Application of combination of triptolide and quercetin in rheumatoid arthritis diseases

Abstract

The invention discloses application of a combination of triptolide and quercetin in rheumatoid arthritis diseases, and belongs to the field of medicines for treating rheumatoid arthritis. The molecular mechanism of XFC for improving RA inflammation symptoms is explored through methods such as network pharmacology, animal experiments, data mining, molecular docking and the like. And finally, establishing an adjuvant arthritis rat model, and verifying the influence of XFC on inflammation-related signal pathways and inflammatory factors. Experiments show that the XFC can effectively improve clinical inflammation indexes of RA. The effective components of the XFC can improve the inflammatory state of RA by adjusting a tlr signal channel. Animal experiments prove that the combination of triptolide and quercetin can be applied to treatment of rheumatoid arthritis diseases.

Description

Technical Field

[0001] The invention belongs to the field of rheumatoid arthritis therapeutic drugs, and specifically relates to an application of a combination of triptolide and quercetin in rheumatoid arthritis. Background Art

[0002] Rheumatoid arthritis is a common systemic autoimmune disease, mainly manifested as invasive arthritis, with an incidence rate that increases between the ages of 25-55, affecting about 0.5-1% of adults worldwide, with a higher prevalence in women than in men. The disease is characterized by symmetrical injuries to single or multiple joints of the hand or wrist, often manifested as swelling and pain, limited mobility, deformed mobility, and even joint dislocation, and can also be complicated by lung, kidney, cardiovascular disease, eye lesions, and other diseases involving multiple organ systems throughout the body.

[0003] The traditional Chinese medicine Xinfeng Capsule (XFC), as an in-hospital preparation of the First Affiliated Hospital of Anhui University of Traditional Chinese Medicine, mainly includes astragalus, coix seed, and tripterygium wilfordii, which can invigorate qi and strengthen the spleen, eliminate dampness and dredge meridians, and has a significant effect on relieving inflammatory symptoms such as joint pain and swelling in patients with rheumatoid arthritis. However, the underlying mechanism of action remains to be elucidated. Summary of the invention

[0004] The present invention intends to explore the molecular mechanism of XFC in improving RA inflammatory symptoms through network pharmacology, animal experiments, data mining, molecular docking and other methods. The Apriori association rule and random walk model were used to evaluate the effect of XFC on clinical inflammatory indicators of RA. The effective ingredients and potential target genes of XFC were obtained from public databases. Based on the STRING database, DAVID database, Cytoscape software and molecular docking method, the molecular mechanism of XFC acting on RA was analyzed. Finally, an adjuvant arthritis rat model was established to verify the effect of XFC on inflammation-related signaling pathways and inflammatory factors.

[0005] The experimental results of the present invention show that XFC significantly reduces the levels of C-reactive protein and vascular endothelial growth factor, and reduces the erythrocyte sedimentation rate. The docking spatial structure of the active ingredients of XFC, triptolide and quercetin, with key targets is stable. Inflammation-related biological processes are considered to be key factors involved in the development of RA, and the regulation of the toll-like receptor (TLR) signaling pathway may be the key link for XFC to improve the inflammatory state of RA. XFC significantly reduces the levels of inflammatory factors interleukin-6 and tumor necrosis factor-α, and inhibits the expression of TRL4, TLR5, and TLR8. Conclusion: XFC can effectively improve the clinical inflammatory indicators of RA. The active ingredients of XFC improve the inflammatory state of RA by regulating the TLR signaling pathway. Animal experiments have confirmed that the drug combination of triptolide and quercetin can be used in the treatment of rheumatoid arthritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0006] Figure 1 Figure 2. The improvement of inflammatory indicators in RA patients after XFC treatment. (A) Apriori association rule model analysis of the correlation between XFC and ESR, CRP, SAA, and VEGF. (B) Comparison of the differences in ESR, CRP, SAA, and VEGF between the XFC group and the control group before and after treatment. (C) Description of the random walk model to evaluate the improvement of XFC on CRP, ESR, and VEGF.

[0007] Figure 2 Screening of XFC active ingredients and targets. (A) PPI network diagram depicting 64 potential targets; (B) Diagram depicting the relationship between TCM, active ingredients and potential target networks. (C) Diagram depicting the top 15 potential network targets.

[0008] Figure 3 Molecular docking of XFC active ingredients with key target genes. (A) FOS and triptolide; (B) JUN and triptolide; (C) RELA and triptolide; (D) STAT1 and triptolide; (E) TNF and triptolide; (F) VEGFA and triptolide; (G) il-1b and quercetin; (H) MMP9 and quercetin.

[0009] Figure 4 KEGG and GO enrichment analysis. (A) The top 15 entries in KEGG enrichment analysis; (B) The top 15 entries in GO BP enrichment analysis; (C) The entries in GO CC enrichment analysis; (D) The top 15 entries in GO MF enrichment analysis.

