The Mechanism On Chinese Medicine Huzhen Tongfeng Formula On Acute Gouty Arthritis

Aim:Combining network pharmacology and experimental verification,the efficacy of Huzhen Tongfeng Formula(HZTF)on acute gouty arthritis(AGA)was evaluated,and the underlying mechanism was explored,providing a theoretical basis for clinical application and formulation development of HZTF.Method:1.Network Pharmacology EvaluationThe network pharmacology is used to analyze the potential mechanism of HZTF on AGA.The specific steps are as follows: First,the effective target and target database of single herbs of HZTF are retrieved and screened through the TCMSP database;then,from TTD,Drug Bank and other databases,screen the targets of AGA-related and build disease database;further,map the targets of the two databases to obtain common targets,use Cytoscape software to build the target network of HZTF on AGA,and use DAVID 6.8 to enrich to get the potential mechanism of HZTF on AGA.2.In vivo and in vitro verification experiments(1)Establishment of AGA ModelC57BL/6 mice were used to establish a therapeutic model of AGA model.Monosodium urate(MSU)was used as the inducer to explore the induction dose and induction time respectively.By comparing the joint swelling degree and proinflammatory factor level,the most appropriate dose and time were found to build a stable model.(2)Efficacy of HZTF on AGAUse HZTF to treat MSU-induced gouty arthritis model,observe the degree of joint swelling,change in gait,and detect mRNA expression of proinflammatory substances in joint tissue,nitric oxide(NO)and metabolic factors in joint washing fluid content,to evaluate the effect of HZTF on AGA.(3)Cell Level ExperimentBased on the mechanism prediction of network pharmacology,the experiment was verified in vitro.Combined with molecular biological techniques,such as MTT,qPCR,ELISA,Western Blot,etc.,the potential mechanism of HZTF was further explored.Result:1.Network Pharmacology EvaluationBased on the conditions of oral availability(OB)≥ 30% and drug-likeness(DL)≥ 0.18,34 active ingredients related to HZTF were screened,corresponding to 181 targets.Thus,5 common databases such as TTD provided 224 targets of AGA-related,and protein-protein interaction network was showed through Cytoscape software.Mutation parameters were obtained through the network analysis function.Based on the "Degree" ≥ 12,"Closeness" ≥ 0.309,and "Betweenness Centrality" ≥ 0.002,77 important targets were obtained.GO enrichment results indicate that multiple targets are involved in biological processes such as transcription-positive regulation of the RNA polymerase II promoter,inflammatory response,extra cellular space,and hemebinding.The enrichment of KEGG pathway suggests that the arachidonic acid pathway is far more relevant than other pathways.2.In vivo and in vitro verification experiments(1)Establishment of AGA ModelThe severity of gouty arthritis caused by different induction time(12,24,48,72 h)and different MSU induction doses(4,8,20 mg/mL)is different,and the phenomenon of attenuation and regression occurs over 24 hours.There is no correlation with the induced dose.In summary,the model induced by 8 mg/mL MSU for 12 h can be used for subsequent efficacy evaluation.(2)Efficacy of HZTF on AGAAfter treatment with HZTF,gouty arthritis caused by MSU was improved,such as reduced joint swelling,improved gait,down-regulated expression of inflammatory substances in joint tissues,and reduced levels of NO and inflammatory factors in joint washing fluid.(3)Cell Level ExperimentHZTF was not found to have significant toxicity to RAW264.7 cells.HZTF has a significant inhibitory effect on the transcription and production of inflammatory substances such as IL-1β,IL-6,TNF-α,and COX-2.And,intracellular reactive oxygen species(ROS)level was down-regulated.WesternBlot showed that the HZTF significantly inhibited the expression of COX-1,COX-2 and 5-LO proteins related to the arachidonic acid pathway.Conclusion:1.The results of network pharmacological analysis indicate that the arachidonic acid metabolic pathway is the most relevant signaling pathway involved in HZTF on AGA.2.Pharmacodynamic results show that HZTF could significantly alleviate gouty arthritis induced by MSU in mice,including significantly improve joint swelling,gait,and inflammation at joint sites.3.We confirmed in vivo and in vitro experiments that HZTF alleviates the inflammatory response in AGA by inhibiting the arachidonic acid pathway and inflammatory cytokines and mediators(such as IL-1β,IL-6,TNF-α,COX-2).