| Objective:In this research,we established a doxorubicin（DOX）-induced myocardial fibrosis rat model to investigate the relationships among the myocardial fibrosis induced by doxorubicin,the autophagy regulated via AMPK-ULK1 and pyroptosis.To observe the influence on the new mitochondria-targeted hydrogen sulfide donor AP39 operating on it,and to explore furtherly that AP39 may upregulate AMPK-ULK1 to promoting autophagy and downregulate pyroptosis to alleviate the myocardial fibrosis induced by doxorubicin or not and its related mechanism.Methods:60 female adult SD rats were stochasticly falled into six groups after one week feeding,which were control group（Control）（n=10）,model group（DOX）（n=10）,AP39 intervention group（AP39+DOX）（n=10）,PAG（DL-Propargylglycine）intervention group（PAG）（n=10）,AP39 control group（AP39）（n=10）,DMSO control group（DMSO）（n=10）.DOX group,AP39+DOX group and PAG group were established by injecting intraperitoneally of doxorubicin in Six sessions over two weeks（3mg/kg/times,prepared temporaryly before using）.Control group,AP39 group and DMSO group were given the same amount of normal saline in the model group by intraperitoneal injection.The rats in DOX group,AP39 group and PAG group were intraperitoneally injected with AP39（50n M/kg/d,dissoved in 0.05%DMSO）,the PAG group were intraperitoneally injected with additional endogenous H2S generating enzyme inhibitor PAG（40mg/kg/d）,and female rats in the DOX group and the Control group were given normal saline intervention,and female rats in the DMSO group were given0.05%DMSO intervention,all lasted for 4 weeks.8 weeks later,the cardiac structure and function were tested by echocardiography.The body weight（BW）and isolated heart weight（HW）of rats in each group were measured,relevant myocardial specimens were retained for subsequent experimental detection after sacrificing the rats:myocardial collagen fiber deposition was detected by Masson staining,expression of CollagenⅢwas tested by immunohistochemistry,Changes of autophagy and mitochondria were observed by transmission electron microscopy,and protein expression levels of CSE、TIMPs/MMPs、CollagenⅢ、Caspase1/4、IL-1β、GSDMD、Beclin-1、Atg16L1、Atg5、AMPK and ULK1 were tested by WB.Results:1.The results of echocardiography are as follows:the LVEDd and the LVESd were significantly increased and the LVFS was distinctly decreased in the DOX group,which compared with the Control group.（P<0.05）.The LVEDd and the LVESd were apparently decreased and the LVFS was obviously increased in the DOX+AP39 group,which compared with the DOX group（P<0.05）.The LVEDd and the LVESd were significantly upregulated and the LVFS were down regulated in the PAG group,which compared with the DOX+AP39 group（P<0.05）.2.The general situation of rats are as follows:Compared with the Control group,BW was Obviously declined and HW/BW were significantly rising in the DOX group（P<0.05）.Compared with the DOX group,BW was increased and HW/BW were apparently decreased in the DOX+AP39group.Compared with the DOX+AP39 group,BW was descended and HW/BW were significantly rising in the PAG group.The comparison of HW in each group shows no statistically significant difference.3.Compared with the Control group,the blue staining area of the myocardium in the DOX group was upregulated and the distribution was uneven,suggesting that the myocardium of the model group occurred fibrosis.Compared with DOX group,the blue staining area in DOX+AP39 group decreased,suggesting that AP39 can lighten myocardial fibrosis in rats.Compared with DOX+AP39 group,the blue staining area of rats in PAG group were increased,suggesting that endogenous hydrogen sulfide can inhibit myocardial fibrosis.4.As is showed in TEM,compared with the Control group,The mitochondrial cristae in the myocardial tissue of DOX group were obviously blurred in the field of electron microscope and the number of autophagosomes was significantly reduced.Compared with the DOX group,the mitochondrial crest in the myocardial tissue was clearly visible in the myocardium of DOX+AP39 group and the number of autophagosomes was increased.5.The results of immunohistochemistry were shown as follows:the expression of type III collagen in DOX group was upregulated compared with the Control group and was obviously decreased compared with DOX+AP39 group（P<0.05）.Compared with the DOX+AP39 group,the myocardial collagen fibers were upregulated in the PAG group.6.The results of WB show:compared with the Control group,the expressions of CollagenⅢ、TIMP1、MMP8、MMP13、Caspase1、Caspase4、IL-1β、GSDMD were evidently growing in the DOX group（P<0.05）,CSE、Beclin-1、Atg5、Atg16L1、AMPK、ULK1 were apparently reduced（P<0.05）.Compared with DOX group,the expressions of CollagenⅢ、TIMP1、MMP8、MMP13、Caspase1、Caspase4、IL-1β、GSDMD were descended in DOX+AP39 group（P<0.05）and the expressions of CSE、Beclin-1、Atg5、Atg16L1、AMPK、ULK1 were distinctly rising（P<0.05）.Compared with the DOX+AP39 group,the expressions of CollagenⅢ、TIMP1、MMP8、MMP13、Caspase1、Caspase4、IL-1β、GSDMD were upregulated in the PAG group（P<0.05）,CSE、Beclin-1、Atg5、Atg16L1、AMPK、ULK1 were declined（P<0.05）.Conclusion:AP39 could ameliorate the doxorubicin-induced myocardial fibrosis.The mechanism could be connected with activating of autophagy through upregulating AMPK-ULK1 pathway and inhibiting pyroptosis. |
