WO2013185339A1 - Application of honeysuckle flower water extract and let-7a microrna in preparation of medicament and health-care product for preventing and treating dengue viruses and dengue fever - Google Patents
Application of honeysuckle flower water extract and let-7a microrna in preparation of medicament and health-care product for preventing and treating dengue viruses and dengue fever Download PDFInfo
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- WO2013185339A1 WO2013185339A1 PCT/CN2012/076983 CN2012076983W WO2013185339A1 WO 2013185339 A1 WO2013185339 A1 WO 2013185339A1 CN 2012076983 W CN2012076983 W CN 2012076983W WO 2013185339 A1 WO2013185339 A1 WO 2013185339A1
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/35—Caprifoliaceae (Honeysuckle family)
- A61K36/355—Lonicera (honeysuckle)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- honeysuckle water extract and let-7a microRNA in the preparation and prevention of dengue virus and dengue fever medicines and health products
- the invention belongs to the technical field of Chinese herbal medicine, and particularly relates to the application of the aqueous extract of honeysuckle in preparing medicines and health care products for preventing and treating dengue virus.
- Dengue fever is a mosquito-borne disease in the tropics and subtropics. There are about 500 million to 1 billion cases of infection worldwide each year, mostly residents living in urban or urban suburbs. The dengue fever from a fever of dengue fever to dengue hemorrhagic fever with severe internal bleeding and shock-dengue shock syndrome caused by severe damage in the body, it is estimated that dengue, which is caused by severe dengue due to infection with dengue fever, is infected in 1 million human cases worldwide each year. Hemorrhagic fever is about 500,000 cases and 20,000 deaths.
- dengue virus dengue virus which causes dengue fever, causes immune-related complex reactions in the body, which has led to the treatment of dengue virus-related dengue diseases to date only in the treatment of 'dose headaches.
- ' 'Therapeutic standard' 'medicine and there is no effective 'can cure the amount of dengue virus activity and proliferation of ''medical' drugs or vaccines. Finding effective treatment for the treatment or prevention of dengue fever is the cause of research and development.
- Lonicera japonica Thunb (honeysuckle) is known to have a therapeutic effect on the symptoms of infection and fever.
- the present invention is to further understand whether drinking water extract of honeysuckle can specifically counter the disease caused by dengue virus, whether the water extract of honeysuckle can regulate the anti-viral microRNA immune mechanism in vivo, and whether the antiviral microRNA regulated by the anti-virus can resist dengue Virus, and its antiviral mechanism.
- honeysuckle Expected to study the establishment of honeysuckle The mechanism and efficacy of treating dengue virus, Jiahui patients worldwide.
- the problem to be solved by the present invention is to establish that honeysuckle is resistant to dengue virus and to control dengue virus by regulating the antiviral microRNA immunity in vivo.
- the technical scheme of the present invention is as follows:
- the preparation method is: Honeysuckle dried flower bud 10g Add 500 ml of water, boil and simmer for 20 minutes. This is a one-time dosage. Each treatment should be taken in the morning and evening for 4 days, then blood is collected. The microRNA expression profile in the blood is measured by Solexa deep sequencing. Change.
- MicroRNA was used for computer analysis to predict its potential for anti-dengue virus RNA genome and its large changes in total. This experiment and analysis prove that the honeysuckle water extract can simultaneously regulate several different microRNAs in the body. Many of these microRNAs can directly target the genes of dengue virus, so it is speculated that the water extract of honeysuckle can be increased at the same time.
- the anti-dengue virus microRNA achieves the simultaneous attack inhibition or regulation of the dengue virus, and greatly reduces the possibility of failure of the drug action caused by the mutation of the virus, ensuring that the honeysuckle water extract is resistant to dengue of different strains. The effectiveness of the virus and the persistence of the treatment.
- the selected 'microRNAs that are regulated by honeysuckle and highly expressed and computerized to predict that they are highly likely to be resistant to the dengue virus RNA genome are replicated in large quantities by RNA synthesis for use in cytological studies.
- let -7a is anti-dengue virus RNA
- the genome of the microRNA represents (biomarker), and computer analysis predicts that it is highly likely to be resistant to the NS1 gene of the dengue virus RNA genome.
- let-7a can inhibit the NS1 gene of the dengue virus RNA genome in the cell and thereby inhibit the dengue virus from producing NS1 protein, which affects the activity of the virus.
- the experiment proves that the honeysuckle water extract can inhibit the dengue virus from producing NS1 gene in the brain, and significantly reduce the NS1 protein produced by it, and reduce the amount of dengue virus in the brain of infected mice.
- Infected mice have milder signs of disease and longer survival.
- the expression of let-7a in infected mice showed tissue specificity: higher in the blood than in the negative control group, and greatly reduced in the brain. Rats who drank honeysuckle water extract without infection with dengue virus increased the expression of let-7a in the blood brain.
- honeysuckle is a "medical” medicine that can effectively inhibit the activity and proliferation of dengue virus.
- the experiment proves that the honeysuckle water extract can inhibit the dengue virus from producing NS1 gene in the brain, which can reduce the amount of dengue virus in the infected mouse brain without significantly reducing the NS1 protein produced by the virus.
- the signs of the disease in infected mice were not significantly milder.
- the expression of let-7a in infected mice showed tissue specificity: comparable to the negative control group in the blood and brain.
- the rats in which the honeysuckle water extract was not infected with the dengue virus had the same let-7a in the blood as the negative control group, and the let-7a in the brain was highly expressed.
- honeysuckle is indeed effective in inhibiting dengue virus activity.
- sexual and proliferative "theral" drugs in the treatment of dengue fever, can further try to match honeysuckle with some of the existing "treatment” drugs to treat symptoms of symptoms to achieve the "standard, the” and the effect of treatment.
- the present invention has the beneficial effects of confirming that the aqueous extract of honeysuckle can regulate the anti-dengue virus microRNA immunity in vivo, and the high expression of the anti-dengue virus microRNA let-7a can inhibit the dengue The activity of the leather virus.
