Exogenous H2s Attenuates The Myocardial Fibrosis In Hyperhomocysteinemia-induced Rats By Down-regulating The Activity Of TGF-β1/Smad3 Signaling Pathway

Objective:This study aimed to observe the effects of exogenous hydrogen suifide（H₂S）on myocardial fibrosis induced by hyperhomocysteinemia（HHcy）via regulating the activity of TGF-β1/Smad3 signaling pathway.Methods:After a week of adjustable feeding,40 male Sprague-Dawley rats were randomly assigned into Control group,HHcy group,HHcy+H₂S group and H₂S group（n=10）.HHcy group and HHcy+H₂S group were induced by adding L-methionine（10g/l）into the drinking water consecutive ten weeks to set up the model of HHcy.After ten weeks,the level of HHcy was detected and the concentration of Hcy no less than 15mmol/l was deemed as HHcy.NaHS（56umol/kg/d,intraperitoneal injection,lasting four weeks）,which was used as an exogenous H₂S,was used in these rats in HHcy+H₂S group and H₂S group.These rats in Control group and HHcy group were injected with the equal volume of normal saline.After fourteen weeks,LVDd,LVSd,LVPWd,LVFS were detected by M ultrasound.Then the rats were weighted（body weight,BW）and killed.The heart of the rats was removed and weighted（heart weight,HW）,The heart weight index（HW/BW,mg/g）was calculated.Collagen deposition was measured by masson staining.The expression of CollagenⅢ was detected by immunohistochemical staining.The expression of CSE,MMPs/TIMPs CollagenⅣ,Bcl-2,Caspase3/9,Bax,NF-κBp65,IL-6,TGF-β1/Smad3 were detected by western blotting.RT-qPCR was used to detect Col1α2 and Col3α1.Results:Compared with the Control group,the concentration of Hcy in the HHcy group and the HHcy+H₂S group was significantly increased（P<0.05）,while there was no significant difference between the Control group and the H₂S group（P>0.05）.Compared with the Control group,HW/BW were significantly increased in the HHcy group（P<0.05）.Relative to the HHcy group,HW/BW of rats in the HHcy+H₂S group were significantly decreased（P<0.05）,while the differences in BW and HW were not statistically significant（P>0.05）.2.The results of M ultrasound showed decrease of cardiac function,compared with the Control group,the values of LVSd and LVDd significantly increased and the value of LVFS significantly decreased in the HHcy group（P<0.05）;In contrast to the HHcy group,the values of LVSd and LVDd significantly decreased and the value of LVFS significantly increased in the HHcy+H₂S group（P<0.05）.3.Compared with the Control group,for rats in the HHcy group,the blue dye collagen fiber was obviously increased（P<0.05）;Compared with the HHcy group,the blue dye collagen fiber was obviously reduced in the HHcy+H₂S group（P<0.05）.4.The results of immunohistochemistry staining revealed that the expression of collagenⅢwas remarkably increased in the HHcy group（P<0.05）,relative to the Control group;Compared with the HHcy group,the expression of CollagenⅢwas remarkably decreased in the HHcy+H₂S group（P<0.05）.5.Western blotting results showed:compared to Control group,the expression of CollagenⅣ,MMP13,TIMP1,Caspase3,Caspase9,Bax,TGF-β1、Smad3、NF-κBp65 and IL-6in myocardial tissue was increased significantly（P<0.05）,while that of CSE,MMP1,Bcl-2 decreased significantly（P<0.05）for rats in HHcy group;Relative to HHcy group,the expression of CollagenⅣ,MMP13,TIMP1,Caspase3,Caspase9,Bax,TGF-β1,Smad3,NF-κBp65 and IL-6 was significantly decreased（P<0.05）,while that of CSE,MMP1,Bcl-2 was significantly increased（P<0.05）for rats in HHcy+H₂S group.7.The results of RT-qPCR analysis revealed that the expression level of Col1α2 and Col3α1 in HHcy group were significantly elevated than Control group（P﹤0.05）.However,compared with HHcy group,the expression level of Col1α2 and Col3α1 were remarkably reduced（P﹤0.05）in HHcy+H₂S group.Conclusion:H₂S can reduce myocardial fibrosis in hyperhomocysteinemia-induced rats,which may be related with down-regulation of TGF-β1/Smad3 signaling pathway to inhibite apoptosis and reduce inflammatory reaction.