CN116004454B - Lactobacillus plantarum J26 and application of composition thereof in relieving chronic and acute alcoholic liver injury - Google Patents
Lactobacillus plantarum J26 and application of composition thereof in relieving chronic and acute alcoholic liver injury Download PDFInfo
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- CN116004454B CN116004454B CN202211674861.9A CN202211674861A CN116004454B CN 116004454 B CN116004454 B CN 116004454B CN 202211674861 A CN202211674861 A CN 202211674861A CN 116004454 B CN116004454 B CN 116004454B
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- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention discloses application of lactobacillus plantarum J26 and a composition thereof in relieving chronic and acute alcoholic liver injury, and belongs to the technical field of microorganisms. Lactobacillus plantarum J26 has good effect on relieving chronic and acute alcoholic liver injury; and the effect is further improved when the composition is compounded with curcumin, kudzuvine root, vine tea, fructo-oligosaccharide and hyaluronic acid.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to application of lactobacillus plantarum J26 and a composition thereof in relieving chronic and acute alcoholic liver injury.
Background
Alcoholic liver injury (Alcoholic liver disease, ALD) is a liver disease caused by excessive alcohol consumption, and its morbidity and mortality are increasing worldwide. The development of ALD includes a range of conditions from mild alcoholic fatty liver, alcoholic hepatitis, to cirrhosis and its complications (including liver cancer). ALD has become one of the leading 30 worldwide causes of death in terms of death and life loss years. In the world, 7.2 cases of death caused by alcoholic cirrhosis are found in every 10 tens of thousands of people, and the death rate caused by ALD in China is about 45%. It is estimated that about one third of the population who is overdrinked will develop alcoholic hepatitis, up to 20% of the population developing cirrhosis, and about 2% of the patients with cirrhosis develop liver cancer. Thus, protection against ALD is urgent.
At present, a plurality of types of anti-alcoholic products exist on the market, but the problems of unstable drug effect and large side effect exist, and only hangover symptoms can be temporarily relieved, and the anti-alcoholic product has no definite curative effect on alcoholic liver injury. More and more studies in recent years have shown that protection against ALD by supplementation with probiotics is feasible. Therefore, it is of great interest to find a probiotic that can alleviate ALD to replace drugs. Because the lactobacillus has the functions of regulating immunity, resisting oxidation, resisting inflammation, maintaining intestinal steady state and the like, a new idea is provided for the protection of ALD.
However, not all probiotics have efficacy in alleviating ALD, so finding a probiotic strain and applying it in alleviating ALD is currently a problem to be solved urgently.
Disclosure of Invention
In order to solve the defects in the prior art, the invention aims to provide the application of lactobacillus plantarum J26 and a composition thereof in relieving chronic and acute alcoholic liver injury; applicant found that lactobacillus plantarum J26 works well in alleviating chronic and acute alcoholic liver injury; and the effect is further improved when the composition is compounded with curcumin, kudzuvine root, vine tea, fructo-oligosaccharide and hyaluronic acid.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
use of lactobacillus plantarum J26 for alleviating chronic and acute alcoholic liver injury. The applicant found that lactobacillus plantarum J26 has the following properties:
(1) Has stronger tolerance to simulated gastrointestinal fluid and good growth in the environments of pH 3.0 and pH 8.0;
(2) The product has strong tolerance to alcohol, and has high survival rate under the condition of 6% alcohol concentration;
(3) Can be effectively adhered to the intestinal wall, and has higher adhesion rate to Caco-2 intestinal epithelial cells;
(4) Can effectively increase the activities of antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-px)) and the content of Glutathione (GSH), can effectively reduce the content of cytochrome CYP2E1 and malondialdehyde, and can effectively weaken oxidative stress injury of liver;
(5) Can effectively reduce the content of MPO and pro-inflammatory factors (tumor necrosis factor alpha (TNF-alpha), interleukin-1 beta (IL-1 beta) and interleukin-6 (IL-6)) and effectively reduce liver inflammatory response;
(6) Can effectively reduce the apoptosis rate of liver cells, improve the ATP content of mitochondria and the copy number of mitochondrial DNA, and effectively reduce the apoptosis and the injury level of the wiregrain of the liver cells which are induced by alcohol;
(7) Can effectively reduce the activity of liver function enzymes (ALT, AST and ALP) and the content of serum fat (TG, TC), and effectively relieve the pathological damage degree of liver;
(8) Can effectively improve the contents of acetic acid, propionic acid and butyric acid and the contents of short-chain fatty acid serving as intestinal metabolites.
Furthermore, the applicant finds that the effect is better when lactobacillus plantarum J26 is used by being compounded with a plurality of medicinal and edible homologous plants with the effects of dispelling the effects of alcohol and protecting the liver; accordingly, the present invention also provides a composition for alleviating chronic and acute alcoholic liver injury, which comprises lactobacillus plantarum J26, curcumin, kudzuvine root, vine tea, fructo-oligosaccharide and hyaluronic acid.
As a further preferred embodiment of the present invention, the composition is prepared from the following raw materials in parts by weight:
further preferably, the composition is prepared from the following raw materials in parts by weight:
in the technical scheme, the curcumin is used as an active ingredient in dried rhizome of curcumae plants such as turmeric, zedoary, turmeric and the like, and has important pharmaceutical effects of resisting cancer, resisting oxidation, reducing lipid, resisting aging and the like; the research shows that curcumin can obviously reduce MDA content in liver tissues of mice suffering from acute alcoholism, obviously raise activities of antioxidant enzyme SOD and GSH-Px, effectively improve antioxidant capacity of organism defense system, reduce damage of active oxygen free radicals to lipid and protein on liver cell membranes, maintain structural and functional integrity of cell membranes, reduce production of lipid peroxide, thereby improving intestinal injury and liver injury of ALD rats and realizing protection of liver injury.
The kudzuvine root is one of the most representative anti-alcoholic drugs in the traditional Chinese medicine, has the pharmacological effects of dispelling the effects of alcohol, preventing drunk, improving cardiovascular and cerebrovascular circulation, reducing myocardial oxygen consumption, reducing blood sugar, preventing hypertension and arteriosclerosis, resisting liver toxicity, resisting inflammation, eliminating phlegm, relieving fever, improving organism immunity, resisting bacteria and viruses and the like, and can effectively improve the activities of liver Alcohol Dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH).
