| Gouty arthritis(GA)is a common inflammatory arthropathy induced by monosodium urate(MSU)crystals deposited in joints and surrounding tissues,and is directly related to hyperuricemia.The incidence and prevalence of gout has steadily increased over the past decades,posing a great threat to people’s health.Although colchicine and NSAIDS are used as clinical first-line drugs in acute gout arthritis,their toxic and side effects cannot be ignored,so it is imperative to find new safe drugs.Traditional Chinese medicine(TCM)has a unique advantage and provides a great contribution to clinical practice.Therefore,it is of great significance to study the Chinese medicine preparation for prevention and treatment of gout.Bike pian(BKP)is a traditional Miao nationality medicinal formula that contain seven herbal medicines.In clinical practice,BKP acts to dispel dampness and remove heat,activate blood and relieve pain,it is generally used to treat on muscle and joint pain,such as rheumatoid arthritis and osteoarthritis.However,whether BKP can improve gouty arthritis and its function and possible mechanism remain unclear.Objective: Study on preclinical pharmacodynamics of BKP in treatment of GA and exploration of related pharmacological mechanism.Methods: 1.Network pharmacology method was used to predict the potential targets and molecular mechanism of BKP in treatment of GA.We searched the targets information corresponding to the active ingredient and the targets related to GA.Constructed a protein interaction network with the common targets.Screened out the core target of BKP in treatment of GA.Finally,we carried out the GO and KEGG enrichment analysis.2.To evaluate the analgesic effect of BKP,we established two kinds of pain models in mice.The hot plate test and the acetic acid-induced abdominal constriction test were set up in the control group and aspirin group,and the low-dose,medile-dose and high-dose groups of BKP were treated.In the hot plate experiment of mice,the pain threshold of mice was detected at four time points: 30 min,60 min,90 min and 120 min.In the acetic acid-induced abdominal constriction test of mice,the writhing times and writhing latency of mice within 15 minutes were recorded.3.To evaluate the anti-inflammatory effect of BKP,we constructed two animal models of GA.The gouty arthritis models of SD rats and New Zealand rabbits were induced by MSU,the changes of foot swelling in rats were detected within 1-8 h and the number of white blood cells in joint fluid of New Zealand rabbits was detected after injection of MSU for 5 h.The HE staining was used to observe the inflammatory infiltration of GA rats and New Zealand rabbits.4.Phorboxylate induced THP-1 cells to differentiate into M0 macrophages,which were stimulated by MSU to construct gout cell model in vitro.SD rats were given BKP for 7 days continuously,and the serum of rats was extracted as medicated serum.Western blotting was used to detect the protein expression levels of NLRP3,ASC and Caspase-1 in the control group,MSU group,5% medicated serum group,10% medicated serum group and 20% medicated serum group,and the content of IL-1β in each group was detected by ELISA.Results: 1.The 106 active ingredients were screened out from BKP and finally 236 targets were screened.The 289 genes related to GA diseases were retrieved from Gene Cards database and Drug Bank database,and it was considered that all these genes were involved of GA.After intersected with drug targets,45 targets of drugs acting on GA were obtained.Subsequently,10 core genes were screened out and enriched,and it was found that they mainly involved in actively regulating inflammatory response and regulating inflammatory pathways,such as IL-17 signaling pathway,TNF signaling pathway,AGERAGE signaling pathway in diabetic complications and NF-Kappa B signaling pathway.2.Hot plate experiment in mice: compared with the control group,the pain threshold of mice in-middle dose and high-dose groups of BKP could be significantly prolonged.Acetic acid-induced abdominal constriction test in mice: compared with the control group,the latency of the first writhing in the in middle-dose group and the high-dose group of BKP was significantly increased.As for the twisting times,the BKP of in middle-dose group and high-dose group were significantly lower than those of control group in 15 min.3.Animal model of gouty arthritis: Rat model: After injected MSU,the foot swelling of rats was obvious increased,the BKP could significantly improve the degree of swelling.With the increase of dose,the improvement effect of BKP on the degree of swelling gradually increased.In the New Zealand rabbit model,the white blood cell count in the knee fluid in low-dose,middle-dose and high-dose groups of BKP was significantly lower than the model group,which suggested that BKP has an obvious inhibitory.4.In vitro cell model: The protein levels of NLRP3,ASC and Caspase-1 were elevated after MSU stimulation,and the expression was down-regulated in a dose-dependent manner after the addition of drug-containing serum.The IL-1β levels increased significantly after MSU stimulation,and decreased in a dose-dependent manner after drug serum preconditioning.The results indicate that medicated serum could inhibit the activation of NLRP3 Inflammasome in THP-1 macrophages induced by MSU.Conclusion: 1.BKP plays an anti-inflammatory role in treatment GA through multiple targets by analysis of network pharmacology.2.Pharmacodynamic results show that BKP has a good anti-inflammatory and analgesic effects.3.BKP can improve the inflammation of gout cell model in vitro by inhibiting the activation of NLRP3 inflammasome. |
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