Therapeutic Effects Of Total Saponin Of Dioscorea On Acute Gouty Arthritisin Rats And Its Influence On NALP3 Inflammasome Signal Pathway

Objective:Investigating the preventive effect of total saponins of Dioscoreae(TSD)on acute gouty arthritis and its possible mechanism,as well as providing experimental basis for clinical application of TSD.Methods:Experiment one:72 healthy male Wistar rats were randomly divided into control group,model group,low-dose group of TSD(30mg·kg-1),middle-dose group(100mg·kg-1),high-dose group(300mg·kg-1)and colchicine group(0.3mg·kg-1).Except for the normal group,the rats in the other groups were injected with monosodium urate into the ankle joint to induce the acute gouty arthritis.Then observing the gait of rates and detecting the swelling of joints.The pathological changes of ankle joints were analyzed through pathology,as well as the expression levels of serum inflammatory factors IL-1β,IL-18 and TNF-a were detected by ELISA method.In addition,Western blot method was used to detect the changes of pro-IL-1β and NALP3 inflammasome signaling pathway related proteins in synovial tissues.Experiment two:THP-1 cells differentiated from monocytes to macrophages induced by different concentration of PMA.The CD11b level was detected by Western blot.THP-1 cells were incubated with different concentration of TSD and MSU,the cell viabilitiy was detected by MTT assay.All cells were randomly devided into 8 groups,included normal group,MSU group,TSD low,middle and high does group(0.3,1,3μg·ml-1),colchicine low and high does group(0.5,5μM).The level of NALP3,ASC,pro-caspase-1 and caspase-1 in MSU induced THP-1 macrophage were detected by Western Blot.The NALP3 mRNA was measured by RT-PCR.The levels of IL-1β,IL-18,and TNF-a in supernatant were measured by ELISA.ROS production were attached with carboxy-H2DCFDA and photographed under fluorescent microscopy.The level of NALP3,ASC,pro-caspase-1 and caspase-1 in Rotenone induced THP-1 macrophages were detected by Western blot,and NALP3 mRNA was detected by RT-PCR.Result:Experiment one:Compared with normal group,limp happened and joint swelling in model group rats,the levels of inflammatory cytokines and the expressions of NALP3,ASC,caspase-1 and cleaved caspase-1 were significantly increased.Compared with model group,all TSD groups and colchicine group changed the gait of rats and TSD 300,100 mg·kg-1 significantly reduced joint swelling and diminished the pathological changes,the expression of NALP3 inflammasome,such as NALP3,ASC,pro-caspase-1 and cleaved caspase-1 protein and the levels of IL-1β,IL-18,and TNF-a were significantly decreased.Experiment two:After incubated with 100ng·ml-1 PMA,THP-1 cells were change to macrophages.The level of CD11b was highly increased.The concentration of TSD from 0 to 3μg·ml-1 and MSU from 0 to 400μg·ml-1 have no effect on cell valiabity in THP-1 macrophages.Pre-treated with 200-400μg·ml-1 MSU,the level of IL-1β were increased.The levels of NALP3 inflammasome and cytokines were significantly increased after MSU(400μg·ml-1)induced,and the levels were decreased by pre-treated with TSD.We found TSD inhibit ROS production,and replace the MSU with Rotenone,the ROS inductive agent,TSD also inhibit the NALP3 inflammasome protein and NALP3 mRNA.Conclusion:TSD has protective effect on acute gouty arthritis and its mechanism may be to inhibit the expression of inflammatory cytokines,as well as to inhibit the assembly and activation of the NALP3 inflammasome.