The vine tea contains rich flavonoid compounds, can obviously reduce MDA content in liver tissues of mice, improve GSH content, relieve hepatic cell steatosis, and has a certain protection effect on liver injury caused by alcohol group; the dihydromyricetin with high content can inhibit liver enzyme release, liver lipid peroxidation and triglyceride deposition caused by ethanol, and regulate protein p62 and autophagy via Keap-1/Nrf2 pathway, thereby relieving liver steatosis and inflammatory reaction.
Fructo-oligosaccharides reduce endotoxin levels in the body and intestinal permeability of the body, reduce fat accumulation and reduce the incidence of fatty liver by affecting the balance of intestinal flora; the probiotics in the intestinal tract ferment fructo-oligosaccharide to produce acid and antibacterial substances to inhibit the growth of other pathogenic bacteria, and the produced biotin is beneficial to the growth of the probiotics.
The invention also provides a preparation method of the composition, which comprises the following steps:
(1) Dissolving curcumin in DMF solvent, then adding hyaluronic acid solution, stirring and reacting for 3-5 h under the action of a catalyst, then dialyzing, and freeze-drying after the dialysis is finished to obtain modified curcumin;
(2) Pulverizing radix Puerariae and Ampelopsis Grossdentata, mixing, adding 8-12 times of water, extracting for 1-3 times, concentrating under reduced pressure to density of 1-2 g/mL, sieving, filtering, and spray drying to obtain mixed dry powder;
(3) Activating lactobacillus plantarum J26 to obtain bacterial liquid, centrifuging, collecting bacterial mud, washing with normal saline for 2-3 times, and performing vacuum freeze drying treatment to obtain bacterial powder;
(4) And (3) uniformly mixing fructo-oligosaccharide with the products obtained in the step (1), the step (2) and the step (3) to obtain the composition.
As a further preferred embodiment of the present invention, the hyaluronic acid solution preparation method in step (1) comprises: dissolving hyaluronic acid in deionized water, adding EDC, and activating for 30-60 min.
As a further preferred embodiment of the present invention, the catalyst used in step (1) is DMAP.
As a further preferred embodiment of the present invention, the dialysis process in step (1) is specifically: dialyzing with DMF for 2-3 h, dialyzing overnight with mixed solution of DMF and water in the volume ratio of 1:1, and finally, dialyzing with water for 6-8 h, wherein the dialysis water is changed every 2h.
Further preferably, the dialysis bag used in the dialysis process has a molecular weight cut-off of 1000 to 8000; more preferably, the molecular weight cut-off is 3500.
As a preferred embodiment of the present invention, the composition obtained has a Lactobacillus plantarum J26 content of 50 to 1000 hundred million/g.
Compared with the prior art, the invention has the following beneficial effects:
1. the lactobacillus plantarum J26 is applied to relieving alcoholic liver injury for the first time; the applicant finds that the strain has good alcohol tolerance and digestive tract characteristics and can be used for developing products related to anti-alcohol and liver protection; the lactobacillus plantarum J26 can reduce the apoptosis level of liver cells caused by alcohol, weaken oxidative stress injury of the liver caused by alcohol, lighten liver inflammatory response caused by alcohol and relieve the intestinal injury degree caused by alcohol, thereby realizing the alleviation of chronic and acute alcoholic liver injury.
2. The lactobacillus plantarum J26 is compounded with the medicine and food homologous plant with the effects of dispelling the effects of alcohol and protecting the liver, so that the effect is further improved; the components such as polysaccharide, polyphenol and the like in the medicinal and edible plants can promote the proliferation and growth of probiotics; the probiotics degrade the active ingredients of the medicine and food homologous plants into micromolecular substances which can be easily absorbed, promote the transportation and absorption of the micromolecular substances and exert the active effect of the micromolecular substances; fructooligosaccharides promote the colonization and growth of probiotics in the intestinal tract.
3. Curcumin can accelerate the decomposition of acetaldehyde by human body, reduce the alcohol content in blood, and has the functions of dispelling the effects of alcohol and protecting liver; the kudzuvine root can reduce blood viscosity by removing free radicals and resisting lipid peroxidation, promote blood circulation, reduce damage to liver and other systems, and play roles in protecting liver, strengthening brain and the like; meanwhile, the active ingredients of the kudzuvine root can become pasty when meeting water, can absorb alcohol in vivo and can slowly release curcumin so as to continuously decompose acetaldehyde.
4. Curcumin has poor self stability and low solubility, is easy to be catabolized when entering a body, has low bioavailability, and cannot fully exert pharmacological activity; based on the above, the applicant introduces hyaluronic acid into the formula, grafts curcumin onto the hyaluronic acid to form a nanoparticle structure, and greatly increases the water solubility and stability of the curcumin.
5. The invention only uses one strain of lactobacillus plantarum J26, which reduces the cost compared with the combined use of a plurality of probiotics in the prior art, but maintains the curative effect; the invention further enhances the effect of lactobacillus plantarum J26 by assistance of medicinal and edible plants, and medicinal materials in the prescription of the composition are safe and nontoxic, can be taken before and after drinking, are beneficial to dispelling effects of alcohol, can be taken for a long time in daily life, and have the health care effects of protecting liver and expelling toxin.
Drawings
FIGS. 1A-1D are the effects of Lactobacillus plantarum J26, compositions on serum ALT, AST, ALP and LPS levels;
FIGS. 2A-2B are the effects of Lactobacillus plantarum J26, composition on serum fat content;
FIGS. 3A-3B are the effects of Lactobacillus plantarum J26, compositions on mitochondrial ATP content and mtDNA number;
FIGS. 4-1A-C, 4-2A-C are the effects of Lactobacillus plantarum J26, composition on oxidative stress injury of the liver;
FIG. 5 shows the number of MPO positive cells in liver;
FIGS. 6A-6C are the effects of Lactobacillus plantarum J26, composition on liver inflammatory factors;
FIGS. 7A-7D are the effects of Lactobacillus plantarum J26, compositions on expression of liver apoptosis-related gene proteins;
FIGS. 8A-8B are the effects of Lactobacillus plantarum J26, compositions on mitochondrial homeostasis related gene expression;
FIGS. 9A-9C are the effects of Lactobacillus plantarum J26, compositions on expression of related gene proteins in the Nrf2 signaling pathway;
FIG. 10 is the effect of Lactobacillus plantarum J26, composition on the expression of related gene proteins in the TLR4 and MAPK signaling pathways;
FIG. 11 is the effect of Lactobacillus on the short chain fatty acid content of the intestinal contents.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the following examples. Of course, the specific embodiments described herein are for purposes of illustration only and are not intended to limit the invention.
