Role And Mechanism Of Valve Interstitial Cell Pyroptosis In Calcified Cardiac Valvular Disease

Objective:With the aging of the population,the rate of the calcific of aortic valve disease(CAVD)in heart valve disease is increasing year by year,and aortic valve replacement is the only effective treatment,it is also accompanied by high economic cost and high surgical risk.Calcified cardiac valvular disease involved many factors,but the mechanism of pyroptosis of Aortic valve interstitial cells in CAVD is currently unknown.Currently,the nosogenesis whether the valve interstitial cells could promote CAVD and regulate CAVD progression through pyroptosis or not is still unknown.Methods:1.To detect the occurrence of pyroptosis in CAVD aortic valves: collect normal aortic valve tissue(control group)and CAVD aortic valve tissue(CAVD group),one part of them were soaked in 4% paraformaldehyde solution for histological testing and the other part were kept in-80℃ refrigerator for molecular biology testing after get those collections.The calcification degree was measured by Alizarin red staining and the expression changes of calcification-related proteins(OPN,Runx2)and pyroptosis-related proteins(NLRP3,GSDMD and Caspase-1)were detected by Western blotting.2.AVICs isolation,identification and in vitro osteogenesis induction model establishment: AVICs were isolated from human aortic valves using type II collagenase and cultured until the third generation,We used cellular immunofluorescence to determine whether the obtained cells were valvular interstitial cells.Then passage 3-8cells was selected to applied to subsequent experiments.The vitro calcification model construction was measured by Alizarin red staining and Western blotting.We divided AVICs into control group(standard medium culture)and calcified group(calcification induced medium culture),and the expression changes of calcification-related proteins(OPN,Runx2)and pyroptosis-related proteins(NLRP3,GSDMD,Caspase-1,GSDMD-N and Cleaved Caspase-1)were re-detected by Western blotting and cellular immunofluorescence.3.Role and mechanism study of cell pyroptosis in calcification of AVICs: according to the treatment conditions,AVICs were divided into VX-765 inhibitor group(Caspase-1inhibitor VX-765 pretreatment for 24 h before induced culture with calcification medium);Caspase-1 overexpression group(overexpression of Caspase-1 in AVICs using cell transfection technology,and then calcification induction),setting up the empty carrier group at the same time;calcified group(AVICs were treated with calcified medium alone).The changes in the calcified nodules of the cells were detected by Alizarin red staining,Western blotting detected the expression changes of calcification-related proteins(OPN,Runx2),pyroptosis-related proteins(NLRP3,GSDMD,Caspase-1,GSDMD-N and Cleaved Caspase-1),and inflammatory factors(IL-1β,IL-18)in AVICs.Results:1.Detection of cell pyroptosis in CAVD aortic valve: In CAVD aortic valve tissue,Alizzarin red staining was obviously positive,indicating that there were deposition of calcium salt to form calcified nodules.The results of immunohistochemistry and Western blotting showed that the expression of Runx2 and OPN and pyroptosis-related proteins NLRP3,GSDMD,Caspase-1 in CAVD aortic valve tissue were significantly increased.The results of Western blotting were consistent with the immunohistochemical testing results.2.Culture and identification of AVICs,calcification model construction in vitro,pyroptosis-related detection: AVICs were phenotyped by cellular immunofluorescence.The results showed that positive staining for Vimentin and α-SMA in AVICs,and negative CD31 staining,indicating that the cultured cells were valve interstitial cells.After AVICs were induced by calcified medium,Alizarin red staining showed orange-red staining,and Western blotting results showed increased expression of calcification marker proteins Runx2 and OPN,indicating the success of calcification model construction.Compared with the control group,the expression of calcified pyroptosis-related proteins NLRP3,GSDMD and Caspase-1were significantly increased,and the immunofluorescence results also showed increased expression of all proteins,which was consistent with the Western blotting results.3.The role and mechanism of pyroptosis regulating osteogenic differentiation of AVICs: Compared with the calcification group,Alizarin red staining showed significantly decreased calcium nodules in the inhibitor group;the Western blotting results showed decreased the expression levels of Runx2,OPN,NLRP3,GSDMD,Caspase-1,GSDMD-N,Cleaved Caspase-1 and inflammatory factors IL-1β and IL-18.The expression levels of Runx2,OPN,NLRP3,GSDMD,Caspase-1,GSDMD-N and Cleaved Caspase-1 and inflammatory-factor IL-1β and IL-18 were significantly increased in the Caspase-1 overexpression group compared with the calcified and the empty groups.Conclusion:Pyroptosis existed in CAVD,and accelerated the progression of CAVD by promoting the transformation of valve interstitial cells into osteoblast-like cells.The results of this study preliminarily explore the mechanism of the development of CAVD,and provide potential targets and therapeutic strategies for the clinical prevention and treatment of CAVD.