cutaneous melanoma. Forkhead transcription factor O1 (FOXO1) is a member of the forkhead box family, which is mainly involved in the regulation of various physiological and biochemical processes such as cellular oxidative stress, cycle arrest, autophagy, and metabolism. Various genes related to cellular metabolism. As a regulator of AMPK, peroxisome proliferator gamma coactivator alpha (PGC-1α) can participate in mitochondrial biosynthesis by regulating adaptive heat production, glucose and lipid metabolism, and blood sugar balance, and improve mitochondrial respiration and oxidation. ability to regulate fatty acid oxidation. PGC-1α can regulate the body's adaptive heat production, glucose and lipid metabolism and blood sugar balance, participate in mitochondrial biosynthesis, and improve mitochondrial respiration and oxidation capacity.
本发明中通过基因表达水平的测定，对NMN联合L.fermentum对AMPK信号通路中的相关因子以及与该信号通过相关因子的水平变化进行展示。实验方法如下：用Trizol试剂对肝脏和皮肤组织进行匀浆和总RNA提取，然后使用cDNA试剂盒将RNA逆转录成cDNA。接着将1μL cDNA、10μL TaqMan
TM Multiplex Master Mix、2μL of 10μM primer和7μL ddH
2O混合均匀后，在实时荧光定量PCR仪上进行扩增检测。扩增条件为：95℃变形15s，55℃退火30s，72℃延伸35s，总共40个循环。最后，通过2
-ΔΔCT计算各目的 基因的相对表达量，其中β-actin作为内参基因。用到的引物序列见表3。
In the present invention, by measuring the level of gene expression, NMN combined with L. fermentum is used to display the related factors in the AMPK signaling pathway and the level changes of the related factors through this signal. The experimental method was as follows: liver and skin tissues were homogenized and total RNA was extracted with Trizol reagent, and then the RNA was reverse transcribed into cDNA using a cDNA kit. Next, 1 μL of cDNA, 10 μL of TaqMan ™ Multiplex Master Mix, 2 μL of 10 μM primer and 7 μL of ddH 2 O were mixed evenly, and amplification detection was performed on a real-time fluorescence quantitative PCR instrument. The amplification conditions were: deformation at 95°C for 15s, annealing at 55°C for 30s, and extension at 72°C for 35s, for a total of 40 cycles. Finally, the relative expression of each target gene was calculated by 2 -ΔΔCT , in which β-actin was used as an internal reference gene. The primer sequences used are shown in Table 3.
表3 实验所用引物序列Table 3 Primer sequences used in the experiment
图4显示了皮肤和肝脏组织中AMPK、NF-κBp65、IκB-α、SOD1和CAT的mRNA表达。由图4可知，与正常组相比，UVB照射增加了小鼠皮肤和肝脏中NF-κBp65的mRNA表达水平，降低了IκB-α、AMPK、SOD和CAT的表达，其中模型组的变化最明显。与模 型组比较，VC组和NMN+L组皮肤和肝脏中的IκB-α、AMPK、SOD和CAT的mRNA表达均有不同程度的升高，而NF-κBp65的表达有所降低，其中NMN+L组以上指标的mRNA表达水平接近于正常组。Figure 4 shows the mRNA expression of AMPK, NF-κB p65, IκB-α, SOD1 and CAT in skin and liver tissues. As can be seen from Figure 4, compared with the normal group, UVB irradiation increased the mRNA expression levels of NF-κBp65 in the skin and liver of mice, and decreased the expressions of IκB-α, AMPK, SOD and CAT, among which the model group had the most obvious changes. . Compared with the model group, the mRNA expressions of IκB-α, AMPK, SOD and CAT in the skin and liver of the VC group and the NMN+L group were increased to varying degrees, while the expression of NF-κBp65 was decreased, among which NMN+ The mRNA expression levels of the above indicators in the L group were close to those in the normal group.
模型组小鼠肝脏和皮肤中AMPK的mRNA和蛋白相对表达量显著低于正常组，说明UVB照射引起了小鼠机体内的能量代谢障碍，增加了氧化应激反应和炎症反应。但是NMN联合L.fermentum灌胃处理能够明显提升肝脏和皮肤组织中AMPK的mRNA表达水平，说明NMN联合L.fermentum能有效促进细胞能量合成，减少机体因氧化应激反应和炎症反应产生的损伤。The relative expression of AMPK mRNA and protein in the liver and skin of mice in the model group was significantly lower than that in the normal group, indicating that UVB irradiation caused energy metabolism disorders in mice, and increased oxidative stress and inflammatory responses. However, NMN combined with L. fermentum gavage treatment can significantly increase the mRNA expression of AMPK in liver and skin tissues, indicating that NMN combined with L. fermentum can effectively promote cellular energy synthesis and reduce the damage caused by oxidative stress and inflammatory reactions.