- the honeysuckle water extract can simultaneously regulate a plurality of different anti-dengue virus microRNAs, and the honeysuckle water extract can directly and indirectly achieve the inhibition of dengue virus activity and quantity and the treatment of dengue fever.
- This study establishes the mechanism and efficacy of honeysuckle in the treatment of dengue virus as a "drug-based" drug that can effectively inhibit the activity and proliferation of dengue virus.
- the present invention requires that the honeysuckle water extract and let-7a microRNA can be prepared into a drug for preventing and treating anti-dengue virus. It can also be used to prepare health foods that are resistant to dengue fever.
- FIG. 1 Honeysuckle regulates microRNA expression profiles in mouse blood. Solexa sequencing data (shown by statistical standard deviation standard deviation data). The microRNAs that are highly expressed and resistant to the target gene of the dengue virus DV2 PL046 RNA genome are specifically noted.
- FIG. 1 Honeysuckle highly expresses the total amount of anti-dengue virus microRNA expression in mouse blood. Solexa sequencing data is presented in a chart.
- Figure 3. Iet-7a is a target gene against the NS1 gene of the dengue virus RNA genome.
- Figure 4. Iet-7a inhibits the NS1 gene of the dengue virus RNA genome and thereby inhibits dengue virus production of NS1 protein.
- (a) Cytological experiments were performed with expression vectors. The higher the amount of let-7a, the greater the inhibition of dengue virus gene expression.
- (b) The left panel shows that let-7a has an inhibitory effect on the normal dengue virus NS1 gene.
- let-7a has no inhibitory effect on the mutated dengue virus NS1 gene; thus, it is proved that let-7a does act on the The virus is on the NS1 gene.
- let-7a When cells infect live dengue virus, let-7a also has the same ability to inhibit viral activity. The higher the let-7a amount, the less NS1 gene can be produced by live virus (c) The amount of NS1 protein produced by it also decreased (d, e). Let-7a also inhibits the expression of Ras protein in abnormal cells caused by dengue virus (d).
- FIG. Honeysuckle regulates the amount of let-7a microRNA in the body against dengue virus in the blood. The amount of let-7a increases on the second day after drinking and changes periodically to 8 days.
- FIG. 6 Using "first injection of dengue virus into the rat brain to infect dengue virus, then drink honeysuckle water extract” 'to study the effect of honeysuckle water extract on dengue virus infection.
- Honeysuckle water extract can inhibit Deng
- the leather virus produces NS1 gene (d) in the brain, reduces the NS1 protein (e) produced by it, and reduces the amount of dengue virus in the brain of infected mice (f).
- the infected mice that drink the honeysuckle water extract have a longer survival period.
- the signs of the disease are relatively mild (b, c).
- the expression of let-7a in infected mice is tissue-specific (g, h), and it is speculated that the immune system regulated by honeysuckle is competitive with viral infection. It is suggested that the active constituents of the honeysuckle water extract and the in vivo regulation mechanism can directly and indirectly achieve the effect of inhibiting dengue virus, but have little effect on the pathogenesis of cytokine storm caused by
- FIG. 7 Using "first drink honeysuckle water extract, then inject dengue virus into the rat brain to make it infected.
- the dengue virus 'to study the efficacy of honeysuckle water extract for the prevention and treatment of dengue virus.
- the honeysuckle water extract inhibits the dengue virus from producing NS1 gene (d) in the brain, and reduces the amount of dengue virus (e, f) in the infected mouse brain without reducing the NS1 protein produced by it. Signs of disease in infected mice were not significantly altered (a, b, c).
- the expression of let-7a in infected mice showed tissue specificity (g, h). It is speculated that the immune system regulated by honeysuckle competes with viral infection. This experiment suggests that the active constituents of honeysuckle water extract and its in vivo regulation mechanism are directly Indirect ways to achieve the efficacy of inhibiting dengue virus. But it does not change the symptoms associated with cytokine storms caused by the virus.
- the ratio of the dose and weight of the person to drink is converted into the dose of the mouse (ie, the average adult weight of about 50kg must be 10 grams of honeysuckle dried flower buds as a one-time dose In the case of mice weighing 25 grams, 0.005 grams of honeysuckle dried flowers should be used as a one-time dose.
- the disposable dose of honeysuckle water extract in mice is 0.2 ml), and then the honeysuckle water is boiled according to the traditional Chinese medicine honeysuckle treatment. Feed the mice according to their medical condition (twice a day, drink for 4 consecutive days).
- MicroRNAs with high expression in the expression profile were first aligned with the miRBase (the microRNA database, www.mirbase.org) database to confirm the same microRNA in humans, and then with ViTa (virus' miRNA target, vita.mbc.nctu.edu. Tw/index.php) database for comparison prediction against dengue virus type 2 PL046 sequence (accession number AJ968413) RNA genome microRNA. Then statistically analyze the changes in the amount of micro-RNAs that promote the anti-dengue virus in the blood by drinking honeysuckle water extract. See Figure 1, 2.
- Immunofluorescence cytochemistry measures the location and expression of viral proteins in cells to demonstrate: (1). Iet-7a inhibits the activity of vectors containing dengue virus NS1 gene, but does not inhibit dengue virus NS1 gene mutants. The activity of the vector thus demonstrates that let-7a directly inhibits the target gene NS1. (2). Iet-7a inhibits the production of NS1 gene and the production of NS1 protein by living dengue virus. See Figure 3, 4.
- Honeysuckle regulates the amount of let-7a microRNA in the body against dengue virus in the blood.
- the honeysuckle water extract was cooked according to the traditional Chinese medicine honeysuckle treatment method and the mice were fed according to their medical treatment (twice a day for 4 consecutive days), and on the 0th, 2nd, 4th, 6th and 8th day.
- Blood samples were taken for real time PCR to measure the expression of let-7a, and the control group was water-fed mice.
- the real time PCR experiment confirmed that the amount of let-7a in the blood of mice fed with honeysuckle water extract was periodically changed to 8 days. See Figure 5.