[0010] Figure 5 Effects of XFC on inflammatory factors and TLR signaling pathways. (A) Correlation analysis of TLR4, TLR5, TLR8 and IL-6; (B) Correlation analysis of TLR4, TLR5, TLR8 and TNF-a; (C) Heat map of inflammatory factors and pathway protein expression; (D) ELISA results describing the levels of IL-6 and TNF-α in each group, ***P<0.001, **P<0.01 (n=10); (E) Western blotting was used to detect the levels of TLR4, TLR5 and TLR8 in each group, ***P<0.001, **P<0.01, *P<0.05 (n=10). (F) Western blotting results of TLR4, TLR5 and TLR8 bands in each group.

[0011] Figure 6 This is a comparison of joint swelling, arthritis index, TNF-α, and IL-1β levels in each group of animal experiments. DETAILED DESCRIPTION

[0012] Example 1

[0013] 1. Using data mining methods to examine the effect of XFC on clinical inflammatory indicators of RA

[0014] 1.1 Apriori association rule analysis between XFC and RA inflammation indicators

[0015] In the Apriori association rule model, the minimum confidence and minimum support are designed to be 50% and 90%, respectively. When the support is >90%, the improvement of XFC on VEGF and ESR increases significantly, which are 1.046 and 1.014, respectively. The improvement of XFC on CRP is lower, which is 0.992, but it reaches the lowest support and confidence. The improvement rate of XFC on SAA is 0.968, which is higher than CRP; however, the support rate and confidence are the lowest. The results of Apriori association rule analysis show that XFC is closely related to the improvement of VEGF, ESR and CRP ( Figure 1 A).

[0016] 1.2 Comparison of clinical inflammatory indicators before and after XFC treatment

[0017] Comparing the differences in CRP, ESR, SAA, and VEGF before and after treatment, the XFC group was superior to the control group in terms of CRP, ESR, and VEGF reduction (P<0.01 or P<0.001) ( Figure 1 B). The random walk model was used to further evaluate the effect of XFC on CRP, ESR, and VEGF in RA patients. The improvement coefficients of inflammatory indicators in the XFC group and the control group were ESR 0.642, 0.566, CRP 0.623, 0.605, VEGF 0.642, 0.612, respectively. The higher the improvement coefficient or improvement coefficient, the more obvious the improvement index of the corresponding group, and the better the treatment effect ( Figure 1 C). The above results suggest that XFC has a good effect on improving clinical inflammatory indicators.

[0018] Construction of 2XFC molecular targets and inflammation-related signaling pathways for the treatment of RA

[0019] 2.1 Screening of XFC active ingredients and potential targets

[0020] Based on the gene chip data of GSE55235 in the GEO gene database, differentially expressed genes in RA were identified. All component data of Tripterygium wilfordii, Astragalus membranaceus, and Coix lachryma-jobi were retrieved from TCMSP. The obtained active ingredients were input into the DrugBank database to obtain the active ingredient binding target genes. Using the intersection of active ingredient binding target genes and differentially expressed genes in RA, a total of 64 potential XFC targets for the treatment of RA were obtained ( Figure 2A)(Supplementary Table S1). The potential targets are 16 active ingredients of Tripterygium wilfordii, 25 active ingredients of Astragalus membranaceus, and 5 active ingredients of Coix lachryma-jobi. A network diagram of traditional Chinese medicine xfc-active ingredients-potential targets was constructed ( Figure 2 B). The topological MCC algorithm was used to analyze the significance of 64 potential targets, of which the top 15 were TNF, IL6, MMP9, VEGFA, PTGS2, JUN, CXCL8, FOS, MYC, EGFR, IL1B, NFKBIA, PTEN, RELA, and STAT1 ( Figure 2 C).

[0021] 2.2 Molecular docking of XFC active ingredients and potential targets

[0022] Active ingredients, such as triptolide, quercetin, and sphenanthene, docked with the top 15 potential targets, namely TNF, MMP9, VEGFA, PTGS2, JUN, FOS, EGFR, IL1B, PTEN, RELA, and STAT1. The binding energy <0 indicates that the potential gene and the active ingredient can bind spontaneously; the lower the binding energy, the more stable the binding conformation. The binding energy <-4.25 kcal / mol indicates a certain binding activity. The binding energy of triptolide for FOS, JUN, RELA, TNF, VEGFA, and STAT1 is <-4.25 kcal / mol, and the binding energy of quercetin for MMP9 and IL1B is <-4.25 kcal / mol. This result shows that the docked structure is stable ( Figure 3 ). Triptolide and quercetin were identified as the active ingredients of XFC. FOS, JUN, RELA, TNF, VEGFA, STAT1, MMP9 and IL1B were identified as the key targets of XFC in the treatment of RA.

