Study On Anti-inflammatory Of Cinnamaldehyde Based On Succinate/HIF-l?/NLRP3 Pathway

Objective: Based on the Re Bi' model rats and RAW 264.7 cell model,focusing on the succinate mediated inflammatory response,to explore the effects of cinnamaldehyde on the level of succinate and succinate/HIF-1?/NLRP3 pathway and confirm the effect and mechanism of cinnamaldehyde antagonism against inflammatory.Methods:1.Effect of Cinnamaldehyde on the synovitis of Re Bi' model rats.The Re Bi' model rats was prepared by complete Freund's adjuvant combined with Damp-heat environment(37 ?,70%-80%).The rat foot volume was measured by the rat foot swelling tester.The foot circumference was measured with a tape.The pathology of the synovial was detected by HE staining,and the level of IL-1? in serum was determined by ELISA kit.The activity of was tested by MTT.The vitro modle was established by LPS and ATP stimulated RAW264.7 cells.The concentration of NO in medium was determined by chemical kit,and the levels of IL-1? and TNF-a in medium were detected by ELISA kit.2.Effects of cinnamaldehyde on succinate/HIF-1?/NLRP3 in RAW264.7 cells induced by LPS and ATPAfter RAW264.7 cells were stimulated by LPS and ATP,The level of succinate was determined by HPLC.The expression of HIF-1? and NLRP3 protein was detected by western blot under different conditions of blocking and blocking HIF-1?.ELISA kit determined the level of IL-1? and TNF-a in the medium,to evaluate the effect of cinnamaldehyde on the succinic/HIF-1?/NLRP3 pathway.The expression of GPR91 was detected by western blot,to study the impact of cinnamaldehyde on the expression of GPR91.HPLC was evaluated fumarate level and chemical kit determined the activity of SDH enzyme,to evaluate the effect of cinnamaldehyde onthe degradation pathway of succinate.3.Effect of cinnamaldehyde on succinate/HIF-1?/NLRP3 pathway in Re Bi'modle.The Re Bi' model was prepared by using complete Freund's adjuvant combined with Damp-heat environment.HPLC was evaluated succinate level and western blot detected the expression of HIF-1? and NLRP3 protein,to evaluate the effect of cinnamaldehyde on the succinic/HIF-1?/NLRP3 pathway.The expression of GPR91 was detected by Immunohistochemistry,to study the effect of cinnamic aldehyde on the expression of GPR91.HPLC was evaluated fumarate level and chemical kit determined the activity of SDH enzyme,to evaluate cinnamaldehyde impact on the degradation pathway of succinate.Result:1.Effect of cinnamaldehyde on synovitis in rats with Re Bi' model ratsCompared with the control,the paw swelling and foot circumference of the rats in the Re Bi' model group were significantly increased(P<0.01),and the synovial tissue of Re Bi' model rats showed obvious synovial cell proliferation and inflammatory cell infiltration(P<0.05),the level of IL-1? in serum was significantly increased(P<0.05).Compared with the Re Bi' model group,the paw swelling and foot circumference of the cinnamaldehyde group were significantly decreased,and the cell proliferation and inflammatory infiltration of the synovial tissue were significantly improved(P<0.05),the content of IL-1? in serum was significantly decreased(P<0.05).Compared with the control group,LPS and ATP treatment significantly promoted the release of NO,IL-1? and TNF-a in cells(P<0.01).With cinnamaldehyde treated 15 h,the levels of NO and IL-1? and TNF-a significantly decreased(P<0.05).2.Effects of cinnamaldehyde on succinate/HIF-1?/NLRP3 in RAW264.7 cells induced by LPS and ATPCompared with the control group,LPS and ATP significantly induced the expression of succinate(P<0.05)and increased the expression of HIF-1? and NLRP3protein(P<0.05).the level of succinate was significantly reduced in the administration group(P< 0.05),the level of HIF-1?/NLRP3 protein was significantly decreased(P<0.05).HY-1 intervention could significantly decrease the expressions of HIF-1?and NLRP3 and the releases of IL-1? and TNF-a(P<0.05),the cinnamaldehyde treatment group had no significant effect on the expressions of HIF-1? and NLRP3(P>0.05).cinnamaldehyde treatment significantly inhibited the secretion of TNF-a by HY-1 treated RAW264.7 cells(P<0.05)and the expression of IL-1? had no statistical difference in the cinnamic aldehyde group(p>0.05).The expression of GPR91 was significantly increased after LPS and ATP stimulated cells(P<0.05).Cinnamaldehyde intervention significantly down-regulated the expression of GPR91 protein(P<0.05).The activity of succinate dehydrogenase was significantly increased(P<0.05),and the level of fumarate in cells was significantly increased(P<0.05)after LPS and ATP stimulated cells.while cinnamaldehyde had no significant effect on SDH activity and fumarate content(P>0.05).3.Effect of cinnamaldehyde on succinate/HIF-1?/NLRP3 pathway in Re Bi model.Compared with the control group,the level of succinate and the expressions of HIF-1? and NLRP3 was significantly increased in the Rebi' model group(P<0.05).The rats were treated with cinnamaldehyde.the concentration of succinate and the expression of HIF-1? and NLRP3 protein in the synovial tissue were decreased(P<0.05).Compared with the control group,the expression of GPR91 in the synovial tissue of the Re Bi model rats was significantly increased(P<0.05),and cinnamaldehyde could significantly down-regulate the expression of GPR91protein(P<0.05).Compared with the control group,the level of fumarate was significantly increased(P<0.01),but the succinate dehydrogenase activity was decreased,but there was no statistically difference(P>0.05).Cinnamaldehyde intervention had no significant effect on SDH enzyme activity and fumarate content(P>0.05).Conclusion: Cinnamaldehyde has the effect of antagonizing the synovialinflammation of Re Bi' model rats,and its mechanism is related to the regulation of succinate/HIF-1?/NLRP3 pathway and inhibition of GPR91 protein expression.