图5显示了皮肤和肝脏组织中PGC-1α、APPL1、mTOR、FOXO1、TNF-α、IL-6、IL-10和GSH的mRNA表达水平。由图5可知，正常组小鼠皮肤和肝脏中PGC-1α、APPL1、FOXO1、IL-10和GSH的mRNA表达是最高的，mTOR、TNF-α和IL-6的表达是最低的，而模型组小鼠皮肤和肝脏中的以上指标的表达水平与正常组相比呈现完全相反的趋势，且两者之间存在显著差异。经过VC以及NMN联合L.fermentum处理之后，皮肤和肝脏中PGC-1α、APPL1、FOXO1、IL-10和GSH表达升高，，而mTOR、TNF-α和IL-6的表达降低，其中NMN+L组的表达水平与正常组接近。Figure 5 shows the mRNA expression levels of PGC-1α, APPL1, mTOR, FOXO1, TNF-α, IL-6, IL-10 and GSH in skin and liver tissues. As can be seen from Figure 5, the mRNA expressions of PGC-1α, APPL1, FOXO1, IL-10 and GSH in the skin and liver of the normal group mice were the highest, and the expressions of mTOR, TNF-α and IL-6 were the lowest, while the model Compared with the normal group, the expression levels of the above indexes in the skin and liver of the mice in the group showed a completely opposite trend, and there were significant differences between the two. After VC and NMN combined with L. fermentum treatment, the expression of PGC-1α, APPL1, FOXO1, IL-10 and GSH in skin and liver increased, while the expression of mTOR, TNF-α and IL-6 decreased, among which NMN+ The expression level of L group was close to that of normal group.
UVB照射能够降低小鼠皮肤和肝脏中APPL1、FOXO1和PGC-1α的mRNA表达，升高mTOR的mRNA表达。但是NMN联合L.fermentum灌胃之后，小鼠皮肤和肝脏中APPL1、LKB1、SIRT1、FOXO1和PGC-1α的mRNA表达明显提高了，而mTOR的mRNA表达水平降低了，说明NMN联合L.fermentum能够通过激活AMPK信号通路，进而改善UVB对小鼠的氧化应激、炎症等损伤。UVB irradiation can reduce the mRNA expression of APPL1, FOXO1 and PGC-1α and increase the mRNA expression of mTOR in mouse skin and liver. However, after NMN combined with L. fermentum gavage, the mRNA expressions of APPL1, LKB1, SIRT1, FOXO1 and PGC-1α in the skin and liver of mice were significantly increased, while the mRNA expression level of mTOR decreased, indicating that NMN combined with L. fermentum can effectively By activating the AMPK signaling pathway, it can improve UVB-induced oxidative stress and inflammation in mice.
综上所述，NMN联合L.fermentum对UVB诱导的小鼠皮肤损伤具有比较明显的改善作用。其可能机制可能与NMN联合L.fermentum通过激活AMPK信号通路，进而抑制NF-κB信号通路的活化，减少炎症介质对小鼠的损伤。此外，激活的AMPK能够通过调节小鼠血清、肝脏和皮肤中相关氧化应激指标的水平，减少皮肤的氧化损伤和提高机体的整体抗氧化能力。本研究对UVB致皮肤损伤的预防和治疗具有重要的参考意义，为开发NMN联合发酵乳杆菌的保健食品提供了理论依据和可用菌种来源。In conclusion, NMN combined with L. fermentum has a relatively obvious improvement effect on UVB-induced skin damage in mice. The possible mechanism may be combined with NMN and L. fermentum by activating the AMPK signaling pathway, thereby inhibiting the activation of the NF-κB signaling pathway, and reducing the damage of inflammatory mediators to mice. In addition, activated AMPK can reduce the oxidative damage of the skin and improve the overall antioxidant capacity of the body by regulating the levels of related oxidative stress indicators in the serum, liver and skin of mice. This study has important reference significance for the prevention and treatment of UVB-induced skin damage, and provides a theoretical basis and available strain sources for the development of NMN combined with Lactobacillus fermentum health food.
