Mechanism Of Reg3? Inhibiting Cardiac Fibrosis In Mice With Viral Myocarditis

Objective:Expression of collagen in mouse cardiac fibroblasts induced by Angiotensin II(ANG II)in vitro,and evaluation of the effect of Regenerating islet-derived protein 3?(Reg3?)on ANG II-induced expression of collagen in cardiac fibroblasts,preliminary study on the mechanism by which Reg3? inhibits collagen secretion by cardiac fibroblasts.The Coxsackie virus B3(CVB3)-induced viral myocarditis model was constructed to evaluate the effect of Reg3? on inhibiting cardiac fibrosis in vivo.Methods:(1)Effect of Reg3? on ANG II-induced collagen expression in cardiac fibroblasts: Mouse cardiac fibroblasts were treated with ANG II and Reg3? for 12 h in vitro.The protein levels of Collagen-I and ?-SMA were detected by Western blot.The mRNA expression levels of MMPs,TIMPs,Collagen-I and Collagen-III were detected by qRTPCR.Immunofluorescence was used to detect Collagen-I and ?-SMA.Human cardiac fibroblasts were treated with ANG II and Reg3? for 12 h.Western blot and immunofluorescence were used to detect the protein levels of Collagen-I and ?-SMA,respectively.(2)Reg3? inhibited ANG II-induced collagen expression in cardiac fibroblasts by EXTL-3: Mouse cardiac fibroblasts were treated with Reg3?,and the protein level of EXTL-3 was detected by Western blot.Mouse cardiac fibroblasts were pretreated with siEXTL-3 and treated with ANG II and Reg3? for 12 h.The protein levels of CollagenI and ?-SMA were detected by Western blot,The mRNA expression levels of MMPs,TIMPs,Collagen-I and Collagen-III were detected by qRT-PCR,and Collagen-I and ?-SMA were detected by immunofluorescence.(3)The molecular mechanism of Reg3? inhibiting the expression of collagen in cardiac fibroblasts induced by ANG II: The cardiac fibroblasts were treated with ANG II for 5 min,15 min,30 min,45 min,and 60 min,respectively.The phosphorylation levels of Smad2/3,Erk1/2,and P38 and the expression of RhoA were detected by Western blot.To verify whether Reg3? downregulates cardiac fibroblasts to express collagen by inhibiting activation of the above pathways.Cardiac fibroblasts were treated with Reg3? for 12 h,and cardiac fibroblasts were treated with ANG II for 30 min.The phosphorylation levels of Smad2/3,Erk1/2 and P38 and the expression of RhoA were detected by Western blot.Cardiac fibroblasts were treated with TGF-?1 receptor inhibitor for 12 h,and cardiac fibroblasts were treated with ANG II for 30 min.The phosphorylation levels of Smad2/3,Erk1/2 and P38 and the expression of RhoA were detected by Western blot.Mouse cardiac fibroblasts were treated with ANG II and Reg3? for 12 h in vitro,and the protein level of Agtr1 was detected by Western blot.qRT-PCR and ELISA were used to detect the mRNA expression level of IL-6 and the concentration of IL-6 in the supernatant,respectively.Cardiac fibroblasts were treated with IL-6 and Reg3? for 12 h,and the protein level of Agtr1 was assessed by Western blot.Cardiac fibroblasts were pretreated with IL-6 neutralizing antibody for 6 h and then treated with ANG II for 12 h.The protein level of Agtr1 was assessed by Western blot.IL-6 can induce phosphorylation of Jak1 and Stat3 in cardiac fibroblasts,and cells treated with IL-6 for 5 min,15 min,30 min,45 min,and 60 min,and the phosphorylation levels of Jak1 and Stat3 were evaluated by Western blot.Cardiac fibroblasts were pretreated with Reg3? for 12 h,then treated with IL-6,cells were harvested at the corresponding points,and phosphorylation levels of Jak1 and Stat3 were assessed by Western blot.(4)Construction of a mouse model with viral myocarditis,the therapeutic effect of Reg3? on cardiac fibrosis in mice with viral myocarditis: Reg3? was administered by tail vein injection before inducing a model of viral myocarditis in mice.And after inducing the model of viral myocarditis in mice,the mice were continuously administered by intravenous injection of Reg3? every other day.The mice were sacrificed on the 7th and 15 th days of cervical dislocation,and Heart tissue was collected and fixed with paraformaldehyde for pathological examination.The protein levels of Collagen-I and ?-SMA were detected by Western blot after protein extraction from fresh heart tissue.Results:(1)The results of qRT-PCR showed that the mRNA levels of Collagen-I and Collagen-III in cardiac fibroblasts of ANG II group were significantly increased compared with Control group,Compared with the ANG II group,the mRNA levels of Collagen-I and Collagen-III in the cardiac fibroblasts of the ANG II+Reg3? group were significantly decreased.Western blot and immunofluorescence showed that Collagen-I and ?