Application of rare ginsenoside in preparation of medicine for improving intestinal flora imbalance and inhibiting rheumatoid arthritis

Abstract

The invention discloses an application of rare ginsenoside in preparation of a medicine for improving intestinal dysbacteriosis and inhibiting rheumatoid arthritis, including an application of rare ginsenoside Rk3 in preparation of a medicine for improving intestinal dysbacteriosis and an application of rare ginsenoside Rk3 in preparation of a medicine for inhibiting rheumatoid arthritis. Rare ginsenoside Rk3 can improve the richness and diversity of intestinal flora of mice with rheumatoid arthritis, enrich probiotics and regulate bile acid metabolism, so that intestinal flora imbalance caused by rheumatoid arthritis is effectively relieved, and intestinal barrier function damage is repaired; the generation of inflammatory factors can be reduced, a TLR4-NF-kappa B signal channel is inhibited, and joint injury in an RA model is treated. Based on investigation of causal association among ginsenoside Rk3, intestinal flora and rheumatoid arthritis, clinical application of rare ginsenoside Rk3 in rheumatoid arthritis is provided, and new strategies and candidate drugs are provided for treatment of rheumatoid arthritis.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the use of a rare ginsenoside in the preparation of a drug for improving intestinal flora imbalance and inhibiting rheumatoid arthritis. Background Art

[0002] Rheumatoid arthritis is one of the most common immune-mediated inflammatory diseases, characterized by symmetrical joint pain, swelling, and stiffness. Globally, approximately 1% of adults suffer from rheumatoid arthritis. The pathophysiology of rheumatoid arthritis is very complex, and there is currently no therapy to treat and eliminate this systemic, multifactorial, inflammatory, and progressive autoimmune disease. Increasing evidence shows that intestinal microbiota dysbiosis is closely related to rheumatoid arthritis. Given the important role of intestinal microorganisms, the complex interaction between the host and the intestinal flora has become a hot area of ​​research on the pathogenic mechanism of rheumatoid arthritis or related coping strategies.

[0003] In recent years, a large number of studies have provided convincing evidence that natural compounds can have a positive impact on the restoration of intestinal homeostasis by affecting the intestinal microbiota and its metabolites. Ginseng belongs to the Araliaceae family and has extremely high medicinal value and good safety. It has been used to treat various diseases and preventive health care. Ginsenosides, as the main active ingredients of ginseng, have multiple pharmacological activities, including anti-inflammatory, antioxidant, anti-tumor and anti-diabetic effects. However, there are no reports on the treatment of rheumatoid arthritis with ginsenoside Rk3. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a rare ginsenoside for the preparation of drugs for improving intestinal flora imbalance and inhibiting rheumatoid arthritis in view of the deficiencies of the above-mentioned prior art. On the one hand, based on the treatment of systemic inflammation and the improvement of intestinal flora in mice with rheumatoid arthritis, experiments show that the rare ginsenoside Rk3 can improve the richness and diversity of intestinal flora in arthritis mice, enrich probiotics, and regulate bile acid metabolism, thereby effectively alleviating intestinal flora imbalance caused by rheumatoid arthritis and repairing intestinal barrier function damage; on the other hand, it is to inhibit rheumatoid arthritis drugs. Experimental results show that the rare ginsenoside Rk3 can reduce the production of inflammatory factors, inhibit the TLR4-NF-κB signaling pathway, and treat joint damage in RA models. Based on the investigation of the causal relationship between ginsenoside Rk3-intestinal flora-rheumatoid arthritis, the present invention provides a clinical application of a rare ginsenoside Rk3 in the treatment of rheumatoid arthritis, which helps to provide new strategies and candidate drugs for the treatment of rheumatoid arthritis.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is: application of a rare ginsenoside Rk3 in the preparation of a drug for improving intestinal flora imbalance.

[0006] Furthermore, the invention also provides a use of a rare ginsenoside Rk3 in the preparation of a drug for inhibiting rheumatoid arthritis.

[0007] The above application is characterized in that the rare ginsenoside Rk3 can change the diversity and richness of the intestinal flora of mice.

[0008] The above application is characterized in that the rare ginsenoside Rk3 can upregulate the beneficial intestinal bacteria Akkmensiamuciniphila and eliminate the harmful intestinal bacteria Desulfovibrionaceae and Neisseria.

[0009] The above application is characterized in that the rare ginsenoside Rk3 can alleviate weight loss, paw swelling and intestinal barrier function damage caused by rheumatoid arthritis.