The test methods described in the following examples and application examples, unless otherwise specified, are all conventional methods; the reagents and biological materials, unless otherwise specified, are commercially available.
In particular, lactobacillus plantarum J26 used in the present invention has been reported in the prior art, for example, in patent document CN113598374A, CN113598375 a; and also in journal literature (YIchao H, xuesong Li, xinyu Liu, yashuo Zhang, weizhang, chaoxin Man, yujun Jiang.2019. Transcriptimic responses of Caco-2cellsto Lactobacillus rhamnosus GG and Lactobacillus plantarum J26 against oxidative stress.Journal of Dairy Science.) the public is available from northeast agricultural university.
Specifically, lactobacillus plantarum J26 used in the invention is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) with a preservation address of Hospital No. 3 of North Chen West Lu 1 of the Korean area of Beijing city and a preservation number of CGMCC NO:5448, the preservation date is 11/08/2011; lactobacillus plantarum J26 was designated NDC 75017 when deposited.
Example 1
A composition capable of relieving chronic and acute alcoholic liver injury is prepared from the following raw materials in parts by weight:
the embodiment also provides a preparation method of the composition, which comprises the following steps:
(1) Curcumin was dissolved in DMF solvent, followed by addition of hyaluronic acid solution (hyaluronic acid in deionized water, followed by EDC, activation for 30 min), stirred for 4h under DMAP, followed by dialysis against 3500 molecular weight cut-off dialysis bags: dialyzing with DMF for 2.5h, dialyzing overnight with mixed solution of DMF and water in a volume ratio of 1:1, dialyzing with water for 6h, and dialyzing with water every 2 h; freeze drying is carried out after the dialysis is finished, so as to obtain modified curcumin;
(2) Pulverizing radix Puerariae and folium Ampelopsis Grossdentata, mixing, adding 10 times of water, extracting for 3 times, concentrating under reduced pressure to density of 2g/mL, sieving, filtering, and spray drying to obtain mixed dry powder;
(3) Activating lactobacillus plantarum J26 to obtain bacterial liquid, centrifuging, collecting bacterial mud, washing with physiological saline for 3 times, and adjusting colony number concentration to 1×10 9 CFU/mL, obtaining bacterial suspension, and then performing vacuum freeze drying treatment to obtain bacterial powder;
(4) And (3) uniformly mixing fructo-oligosaccharide with the products obtained in the step (1), the step (2) and the step (3) to obtain the composition.
Example 2
A composition capable of relieving chronic and acute alcoholic liver injury is prepared from the following raw materials in parts by weight:
the embodiment also provides a preparation method of the composition, which comprises the following steps:
(1) Curcumin was dissolved in DMF solvent, followed by addition of hyaluronic acid solution (hyaluronic acid in deionized water, followed by EDC, activation for 30 min), stirred for 4h under DMAP, followed by dialysis against 3500 molecular weight cut-off dialysis bags: dialyzing with DMF for 2.5h, dialyzing overnight with mixed solution of DMF and water in a volume ratio of 1:1, dialyzing with water for 6h, and dialyzing with water every 2 h; freeze drying is carried out after the dialysis is finished, so as to obtain modified curcumin;
(2) Pulverizing radix Puerariae and folium Ampelopsis Grossdentata, mixing, adding 10 times of water, extracting for 3 times, concentrating under reduced pressure to density of 2g/mL, sieving, filtering, and spray drying to obtain mixed dry powder;
(3) Activating lactobacillus plantarum J26 to obtain bacterial liquid, centrifuging, collecting bacterial mud, washing with physiological saline for 3 times, and adjusting colony number concentration to 1×10 9 CFU/mL to obtain bacterial suspension, and vacuum freeze dryingObtaining bacterial powder;
(4) And (3) uniformly mixing fructo-oligosaccharide with the products obtained in the step (1), the step (2) and the step (3) to obtain the composition.
Example 3
A composition capable of relieving chronic and acute alcoholic liver injury is prepared from the following raw materials in parts by weight:
the embodiment also provides a preparation method of the composition, which comprises the following steps:
(1) Curcumin was dissolved in DMF solvent, followed by addition of hyaluronic acid solution (hyaluronic acid in deionized water, followed by EDC, activation for 30 min), stirred for 4h under DMAP, followed by dialysis against 3500 molecular weight cut-off dialysis bags: dialyzing with DMF for 2.5h, dialyzing overnight with mixed solution of DMF and water in a volume ratio of 1:1, dialyzing with water for 6h, and dialyzing with water every 2 h; freeze drying is carried out after the dialysis is finished, so as to obtain modified curcumin;
(2) Pulverizing radix Puerariae and folium Ampelopsis Grossdentata, mixing, adding 10 times of water, extracting for 3 times, concentrating under reduced pressure to density of 2g/mL, sieving, filtering, and spray drying to obtain mixed dry powder;
(3) Activating lactobacillus plantarum J26 to obtain bacterial liquid, centrifuging, collecting bacterial mud, washing with physiological saline for 3 times, and adjusting colony number concentration to 1×10 9 CFU/mL, obtaining bacterial suspension, and then performing vacuum freeze drying treatment to obtain bacterial powder;
(4) And (3) uniformly mixing fructo-oligosaccharide with the products obtained in the step (1), the step (2) and the step (3) to obtain the composition.
Application example 1
The application example verifies that lactobacillus plantarum J26 has stronger tolerance to alcohol:
inoculating frozen lactobacillus plantarum J26 with an inoculum size of 5% to an MRS liquid culture medium, culturing for 18 hours under anaerobic condition at 37 ℃, inoculating to a solid MRS culture medium by adopting a three-area line drawing mode, culturing for 48 hours under anaerobic condition at 37 ℃, and then picking single colony to the MRS liquid culture medium for continuous culturing for 18 hours.