-SMA were significantly increased in cardiac fibroblasts of ANG II group compared with Control group,Compared with the ANG II group,Collagen-I and ?-SMA were significantly decreased in the cardiac fibroblasts of the ANG II+Reg3? group.Reg3? treatment of human cardiac fibroblasts also showed the same results compared to mouse cardiac fibroblasts,and these results clearly indicate that Reg3? can inhibit ANG II induced cardiac fibroblast expression of collagen.(2)The results of qRT-PCR and Western blot showed that the expression of EXTL-3 mRNA and protein in Reg3? treatment group was significantly higher than that in Control group.Western blot and immunofluorescence showed that there was no significant change in the expression of Collagen-I and ?-SMA in the siEXTL-3 treated group compared with the ANG II group.The results of qRT-PCR showed that there was no significant change in MMP1 and TIMP1 mRNA levels in the siEXTL-3 treated group compared with the ANG II group.(3)The ELISA results showed that the content of TGF-?1 in the MFB group was significantly higher than that in the MCF group.Western blot results showed that the levels of p-Smad2/3,p-Erk1/2,p-P38 and RhoA protein were significantly increased in ANG II treated cardiac fibroblasts for 30 min compared with 0 min.The results of Western blot showed that the levels of p-Smad2/3,p-Erk1/2,p-P38 and RhoA in ANG II group were increased compared with Control group.Compared with the ANG II group,the levels of p-Smad2/3,p-Erk1/2,p-P38 and RhoA were decreased in cardiac fibroblasts of the ANG II+Reg3? group.Compared with the ANG II group,the levels of p-Smad2/3,p-Erk1/2,p-P38 and RhoA were decreased in cardiac fibroblasts of the ANG II+TGF-?1 receptor inhibitor group.The results of qRT-PCR showed that the expression of IL-6 mRNA in the cardiac fibroblasts of ANG II group was increased compared with Control group.Compared with ANG II group,the expression of IL-6 mRNA in the cardiac fibroblasts of ANG II+Reg3? group was decreased.The ELISA results showed that the concentration of IL-6 in the supernatant of cardiac fibroblasts in ANG II group was increased compared with the Control group,and the concentration of IL-6 in the supernatant of cardiac fibroblasts in ANG II+Reg3? group was decreased compared with the ANG II group.The results of Western blot showed that the level of Agtr1 protein in cardiac fibroblasts of ANG II group was significantly higher than that of Control group.Compared with ANG II group,the level of Agtr1 protein in cardiac fibroblasts of ANG II+Reg3? group was decreased.Compared with the Control group,Agtr1 protein levels were increased in cardiac fibroblasts of IL-6 group,and Agtr1 protein levels were decreased in cardiac fibroblasts of IL-6+Reg3? group compared with IL-6 group.Compared with the Control group,the Agtr1 protein level was increased in the cardiac fibroblasts of ANG II group,and the Agtr1 protein level was decreased in the cardiac fibroblasts of ANG II+ IL-6 neutralizing antibody group compared with the ANG II group.Western blot results showed that p-STAT3 began to increase at 5 min until 30 min began to decrease,and p-JAK1 began to increase at 30 min.Compared with the Control group,the levels of p-STAT3 and p-JAK1 in the cardiac fibroblasts of IL-6 group were increased.Compared with the IL-6 group,the levels of p-STAT3 and p-JAK1 protein in the cardiac fibroblasts of the IL-6+Reg3? group were decreased.(4)On the 15 th day of establishing a viral myocarditis model,HE staining results showed that inflammatory cell infiltration was alleviated in the 15d+Reg3? group compared with the 15 d group.The results of Sirius red staining showed that the heart entered the fibrosis stage on the 15 th day of establishing the viral myocarditis model.Compared with the 15 d group,the collagen deposition in the 15d+Reg3? group was significantly reduced.Western blot results showed that the protein levels of Collagen-I and ?-SMA in the CVB3+Reg3? group were significantly lower than those in the CVB3 group.Conclusion:(1)Reg3? can reduce the expression of Agtr1 in mouse cardiac fibroblasts and inhibit ANG II-induced expression of collagen in mouse cardiac fibroblasts.(2)ANG II promotes the expression of collagen in mouse cardiac fibroblasts via TGF-?1-Smad2/3,Erk1/2,P38 and RhoA pathways.Reg3? inhibits the expression of collagen in mouse cardiac fibroblasts by blocking the TGF-?1-Smad2/3,Erk1/2,P38 and RhoA pathways.Reg3? reduces collagen expression in mouse cardiac fibroblasts by blocking activation of the IL-6 / Jak1 / Stat3 pathway.(3)Reg3? alleviated the progression of cardiac fibrosis in mice with viral myocarditis.