- mice of the ICR strain received brain injection of a dose of dengue virus sufficient to cause lethal hemorrhagic dengue fever and shock-type dengue fever to cause infection.
- the honeysuckle water extract was started for 4 consecutive days;
- the NS1 and let-7a gene expression levels were measured by real time-PCR on the 12th day after birth, the NS1 protein expression was measured by Western blot, and the amount of dengue virus released from the brain of infected mice was measured by PFU forming assay. .
- the body weight, mortality, and disease symptoms of the rats were measured on the 6th to 13th day after birth. See Figure 6.
- NS1 and let-7a genes were measured by real time-PCR on the 13th day after birth, the expression of NS1 protein was detected by Western blot, and the amount of dengue virus released from the brain of infected mice was measured by PFU forming assay. On the 7-14th day, the body weight, mortality, and disease symptoms of the rats were measured. See Figure 7.
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Abstract
The invention belongs to the technical field of Chinese medicine herb pharmacy, and particularly relates to a honeysuckle flower water extract and an application thereof in preparation of a medicament and a health-care product for preventing and treating dengue viruses and dengue fever. According to the new application of the honeysuckle flower for improving the in-vitro microRNA immune function described in the present invention, the honeysuckle flower can be used for treating organism dengue virus infection. The invention further relates to an application of anti-dengue virus let-7amicroRNA regulated and controlled by using the honeysuckle flower in a medicament and a health-care product for treating dengue fever.
Description
金银花水提物和 let-7a microRNA在制备预防及治疗登革病毒和登革 热药物与保健品中的应用 Application of honeysuckle water extract and let-7a microRNA in the preparation and prevention of dengue virus and dengue fever medicines and health products
一：技术领域 One: technical field
本发明属中草药制药技术领域，具体涉及金银花水提物其在制备预防 和治疗登革病毒的药物与保健品中的应用。 The invention belongs to the technical field of Chinese herbal medicine, and particularly relates to the application of the aqueous extract of honeysuckle in preparing medicines and health care products for preventing and treating dengue virus.
二： 背景技术 Two: Background technology
登革热是热带与亚热带地区的蚊虫传染病， 每年全球约有 5亿至 10 亿人感染病例， 大多为居住在城市或城镇郊区的居民。其感染病症从 发烧疼痛的 dengue fever到严重内出血的 dengue hemorrhagic fever 与体内受损重创导致的休克型 dengue shock syndrome,估计在每年全 球 10亿人感染病例中因感染登革热而衍发为严重内出血的 dengue hemorrhagic fever约为 50万病例及 2万的死亡病例。 然而， 引起登 革热的登革病毒 dengue virus 其在体内会引起免疫相关的复杂反应， 因而导致至今治疗登革病毒相关的登革热疾病只有' '头痛医头， 脚痛 医脚' '的治疗症状的''治标' '药， 而没有确实有效能抑制登革病毒活性 与增生数量的''治本"药或是疫苗。寻找治疗或预防登革热的有效治疗 药为本研发的起因。 Dengue fever is a mosquito-borne disease in the tropics and subtropics. There are about 500 million to 1 billion cases of infection worldwide each year, mostly residents living in urban or urban suburbs. The dengue fever from a fever of dengue fever to dengue hemorrhagic fever with severe internal bleeding and shock-dengue shock syndrome caused by severe damage in the body, it is estimated that dengue, which is caused by severe dengue due to infection with dengue fever, is infected in 1 million human cases worldwide each year. Hemorrhagic fever is about 500,000 cases and 20,000 deaths. However, dengue virus dengue virus, which causes dengue fever, causes immune-related complex reactions in the body, which has led to the treatment of dengue virus-related dengue diseases to date only in the treatment of 'dose headaches.' 'Therapeutic standard' 'medicine, and there is no effective 'can cure the amount of dengue virus activity and proliferation of ''medical' drugs or vaccines. Finding effective treatment for the treatment or prevention of dengue fever is the cause of research and development.
中草药金银花 (Lonicera japonica Thunb; honeysuckle)已知其具有治疗 感染发热症状的疗效。本发明在于进一歩的了解饮用金银花水提物是 否能专门对抗 dengue virus 引起的疾病，金银花水提物是否能调控体 内抗病毒的 microRNA 免疫机制， 其调控的体内抗病毒的 microRNA 是否能抗登革病毒， 以及其抗病毒的机制。期望因此研究确立金银花
治疗登革病毒的机制与功效， 佳惠全世界的患者。 Lonicera japonica Thunb (honeysuckle) is known to have a therapeutic effect on the symptoms of infection and fever. The present invention is to further understand whether drinking water extract of honeysuckle can specifically counter the disease caused by dengue virus, whether the water extract of honeysuckle can regulate the anti-viral microRNA immune mechanism in vivo, and whether the antiviral microRNA regulated by the anti-virus can resist dengue Virus, and its antiviral mechanism. Expected to study the establishment of honeysuckle The mechanism and efficacy of treating dengue virus, Jiahui patients worldwide.
三： 发明内容 Three: Summary of the content
本发明需要解决的问题是确立金银花可抗登革病毒及其是以调控体 内抗病毒的 microRNA 免疫机制 (microRNA immunity)来对抗登革病 毒， 本发明的技术方案如下： The problem to be solved by the present invention is to establish that honeysuckle is resistant to dengue virus and to control dengue virus by regulating the antiviral microRNA immunity in vivo. The technical scheme of the present invention is as follows:
1. 以小鼠为实验动物模式， 按中医治疗方式喂食金银花水提物 (即中 医治病时病人所喝的金银花水提物， 俗称' '金银花茶 "； 制备方式为： 金银花干花苞 10克加水 500毫升， 大火煮开后文火煮 20分钟即可。 此为一次性服用剂量。 治疗期须早晚各服一剂， 连续服用 4天)， 然 后采血， 以 Solexa deep sequencing 测血中 microRNA表达谱的改变。 1. Using mouse as the experimental animal model, feeding the honeysuckle water extract according to the traditional Chinese medicine treatment method (that is, the honeysuckle water extract that the patient drinks when the Chinese medicine cures the disease, commonly known as ''Honeysuckle Tea'); the preparation method is: Honeysuckle dried flower bud 10g Add 500 ml of water, boil and simmer for 20 minutes. This is a one-time dosage. Each treatment should be taken in the morning and evening for 4 days, then blood is collected. The microRNA expression profile in the blood is measured by Solexa deep sequencing. Change.