[0010] The above application is characterized in that the rare ginsenoside Rk3 can reduce the production of inflammatory factors, inhibit the TLR4-NF-κB signaling pathway, and treat joint damage in the RA model.

[0011] The above application is characterized in that the drug includes pharmaceutically acceptable excipients.

[0012] The above application is characterized in that the dosage form of the drug includes tablets, capsules, granules, pills, oral liquids, injections or infusions.

[0013] Compared with the prior art, the present invention has the following advantages:

[0014] 1. The present invention is based on the treatment of systemic inflammation and improvement of intestinal flora in mice with rheumatoid arthritis, and provides an application of a rare ginsenoside Rk3 in the preparation of a drug for improving intestinal flora imbalance. The rare ginsenoside Rk3 can change the diversity and richness of the intestinal flora of mice, upregulate the beneficial intestinal bacteria Akkmensia muciniphila, eliminate the harmful intestinal bacteria Desulfovibrionaceae and Neisseria, enrich probiotics, regulate bile acid metabolism, and significantly promote the production of beneficial secondary bile acid taurodeoxycholic acid (THDCA), thereby alleviating the intestinal flora imbalance caused by rheumatoid arthritis and repairing the damage to the intestinal barrier function.

[0015] 2. Based on the investigation of the causal relationship among ginsenoside Rk3, intestinal flora and rheumatoid arthritis, the present invention provides an application of a rare ginsenoside Rk3 in the preparation of a drug for inhibiting rheumatoid arthritis. The rare ginsenoside Rk3 can relieve weight loss, paw swelling and intestinal barrier function damage caused by rheumatoid arthritis, reduce the production of inflammatory factors, inhibit the TLR4-NF-κB signaling pathway, and treat joint damage in the RA model, which helps to provide new strategies and candidate drugs for the treatment of rheumatoid arthritis.

[0016] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 (AC) Alpha diversity analysis represented by Chao, Shannon and Ace indices.

[0018] Figure 2 (AC) β-diversity analysis of gut microbiota using NMDS, PCoA and PCA.

[0019] Figure 3 Linear discriminant analysis effect size (LEfSe) analysis was performed.

[0020] Figure 4 Species differences analysis of Akkmensiamuciniphila.

[0021] Figure 5 Heat map analysis of differential metabolites.

[0022] Figure 6 (AC) Body weight, paw thickness, and arthritis score of CIA mice.

[0023] Figure 7 These are H&E staining and safranin fast green staining images of mouse joints.

[0024] Figure 8 Immunoblotting was used to analyze the expression of intestinal tight junction proteins ZO-1 and Occludin.

[0025] Fig. 9 is the level of IL-4, IL-6, IL-17α and TNF-α in serum.

[0026] Fig.10 Immunoblotting was used to analyze the expression of proteins related to the TLR4-NF-κB signaling pathway. DETAILED DESCRIPTION

[0027] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail in combination with the examples below. The following examples use commercially available rare ginsenoside Rk3 (purity> 97%). Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used, unless otherwise specified, can all be obtained from commercial channels.

[0028] Example 1

[0029] The present invention provides an animal experiment to detect the application of ginsenoside Rk3 in improving the intestinal flora of rheumatoid arthritis mice (CIA) and its metabolites in inhibiting the development of rheumatoid arthritis. The specific experimental process includes: mixing 2 mg / mL bovine type II collagen with 4 mg / mL complete Freund's adjuvant or incomplete Freund's adjuvant in a ratio of 1:1. On day 0, mice were immunized initially at the tail with 100 μL CII / CFA emulsion, and on day 21, they were immunized again at the tail with 100 μL CII / IFA emulsion. They were divided into Rk3-H group, Rk3-L group, normal group, model group and positive drug MTX group. Mice in Rk3-H group were gavaged with ginsenoside Rk3 once a day at a dose of 80 mg / kg Rk3, and mice in Rk3-L group were gavaged with 40 mg / kg Rk3 for 21 consecutive days. Mice in the normal group and model group were gavaged with normal saline, and mice in the positive drug MTX group were gavaged once every 3 days at a dose of 1.5 mg / kg. After 21 days, the animals were analyzed after the end of the experiment. The analysis methods included H&E staining, SO / FG staining, 16S rRNA sequencing analysis, metabolomics analysis, immunoblotting analysis and enzyme-linked immunosorbent assay.

[0030] H&E staining: The tissues were fixed with paraformaldehyde and then embedded in paraffin, cut into 5 μm sections and mounted on slides, and then stained with hematoxylin-eosin.

[0031] SO / FG staining: The paraffin sections of the joints were dewaxed to water, then stained with Safranin O and Fast Green, and finally dehydrated and mounted.