Centrifuging at 8000r/min at 4deg.C for 10min at the end of logarithmic growth phase, collecting bacterial precipitate, washing with sterile PBS for 3 times, and re-suspending in MRS culture medium with equal volume and different alcohol concentrations of 0%, 2%, 4% and 6%, respectively, and stress for 2 hr. Washing the stressed bacterial suspension twice and re-suspending in PBS (phosphate buffer solution) with equal volume, taking 1mL of heavy suspension for gradient dilution, selecting proper dilution gradient to be coated on MRS solid culture medium, carrying out anaerobic culture for 18h at 37 ℃, and measuring the survival rate.
The survival rate (%) is calculated as the ratio of the number of viable bacteria after alcohol stress to the number of viable bacteria without alcohol stress, expressed as%. The experimental results are shown in Table 1, and the results show that the lactobacillus plantarum J26 has stronger tolerance to alcohol.
TABLE 1 tolerance of Lactobacillus plantarum J26 to alcohol
| Alcohol concentration 2% | Alcohol concentration 4% | Alcohol concentration 6% | |
| Survival (%) | 59.41±0.53 | 30.31±0.31 | 20.28±0.24 |
Application example 2
The application example verifies that the lactobacillus plantarum J26 has stronger tolerance to simulated gastrointestinal fluids:
pepsin (1:10000) is dissolved in sterile PBS, the concentration is adjusted to 3g/L, the pH is adjusted to 3.0, and the gastric juice is filtered and sterilized for standby use, so that gastric juice is simulated. Trypsin (1:250) was dissolved in sterile PBS, adjusted to a concentration of 1g/L, then 0.3% bile salts were added, and the pH was adjusted to 8.0, filtered and sterilized for use to simulate intestinal fluid.
Lactobacillus plantarum J26 is activated for two generations, centrifuged for 10min at 8000r/min at 4 ℃ at the end of the logarithmic growth phase, the bacterial precipitate is collected, washed 3 times by sterile PBS and resuspended in simulated gastric fluid and mixed uniformly, anaerobic culture is carried out at 37 ℃ for 3 hours, and a proper gradient is selected for plate coating counting. After culturing in a simulated intestinal fluid in a constant temperature incubator at 37 ℃ for 8 hours, viable bacteria were counted by the same method. The viability of lactobacillus plantarum J26 was calculated separately.
Survival (%) was calculated as the ratio of the number of viable bacteria after treatment to the number of viable bacteria before treatment, expressed as%. The experimental results are shown in Table 2, and the results show that the lactobacillus plantarum J26 has stronger tolerance to simulated gastrointestinal fluids.
TABLE 2 Lactobacillus plantarum J26 tolerance to simulated gastrointestinal fluids
| Simulated gastric fluid | Simulated intestinal juice | |
| Treatment time (h) | 3 | 8 |
| Survival (%) | 91.44±0.30 | 89.27±0.27 |
Application example 3
The application example verifies that the lactobacillus plantarum J26 has stronger adhesion capability to Caco-2 intestinal epithelial cells:
inoculating recovered Caco-2cells into DMEM high sugar culture solution containing 10% foetal calf serum, and placing at 37deg.C and 5% CO 2 The culture is carried out in an incubator of (2), the liquid is changed when the cell culture state is observed, and when the cell is paved on the bottle bottom and more than 80% is in an adherent state, the passage operation is carried out. Discarding the culture solution, washing for 3 times, adding pancreatin for digestion for 2min, adding DMEM for stopping digestion, blowing cells to completely fall off the bottle bottom, centrifuging for 5min at 1000r/min, collecting precipitate, adding a proper amount of DMEM, blowing and mixing uniformly, and carrying out passage in a ratio of 1:3 for subsequent experiments.
Lactobacillus plantarum J26 is activated for two generations, centrifuged for 10min at 8000r/min at 4deg.C at the end of logarithmic growth phase, and the bacterial precipitate is collected, washed 3 times with sterile PBS, resuspended in DMEM high sugar culture solution containing 10% foetal calf serum, and the bacterial concentration is adjusted to 1×10 8 CFU/mL. Inoculating Caco-2cells transferred to the fourth generation in a 6-hole plate cell culture plate, culturing for 24 hours by using a DMEM high-sugar culture medium without antibiotics after the cells are attached to the wall and are fully paved on a bottom plate by more than 80%, observing the cell state, and re-suspending the cells in the polarized state in the high-sugar culture solution to incubate the cells for 2 hours.
After the co-incubation was completed, the cells were gently washed 3 times with sterile PBS to remove non-adherent cells. Cells from each well were then digested and collected using pancreatin, then subjected to gradient dilution, and a suitable gradient was selected and spread on MRS agar medium, and colony counting was performed after 48 hours, to calculate lactobacillus plantarum J26 adhesion rate.
The adhesion rate (%) was calculated as a ratio of the number of colonies after adhesion to the number of colonies before adhesion, expressed as%. Experimental results show that the lactobacillus plantarum J26 has stronger adhesion capability to Caco-2 intestinal epithelial cells, and the adhesion rate reaches 21.40%.
Application example 4
The application example verifies that lactobacillus plantarum J26 and the composition (named PCP) can relieve alcoholic liver injury of mice.
1. Experimental method
(1) Animal model building and grouping
60 clean 6-week-old male C57BL/6J mice are selected, the weight is 20-25 g, the mice are fed into animal houses, the temperature is controlled to be 22+/-2 ℃, and the mice are fed with standard feed adaptively for 1 week. They were randomly divided into 6 groups of 10: control, alcohol, low dose l.plantarum J26, medium dose l.plantarum J26, high dose l.plantarum J26 and combination. Wherein the alcohol group, the low dose L.plantarum J26 group, the medium dose L.plantarum J26 group, the high dose L.plantarum J26 group and the composite group are subjected to Lieber-DeCarli alcohol liquid feed transient adaptation feeding for 1 week (the alcohol concentration is from 0 to 5% (w/v)), and the control group is fed with an isocaloric control liquid feed.