2. 将血中 microRNA表达谱中因喝金银花水提物而高表达的 2. High expression of microRNA expression in blood due to drinking water extract of honeysuckle
microRNA做计算机分析预测其抗登革病毒 RNA genome的可能性及 其总量上的大幅度变化。此实验与分析证明金银花水提物可同时调控 体内数个不同的 microRNA, 这些 microRNA 中有许多可直接以登革 病毒的各个基因为目标，因而推测喝金银花水提物可因同时调高多种 抗登革病毒的 microRNA达到同时攻击抑制或调控登革病毒的功效， 并大大减少因病毒发生突变而产生的药作用点失效的可能性，保证了 此金银花水提物在对抗不同品系的登革病毒的有效性与疗方的持久 性。 MicroRNA was used for computer analysis to predict its potential for anti-dengue virus RNA genome and its large changes in total. This experiment and analysis prove that the honeysuckle water extract can simultaneously regulate several different microRNAs in the body. Many of these microRNAs can directly target the genes of dengue virus, so it is speculated that the water extract of honeysuckle can be increased at the same time. The anti-dengue virus microRNA achieves the simultaneous attack inhibition or regulation of the dengue virus, and greatly reduces the possibility of failure of the drug action caused by the mutation of the virus, ensuring that the honeysuckle water extract is resistant to dengue of different strains. The effectiveness of the virus and the persistence of the treatment.
3. 将筛选出的' '受金银花调控而高表达且计算机分析预测其极有可能 可抗登革病毒 RNA genome的 microRNA" 以 RNA 合成方式大量复制 以便运用于细胞学研究。 在此我们采用 let-7a 为抗登革病毒 RNA
genome的 microRNA代表 (biomarker) ,计算机分析预测其极有可能可 抗登革病毒 RNA genome的 NS1 gene 为目标基因。 3. The selected 'microRNAs that are regulated by honeysuckle and highly expressed and computerized to predict that they are highly likely to be resistant to the dengue virus RNA genome" are replicated in large quantities by RNA synthesis for use in cytological studies. Here we use let -7a is anti-dengue virus RNA The genome of the microRNA represents (biomarker), and computer analysis predicts that it is highly likely to be resistant to the NS1 gene of the dengue virus RNA genome.
4. 细胞学研究中， 我们以 luciferase assay, real time-PCR, Western blot, immunofluorescence cytochemistry分另 iji正明 let-7a可抑制含有登革病 毒 NS1 gene的载体的活性，可抑制活体登革病毒制造 NS1 gene 及生 成 NS1 蛋白质。 因而证实 let-7a确实可在细胞内抑制登革病毒 RNA genome的 NSl gene并从而抑制登革病毒制造 NS1 蛋白质， 影响病 毒的活性。 4. In cytology studies, we can inhibit the activity of the vector containing dengue virus NS1 gene by luciferase assay, real time-PCR, Western blot, immunofluorescence cytochemistry, and inhibit the production of NS1 by living dengue virus. Gene and generate NS1 protein. Thus, it was confirmed that let-7a can inhibit the NS1 gene of the dengue virus RNA genome in the cell and thereby inhibit the dengue virus from producing NS1 protein, which affects the activity of the virus.
5. 小鼠模式研究中， real time PCR 证实喝金银花水提物的小鼠其血 液中 let-7a会在开始喝后的第 2天呈现高表达，并呈周期改变至 8天。 5. In the mouse model study, real time PCR confirmed that let-7a in the blood of mice fed with honeysuckle water extract showed high expression on the 2nd day after the start of drinking, and changed periodically to 8 days.
6. 小鼠模式研究中， 我们采用' '先注射登革病毒入鼠大脑内使其感染 登革病毒， 再喝金银花水提物"以研究金银花水提物对感染登革病毒 的疗效。 6. In the mouse model study, we used ''first injection of dengue virus into the rat brain to infect dengue virus, then drink honeysuckle water extract" to study the efficacy of honeysuckle water extract on dengue virus infection.
实验证明金银花水提物可抑制登革病毒在脑内制造 NSl gene ,并明显 降低其所生成的 NS1 蛋白质， 与减少感染鼠脑内的登革病毒数量。 感染鼠的疾病征兆比较轻微， 存活期较长。 感染鼠体内 let-7a表达量 呈现组织特异性： 在血液中比阴性对照组高， 在脑中大为减少。 喝金 银花水提物而没感染登革病毒的鼠其血中脑中 let-7a 表达量提高。感 染鼠喝金银花水提物者虽然其脑内 NS1 与其脑内的登革病毒数量下 降， 但其体内 let-7a表达量在血与脑中和感染鼠没喝金银花水提物者 相当。我们推测此为金银花调控的各个免疫***与病毒感染的竞争关 系。此实验建议金银花水提物其活性成份与体内调控机制以直接与间
接的方式达到抑制登革病毒的疗效。其可作用于抑制足以引起致命性 出血型登革热与休克型登革热的登革病毒剂量，但其对改变病毒引起 的细胞素风暴 (cytokine storm)相关的病兆影响较微。 我们依此认为金 银花为能确实有效能抑制登革病毒活性与增生数量的"治本' '药，在治 疗登革热时可进一步尝试将金银花与现有的一些治疗病兆症状的"治 标"药配伍以达到 "标， 本"兼治的效果。 The experiment proves that the honeysuckle water extract can inhibit the dengue virus from producing NS1 gene in the brain, and significantly reduce the NS1 protein produced by it, and reduce the amount of dengue virus in the brain of infected mice. Infected mice have milder signs of disease and longer survival. The expression of let-7a in infected mice showed tissue specificity: higher in the blood than in the negative control group, and greatly reduced in the brain. Rats who drank honeysuckle water extract without infection with dengue virus increased the expression of let-7a in the blood brain. Infected rats with honeysuckle water extracts, although the number of dengue viruses in the brain and NS1 in the brain decreased, but the expression of let-7a in the body was comparable in blood and brain to the infected rats who did not drink honeysuckle water extract. We speculate that this is the competitive relationship between various immune systems regulated by honeysuckle and viral infection. This experiment suggests that the active constituents of the honeysuckle water extract and the in vivo regulation mechanism are directly and indirectly The way to connect is to inhibit the efficacy of dengue virus. It acts to inhibit dengue virus doses sufficient to cause lethal hemorrhagic dengue and shock-type dengue fever, but it has little effect on the symptoms associated with changing the cytokine storm caused by the virus. We believe that honeysuckle is a "medical" medicine that can effectively inhibit the activity and proliferation of dengue virus. In the treatment of dengue fever, we can further try to match honeysuckle with some existing "medicated" drugs for treating symptoms. Achieve the effect of "standard, this" and cure.