[0032] 16S rRNA sequencing analysis: 16S rRNA sequencing was used to analyze the changes in intestinal microorganisms, including: extracting total DNA from feces using an OmegaBio-Tek commercial kit, identifying the purity and concentration of DNA by electrophoresis using a NanoDrop ND-1000 spectrophotometer, a NanoDrop ND-1000 spectrophotometer (Thermo Fisher), and agarose gel; PCR amplification of bacterial genomic DNA using a fusion primer reaction system, and sequencing of the highly variable V3-V4 region of the bacterial 16SrRNA gene. PCR amplification was performed using primers 338F (5'barcode+ACTCCTACGGGAGGCAGCA-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3') specific to the V3-V4 region. The collected results were analyzed for sequence analysis according to the library construction standard instructions on the Illumina MiSeq platform.

[0033] Metabolomics analysis: Liquid chromatography-tandem mass spectrometry (LC-MS / MS) technology is used to separate and identify metabolites, specifically including: first using liquid chromatography (LC) to separate the sample to obtain metabolites, using tandem mass spectrometry (MS / MS) for detection and identification, converting the obtained data into relative abundance and mass, comparing with the standard metabolite database to determine the identity of the metabolites, and using bioinformatics tools for analysis to determine their functions and roles in biological processes.

[0034] Analysis of immunoblotting experiments: The colon tissue was completely lysed with RIPA buffer, and then separated by electrophoresis using sodium dodecyl sulfate polyacrylamide gel. The obtained proteins were transferred to polyvinylidene fluoride membrane (PVDF membrane), incubated with the primary antibody at 4°C overnight, and then incubated with the secondary antibody at room temperature for 1 hour. The PVDF membrane after the secondary incubation was immersed in the prepared ECL luminescent developer, and then placed in the developer for development. The obtained protein quantitative blotting images were quantitatively analyzed using ImageJ software.

[0035] Enzyme-linked immunosorbent assay: The levels of IL-4, IL-6, IL-17α and TNF-α in the serum of different groups of mice were detected according to the standard method of the enzyme-linked immunosorbent (ELISA) kit instructions.

[0036] Experimental results: (1) Ginsenoside Rk3 can improve the intestinal flora and its metabolites in rheumatoid arthritis mice.

[0037] The present invention uses CII / CFA to construct a rheumatoid arthritis model, and conducts a ginsenoside Rk3 administration experiment to analyze the effects of ginsenoside Rk3 on the intestinal flora and its metabolites of rheumatoid arthritis mice.

[0038] Figure 1 Ginsenoside Rk3 improves the intestinal flora of mice with rheumatoid arthritis. Among them, the Chao, Shannon and Ace indices represented by alpha diversity analysis showed that the Chao, Shannon and Ace indices of mice in the model group were significantly higher than those in the normal group and the drug-treated group.

[0039] Figure 2 NMDS, PCoA and PCA were used to analyze the beta diversity of the intestinal microbiota, and the results showed that there was a significant separation among the control group, CIA group and Rk3 group.

[0040] Combination Figure 1 and Figure 2 It can be seen that ginsenoside Rk3 treatment has a regulatory effect on the diversity of intestinal flora in mice, indicating that ginsenoside Rk3 treatment can improve the diversity and richness of intestinal flora.

[0041] Figure 3 The linear discriminant analysis effect size (LEfSe) analysis showed that ginsenoside Rk3 could enrich the beneficial bacteria Akkmensia and Verrucomicrobia, and effectively eliminate the harmful intestinal bacteria Desulfovibrionaceae and Neisseria.

[0042] Figure 4 This is a species difference analysis of Akkmensia muciniphila in each group of mice. It can be seen that ginsenoside Rk3 can significantly upregulate the abundance of the beneficial bacteria Akkmensia muciniphila.

[0043] Figure 5 The differential metabolite expression levels were used to measure the improvement effect of ginsenoside Rk3 on the intestinal flora metabolites of rheumatoid arthritis mice. Figure 5 The cluster analysis results showed that ginsenoside Rk3 significantly upregulated the beneficial metabolites Taurochenodeoxycholic acid (TCDCA) and Taurohyodeoxycholic Acid (THDCA).

[0044] Combination Figure 1 , Figure 2 , Figure 3 , Figure 4 and Figure 5 It can be seen that treatment with ginsenoside Rk3 can improve the composition of intestinal microbiota, especially significantly upregulate the abundance of beneficial bacteria Akkmensia muciniphila, and further regulate intestinal microbiota metabolites, thereby improving the imbalance of intestinal microbiota in CIA mice.