After the end of the transitional period, the alcohol group, the low-dose L.plantarum J26 group, the medium-dose L.plantarum J26 group, the high-dose L.plantarum J26 group and the compound group continue to feed the alcohol liquid feed for 8 weeks (alcohol concentration 5% (w/v)), wherein the alcohol group is filled with 0.2mL of sterile PBS every morning; low dose L.Plantarum J26, medium dose L.Plantarum J26, high dose L.Plantarum J26 and combination groups were each gavaged daily at morning with 0.2mL of low dose L.Plantarum J26 (1×10) 7 CFU/mL), medium dose L.Plantarum J26 (1×10) 8 CFU/mL), high dose L.plantarum J26 (1X 10) 9 CFU/mL) and 0.2mL of the composition (L.plantarum J26 concentration 1X 10) 9 CFU/mL), 1 high concentration alcohol (31.5% (v/v)) gavage was performed around 7 hours a day of the 10 th weekend, at a dose of 5g/kg, and sacrificed after 9 hours of fasted. The control group was fed with a dedicated control maltodextrin liquid feed, etc. caloric for 8 weeks (daily feed amount was the average feed intake of the other 5 groups on the previous day) and was filled with 0.2mL of sterile PBS daily at noon. Maltodextrin, which is the same caloric equivalent to high concentration alcohol (31.5% (v/v)) was infused at 7 hours on the next day on the 10 th weekend, at the same dose of 5g/kg, and sacrificed after 9 hours of fasting. The specific grouping operation is as follows.
TABLE 3 grouping and treatment of mice
(2) Sample collection and processing
After the animal test is finished, the mice are sacrificed after blood collection through capillary eyeballs and neck breaking, and body surfaces are dissected after disinfection.
(1) Serum samples: taking blood from capillary eyeball, centrifuging at 4deg.C for 10min at 3000r/min, and collecting supernatant, and storing in refrigerator at-80deg.C for biochemical index detection.
(2) Liver tissue samples: dissecting the mouse in a low-temperature environment to extract the liver, flushing with pre-cooled normal saline to remove surface connective tissue, and sucking excessive liquid with filter paper. The liver is divided into 3 parts, and one part of the liver is fixed in 2.5% glutaraldehyde-PBS for ultrastructural observation; a portion of liver tissue was fixed in 10% formaldehyde solution for pathology detection, immunohistochemistry and apoptosis staining; the rest of the tissue is preserved at-80deg.C in sterile cryopreservation tube without RNase for biochemical index detection and extraction of RNA and protein.
(3) Colon tissue samples: the mice were dissected in a low temperature environment to remove the ileum, rinsed with pre-chilled saline, and the excess fluid was removed by filter paper. The colon was divided into 2 parts, and a part of liver tissue was fixed in 10% formaldehyde solution for pathology detection; the rest of tissues are put in a sterile freezing tube without RNase for preservation at-80 ℃ and used for biochemical index detection and RNA extraction.
(4) Stool and cecum content samples: collecting the cecal content of mice on ice under aseptic condition, and wrapping with tinfoil, and storing at-80deg.C in sterile frozen tube without RNase for short chain fatty acid determination.
(3) Determination of physiological index
The activity of ALT, AST and ALP in serum is measured by a full-automatic biochemical analyzer; LPS is detected according to the operation on the ELSA kit instruction; TG and TC were measured according to biochemical detection kit instructions.
Weighing a proper amount of liver, fully grinding the liver by using an electric tissue grinder under ice water bath according to the ratio of physiological saline to the liver of 9:1, and after grinding, placing the homogenate under the condition of 4 ℃ to measure relevant indexes of antioxidant and mitochondrial functions, immune-related cytokines and the like. SOD, CAT, GSH-px, MDA, GSH and ATP are assayed according to biochemical test kit instructions; CYP2E1, TNF- α, IL-1β, IL-6 and interleukin-10 (IL-10) were assayed according to ELISA kit instructions. The total protein content in liver tissue was determined according to the kit instructions.
Determination of liver mtDNA copy number: extracting whole genome DNA of liver tissue according to the reagent kit, measuring concentration, and detecting and calculating mtDNA copy number according to a real-time fluorescence quantification method by taking mitochondrial conserved gene cytochrome B (Cytb) as a representative.
(4) Immunohistochemical detection
Liver tissue fixed with 10% methanol solution was cut into paraffin-embedded slices, and after dewaxing and hydration of the slices, MPO was measured according to the following procedure.
(1) And (3) carrying out microwave antigen retrieval on the citric acid antigen retrieval liquid, and cooling to room temperature.
(2) Adding 3% H 2 O 2 Incubate for 15min at room temperature and repeat the wash 3 times.
(3) The mixture was blocked at room temperature for 1h by adding 3% goat serum.
(4) The diluted MPO antibody was added and incubated overnight at 4 ℃.
(5) The PBS was washed 3 times, 5 min/time. The biotinylated secondary antibody was added and incubated for 1h at room temperature, and after the end, the solution was washed 3 times with PBS again.
(6) Horseradish enzyme-labeled avidin was added and incubated at 37℃for 30min, and after completion, washed 3 times with PBS.
(7) Adding DAB color development liquid for 1-5 min, and then carrying out hematoxylin counterstaining.
(8) And (5) dehydrating and transparentizing the gradient ethanol solution and the dimethylbenzene, sealing the resin, and airing.
(9) The record was observed under a microscope.
(5) Detection of mRNA expression in tissues
(1) Extraction and reverse transcription of total RNA from liver/colon tissue
The appropriate amount of liver/colon tissue was weighed into a pre-chilled mortar, added with liquid nitrogen for full use, and the powder was rapidly transferred into a 1.5mL RNase-free centrifuge tube. Extracting total RNA of the liver tissue of the mouse according to the instruction of the kit, performing light-shielding operation in the whole process, rapidly subpackaging the extracted total RNA into a centrifuge tube of pentarnase, and placing the centrifuge tube at the temperature of-80 ℃ for standby. Subsequently calculated from the concentration and purity of the extracted RNA samples, according to PrimeScript TM The RT kit instruction is subjected to reverse transcription reaction to synthesize cDNA, and the cDNA is stored at-20 ℃ for standby.
(2) Real-time fluorescent quantitative RT-PCR (qRT-PCR) reaction
Specific primers for the genes were designed according to the gene sequences in the NCBI website using Primer 5.0 software and were synthesized by Shanghai Biotechnology. And primer information is shown in Table 4.
