Study On The Mechanism Of Total Flavonoids Of Smilax Glabra Roxb. In Treatment Of Gouty Arthritis Based On NLRP3/ASC/Caspase-1 Axis

Objective To study the effect of Total Flavonoids of Smilax glabra Roxb.(TFSG)on the expression of inflammatory factors IL-1β,IL-6 and TNF-α in THP-1 cells and mice synovium induced by monosodium urate(MSU);to explore the protective mechanism of TFSG on the gout arthritis by measuring the expression with NLRP3 inflammasome axis of NLRP3,ASC,Caspase-1 protein and m RNA.Methods(1)THP-1 cells were induced into macrophages by PMA.The effects of different concentrations of colchicine,MSU and TFSG on the cytotoxicity were determined by MTT assay.The cells were divided into the normal group,the LPS group,the LPS + MSU group,the colchicine group(100 ng/m L),the low-dose,middle-dose,and high-dose TFSG groups(100,200,400 μg/m L).In addition to the normal group,LPS treatment(the final concentration of 1 μg/m L)was added,and the incubator was placed after 2 h and added the MSU to continue for 24 h.The levels of IL-1β,IL-6 and TNF-α were measured by enzyme-linked immunosorbent assay.The protein expressions of NLRP3,ASC and Caspase-1 were detected by Western blot.(2)The 120 C57BL/6 mice were randomly divided into the normal group,the model group,the colchicine group(0.65 mg/kg),the low-dose,middle-dose,and high-dose TFSG groups(100,300,500 mg/kg),with 20 mice in each group.The normal group and the model group were given distilled water by gavage,and the other groups were given the corresponding drugs for 7 days.In addition to the normal group,the other groups were treated with MSU to replicate the mouse gouty arthritis model 6 days after gavage,and the normal group injected with normal saline.At the end of the experiment,the swelling degree of the ankle was measured.Mice synovium tissues were collected to detect the inflammatory mediators IL-1β,IL-6 and TNF-α with enzyme-linked immunosorbent assay.The protein expressions of NLRP3,ASC,Caspase-1 were detected by Western blot,and the expressions of NLRP3,ASC,Caspase-1 messenger RNA in synovium tissues were detected with quantitative Real-time PCR method.And the histological observation of the sacroiliac joints of mice was performed.Results(1)In cell experiments,compared with the normal group,the levels of IL-1β,IL-6 and TNF-α in LPS+MSU group were significantly increased(P<0.01),and the protein expressions of NLRP3,ASC and Caspase-1 were significantly increased(P<0.01).Compared with the LPS+MSU group,the levels of IL-1β,IL-6 and TNF-α in the middle-dose and high-dose TFSG groups were significantly reduced(P<0.01),as well as the protein expressions of NLRP3,ASC and Caspase-1(P<0.01).(2)In mouse experiments,compared with the normal group,after modeling six hours,mice ankle swelling degree of the model group increased significantly(P<0.01),the TFSG groups could significantly reduce ankle swelling degree(P<0.05).Compared with the normal group,the levels of IL-1β,IL-6,TNF-α and the protein and m RNA expressions of NLRP3,ASC,Caspase-1 with the model group were significantly increased(P<0.01).Compared with the model group,the TFSG group could significantly reduce IL-1β,IL-6,TNF-α levels(P<0.01),as well as NLRP3,ASC,Caspase-1 protein levels and m RNA expressions(P<0.01).TFSG can effectively improve the pathological changes of the ankle joint in model mice.Conclusion TFSG could decrease the release of inflammatory cytokines induced by sodium urate crystals,regulate the expression of related proteins and m RNA on NLRP3/ASC/ Caspase-1 axis,and show a certain degree of anti-gouty arthritis.It has great potential in treating the acute gouty arthritis.