7. 小鼠模式研究中， 我们采用''先喝金银花水提物， 再注射登革病毒 入鼠大脑内使其感染登革病毒"以研究金银花水提物对预防防治登革 病毒的疗效。 7. In the mouse model study, we used ''first drink honeysuckle water extract, then injected dengue virus into the rat brain to make it infected with dengue virus" to study the efficacy of honeysuckle water extract on prevention and treatment of dengue virus.
实验证明金银花水提物可抑制登革病毒在脑内制造 NSl gene ,其在没 有明显降低其所生成的 NS1 蛋白质状况下， 可减少感染鼠脑内的登 革病毒数量。 感染鼠的疾病征兆没有明显的比较轻微。 感染鼠体内 let-7a表达量呈现组织特异性： 在血液和脑中与阴性对照组相当。 喝 金银花水提物而没感染登革病毒的鼠其血中 let-7a 与阴性对照组相 当， 其脑中 let-7a被高表达。 感染鼠喝金银花水提物者虽然其脑内 NSl gene 与其脑内的登革病毒数量下降，但其体内 let-7a表达量在血 中呈现被抑制状态， 在脑中和感染鼠没喝金银花水提物者相当。我们 推测此为金银花调控的各个免疫***与病毒感染的竞争关系。此实验 建议金银花水提物其活性成份与体内调控机制以直接与间接的方式 达到抑制登革病毒的疗效。其可作用于抑制足以弓 I起致命性出血型登 革热与休克型登革热的登革病毒剂量，但其不改变病毒引起的细胞素 风暴相关的病兆。我们依此认为金银花为能确实有效抑制登革病毒活
性与增生数量的"治本"药，在治疗登革热时可进一步尝试将金银花与 现有的一些治疗病兆症状的 "治标 "药配伍以达到"标， 本"兼治的效 呈。 本发明与现有技术相比，其有益效果是证实喝金银花水提物可调控体 内抗登革病毒 microRNA 免疫*** （microRNA immunity)，其中的抗登 革病毒 microRNA let-7a的高表达可抑制登革病毒的活性。金银花水提 物可同时调控多个不同的抗登革病毒 microRNA, 金银花水提物从而 以直接与间接的方式达到抑制登革病毒的活性与数量及治疗登革热 的疗效。此研究确立金银花治疗登革病毒的机制与功效为能确实有效 抑制登革病毒活性与增生数量的"治本' '药。依此我们认为在治疗登革 热时可进一歩尝试将金银花与现有的一些治疗病兆症状的''治标' '药 配伍以达到' '标，本''兼治的效果。本发明所要求金银花水提物和 let-7a microRNA可制备成预防和治疗抗登革病毒的药物， 也可治备成抗登 革热的保健食品。 The experiment proves that the honeysuckle water extract can inhibit the dengue virus from producing NS1 gene in the brain, which can reduce the amount of dengue virus in the infected mouse brain without significantly reducing the NS1 protein produced by the virus. The signs of the disease in infected mice were not significantly milder. The expression of let-7a in infected mice showed tissue specificity: comparable to the negative control group in the blood and brain. The rats in which the honeysuckle water extract was not infected with the dengue virus had the same let-7a in the blood as the negative control group, and the let-7a in the brain was highly expressed. Infected rats with honeysuckle water extracts, although the number of dengue viruses in the brain and the number of dengue viruses in the brain decreased, but the expression of let-7a in the body was inhibited in the blood, and the rats in the brain did not drink honeysuckle water. The extractor is quite. We speculate that this is the competitive relationship between various immune systems regulated by honeysuckle and viral infection. This experiment suggests that the active constituents of the honeysuckle water extract and its in vivo regulation mechanism can directly and indirectly achieve the efficacy of inhibiting dengue virus. It acts to inhibit dengue virus doses sufficient to cause lethal hemorrhagic dengue and shock-type dengue, but it does not alter the cytokine storm-related signs caused by the virus. We believe that honeysuckle is indeed effective in inhibiting dengue virus activity. Sexual and proliferative "theral" drugs, in the treatment of dengue fever, can further try to match honeysuckle with some of the existing "treatment" drugs to treat symptoms of symptoms to achieve the "standard, the" and the effect of treatment. Compared with the prior art, the present invention has the beneficial effects of confirming that the aqueous extract of honeysuckle can regulate the anti-dengue virus microRNA immunity in vivo, and the high expression of the anti-dengue virus microRNA let-7a can inhibit the dengue The activity of the leather virus. The honeysuckle water extract can simultaneously regulate a plurality of different anti-dengue virus microRNAs, and the honeysuckle water extract can directly and indirectly achieve the inhibition of dengue virus activity and quantity and the treatment of dengue fever. This study establishes the mechanism and efficacy of honeysuckle in the treatment of dengue virus as a "drug-based" drug that can effectively inhibit the activity and proliferation of dengue virus. We believe that in the treatment of dengue fever, try to compare honeysuckle with existing ones. The ''therapeutic standard' for the treatment of symptoms of the disease's drug compatibility to achieve the effect of 'standard, present' and concurrent treatment. The present invention requires that the honeysuckle water extract and let-7a microRNA can be prepared into a drug for preventing and treating anti-dengue virus. It can also be used to prepare health foods that are resistant to dengue fever.
