| Objective:To investigate the role of purinergic receptor P2 X ligand-gated ion channel 7(P2X7R)in the pathogenesis of acute gouty arthritis in rats and the ratio of regulatory T cell(Treg),Th17 cell and Treg / Th17 Impact of change.Methods:Male Sprague-Dawley rats were randomly divided into 3 groups: P2X7 R agonist ATP(ATP group),P2X7 R inhibitor BBG(BBG group)and PBS(control group)were administered after injection of urate crystals(MSU)into the ankle joint cavity of rats to establish an acute gouty arthritis model.The clinical manifestations of the right ankle joint were evaluated at different time points(6h,12 h,24h,48 h and 72h)of the acute onset process;After evaluation the rats were sacrificed,and the degree of inflammatory cell infiltration in the synovial tissue of the right ankle joint was evaluated by H&E staining;The expression of P2X7 R,the uptake of YO-PRO-1 in rat spleen macrophages,the Treg and Th17 cells were detected by flow cytometry;The levels of IL-1?,IL-6,TNF-?,IL-17,IL-10 and TGF-? in rat serum were detected by ELISA kits;Detection of P2X7 R,NLRP3 and IL-1? expression levels by q RT-PCR;The experimental results were analyzed by SPSS21.0 software,and one-way ANOVA or two independent sample t-tests were used for statistical analysis.Results:1 Clinical and pathological changes in the activation of acute gouty arthritis in rats with P2X7R: At 12 h,the clinical scores of ATP group were significantly higher than those of BBG group and control group(P=0.001,0.042),and the control group was higher than BBG group(P=0.034);At 12 h and 24 h,the swelling index of ATP groupwas the highest(P = 0.000,0.001;P = 0.000,0.003),followed by the control group(P =0.009,0.001)and the difference between the three groups were statistically significant;2 At 12 hand 24h,the infiltration of mononuclear cell in ATP group was significantly higher than that in BBG group and control group(P=0.000,0.007;P=0.000,0.001);The neutrophils infiltration in ATP group was the highest among the three groups at 24h(P=0.001,0.04),and the control group was higher than BBG group(P=0.04).3 The expression of P2X7 R on macrophages of each group increased first and then decreased,and reached their highest at 12h: At 12 h and 24 h,the expression of P2X7 R in ATP group was obviously higher than that in BBG group and Control group(P=0.000,0.019;P=0.000,0.001),and BBG group was lower than control group(P=0.012,0.004).The ability of macrophages to take up YO-PRO-1 reached the highest in both ATP group and control group at 12 h,while the BBG group was reversed;At 12 h and 24 h,the ability of ATP group macrophages to uptake YO-PRO-1 was significantly higher than that of BBG group and control group(P=0.000,0.023;P=0.001,0.031),followed by the control group(P=0.01),but at 24 h,there was no difference between BBG group and Control group(P>0.05).4 P2X7 R promotes IL-1?release by activating NLRP3 inflammasome: At 12 h,the expression of P2X7 R,NLRP3and IL-1? m RNA in ATP group were significantly higher than those in BBG group and control group(P=0.001,0.008;P=0.000,0.012;P=0.002,0.034);At 24 h,the expression of IL-1? was obviously higher in ATP group than in BBG group and control group(P=0.000,0.001),followed by the control group(P=0.007),and the difference among the three groups were significant.5 P2X7 R regulates pro-inflammatory cytokines production: At24 h,ATP group was significantly higher than BBG group and control group(P=0.001,0.003);At 6h,12 h and 24 h,higher level of IL-6 in ATP group compared with BBG group and control group(P=0.004,0.04;P=0.000,0.002;P=0.001,0.012);The level of TNF-? was obviously higher in ATP group than in BBG group and control group at 6h and 24h(P=0.007,0.011;P=0.001,0.018).6 P2X7 R affects thechanges of Treg,Th17 cell and Treg / Th17 ratio: At 12 h,The expression levels of Treg and Th17 cell in the ATP group were significantly higher than that in the BBG group and control group(P=0.001,0.021;P=0.01,0.025);The expression levels of Treg and Th17 cell in control group were higher than that in BBG group(P=0.021,0.044).The expression trends of Treg and Th17 cell at different time points: the level of Treg was increased at 6h,but decreased gradually at12 h,24h and then increased at 48 h again;The level of Th17 was increased at 6h,12 h and 24 h,but decreased gradually at 48 h,72h.The ratio of Treg / Th17 gradually decreased in the first three time points,increased at 48 h and 72 h among the three groups,the ratio of Treg / Th17 in ATP group was lower than that in BBG group and control group at 12 h,with significant difference(P <0.05),but at other four time poins,the ratios were no significant differences among the three groups.Conclusion: P2X7 R may play a key role in MSU crystal-induced acute gouty arthritis in rats and regulate the progression of inflammation by affecting changes in of Treg,Th17 cells and the ratio Treg/Th17. |
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