The Mechanisms Of NLRP3 Inflammatory Signaling Pathway Activated By Uremic Toxins In Vascular Neointimal Hyperplasia

Objective:1.Through the construction of mouse CKD model and arteriovenous fistula(AVF)model,to explore whether CKD inflammation through NLRP3 signal pathway mediated by inflammation promotes vascular smooth muscle cell proliferation and neointimal hyperplasia(NH).2.Make it clear that NLPP3 knockout can slow down the neointimal proliferation of CKD mice,and explore whether the NLRP3 inhibitor glibenclamide has therapeutic effect on CKD related neointimal hyperplasia.Methods:1.Experimental group: 24 healthy male C57 mice were randomly divided into CKD+AVF operation group,CKD sham operation group,glibenclamide group and drug control group.NLRP3 knockout male C57 mice were divided into NLRP3 knockout group.CKD mouse model was established by 5/6 nephrectomy,and blood samples were obtained after a week of CKD.The mouse model of AVF was established by the anastomosis of the right common carotid artery and the internal jugular vein,and vascular samples were obtained after 3 weeks of operation.2.Use picric acid method to determin serum creatinine(SCr)level,use the urease method to determin blood urea nitrogen(BUN)levei,use the biochemical method to determin the Caspase-1 activity of vascular tissue.3.HE staining was used to observe the pathological changes of blood vessels,and to measure and analyze the neointimal hyperplasia of vessels in each group.4.The expression levels of NLRP3,α-SMA and NLRP3 downstream signal molecules IL-1β and IL-18 in vascular tissue were analyzed by immunofluorescence double labeling method.5.RT-PCR method was used to analyze the expression of NRLP3 mRNA in vascular tissues of each group.Results:1.Compared with CKD group,the serum SCr and BUN levels were significantly higher in CKD+AVF group,glibenclamide group,drug control group and NLRP3 knockout group(P<0.05).2.The vascular tissue HE staining showed that compared with the CKD sham operation group,the vascular intima in CKD+AVF group was obviously thickened(P<0.05),the vascular intimal thickness in glibenclamide group and NLRP3 knockout group had no significant change(P>0.05).3.Compared with the CKD sham operation group,the expression of NLRP3 mRNA in CKD+AVF surgery group mice vascular tissue was significantly increased,the Caspase-1 activity increased significantly,the NLRP3,IL-1 beta and IL-18 protein in vascular smooth muscle cells was significantly increased;and compared with CKD sham group,the NLRP3 mRNA expression,Caspase-1 activity and NLRP3,IL-1 beta,IL-18 protein in vascular smooth muscle cell of glibenclamide treatment group and NLRP3 knockout group had no statistical difference(P>0.05).4.There was no significant difference between the CKD+AVF group and the control group(P>0.05).Conclusion:1.Uremic toxins produced by CKD can induce arteriovenous fistula vascular smooth muscle cells expression of inflammatory cytokines,such as Caspase-1,IL-1 beta,IL-18,induced the vascular smooth muscle cell proliferation and migration to vascular intimal,cause the arteriovenous fistula stenosis;2.NLRP3 knockout could significantly inhibit the production of inflammatory factors such as Caspase-1,IL-1 beta,IL-18 and so on in the NLRP3 inflammatory pathway,and inhibit the proliferation of CKD related angiogenesis.3.The protective effect of glibenclamide on vascular intimal and inhibiting NLRP3 signal pathway activation of inflammation,reduce inflammatory cytokines Caspase-1,the expression of IL-1 beta and IL-18,and inhibit the proliferation of vascular smooth muscle cells and migration to vascular intimal,and has inhibitory effect for CKD related arteriovenous fistula stenosis.