以上显示和描述了本发明的基本原理、主要特征和本发明的优点。本行业的技术人员应该了解，本发明不受上述实施例的限制，上述实施例和说明书中描述的只是说明本发明 的原理，在不脱离本发明精神和范围的前提下本发明还会有各种变化和改进，这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等同物界定。The foregoing has shown and described the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above-mentioned embodiments. The above-mentioned embodiments and descriptions only illustrate the principle of the present invention. Such changes and improvements fall within the scope of the claimed invention. The claimed scope of the present invention is defined by the appended claims and their equivalents.
Claims (10)
- 烟酰胺单核苷酸与发酵乳杆菌Lactobacilus fermentum联合在制备缓解皮肤光老化制剂中的应用，其中，发酵乳杆菌Lactobacilus fermentum保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC No.18222。The application of nicotinamide mononucleotide in combination with Lactobacillus fermentum Lactobacilus fermentum in the preparation of preparations for relieving skin photoaging, wherein, Lactobacillus fermentum Lactobacilus fermentum is preserved in the General Microbiology Center of China Microorganism Culture Collection Management Committee, and the preservation number is CGMCC No. 18222.
- 根据权利要求1所述的烟酰胺单核苷酸与发酵乳杆菌Lactobacilus fermentum联合在制备缓解皮肤光老化制剂中的应用，其特征在于：The combined application of nicotinamide mononucleotide and Lactobacillus fermentum according to claim 1 in the preparation of preparations for relieving skin photoaging, is characterized in that:其中，所述制剂包括缓解皮肤光老化的药物、保健品、护肤品或食品添加剂。Wherein, the preparation includes medicines, health products, skin care products or food additives for relieving skin photoaging.
- 根据权利要求2所述的烟酰胺单核苷酸与发酵乳杆菌Lactobacilus fermentum联合在制备缓解皮肤光老化制剂中的应用，其特征在于：The application of nicotinamide mononucleotide according to claim 2 in combination with Lactobacillus fermentum in the preparation of preparations for relieving skin photoaging, it is characterized in that:其中，当所述制剂为药物、保健品或食品添加剂时，发酵乳杆菌Lactobacilus fermentum的形式为间歇灭菌的发酵乳杆菌菌体或溶胞产物；Wherein, when the preparation is a medicine, a health product or a food additive, the form of Lactobacillus fermentum Lactobacilus fermentum is the Lactobacillus fermentum thalline or lysate of intermittent sterilization;当所述制剂为护肤品时，发酵乳杆菌Lactobacilus fermentum的形式为发酵乳杆菌Lactobacilus fermentum的代谢产物、细胞质片段、细胞壁组分或多糖复合体。When the formulation is a skin care product, Lactobacillus fermentum Lactobacilus fermentum is in the form of a metabolite, cytoplasmic fragment, cell wall component or polysaccharide complex of Lactobacillus fermentum.
- 根据权利要求3所述的烟酰胺单核苷酸与发酵乳杆菌Lactobacilus fermentum联合在制备缓解皮肤光老化制剂中的应用，其特征在于：The combined application of nicotinamide mononucleotide and Lactobacillus fermentum according to claim 3 in the preparation of preparations for relieving skin photoaging, characterized in that:其中，所述间歇灭菌的发酵乳杆菌菌体为冷冻干燥的粉末形式，烟酰胺单核苷酸与间歇灭菌的发酵乳杆菌菌体之间的浓度比为1:1。Wherein, the intermittently sterilized Lactobacillus fermentum cells are in the form of freeze-dried powder, and the concentration ratio between the nicotinamide mononucleotide and the intermittently sterilized Lactobacillus fermentum cells is 1:1.
- 根据权利要求1所述的烟酰胺单核苷酸与发酵乳杆菌Lactobacilus fermentum联合在制备缓解皮肤光老化制剂中的应用，其特征在于：The combined application of nicotinamide mononucleotide and Lactobacillus fermentum according to claim 1 in the preparation of preparations for relieving skin photoaging, is characterized in that:其中，所述皮肤光老化为紫外线辐射引起的皮肤老化。Wherein, the skin photoaging is skin aging caused by ultraviolet radiation.
