Role And Mechanism Of The Specific Knockout Of Lgr5 In Myocardial Tissue In An ISO-induced Mouse Myocardial Fibrosis Model

BACKGROUND and OBJECTIVES:Myocardial fibrosis(MF)is a process of myocardial adaptive remodeling when the myocardium is subjected to various external insults,such as mechanical pressure,inflammatory stimuli,and ischemia.One of the distinctive pathological change of MF is the accumulation of related iBAnflammatory cells and activated cardiac fibroblasts(CFs),which in turn causes a dramatic increase in the production of extracellular matrix(ECM).Many evidences have shown that heart failure(HF),characterized by ventricular systolic and diastolic dysfunction,exhibits severe MF.Due to the poor prognosis of HF,it poses a great threat to the health of most patients with cardiovascular disease.Therefore,it is of great significance to actively search for effective interventions for MF to delay the disease process of HF.The development of MF is influenced by a variety of factors,of which both oxidative stress and inflammatory response are involved,and inflammatory factors,such as tumor necrosis factor-?(TNF-?),interleukin-6(IL-6)and IL-1?,are closely related to its development.Lgr5 is a G protein-coupled receptor,and it has been found that R-spondin is able to bind to Lgr5,which causes the activation of Wnt/?-Catenin signaling.At the same time,several studies have reported that Wnt/?-catenin signaling plays a very important role in the disease progression of MF.In previous cellular experiments,our team have found that the molecular mechanism by which Lgr5 affects myocardial fibrosis may be achieved through the Wnt/?-Catenin signaling pathway.The aim of this study is to explore the role and mechanism of specific knockout of Lgr5 in myocardial tissue in an animal model of isoproterenol(ISO)-induced myocardial fibrosis in mice.In this study,Lgr5-specific knockout mice in myocardial tissue were used to create an animal model of myocardial fibrosis by subcutaneous injection of ISO,to look for the effect of specific knockout of Lgr5 in myocardial tissue on MF.At the same time,the changes of wnt1 and ?-Catenin were also detected to verify whether the molecular mechanism of specific knockout of Lgr5 in myocardial tissue affecting myocardial fibrosis was achieved through the Wnt/?-Catenin signaling pathway.MATERIALS AND METHODS:1.Establishment of myocardial fibrosis animal model induced by ISO in miceIn the experiment,wild type mice with genetic background of C57BL/6J(20healthy mice weighing 20-25 g,age 8-10 weeks)were divided into two groups,and Vehicle/ISO 30mg/kg/d was injected subcutaneously for 10 consecutive days to produce an animal model of myocardial fibrosis,and the weight,heart rate,and blood pressure of the mice were measured.Massson staining was used to determine whether the myocardial fibrosis model was successfully manufactured.RT-PCR and Western blotting were used to determine the expression of Lgr5,collagen I and collagen III.2.Effects of the specific knockout of Lgr5 in myocardial tissue on myocardial fibrosis in miceThe experiment used the C57BL/6J genetic background Lgr5-specific knockout mice and littermate wild type mice(body weight 20-25 g,20 healthy mice aged 8-10weeks),divided into 4 groups:WT + Veh Group,WT + ISO group,Lgr5 cKO + Veh group,Lgr5 cKO + ISO group for modeling to observe the effects of the specific knockout of Lgr5 in myocardial tissue on mouse body weight,heart rate,blood pressure,heart function,myocardial fibrosis,and myocardial inflammatory response.RT-PCR and Western blotting were used to detect the expression of the collagen I,collagen ?,MMP1,TIMP1,and IL-6,TNF-? to study the effect of the specific knockout of Lgr5 in myocardial tissue on mouse fibrosis indicators.3.Mechanism of the specific knockout of Lgr5 in myocardial tissue in myocardial fibrosis in miceThe experiment used RT-PCR and Western blotting were used to determine the expression of Lgr5,wnt1,and ?-Catenin to verify whether the specific knockout of Lgr5 in myocardial tissue affected myocardial fibrosis in mice was through the Wnt/?