[0023] 2.3 KEGG and GO enrichment analysis

[0024] The DAVID database was used to perform GO and KEGG enrichment analysis on key targets. The screening condition P<0.05, and the top 15 entries were selected for display. KEGG analysis obtained a total of 63 signaling pathways, including the TLR signaling pathway ( Figure 4 A). A total of 101 entries were obtained from GO BP analysis, including responses to cytokines and inflammatory responses ( Figure 4 B). A total of 8 entries were obtained from the graphene oxide CC analysis, including the extracellular region and secretory granules ( Figure 4 C). A total of 17 entries were obtained from the graphene oxide MF analysis, including tumor necrosis factor receptor binding and cytokine activity ( Figure 4D) This result suggests that affecting inflammation-related biological processes and TLR signaling pathways may be an important mechanism for XFC to treat RA.

[0025] Effects of 3XFC on TLR signaling pathway and inflammatory factors in rats with adjuvant arthritis

[0026] Correlation analysis showed that TLR4 was positively correlated with IL-6 and TNF-α, TLR5 was positively correlated with TNF-α, and TLR8 was positively correlated with IL-6 and TNF-α ( Figure 5 AB). This finding suggests that excessive activation of the TLR signaling pathway may aggravate the inflammatory response. ELISA results showed that the levels of inflammatory factors IL-6 and TNF-α in the MC group were significantly higher than those in the NC group (P<0.001). This result suggests that the inflammatory factors in the joints of the model rats are unbalanced. After XFC treatment, the levels of IL-6 and TNF-α were significantly reduced (P<0.01 or P<0.001)( Figure 5 CD). Western blotting results showed that the expression of TLR4, TLR5, and TLR8 proteins in the MC group were significantly or extremely significantly higher than those in the NC group (P<0.01 or P<0.001). This observation indicated that the TLR signaling pathway was overactivated in the joints of the model rats. After XFC intervention, the expression of TLR4, TLR5, and TLR8 proteins was significantly decreased (P<0.05 or P<0.01) ( Figure 5 EF). These results suggest that XFC may inhibit the TLR signaling pathway through its activation state and further reduce the level of inflammatory factors.

[0027] Example 2

[0028] Animal experiments

[0029] Twelve clean-grade male Wistar rats with an average body weight of 206.14±22.75g were selected and randomly divided into 4 groups. There were 3 rats in each of the model group, quercetin group, triptolide group, and triptolide + quercetin group. The rats were injected with 0.1mL of Freud's complete adjuvant from the left hind foot pad to the lateral side of the ankle joint to establish an arthritis model. Based on the clinical human once-daily dose, triptolide and quercetin (100mg / kg) were converted to rat dosing. At the same time, normal saline was given according to the same scheme. Each group was treated with medication for 14 consecutive days, once a day. Triptolide (batch number: PHL80555) and quercetin (batch number: PHR1488) were purchased from Merck. This animal experiment was approved by the Animal Ethics Committee of the First Affiliated Hospital of Anhui University of Traditional Chinese Medicine (No.: AHUCM-rats-2021022).

[0030] IL-1β (JYM0419Ra) and TNF-α (JYM0635Ra) levels were diluted according to the kit instructions (Genemei Biotechnology Corp., Wuhan, China). The absorbance values ​​were determined using a microplate reader (Ledo, Shenzhen, China). The standard curve was drawn using the standard concentration and the corresponding absorbance value, and the final concentrations of TNF-α and IL-1β in the serum samples were calculated after considering the dilution factor.

[0031] The experimental results are as follows Figure 6 As shown in the figure, compared with the model group, the joint swelling, arthritis index, TNF-α, and IL-1β levels of the triptolide group, quercetin group, and triptolide + quercetin group were significantly reduced; this indicates that the intervention of Chinese medicine monomer components can effectively reduce the level of inflammation. Compared with the quercetin group, the joint swelling, arthritis index, and TNF-α level of the triptolide group were significantly reduced; this indicates that the anti-inflammatory effect of triptolide is greater than that of quercetin. Compared with the triptolide and quercetin groups, the joint swelling, arthritis index, TNF-α, and IL-1β levels of the triptolide + quercetin group were significantly reduced; this indicates that the simultaneous use of triptolide and quercetin can exert a stronger anti-inflammatory effect.

[0032] The above contents are merely examples and explanations of the concept of the present invention. The technicians in this technical field may make various modifications or additions to the specific embodiments described or replace them in a similar manner. As long as they do not deviate from the concept of the invention or exceed the scope defined by the claims, they should all fall within the protection scope of the present invention.

Claims

1. An application of a combination of triptolide and quercetin in rheumatoid arthritis, characterized in that: A drug combination containing triptolide and quercetin can be used as a therapeutic drug for rheumatoid arthritis.

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