| ObjectiveBy constructing a new clinical mouse model of gouty arthritis,and using this model to further explore the efficacy and mechanism of Simiao decoction in the treatment of gouty arthritis,and to clarify the role of gut microbiota in the pathogenesis of gouty arthritis and the treatment of Simiao decoction in gout.Methods1.Construction and evaluation of compound gout modelTwenty-eight specific pathogen-free(SPF)C57BL/6 mice(male,4-week-old,15±3g)were randomly divided into four groups after 7 days of acclimatization:(1)control group(CT,fed a normal diet and injected with PBS solution);(2)hyperuricemia model group(HFD,fed a high-fat diet(10%yeast extract)and injected with PBS solution);(3)acute gout model(MSU,fed a normal diet and injected with monosodium urate(MSU)crystals(1 mg MSU crystals in 40 μL PBS/mouse every 10 days);(4)compound gout model(CGM,fed a high-fat diet(10%yeast extract)and injected with MSU crystals(1 mg MSU crystals in 40μL PBS/mouse every 10 days).The levels of serum uric acid,the swelling,and the pain of the footpad were assessed on days 22,32,and 42.To explore a suitable gout mouse model,the severity of the disease induced by three different modeling methods was evaluated by detecting serum uric acid,foot swelling,pain threshold,inflammatory factors,gut microbiota,and other indicators of gout disease.2.Efficacy and mechanism of conventional administration of Simiao decoction in the treatment of goutForty-two SPF C57BL/6 mice(male,4-week-old,15±3g)were randomly divided into two groups after 7 days of acclimatization:(1)control group(CT,fed a normal diet and injected with PBS solution,N=7);(2)compound gout model group(fed a high-fat diet and injected with MSU,N=35).After 7 days,the model group was further divided into five subgroups(7 mice/group):(Ⅰ)model group(Model,oral gavage with sterile water every day);(Ⅱ)low-dose of Simiao group(Low-SM,Simiao decoction was given by oral gavage daily at 4.0g/kg);(Ⅲ)middle-dose of Simiao group(Middle-SM,Simiao decoction was given by oral gavage daily at 8.0g/kg);(Ⅳ)high-dose of Simiao group(High-SM,Simiao decoction was given by oral gavage daily at 16.0g/kg every day);(Ⅴ)febuxostat group(Febuxostat,febuxostat was given by oral gavage daily at 5.0mg/kg).The swelling and the pain of the footpad were assessed 48 h after each injection of MSU.To explore the efficacy and mechanism of Simiao decoction in treating gouty arthritis,after the intervention of Simiao decoction with different concentrations,the changes in serum uric acid,foot swelling,pain threshold,inflammatory factors,NLRP3 inflammasome,gut microbiota,and other indicators of gout disease were detected.The network pharmacology combined with animal experiments was also applied to verify the potential targets of Simiao decoction.3.Efficacy and mechanism of Simiao FMT in the treatment of goutForty-nine SPF C57BL/6 mice(male,4-week-old,15±3g)were randomly divided into two groups after 7 days of acclimatization:(1)control group(CT,fed a normal diet and injected with PBS solution,N=7);(2)compound gout model group(fed a high-fat diet and injected with MSU);(3)Simiao group(SM,after the gout model was established,Simiao decoction was given by oral gavage daily at 8.0g/kg);(4)Simiao FMT group(MTFSM,after SM group was treated for 3 days,the feces were obtained from Simiao group.The fecal supernatant was collected and 200 μL was administered by oral gavage to MT mice);(5)CT+ MT FMT group(CTFMT,the feces were obtained from MT group.The fecal supernatant was collected and 200μL was administered by oral gavage to CT mice);(6)CT+SM FMT group(CTFSM,the feces were obtained from SM group.The fecal supernatant was collected and 200 μL was administered by oral gavage to CT mice).The swelling and the pain of the footpad were assessed 48 h after each injection of MSU.To clarify the effect of Simiao group’s fecal bacteria transplantation on gouty arthritis and the role of gut microbiota in Simiao decoction in the treatment of gout,the feces of the Simiao group were transplanted to gout model mice and normal mice,respectively.And the indicators of gout disease such as serum uric acid,foot swelling,pain threshold,inflammatory factors,NLRP3 inflammasome,gut microbiota,and intestinal mucosal barrier were measured.Results1.Compared with the hyperuricemia model or acute gouty arthritis model,the compound gout model can significantly up-regulate serum IL-1α,IL-4,IL-6,IL-10,IL-12p40,IL-12p70,IFN-y,KC,MCP-1,and MIP-1 β levels(P<0.05).In addition,the compound gout model caused a significant increase in bacteria such as Desulfovibrio,Prevotella and Parasutterella(P<0.05).2.After the treatment of Simiao decoction in compound gout model mice,the swelling and the pain of the footpad were significantly relieved(P<0.05),and the serum uric acid,the MPO level in the footpad,and XOD and ADA enzyme activities in the liver were also significantly reduced(P<0.05).In addition,Simiao decoction could down-regulate serum pro-inflammatory cytokines(IL-1β,IL-9,IFN-γ,MIP-1αand MIP-1 β),alleviate gut inflammation(NLRP3),inhibit apoptosis(TNF-α,Caspase 8 and AIFM1),improve lipid metabolism(APOB,LPL and PPARα)and inhibited pathogenic gut bacteria(Klebsiella,Blautia,Escherichia-Shigella and Enterococcus)(P<0.05).3.In the MTFSM and MTFCT group,serum uric acid was significantly decreased,uric acid in the urine was specifically significantly increased(P<0.01),and the symptoms of gouty arthritis such as joint swelling and pain were also relieved(P<0.01).After fecal microbiota transplantation of Simiao group,the serum inflammatory factors TNF-α and IL-1β of gouty arthritis model mice were significantly down-regulated(P<0.01),and the expressions of inflammatory factors TNF-α,IL-1β and IL-6,NLRP3 inflammasome and related proteins in colon tissue were down-regulated(P<0.05),and the thickness of the mucosal layer and the number of goblet cells in the colon tissue were significantly increased(P<0.05).Conclusion1.The compound gout model constructed by the combination of high-fat diet feeding and MSU injection induced more severe serum inflammatory cytokines and the dysbiosis gut microbiota was similar to the characteristics of gout patients.2.Both the Simiao and Simiao FMT groups have anti-inflammatory and analgesic effects,by the means of inhibiting the expression of serum proinflammatory factors;regulating the NLRP3 inflammasome;relieveing the inflammation in the joint and intestine,and repairing the intestinal mucosal barrier.However,the mechanisms of lowering uric acid between the two groups are different.The Simiao group works by inhibiting the activity of the XOD enzyme,while the Simiao FMT group can promote uric acid excretion.3.The disturbed gut microbiota in gouty arthritis mice could promote intestinal inflammatory response,while the restored gut microbiota after Simiao decoction treatment not only had no obvious pro-inflammatory effect,but also has antiinflammatory and uric acid-lowering effects.Therefore,the gut microbiota plays a pro-inflammatory and synergistic role in the occurrence and development of gout and the treatment of Simiao decoction,respectively. |