-Catenin signaling pathway.RESULTS:1.ISO-induced animal model of myocardial fibrosis in miceThe results showed that compared with the Veh group,the weight in the ISO group was lower,heart rate and heart index increased(P<0.05).The systolic blood pressure inthe ISO group was almost unchanged compared to the Veh group,and the diastolic blood pressure was slightly lower than that in the Veh group,but the difference is not statistically significant;Masson staining found that the degree of myocardial fibrosis in the ISO group was more severe than that in the Veh group,and the collagen area increased significantly(P<0.05).These results indicated that the selected dose of ISO successfully induced an animal model of myocardial fibrosis.The results of PCR and Western blotting showed that the expressions of Lgr5,collagen I,and collagen ? in the ISO group were increased compared with the Veh group(P<0.05).The above results show that the selected dose of ISO successfully induced an animal model of myocardial fibrosis,and the expression of Lgr5 increased in myocardial fibrosis,so the Lgr5 may participate in myocardial fibrosis in mice.2.Effects of he specific knockout of Lgr5 in myocardial tissue on myocardial fibrosis in miceThe results showed that there was no significant change in blood pressure in each group;compared with the Veh group,the body weight of the ISO group decreased(P<0.05),and the WT + ISO group had a more significant decrease than the Lgr5 cKO +ISO group,but the difference was not statistically significant.The heart rate and heart index of the ISO group was increased,and the increase of the WT + ISO group was more significant than that of the Lgr5 cKO + ISO group(P<0.05);the EF and FS values of the WT + ISO group were decrease compared to the WT + Veh group,LVIDs value were increased compared with WT + Veh group(P<0.05),LVIDd value also showed an upward trend,but the difference was not statistically significant;compared with Lgr5 cKO + Veh group,Lgr5 cKO+ ISO group had lower EF value(P<0.05),FS value also became lower,and LVIDs and LVIDd values increased,but the difference was not statistically significant;compared with Lgr5 cKO + ISO group,The EF value of the WT+ ISO group became lower(P<0.05),and the FS value also tended to decrease,while the LVIDs and LVIDd values increased,but the differences were not statistically significant.Masson and Sirius Red staining revealed that the collagen volume fraction of the ISO group increased.The increase in WT + ISO group was more significant than that in Lgr5cKO+ ISO group(P<0.05).The heart tissue of the Veh group in the HE staining wasbasically normal,and the inflammatory cell infiltration in the heart tissue of the ISO group occurred to different degrees.The WT + ISO group was more severe than the Lgr5 cKO+ ISO group,and fibrous tissue proliferation occurred;the results of RT-PCR and Western blotting showed that compared with Veh group,The expression of collagen I,collagen ?,TIMP1,IL-6 and TNF-? were increased in ISO group,MMP1 in the ISO group were decreased,and The degree of change in WT + ISO group was more significant than that in Lgr5 cKO+ ISO group(P<0.05).The above results indicate that the specific knockout of Lgr5 in myocardial tissue can reduce myocardial fibrosis in mice.3.Mechanism of the specific knockout Lgr5 in myocardial fibrosis in miceThe results showed that compared with the WT + Veh group,Lgr5 mRNA and protein expression increased in the WT + ISO group(P<0.05),and there were almost no Lgr5 mRNA or protein expression in the Lgr5 cKO+ Veh group and Lgr5 cKO+ ISO group;compared with the Veh group,the mRNA and protein expressions of wnt1 and?-Catenin in the ISO group were increased,and the increase in the WT + ISO group was more significant than that in the Lgr5 cKO+ ISO group(P< 0.05),which suggested that the molecular mechanism of the specific knockout of Lgr5 in myocardial fibrosis was achieved through down-regulation of Wnt/?-Catenin signaling pathway.CONCLUSIONS:1.Specific knockout of Lgr5 in myocardial tissue can reduce myocardial fibrosis.2.The molecular mechanism of specific knockout of Lgr5 in myocardial tissues to reduce myocardial fibrosis was achieved through down-regulation of Wnt/?-Catenin signaling pathway.