Hydrogen Sulfide Attenuates Myocardial Fibrosis In Metabolic Syndrome Rats Through PI3K-AKT Signaling Pathway Regulating Autophagy

Objective:To establish a model of myocardial injury induced by high-fat/high-sucrose diet（HFSD） in metabolic syndrome（MS）,and to understand the effect of HFSD on myocardial fibrosis,exogenous hydrogen sulfide（H₂S） was injected intraperitoneally to reveal it’s role and regulatory mechanism in ameliorating the metabolic syndrome rat myocardial fibrosis.It may be related to up-regulation of autophagy by involving into the activity of the PI3K-AKT signaling pathway.Methods:Forty healthy male Sprague-Dawley rats were randomly divided into Control group（n=10）,MS group（n=10）,MS+H₂S group（n=10） and H₂S group（n=10）.All of them were fed separately.To establish the model of myocardial injure,the MS group and MS+H₂S group were fed with HFSD,and the control group as well as H₂S group were fed with the same amount of common feed.Seven months later,we administered NaHS（56μmol/kg.d,）to the rats in the MS+H₂S group and the H₂S group by intraperitoneal injection,while the control group and the MS group received the same volume of normal saline.At the beginning of the experiment and 7 months after the experiment,we measured the height,body weight,abdominal circumference（AC） and blood pressure of the rats,and calculated Lee’s index.The fasting plasma glucose（FPG）,insulin,triglyceride（TG）,total cholesterol（TC）,uric acid（UA） and homeostasis model assessment insulin resistance（HOMA-IR） were detected after 7 months of the experiment.After 6 weeks of NaHS intervention,we measured cardiac function of each rat by echocardiography,the body weight（BW） and the heart weight（HW） by electronic scale,and calculated the heart weight index（HW/BW）.We also observed the collagen deposition by Masson staining,the expression of Collagen Ⅲ by immunohistochemistry,the gene expression of Col1α2 and Col3α1by Real-time fluorescent quantitative PCR（RT-qPCR）.The expressions of Beclin-1、Atg3、Atg5、AKT、PI3K、MMP3、MMP1、TIMP2、CSE were detected by Western blotting analysis.Results:1.There was no significant difference in the height,BW,AC of rats between each groups before the experiment.Seven month later,the height,body weight,AC and Lee’s index of MS group and H₂S+MS group increased significantly than Control group（P<0.05）.But there was no significant difference between MS group and H₂S+MS group（P>0.05）.2.Biochemical results suggest that,the FPG,insulin,TG,TC,UA and HOMA-IR were significantly higher than Control group（P<0.05）.However,there was no significant difference between MS group and H₂S+MS group（P>0.05）.3.Compared with the control group,the blood pressure in MS group and H₂S+MS group increased significantly（P<0.05）,and there was no significant difference in blood pressure between MS group and H₂S+MS group.4.Compared with the control group,the left ventricular end-systolic diameter（LVESD）and left ventricular end-diastolic diameter（LVEDD）of the MS group were significantly increased,and the value of EF and FS were decreased.The difference was statistically significant（P<0.05）.After the H₂S intervention for 6 weeks,the LVEDD and LVESD were significantly lower than those in the MS group,and the value of EF and FS were increased（P<0.05）.5.Compared with the Control group,the HW value and the BW value of the MS group were significantly increased（P<0.05）;Compared with MS group,the HW value of the MS+H₂S group were decreased（P<0.05）,and the BW value of the MS+H₂S group was also decreased,but no significant difference between them（P>0.05）;The comparison of the values of HW/BW between any two groups showed that the difference was not statistically significant（P>0.05）.6.The result of Masson staining showed that,the blue-stained fibers in the MS group were significantly coarser,unevenly distributed,and the deposition increased,while the myocardial cells were significantly reduced and disordered.And the blue-stained fibers were significantly relieved in MS+H₂S group than that in MS group.7.As is shown in transmission electron microscopy results,compared with the Control group and the H₂S group,the vacuolation in mitochondria and autophagosomes,disordered myocardium fibers,and the mitochondrial ridges were observed in the MS group.Compared with the MS group,we can see autophagosomes in MS+H₂S group without obvious vacuolization,and the myocardium fibers were arranged neatly.8.The result of immunohistochemistry showed that compared with Control group,the expression of Collagen Ⅲ in MS group was significantly higher（P<0.05）.However,compared with MS group,the expression of Collagen Ⅲ was significantly reduced in MS+H₂S group（P<0.05）.9.As shown by RT-qPCR results,the expression of Col1α2 and Col3α1 in MS group were more higher than Control group（P<0.05）;However,compared with MS group,the expression of Col1α2 and Col3α1 were significantly reduced in MS+H₂S group（P<0.05）.10.Compared with the Control group,the expressions of MMP3、MMP1、AKT、PI3K were significantly increased and the expression levels of Beclin-1、Atg3、Atg5、TIMP2、CSE were significantly decreased in MS group（P<0.05）.Compared with MS group,the changes mentioned above were significantly opposite in the MS+H₂S group（P<0.05）.Conclusion:Myocardial fibrosis was observed significantly in MS rats.The mechanism may be related to the down-regulation of autophagy and the up-regulation of PI3K pathway.H₂S could improve myocardial fibrosis in MS rats,and its mechanism may be related to the regulation of PI3K-AKT pathway to promote autophagy.