TABLE 4 primer information
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Note that: f represents a forward sequence, R represents a reverse sequence
According to TBPremix Ex Taq TM II kit instructions, real-time RT-PCR reactions were performed. Beta-actin is used as an internal reference, 2 is utilized -ΔΔCt The method calculates the relative expression of the related genes.
(6) Determination of related protein expression in tissues
Western blotting is used to detect the expression level of Bax, bcl2, C-Caspase-9C-Caspase-3, nrf2, HO-1, NQO1, TLR4 proteins, p38, JNK and ERK proteins and their phosphorylated protein. Whole proteins in liver tissue were extracted according to the procedure of the present kit and tissue protein concentration was measured using the BCA protein concentration measurement kit. After SDS-PAGE electrophoresis, 100V constant pressure transfer film is carried out for 1-2h, and then room temperature sealing is carried out for 1h. Removing the blocking solution after the completion, adding the diluted primary antibody at 4 ℃ overnight, washing 3 times by TBST for 5 min/time in the next day, continuously adding the secondary antibody, incubating for 30min at room temperature, washing four times by TBST, and finally dripping ECL mixed solution for chemiluminescence detection. The optical density values of the target bands were analyzed using an Image J software processing system.
(7) Short chain fatty acid assay
The cecum content was weighed 0.50.+ -. 0.01g, 1mL of purified water was added, and vortexed for 10s. The suspension with the concentration of 10 percent is prepared by the treatment of a fecal treatment instrument. 0.5mL of the suspension and 100. Mu.L of the crotonic acid metaphosphoric acid solution were taken in a 1.5mL sterile centrifuge tube and frozen at-30℃for 24 hours. Thawing, centrifuging at 4deg.C, collecting supernatant (8000 r/min,3 min), filtering with 0.22 μm water system filter membrane, and measuring by gas chromatograph. The sample loading was 1. Mu.L, and the column HP-FFAP (30 m. Times.250. Mu.m. Times.0.25 μm).
2. Experimental results
(1) Changes in liver function enzyme, LPS and fat content in serum
(1) Liver function enzyme Activity and LPS content
The liver function enzyme and LPS levels in serum were measured as shown in FIG. 1. Compared with the control group, the alcohol induction increases the activity of liver function enzyme, and the ratio of AST to ALT in the alcohol group is more than 2, which indicates that the liver function is seriously damaged. The liver function enzyme activities of each group of pre-ingested l.plantarum J26 were all in a decreasing trend. The low dose L.plantarum J26 group ALT, AST and ALP activities were reduced by 10.38%, 13.08% and 3.69% respectively compared with the alcohol group; the activities of ALT, AST and ALP in the medium-dose L.plant J26 group are respectively reduced by 16.69 percent, 27.58 percent and 8.31 percent; the high dose L.Plantarum J26 group ALT, AST and ALP activities were reduced by 44.61%, 58.79% and 16.84% respectively, and the complex groups ALT, AST and ALP activities were reduced by 53.61%, 69.79% and 25.84% respectively, all in very significant differences. This shows that l.plantarum J26 can effectively ameliorate impaired liver function and that the effect exhibits some dose dependence; the effect of the composition containing high dosage of L.plantarum J26 on improving liver function is further improved.
As shown in fig. 1D, the LPS content in the serum of mice in the alcohol group was significantly higher than that of the control group. Compared with the alcohol group, the LPS content of the medium-dose L.plantarum J26 group is reduced by 51.37%; the LPS content of the high-dose L.plantarum J26 group is reduced by 60.1 percent, the LPS content of the compound group is reduced by 69.3 percent, and the reduction of the LPS content of the low-dose L.plantarum J26 group has no obvious difference. The results show that the high dose of L.plantarum J26 can more effectively reduce the LPS content in the serum of the mice; the effect of the composition containing high dose L.plantarum J26 is further improved.
(2) TG, TC content
Alcohol's calories are not converted to fat but can disrupt fat metabolism in the body and thus raise fat content in serum, TG, TC being the predominant fat accumulation form. As shown in fig. 2, the TG and TC levels in the serum of mice in the alcohol group were elevated compared to the control group, and the fat levels in the serum of both the gastric lavage composition and the different doses of l.plantarum J26 were reduced, but the effect of low doses of l.plantarum J26 on fat levels was not significantly different. Compared with the alcohol group, the TC content of the medium-dose L.plantarum J26 group is reduced by 23.75%, the TG and TC contents of the high-dose L.plantarum J26 group are respectively reduced by 28.60% and 25.70%, and the TG and TC contents of the compound group are respectively reduced by 30.50% and 27.90%. Indicating that L.plantarum J26 can reduce fat accumulation caused by alcohol induction, and the composition containing high dosage of L.plantarum J26 has more remarkable effect.
(2) Changes in apoptosis levels in liver
Mitochondrial ATP and mtDNA content
The ATP content and mtDNA content of the mitochondria can be reduced in the apoptosis process, and the ATP content and mtDNA copy number of the alcohol group are lower than those of the control group as shown in the figure 3, and the ATP content and mtDNA copy number of the alcohol group can be inhibited by pre-intake of each dose of L.plantarum J26 or the composition, the dose dependency relationship is shown, and the effect of the composition is better than that of independently taking the L.plantarum J26.
(3) Changes in oxidative stress injury in the liver
(1) SOD, CAT and GSH-Px Activity
Alcohol is metabolized in the liver to produce a large amount of ROS, which is liable to cause oxidative stress in the liver. The levels of oxidative stress were measured as shown in FIG. 4-1, and the SOD, CAT, GSH-Px activity was decreased in the livers of mice in the alcohol group as compared with the control group. Although there was no significant difference in SOD activity between each dose l.plantarum J26 group and the complex group, the mid-dose l.plantarum J26 group, the high-dose l.plantarum J26 group and the complex group showed 17.01%, 20.21% and 22.33% increase in SOD activity, respectively, compared to the alcohol group. In terms of CAT activity, there was no significant difference in the increase in low dose l.plantarum J26 compared to the alcohol group, with the medium dose l.plantarum J26, high dose l.plantarum J26 and the complex group increased by 11.79%, 13.77% and 15.01%, respectively. The individual doses of L.plantarum J26 and complex groups increased GSH-px activity by 5.75%, 7.98%, 8.19% and 9.23%, respectively, compared to the alcohol group. The L.plantarum J26 is shown to prevent the reduction of the liver antioxidant enzyme activity induced by alcohol and shows a certain dose dependency, and the effect of the composition is better than that of the L.plantarum J26 singly.