四： 附图说明 Four: BRIEF DESCRIPTION OF THE DRAWINGS
图 1. 金银花可调控小鼠血中 microRNA表达谱。 Solexa sequencing 数 据 (以统计学的 standard deviation 标准偏差数据显示)。其中各个高表 达且能抗登革病毒 DV2 PL046 RNA genome的目标基因的 microRNA有 特别注明。 Figure 1. Honeysuckle regulates microRNA expression profiles in mouse blood. Solexa sequencing data (shown by statistical standard deviation standard deviation data). The microRNAs that are highly expressed and resistant to the target gene of the dengue virus DV2 PL046 RNA genome are specifically noted.
图 2. 金银花可高表达小鼠血中抗登革病毒 microRNA表达量总量。 Solexa sequencing 数据以统计图呈现。
图 3. Iet-7a 可抗登革病毒 RNA genome的 NS1 gene 为目标基因。 图 4. Iet-7a可在细胞内抑制登革病毒 RNA genome的 NS1 gene并从而 抑制登革病毒制造 NS1 蛋白质。 （a).以表达量载体做细胞学实验， let-7a 的量越高，对登革病毒基因表达抑制力越大。 (b).左图显示 let-7a 对正常的登革病毒 NS1基因有抑制力，右图显示 let-7a对突变的登革 病毒 NS1基因失去抑制力； 因此证明 let-7a确实是作用在登革病毒 NS1基因上。 （c, d, e).当细胞感染活的登革病毒时， let-7a也有同样的 抑制病毒活性的能力， let-7a量越高， 活病毒能制造的 NS1 gene就越 少 (c)， 其制出的 NS1 protein 量也下降 (d, e)。 let-7a对登革病毒引起 的不正常细胞 Ras protein表达也具有抑制作用 (d)。 Figure 2. Honeysuckle highly expresses the total amount of anti-dengue virus microRNA expression in mouse blood. Solexa sequencing data is presented in a chart. Figure 3. Iet-7a is a target gene against the NS1 gene of the dengue virus RNA genome. Figure 4. Iet-7a inhibits the NS1 gene of the dengue virus RNA genome and thereby inhibits dengue virus production of NS1 protein. (a). Cytological experiments were performed with expression vectors. The higher the amount of let-7a, the greater the inhibition of dengue virus gene expression. (b). The left panel shows that let-7a has an inhibitory effect on the normal dengue virus NS1 gene. The right panel shows that let-7a has no inhibitory effect on the mutated dengue virus NS1 gene; thus, it is proved that let-7a does act on the The virus is on the NS1 gene. (c, d, e). When cells infect live dengue virus, let-7a also has the same ability to inhibit viral activity. The higher the let-7a amount, the less NS1 gene can be produced by live virus (c) The amount of NS1 protein produced by it also decreased (d, e). Let-7a also inhibits the expression of Ras protein in abnormal cells caused by dengue virus (d).
图 5. 金银花调控体内抗登革病毒的 let-7a microRNA在血中的量呈周 期改变: let-7a的量于喝后第 2天上升， 呈周期性改变至 8天。 Figure 5. Honeysuckle regulates the amount of let-7a microRNA in the body against dengue virus in the blood. The amount of let-7a increases on the second day after drinking and changes periodically to 8 days.
图 6. 采用"先注射登革病毒入鼠大脑内使其感染登革病毒，再喝金银 花水提物' '以研究金银花水提物对感染登革病毒的疗效。 金银花水提 物可抑制登革病毒在脑内制造 NSl gene (d) , 降低其所生成的 NS1 蛋 白质 （e)，减少感染鼠脑内的登革病毒数量 （f)。喝金银花水提物的感 染鼠其存活期较长 （a)， 疾病征兆比较轻微 （b, c)。 感染鼠体内 let-7a 表达量呈现组织特异性 （g, h)， 据此推测金银花调控的各个免疫*** 与病毒感染呈竞争关系，此实验建议金银花水提物其活性成份与体内 调控机制以直接与间接的方式达到抑制登革病毒的疗效。但对改变病 毒引起的细胞素风暴相关的病兆影响较微。 Figure 6. Using "first injection of dengue virus into the rat brain to infect dengue virus, then drink honeysuckle water extract" 'to study the effect of honeysuckle water extract on dengue virus infection. Honeysuckle water extract can inhibit Deng The leather virus produces NS1 gene (d) in the brain, reduces the NS1 protein (e) produced by it, and reduces the amount of dengue virus in the brain of infected mice (f). The infected mice that drink the honeysuckle water extract have a longer survival period. (a), the signs of the disease are relatively mild (b, c). The expression of let-7a in infected mice is tissue-specific (g, h), and it is speculated that the immune system regulated by honeysuckle is competitive with viral infection. It is suggested that the active constituents of the honeysuckle water extract and the in vivo regulation mechanism can directly and indirectly achieve the effect of inhibiting dengue virus, but have little effect on the pathogenesis of cytokine storm caused by virus.
图 7. 采用"先喝金银花水提物，再注射登革病毒入鼠大脑内使其感染
登革病毒' '以研究金银花水提物对预防防治登革病毒的疗效。 金银花 水提物可抑制登革病毒在脑内制造 NS1 gene (d)， 在不降低其所生成 的 NS1 蛋白质状况下减少感染鼠脑内的登革病毒数量 （e, f)。 感染鼠 的疾病征兆改变不明显 （a, b, c)。 感染鼠体内 let-7a表达量呈现组织 特异性 （g, h)， 据此推测金银花调控的各个免疫***与病毒感染呈竞 争关系，此实验建议金银花水提物其活性成份与体内调控机制以直接 与间接的方式达到抑制登革病毒的疗效。但不改变病毒引起的细胞素 风暴相关的病兆。 Figure 7. Using "first drink honeysuckle water extract, then inject dengue virus into the rat brain to make it infected. The dengue virus 'to study the efficacy of honeysuckle water extract for the prevention and treatment of dengue virus. The honeysuckle water extract inhibits the dengue virus from producing NS1 gene (d) in the brain, and reduces the amount of dengue virus (e, f) in the infected mouse brain without reducing the NS1 protein produced by it. Signs of disease in infected mice were not significantly altered (a, b, c). The expression of let-7a in infected mice showed tissue specificity (g, h). It is speculated that the immune system regulated by honeysuckle competes with viral infection. This experiment suggests that the active constituents of honeysuckle water extract and its in vivo regulation mechanism are directly Indirect ways to achieve the efficacy of inhibiting dengue virus. But it does not change the symptoms associated with cytokine storms caused by the virus.