- 根据权利要求5所述的烟酰胺单核苷酸与发酵乳杆菌Lactobacilus fermentum联合在制备缓解皮肤光老化制剂中的应用，其特征在于：The combined application of nicotinamide mononucleotide and Lactobacillus fermentum according to claim 5 in the preparation of preparations for relieving skin photoaging, characterized in that:其中，所述紫外线辐射引起的皮肤老化为中波紫外线辐射诱导的皮肤光老化。Wherein, the skin aging caused by ultraviolet radiation is skin photoaging induced by medium-wave ultraviolet radiation.
- 根据权利要求1所述的烟酰胺单核苷酸与发酵乳杆菌Lactobacilus fermentum联合在制备缓解皮肤光老化制剂中的应用，其特征在于：The combined application of nicotinamide mononucleotide and Lactobacillus fermentum according to claim 1 in the preparation of preparations for relieving skin photoaging, is characterized in that:其中，所述缓解皮肤光老化制剂为缓解因紫外线照射诱发的氧化应激损伤以及降低机体炎症损伤的制剂。Wherein, the preparation for alleviating skin photoaging is a preparation for alleviating oxidative stress damage induced by ultraviolet radiation and reducing inflammatory damage in the body.
- 根据权利要求7所述的烟酰胺单核苷酸与发酵乳杆菌Lactobacilus fermentum联合在制备缓解皮肤光老化制剂中的应用，其特征在于：The application of nicotinamide mononucleotide according to claim 7 in combination with Lactobacillus fermentum in the preparation of preparations for relieving skin photoaging, characterized in that:其中，所述缓解因紫外线照射诱发的氧化应激损伤以及降低机体炎症损伤的制剂为激活AMPK信号通路的试剂。Wherein, the preparation for alleviating oxidative stress damage induced by ultraviolet irradiation and reducing inflammatory damage in the body is an agent for activating the AMPK signaling pathway.
- 根据权利要求2～8任一项所述的烟酰胺单核苷酸与发酵乳杆菌Lactobacilus fermentum联合在制备缓解皮肤光老化制剂中的应用，其特征在于：The application of nicotinamide mononucleotide according to any one of claims 2 to 8 in combination with Lactobacillus fermentum in the preparation of preparations for relieving skin photoaging, characterized in that:其中，所述缓解皮肤光老化制剂的活性成分仅包括烟酰胺单核苷酸和发酵乳杆菌Lactobacilus fermentum。Wherein, the active ingredients of the preparation for relieving skin photoaging only include nicotinamide mononucleotide and Lactobacilus fermentum.
- 根据权利要求2所述的烟酰胺单核苷酸与发酵乳杆菌Lactobacilus fermentum联合在制备缓解皮肤光老化制剂中的应用，其特征在于：The application of nicotinamide mononucleotide according to claim 2 in combination with Lactobacillus fermentum in the preparation of preparations for relieving skin photoaging, it is characterized in that:其中，缓解皮肤光老化的药物为胶剂、膏剂、乳剂、气雾剂、注射剂、合剂、口服安瓿剂、片剂或胶囊剂；Among them, the drug for relieving skin photoaging is glue, ointment, emulsion, aerosol, injection, mixture, oral ampoule, tablet or capsule;缓解皮肤光老化的保健品为胶囊剂、片剂、口服液或固体饮料；Health care products for relieving skin photoaging are capsules, tablets, oral liquids or solid beverages;缓解皮肤光老化的护肤品为液状、面霜、湿粉或面膜。Skin care products to relieve skin photoaging are liquids, creams, moisturizing powders or masks.
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| PCT/CN2020/120924 WO2022077276A1 (en) | 2020-10-14 | 2020-10-14 | Application of combination of nicotinamide mononucleotide and lactobacillus fermentum in preparation of formulation for relieving skin photoaging |
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| CN (1) | CN117377480A (en) |
| WO (1) | WO2022077276A1 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115960742A (en) * | 2022-07-12 | 2023-04-14 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | Lactobacillus fermentum XJC48 with functions of prolonging service life of dermal fibroblasts and resisting skin aging and application thereof |
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| CN115960742A (en) * | 2022-07-12 | 2023-04-14 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | Lactobacillus fermentum XJC48 with functions of prolonging service life of dermal fibroblasts and resisting skin aging and application thereof |
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| CN117377480A (en) | 2024-01-09 |
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