(2) CYP2E1, MDA and GSH content
CYP2E1 is a key enzyme mainly producing ROS pathway in alcohol metabolism, and the levels of MDA and GSH can also reflect the level of oxidative stress in the body. As shown in fig. 4-2, the induction of alcohol extremely increased the content of CYP2E1, whereas the low dose l.plantarum J26 group, the medium dose l.plantarum J26 group, the high dose l.plantarum J26 group and the complex group showed a decrease in the content of CYP2E1 by 27.32%, 32.24%, 36.80% and 38.56%, respectively, compared to the alcohol group. The MDA content in the liver of mice in the alcohol group was increased compared to the control group, whereas the MDA content in the high dose L.plantarum J26 group and the compound group was reduced by 16.69% and 17.89%, respectively, compared to the alcohol group. The GSH content in the alcohol group was significantly reduced compared to the control group, while l.plantarum J26 restored GSH levels in the liver and exhibited some dose dependence. These results indicate that high doses of l.plantarum J26 are more effective in elevating the antioxidant level of the liver of the body, attenuating oxidative stress damage, and that the composition is more effective than l.plantarum J26 alone.
(4) Changes in MPO and inflammatory factors in the liver
(1) MPO content
The degree of neutrophil infiltration in the liver is generally positively correlated with the severity of liver inflammation, whereas MPO is a functional and activating marker for neutrophils, and the level of inflammatory factors also reflects liver inflammation. The research analyzes infiltration condition of liver neutrophil MPO by an immunohistochemical method, and detects levels of pro-inflammatory factors TNF-alpha, IL-1 beta and IL-6 in the liver by an ELISA method. As shown in fig. 5, the number of MPO positive cells in the liver of mice in the alcohol group was extremely significantly increased as compared to the control group. The number of MPO positive cells in the livers of mice in the low, medium, high, and compound groups of l.plantarum J26 groups were reduced by 19.21%, 30.10%, 36.23%, and 39.79%, respectively, compared to the alcohol group.
(2) Inflammatory factor content
As shown in FIG. 6, the amounts of the pro-inflammatory factors TNF- α, IL-1β, and IL-6 in the alcohol group were increased as compared to the control group. The low dose l.plantarum J26 group had reduced but no significant differences in the content of the three pro-inflammatory factors compared to the alcohol group. The levels of the three pro-inflammatory factors were reduced in the medium and high dose L.plantarum J26 groups, wherein the levels of TNF- α, IL-1β and IL-6 in the high dose L.plantarum J26 group were reduced by 35.38%, 34.27% and 31.14% respectively compared to the alcohol group, and the levels of TNF- α, IL-1β and IL-6 in the complex group were reduced by 37.69%, 37.03% and 34.58% respectively compared to the alcohol group. The results show that the inflammation of the liver of mice can be reduced to different degrees by each dose of L.plantarum J26, and the compound group containing high doses of L.plantarum J26 has the best effect.
(5) Detection of apoptosis-related marker expression
(1) Apoptosis of cells
The expression of apoptosis-related proteins, as shown in FIG. 7, was up-regulated in the alcohol group pro-apoptotic protein Bax, sheared Caspase-9 and Caspase-3, and down-regulated in the anti-apoptotic protein Bcl2, compared to the control group, indicating that the induction of alcohol increased the apoptotic state of the cells. The apoptotic status of the cells of the low dose L.plantarum J26, the medium dose L.plantarum J266, the high dose L.plantarum J26 and the complex group are reduced compared with the alcohol group, wherein the reduction degree of the high dose L.plantarum J26 and the complex group is the greatest, and the Bax and C-Caspase-3 expression is extremely obviously reduced compared with the alcohol, the C-Caspase-9 protein expression is obviously reduced, and the Bcl2 expression is extremely obviously up-regulated. The L.plantarum J26 is shown to reduce the apoptosis state of the liver of the mice to different degrees, a certain dose dependency relationship is shown, and the effect of the composition is better than that of the single use of the L.plantarum J26.
(2) Mitochondrial homeostasis
Mitochondrial homeostasis plays an important role in apoptosis, and to further study the role of mitochondria in the process of apoptosis, the present study examined the expression levels of mitochondrial dynamics and autophagy-related genes. As shown in fig. 8A, the expression levels of mitochondrial fission related genes Drp and MFF were up-regulated in the alcohol group and the expression levels of mitochondrial fusion related genes Mnf2 and Opa1 were extremely down-regulated in the alcohol group as compared to the control group. The expression of Drp1 gene was down-regulated with increasing l.plantarum J26 dose compared to the alcohol group; the expression level of MFF gene was down-regulated with no significant difference after pre-uptake of l.plantarum J26 at each dose, mnf gene was up-regulated at the high dose l.plantarum J26 group; the Opa1 gene was up-regulated in the high dose l.plantarum J26 group. Wherein pretreatment of L.plantarum J26 has the most significant effect on Drp1 gene expression, and the high dose L.plantarum J26 group has 41.72% down-regulated Drp gene expression compared to alcohol group, while the complex group has 43.87% down-regulated.
The results of autophagy-related gene expression are shown in FIG. 8B, and compared with the control group, the expression levels of the Beclin1, PINK1 and Parkin genes in the alcohol group are up-regulated, the expression of the LC3 gene is significantly up-regulated, and the expression of the P62 gene is down-regulated. Compared with the alcohol group, the expression of the Beclin1 gene is down-regulated with the increase of the dosage of L.plantarum J26, and shows a significant difference; the P62 gene is up-regulated in the high-dose L.plantarum J26 group; the expression of LC3 gene showed a decreasing trend but no significant difference at each dose of l.plantarum J26 group; the PINK1 gene is down-regulated in the high-dose L.plantarum J26 group expression; the Parkin gene was down-regulated in medium and high dose L.plantarum J26 groups. The results show that the L.plantarum J26 can reduce the level of alcohol-induced liver autophagy, and has a certain dose-dependent relationship, and the effect of the composition is better than that of the L.plantarum J26 used alone.