五： 具体实施方式 Five: specific implementation
1. 金银花调控小鼠血中 microRNA表达谱与其计算机预测。 1. Honeysuckle regulates microRNA expression profiles in mice and its computer prediction.
参考中医金银花治发烧感染的医方与中华人民共和国药典，将人喝的 剂量与体重之比例换算成小鼠喝的剂量 (即以一般成人约 50kg的体重 须 10克金银花干花苞为一次性剂量，换算成体重为 25克的小鼠就须 0.005克金银花干花苞为一次性剂量。 小鼠喝的金银花水提物的一次 性剂量为 0.2毫升)，然后按照中医的金银花治疗方式煮金银花水提物 并依其医疗方式喂食小鼠 (一日两次， 连续喝 4天)。然后采血，抽 RNA, 交予深圳华大基因公司（BGI, www.genomics.cn)进行 microRNA的 Solexa deep sequencing与数据分析形成表达谱。 表达谱中高表达的 microRNA 先与 miRBase (the microRNA database, www.mirbase.org) 数据库做比对以确认鼠与人相同的 microRNA,再与 ViTa (virus' miRNA target, vita.mbc.nctu.edu.tw/index.php)数据库做比对预测可抗登革病 毒 dengue virus type 2 PL046 sequence (accession number AJ968413)
RNA genome的 microRNA。然后以统计方式分析了解喝金银花水提物 对提高血中抗登革病毒的 microRNA其量上的改变。 见附图 1, 2。Refer to the Chinese medicine honeysuckle treatment of fever infection of the doctor and the Pharmacopoeia of the People's Republic of China, the ratio of the dose and weight of the person to drink is converted into the dose of the mouse (ie, the average adult weight of about 50kg must be 10 grams of honeysuckle dried flower buds as a one-time dose In the case of mice weighing 25 grams, 0.005 grams of honeysuckle dried flowers should be used as a one-time dose. The disposable dose of honeysuckle water extract in mice is 0.2 ml), and then the honeysuckle water is boiled according to the traditional Chinese medicine honeysuckle treatment. Feed the mice according to their medical condition (twice a day, drink for 4 consecutive days). Then, blood was collected, RNA was extracted, and submitted to Shenzhen Huada Gene Company (BGI, www.genomics.cn) for Solexa deep sequencing and data analysis of microRNA to form an expression profile. MicroRNAs with high expression in the expression profile were first aligned with the miRBase (the microRNA database, www.mirbase.org) database to confirm the same microRNA in humans, and then with ViTa (virus' miRNA target, vita.mbc.nctu.edu. Tw/index.php) database for comparison prediction against dengue virus type 2 PL046 sequence (accession number AJ968413) RNA genome microRNA. Then statistically analyze the changes in the amount of micro-RNAs that promote the anti-dengue virus in the blood by drinking honeysuckle water extract. See Figure 1, 2.
2. 细胞学实验证明抗登革病毒的 microRNA可抑制病毒活性。 2. Cytological experiments demonstrate that anti-dengue virus microRNAs inhibit viral activity.
我们以可受金银花调控而高表达且计算机预测可抗登革病毒的 microRNA let-7a为研究对象， 以其预测的登革病毒的 NS1 gene 为目 标基因做细胞学研究。我们以 Huh7 肝癌细胞株为细胞模式，运用 real time-PCR 测基因表达量， Western blot 测蛋白质表达量， We studied microRNA let-7a, which is highly regulated by honeysuckle and computer-predicted against dengue virus, and cytological studies using its predicted NS1 gene of dengue virus as the target gene. We used Huh7 hepatocellular carcinoma cell line as the cell model, measured the gene expression by real time-PCR, and measured the protein expression by Western blot.
immunofluorescence cytochemistry测病毒蛋白质在细胞中的分布位置 与是否被表达来分别证明： (1). Iet-7a可抑制含有登革病毒 NSl gene的 载体的活性， 但不能抑制含有登革病毒 NSl gene突变体的载体的活 性， 因而证明 let-7a 是直接抑制目标基因 NS1。 （2). Iet-7a 可抑制活 体登革病毒制造 NSl gene 及生成 NS1 蛋白质。 见附图 3, 4。 Immunofluorescence cytochemistry measures the location and expression of viral proteins in cells to demonstrate: (1). Iet-7a inhibits the activity of vectors containing dengue virus NS1 gene, but does not inhibit dengue virus NS1 gene mutants. The activity of the vector thus demonstrates that let-7a directly inhibits the target gene NS1. (2). Iet-7a inhibits the production of NS1 gene and the production of NS1 protein by living dengue virus. See Figure 3, 4.
3. 金银花调控体内抗登革病毒的 let-7a microRNA在血中的量呈周期 改变。 3. Honeysuckle regulates the amount of let-7a microRNA in the body against dengue virus in the blood.
小鼠模式研究中，按照中医的金银花治疗方式煮金银花水提物并依其 医疗方式喂食小鼠 (一日两次， 连续喝 4天)， 并于第 0, 2, 4, 6, 8 日采 血以 real time PCR 测 let-7a 的表达量， 对照组为喝水的小鼠。 real time PCR 实验证实喝金银花水提物的小鼠其血液中 let-7a的量呈周 期改变至 8天。 见附图 5。 In the mouse model study, the honeysuckle water extract was cooked according to the traditional Chinese medicine honeysuckle treatment method and the mice were fed according to their medical treatment (twice a day for 4 consecutive days), and on the 0th, 2nd, 4th, 6th and 8th day. Blood samples were taken for real time PCR to measure the expression of let-7a, and the control group was water-fed mice. The real time PCR experiment confirmed that the amount of let-7a in the blood of mice fed with honeysuckle water extract was periodically changed to 8 days. See Figure 5.