[0045] (2) Ginsenoside Rk3 can treat severe cases of rheumatoid arthritis

[0046] The present invention adopts CII / CFA to construct a rheumatoid arthritis model, and conducts a ginsenoside Rk3 administration experiment to analyze the effect of ginsenoside Rk3 on the development of rheumatoid arthritis.

[0047] Figure 6 The inhibitory effect of ginsenoside Rk3 on the occurrence of rheumatoid arthritis was demonstrated. Figure 6 (AC) are graphs of mouse body weight, paw thickness and AI score. It can be seen that ginsenoside Rk3 can significantly increase mouse body weight, reduce paw swelling and reduce arthritis scores.

[0048] Figure 7 These are H&E staining and safranin fast green staining pictures of mouse joints. The results show that ginsenoside Rk3 can reduce the inflammatory infiltration and bone destruction of rheumatoid arthritis, and the condition is significantly improved compared with the CIA group.

[0049] Figure 8 Immunoblotting analysis of the expression of intestinal tight junction proteins ZO-1 and Occludin showed that ginsenoside Rk3 upregulated the expression of intestinal tight junction proteins ZO-1 and Occludin.

[0050] Fig. 9 (AD) ELISA detected the expression levels of inflammatory factors such as IL-4, IL-6, IL-17α and TNF-α. It can be seen that ginsenoside Rk3 can reduce the content of pro-inflammatory cytokines and increase the level of anti-inflammatory cytokines.

[0051] Fig.10 The expression of proteins related to the TLR4-NF-κB signaling pathway was analyzed by immunoblotting. It can be seen that ginsenoside Rk3 inhibits the TLR4-NF-κB signaling pathway, indicating that ginsenoside Rk3 in the present invention can inhibit the development of rheumatoid arthritis by regulating intestinal flora and reducing the release of inflammatory factors.

[0052] Combination Figure 6 , Figure 7 , Figure 8 , Fig. 9 and Fig.10 It can be seen that ginsenoside Rk3 can significantly alleviate the symptoms of rheumatoid arthritis such as weight loss, paw swelling and intestinal barrier function damage, and can further reduce the production of inflammatory factors, inhibit the TLR4-NF-κB signaling pathway, and inhibit the development of rheumatoid arthritis.

[0053] In summary, on the one hand, rare ginsenoside Rk3 can change the diversity and richness of the intestinal flora of mice, upregulate the beneficial intestinal bacteria Akkmensia muciniphila, and eliminate the harmful intestinal bacteria Desulfovibrionaceae and Neisseria, and can be used to prepare drugs for improving intestinal flora imbalance; on the other hand, rare ginsenoside Rk3 can relieve weight loss, paw swelling and intestinal barrier function damage caused by rheumatoid arthritis, reduce the production of inflammatory factors, inhibit the TLR4-NF-κB signaling pathway, and treat joint damage in RA models, and can be used to prepare drugs for inhibiting rheumatoid arthritis, providing new strategies and candidate drugs for the inhibitory treatment of rheumatoid arthritis; the above-mentioned drugs include pharmaceutically acceptable excipients, and the dosage forms of the drugs include tablets, capsules, granules, pills, oral solutions, injections or infusions.

[0054] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent structural change made to the above embodiment according to the technical essence of the invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. Application of a rare ginsenoside Rk3 in the preparation of drugs for improving intestinal flora imbalance.

2. Application of a rare ginsenoside Rk3 in the preparation of drugs for inhibiting rheumatoid arthritis.

3. The use according to claim 1, characterized in that: The rare ginsenoside Rk3 can change the diversity and richness of the intestinal flora of mice.

4. The use according to claim 1, characterized in that: The rare ginsenoside Rk3 can upregulate the beneficial intestinal bacteria Akkmensia muciniphila and eliminate the harmful intestinal bacteria Desulfovibrionaceae and Neisseria.

5. The use according to claim 2, characterized in that: The rare ginsenoside Rk3 can alleviate weight loss, paw swelling and intestinal barrier function damage caused by rheumatoid arthritis.

6. The use according to claim 2, characterized in that: The rare ginsenoside Rk3 can reduce the production of inflammatory factors, inhibit the TLR4-NF-κB signaling pathway, and treat joint damage in RA models.

7. The use according to any one of claims 1 to 6, characterized in that: The drug includes pharmaceutically acceptable excipients.

8. The use according to any one of claims 1 to 6, characterized in that: The dosage form of the drug includes tablets, capsules, granules, pills, oral liquids, injections or infusions.

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