TLR4-mediated NLRP3 Inflammasome On Ang Ⅱ-induced Proliferation Of Cardiac Fibroblasts

Background and Purpose:Myocardial fibrosis（MF）is the common pathophysiological changes of a variety of cardiovascular diseases such as ischemic cardiomyopathy,cardiomyopathy,and valvular heart disease in the final stage of development.Among them,Cardiac fibroblasts（CFs）are responsible for the fibrosis of the heart.The excessive proliferation of CFs and the accumulation of extracellular matrix（ECM）are one of the pathological bases of myocardial fibrosis.Myocardial fibrosis can not only affect the systolic and diastolic functions of the heart,but also cause the structural remodeling of the heart,which brings psychological and economic burden to patients and their families.Studies at home and abroad have shown that the activation of NLRP3 inflammasome is closely related to myocardial fibrosis.TLR4 is an upstream factor of NLRP3inflammasome,which can induce inflammatory molecular gene coding through the My D88 pathway.Therefore,this study proposed the following theoretical hypothesis:during the process of CFs proliferation to myocardial fibrosis,TLR4may participate in the regulation of CFs proliferation and fibrosis by regulating the activation of NLRP3 inflammasome.In this study,the fibrosis model of CFs was induced by AngiotensinⅡin vitro.The CFs was transfected with TLR4-shRNA to observe the effect of TLR4-shRNA on the proliferation and collagen synthesis of CFs,and the expression of NLRP3 inflammasome was detected.To investigate the role of TLR4-shRNA in regulating NLRP3inflammasome proliferation in CFs,and the results are expected to provide a theoretical basis for the treatment of MF.Methods:1.Isolation,culture and identification of primary CFs in SD Suckling miceThe hearts of 1-3d SD Suckling rats were isolated,the heart tissues were digested with trypsin andⅡcollagenase,and the cardiac fibroblasts were obtained by differential adhesion method.The cardiac fibroblasts were identified by ordinary light microscope observation and immunofluorescence.2.AngⅡstimulated CFs to construct myocardial fibrosis model in vitroCCK-8 kit was used to detect the proliferation effect of different concentrations of AngⅡ(0mol/L,10^-5 mol/L,10^-6 mol/L,10^-7 mol/L,10^-8 mol/L)on CFs.The m RNA and protein expressions of CollagenⅠ/nucleus were detected by real-time fluorescence quantitative PCR and Western blot.3.Effect of TLR4-shRNA on AngⅡ-induced proliferation of cardiac fibroblastsThe experiment was divided into four groups:Control group,AngⅡgroup,AngⅡ+vehicle,AngⅡ+TLR4-shRNA group.The Control group was not treated.The AngⅡgroup was treated with 10^-6 mol/L Ang stimulation,and the AngⅡ+vehicle and AngⅡ+TLR4-shRNA groups were transfected with vehicle and TLR4-shRNA for 3 days,respectively,and then treated with AngⅡstimulation.CCK-8 kit was used to detect the proliferation rate of cells in each group.4.The effect of TLR4-shRNA on the expression of NLRP3 inflammasome and collagen in cardiac fibroblastsThe changes of organelles in each group were observed by electron transmission microscopy.The m RNA and protein expressions of TLR4,NLRP3,Caspase-1,TGF-βand CollagenⅠin each group were detected by real-time fluorescence quantitative PCR and Western blot.Results:1.Isolation,culture and identification of primary CFs in SD Suckling miceUnder ordinary light microscope,CFs is mostly triangular or star-shaped,usually containing 2 to 3 nuclei,oval in shape,large and mostly centered,and transparent cytoplasm.The cells adhered to the wall radially without spontaneous pulsation,and gradually became fusiform with the increase of the degree of cell fusion.Immunofluorescence showed vimentin positive.2.AngⅡstimulated CFs to construct myocardial fibrosis model in vitroCCK-8 results showed that AngⅡstimulated the proliferation of cardiac fibroblasts most obviously at the concentration of 10^-6mol/L after 24h treatment（P<0.05）.Real-time quantitative fluorescence PCR and Western blot results showed that m RNA and protein expressions of CollagenⅠand CollagenⅢin AngⅡgroup were increased compared with the Control group（P<0.05）,which confirmed that the model of myocardial fibrosis in vitro was successfully established.3.Effect of TLR4-shRNA on AngⅡ-induced proliferation of cardiac fibroblastsCCK-8 results showed that compared with the Control group,the proliferation of cardiac fibroblasts in AngⅡgroup,AngⅡ+vehicle group and AngⅡ+TLR4-shRNA group was significantly（P<0.05）.Compared with AngⅡ+vehicle group,the proliferation of cardiac fibroblasts in AngⅡ+TLR4-shRNA group was decreased（P<0.05）.4.The effect of TLR4-shRNA on the expression of NLRP3 inflammasome and collagen in cardiac fibroblastsThe results of transmission electron microscopy showed that the differences among the four groups of cells were mainly reflected in the degree of mitochondrial endoplasmic reticulum swelling and expansion,and the number of intracellular autophagy lysosomal related structures.Among them,AngⅡgroup had fewer intracytoplasmic organelles,a large number of vacuoles suspected to be formed by autophagy,and mitochondria had mild to moderate swelling.Compared with other groups,the damage was the most serious.Compared with AngⅡgroup,the damage of AngⅡ+TLR4-shRNA group was reduced.Real-time quantitative PCR and Western blot results showed that compared with the Control group,m RNA and protein expressions of TLR4,NLRP3,Caspase-1,TGF-βand CollagenⅠin AngⅡgroup were increased（P<0.05）.Compared with AngⅡ+vehicle group,the m RNA and protein expressions of TLR4,NLRP3,Caspase-1,TGF-βand Collagen II in Ang+TLR4-shRNA group were decreased（P<0.05）.Conclusions:Silencing TLR4 can reduce the level of NLRP3 inflammasomes and the expression of TGF-βand CollagenⅠ,then inhibit the proliferation of cardiac fibroblasts induced by AngⅡ.