4. 研究金银花水提物对感染登革病毒的疗效。 4. To study the efficacy of honeysuckle water extract on dengue virus infection.
小鼠模式研究中， 采用 "先注射登革病毒入鼠大脑内使其感染登革病 毒， 再喝金银花水提物' '的方式以研究金银花水提物对感染登革病毒
的疗效。 ICR 品系的小鼠于出生后第 6日接受大脑注射足以引起致命 性出血型登革热与休克型登革热的登革病毒剂量以使其感染，第 7日 开始喝金银花水提物，连续喝 4天；于出生后第 12日以 real time-PCR 测 NS1 与 let-7a 基因表达量， Western blot 测 NS1 蛋白质表达量， PFU forming assay (空斑形成单位） 测感染鼠其脑中释放的登革病毒 数量。 于出生后第 6-13日测鼠的体重， 死亡率， 疾病症状指标。 见 附图 6。 In the mouse model study, the method of "injecting dengue virus into the brain of the rat to infect the dengue virus and then drinking the honeysuckle water extract" was used to study the dengue virus infection of the honeysuckle water extract. Efficacy. On the 6th day after birth, mice of the ICR strain received brain injection of a dose of dengue virus sufficient to cause lethal hemorrhagic dengue fever and shock-type dengue fever to cause infection. On the 7th day, the honeysuckle water extract was started for 4 consecutive days; The NS1 and let-7a gene expression levels were measured by real time-PCR on the 12th day after birth, the NS1 protein expression was measured by Western blot, and the amount of dengue virus released from the brain of infected mice was measured by PFU forming assay. . The body weight, mortality, and disease symptoms of the rats were measured on the 6th to 13th day after birth. See Figure 6.
5. 研究金银花水提物对预防防治登革病毒的疗效。 5. To study the efficacy of water extract of honeysuckle on preventing and controlling dengue virus.
小鼠模式研究中， 采用"先喝金银花水提物， 再注射登革病毒入鼠大 脑内使其感染登革病毒' '的方式以研究金银花水提物对预防防治登革 病毒的疗效。 ICR 品系的小鼠于出生后第 4日开始喝金银花水提物， 连续喝 4天；于出生后第 7日接受大脑注射足以引起致命性出血型登 革热与休克型登革热的登革病毒剂量以使其感染， 于出生后第 13日 以 real time-PCR 测 NS1 与 let-7a 基因表达量， Western blot 测 NS1 蛋白质表达量， PFU forming assay 测感染鼠其脑中释放的登革病毒数 量。 于出生后第 7-14日测鼠的体重， 死亡率， 疾病症状指标。 见附 图 7。
In the mouse model study, the effect of extracting honeysuckle water extract on the prevention and treatment of dengue virus was studied by "drinking honeysuckle water extract and then injecting dengue virus into the rat brain to infect dengue virus". The mice of the strain began to drink the honeysuckle water extract on the 4th day after birth, and continued to drink for 4 days. On the 7th day after birth, the brain was injected with a dose of dengue virus sufficient to cause lethal hemorrhagic dengue fever and shock-type dengue fever. Infection, the expression of NS1 and let-7a genes was measured by real time-PCR on the 13th day after birth, the expression of NS1 protein was detected by Western blot, and the amount of dengue virus released from the brain of infected mice was measured by PFU forming assay. On the 7-14th day, the body weight, mortality, and disease symptoms of the rats were measured. See Figure 7.
Claims
1. 一种金银花水提物或其影响的 let-7a microRNA，其特征是金银花干 花苞 10克加水 500毫升， 大火煮开后文火煮 20分钟。 1. A honeysuckle water extract or its affected let-7a microRNA, characterized by 10 grams of honeysuckle dried flower buds and 500 ml of water, boiled for 20 minutes after simmering.
2. 权利要求 1所述金银花水提物在制备预防或治疗登革病毒或登革 热药物中的应用。 2. The use of the aqueous extract of honeysuckle according to claim 1 for the preparation of a medicament for preventing or treating dengue virus or dengue fever.
3. 权利要求 1所述金银花水提物在制备预防或治疗登革病毒或登革 热保健品中的应用。 3. The use of the aqueous extract of honeysuckle according to claim 1 for the preparation or prevention of dengue virus or dengue health care products.
4. 权利要求 1所述金银花水提物影响的 let-7a microRNA 在制备预防 或治疗登革病毒或登革热药物中的应用。 4. Use of let-7a microRNA affected by water extract of honeysuckle according to claim 1 for the preparation of a medicament for preventing or treating dengue virus or dengue fever.
5. 权利要求 1所述金银花水提物影响的 let-7a microRNA 在制备预防 或治疗登革病毒或登革热保健品中的应用。
5. The use of let-7a microRNA affected by the honeysuckle water extract of claim 1 for the preparation of a prophylactic or therapeutic dengue virus or dengue health product.
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| CN101352475A (en) * | 2008-04-01 | 2009-01-28 | 南京大学 | Use of honeysuckle aqueous extract in preparing medicament for improving insulin resistance |
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| Title |
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| XIONG, XIN ET AL.: "The Research of Gastric Cancer-related MicroRNA", PROGRESS IN MODERN BIOMEDICINE, vol. 9, no. 2, January 2009 (2009-01-01), pages 395 - 396 AND 400 * |
| ZHOU, JUEYU ET AL.: "Time course of let-7a expression in the cell cycle of human cervical carcinoma HeLa cells", JOURNAL OF SOUTHERN MEDICAL UNIVERSITY, vol. 29, no. 3, March 2009 (2009-03-01), pages 442 - 445 * |
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