(7) Detection of Nrf2 Signal pathway-associated protein expression
Nrf2 is an important endogenous antioxidant factor and is normally associated with Keap1 in the cytoplasm. When Nrf2 dissociates from Keap1 for nuclear transfer, it is shown that Nrf2 is activated, which activation can protect and treat diseases associated with oxidative damage, such as alcohol-induced liver damage. To explore the mechanism by which L.plantarum J26 reduces the oxidative stress level of the organism, the expression level of the Nrf2 protein in the nucleus and the NQO1 and HO-1 proteins downstream thereof were examined. The results are shown in FIG. 9, where the expression of Nrf2, NQO1 and HO-1 was down-regulated in the nuclei of the alcohol group compared to the control group. Compared with the alcohol group, the expression of Nrf2 and HO-1 in the medium-dose L.plantarum J26 group is up-regulated, and the expression of Nrf2, NQO1 and HO-1 in the high-dose L.plantarum J26 group and the compound group are up-regulated, so that the result shows that the inhibition effect of the high-dose L.plantarum J26 and the composition is more obvious. The comprehensive results show that the L.plantarum J26 can relieve alcohol-induced liver oxidative stress and liver injury by activating an Nrf2 signal pathway, and the composition has better action and effect than the L.plantarum J26 singly.
(8) Detection of MAPK signal pathway related protein expression
TLRs are receptors of pathogen-associated molecular patterns, in particular TLR4 is a specific receptor for LPS, playing an important role in alcohol-induced liver injury, which can activate the MAPK signaling pathway, thus eliciting an immune response, producing inflammatory factors. To explore the mechanism of inhibition of inflammatory levels by l.plantarum J26, the expression levels of TLR4 proteins and the expression levels of the related proteins (p 38, JNK and ERK) and their phosphorylation in the MAPK signaling pathway were examined. The results are shown in fig. 10, where TLR4 protein expression and the levels of phosphorylation of p38, JNK and ERK proteins were upregulated in the alcohol group compared to the control group, indicating activation of the MAPK pathway. The pre-ingestion of each dose of l.plantarum J26 can significantly reduce the expression of TLR4 protein, and exhibits a certain dose-dependent relationship. The pre-intake of each dose of l.plantarum J26 was able to significantly down-regulate the phosphorylation level of p38 compared to the alcohol group; JNK phosphorylation levels were down-regulated in medium-dose l.plantarum J26 group, high-dose l.plantarum J26 group and complex group; phosphate downregulation of ERK in medium dose l.plantarum J26, high dose l.plantarum J26 and complex groups, and there was a reduction but no significant difference in phosphorylation of three proteins in low dose l.plantarum J26. From the comprehensive results, L.plantarum J26 can reduce liver inflammation level by inhibiting the TLR4 mediated MAPK signal pathway, reduce liver injury, and have better action effect.
(9) Determination of short chain fatty acid content in intestinal tract content
Changes in intestinal flora lead to changes in their metabolites, and the study uses GC-MS to determine short chain fatty acids in intestinal microbial metabolites. As shown in fig. 11, the acetic acid, propionic acid and butyric acid contents in the intestinal tracts of the mice in the alcohol group were reduced as compared to the control group. The pre-intake of L.plantarum J26 or the composition can improve the content of short-chain fatty acid to a certain extent, compared with an alcohol group, the low-dose L.plantarum J26 group and the medium-dose L.plantarum J26 group have improved acetic acid and acrylic acid content without obvious difference, the high-dose L.plantarum J26 group has improved acetic acid and acrylic acid content, the propionic acid improving effect is the best, the propionic acid content exceeds the propionic acid content in a control group, and only the improvement of the butyric acid content in the medium-dose L.plantarum J26 group has statistical significance.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (6)
1. The application of the lactobacillus plantarum J26 in preparing a product for relieving chronic and acute alcoholic liver injury is characterized in that the lactobacillus plantarum J26 is preserved in the China general microbiological culture Collection center with the preservation address of Beijing area North Chen Xie Lu No. 1, 3 and the preservation number of CGMCC NO:5448, the preservation date is 11/08/2011.
2. The use according to claim 1, wherein the lactobacillus plantarum J26 and curcumin, kudzuvine root, vine tea, fructo-oligosaccharides, hyaluronic acid constitute a composition for alleviating chronic and acute alcoholic liver injury.
3. Use according to claim 2, characterized in that the composition is prepared from the following raw materials in parts by weight:
4. use according to claim 2, characterized in that the composition is prepared from the following raw materials in parts by weight:
5. the use according to claim 2, wherein the preparation of the composition comprises the steps of:
(1) Dissolving curcumin in DMF solvent, then adding hyaluronic acid solution, stirring and reacting for 3-5 h under the action of a catalyst, then dialyzing, and freeze-drying after the dialysis is finished to obtain modified curcumin;
(2) Pulverizing radix Puerariae and Ampelopsis Grossdentata, mixing, adding 8-12 times of water, extracting for 1-3 times, concentrating under reduced pressure to density of 1-2 g/mL, sieving, filtering, and spray drying to obtain mixed dry powder;
(3) Activating lactobacillus plantarum J26 to obtain bacterial liquid, centrifuging, collecting bacterial mud, washing with normal saline for 2-3 times, and performing vacuum freeze drying treatment to obtain bacterial powder;
(4) And (3) uniformly mixing fructo-oligosaccharide with the products obtained in the step (1), the step (2) and the step (3) to obtain the composition.
6. The use according to claim 5, wherein the composition comprises lactobacillus plantarum J26 in an amount of 50 to 1000 v/g.
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| CN115089619A (en) * | 2022-07-18 | 2022-09-23 | 南昌大学 | Application of lactobacillus plantarum ZDY2013 in preparation with function of relieving non-alcoholic fatty liver disease |
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| CN111437294A (en) * | 2020-05-28 | 2020-07-24 | 东北农业大学 | Lactic acid bacteria formula for preventing acute and chronic alcoholic liver injury and application thereof |
| CN114569641A (en) * | 2022-04-22 | 2022-06-03 | 上海朗泰凯尔生物技术有限公司 | Composition for relieving acute alcoholic liver injury and preparation method and application thereof |
| CN115089619A (en) * | 2022-07-18 | 2022-09-23 | 南昌大学 | Application of lactobacillus plantarum ZDY2013 in preparation with function of relieving non-alcoholic fatty liver disease |
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