Hydrogen Sulfide Attenuates Myocardial Fibrosis Induced By Doxorubicin Through Keap1-Nrf2 Signaling Pathway

Objective:This study was aimed to investigate the role and regulatory mechanism of the hydrogen sulfide（H₂S）in amelioration of rat myocardial fibrosis induced by doxorubicin（DOX）via regulating the activity of Keap1/Nrf2 signaling pathway.Methods:Forty-four SD rats were randomly divided into Control group,DOX group,DOX+H₂S group and H₂S group（n=10）.DOX group and DOX+H₂S group were induced by intraperitoneal injection of doxorubicin（3mg/kg,3 times per week with a total cumulative dose of Dox 18 mg/kg）to establish the model of myocardial injure.NaHS（56 umol/kg.d,i.p）,which was used as an exogenous donor of H₂S,was given to rats in DOX+H₂S group and H₂S group.Rats in Control group and DOX group were injected the same volume of saline.After 8 weeks of the first injection of doxorubicin,LVEDD,LVESD,LVEF and LVFS were measured by echocardiography.Then the body weight（BW）of each rat was weighed and the rats were sacrificed.The heart of the rats was taken out and the heart weight（HW）was measured.The heart weight index（HW/BW）was calculated.The pathological morphological changes in myocardial fiber were observed by Masson staining.Immunohistochemistry was used to determining the expression of collagen III.Oxidative stress was evaluated through detecting the contents of malondialdehyde（MDA）,glutathione（GSH）and superoxide dismutase（SOD）in the myocardial cells.ELISA method was also employed to determine the H₂S content.The expressions of CSE,CollagenIII,MMPs/TIMPs,TGF-β1,caspase3,caspase9,Bax,Bcl2,TNF-α,IL1β,IL6,COX2,Keap1,Nrf2,NQO1,HO-1,GCLC and SOD were detected by Western blotting analysis.Results:1.Compared with the Control group,the HW/BW value of the DOX group was significantly increased and the BW value of the DOX group was significantly decreasd（P<0.05）;Compared with DOX group,the HW/BW value of the DOX group was significantly decreased and the BW value of the DOX group was significantly increased（P<0.05）;No significant difference was observed in the values of HW between any two groups（P>0.05）.2.Compared with the Control group,the values of LVEDD and LVESD were increased significantly and the values of LVEF and LVFS were decreased significantly in DOX group（P<0.05）;While compared with the DOX group,the values of LVEDD and LVESD were decreased significantly and the values of LVEF and LVFS were increased significantly in DOX group（P<0.05）.3.Masson staining showed that myocardial fibrosis was barely observed in the Control group.In the DOX group,the myocardial cells showed a disorderly arrangement,and myocardial fibres significantly increased.Unlike in the DOX group,the myocardial fibrosis was obviously alleviated in the DOX+H₂S group.4.Immunohistochemistry result indicated that the expression of collagen III in DOX group was significantly higher than Control group.However,compared with Control group,the expression of collagen III was significantly reduced in DOX+H₂S group.5.The results of transmission electron microscopy showed that the myocardium fibers and myocardial mitochondria in DOX group appeared edema,disorder and focal necrosis.While H₂S could reduce myocardial fibrosis and alleviate the edema,disorder and focal necrosis.6.The results of ELISA showed that the contents of GSH,SOD and H₂S in DOX group were decreased and the content of MDA was increased obviously compared with the Control group（P<0.05）,but this was reversed by NaHS treatment in DOX+H₂S group（P<0.05）.7.Compared with the Control group,the expressions of TGF-β1,CollagenIII,MMP8,MMP16,TIMP1,caspase-3,caspase-9,Bax,TNF-α,IL1β,IL6,COX2 and Keap1 were significantly increased and the expression levels of CSE,Bcl-2,Nrf2,SOD,NQO1,HO-1 and GCLC were significantly decreased in DOX group.Compared with DOX group,these above-mentioned changes were obviously reversed in DOX+H₂S group（P<0.05）.Conclusion:H₂S can improve the myocardial fibrosis in rats induced by doxorubicin.The mechanism of which may be related to the regulation of Keap1/Nrf2 signaling pathway,thereby suppressing oxidative stress,inflammatory reaction and apoptosis.