| Objective:To explore the role of Bmal1-Sirt3 pathway in diabetes MI/RI susceptibility and its relationship with mitochondrial autophagy rhythm.Methods:Choose healthy male SD rats of clean grade for 4-6 weeks,In terms of weight,about 200～220 g.After fasted 12 hours,rats were administrated by intraperitoneal injection 60 mg/kg streptozotocin(STZ)to establish diabetes model as described previously The fasting blood glucose(after fasting for 6 hours)was measured after 72 h.The hallmarks of successful establishment of diabetes model are blood glucose levels16.7 mmol/L with increased consumption of food and water and increased urination of rats.After that,all rats are continue raised for 8 weeks.According to the random number table method,30 selected type 2 diabetes(T2DM)rats were equally allocated to the following three groups: DM-S group(DM-S group only sham operation group without ligation),DM-I/R group(DM-I/R group)Ischemia-reperfusion group),DMS-I/R group(DM+SR8278 ischemia-reperfusion group).In addition,30 healthy rats were selected,and they were equally distributed to the following 3 groups by random number table method: nondiabetes mellitus sham operation group(N-S group);nondiabetes mellitus I/R group(N-I/R group);N-I/R SR8278 conditioning group(NS-I/Rgroup).Myocardial I/R was induced of the left anterior descending branch of coronary artery(after 45 min of equilibration)followed by 24 h of reperfusion.DMS-I/R group and NS-I/R group were intraperitoneally injected with SR8278(Rev-erbα antagonist)50mg/kg at T8 before surgery for 7 days.After 24 hours of myocardial reperfusion,HE staining was performed and the pathological changes were observed under light microscope;the myocardial infarction volume was detected by TTC staining;the expression of Bmal1,Sirt3,Nlrp3 and Bnip3 in myocardial tissue was detected by Western blot.Results:Compared with the NS group,the MI volume of the other 4 groups(N-I/R group,DMS-I/R group,NS-I/R group and DM-I/R group)excluding DM-S group was significantly increased(P<0.05).Compared with the N-I/R group,the myocardial infarction volume of the non-diabetic rats treated with SR8278,namely the NS-I/R group,was reduced(P<0.05).Compared with DM-S group,DM-I/R group and DMS-I/R group The volume of myocardial infarction increased(P<0.05).Compared with the DM-I/R group,the volume of myocardial infarction decreased in the DMS-I/R group(P<0.05).Compared with N-S group,BMAL1 and SIRT3 protein expressions were decreased in N-I/R group,NS-I/R group,DM-S group,DM-I/R group and DMS-I/R group,while BNIP3 and NLRP3 protein expressions were increased(P<0.05).Compared with the N-I/R group,the protein expression levels of BNIP3,SIRT3 and BMAL1 in NS-I/R group were significantly up-regulated,while the protein expression levels of NLRP3 were significantly decreased(P<0.05).Compared with N-I/R group,the protein expressions of SIRT3 and BMAL1 were significantly decreased in DM-I/R group,while the protein expressions of NLRP3 and BNIP3 were up-regulated(P<0.05).Compared with the DM-S group,the protein expressions of SIRT3 and BMAL1 in the DM-I/R group and the DMS-I/R group were significantly decreased,while the protein expressions of BNIP3 and NLRP3 were significantly increased(P<0.05).Compared with the DMS-I/R group,the protein expressions of BNIP3,SIRT3 and BMAL1 in DMS-I/R group were significantly increased,while the protein expressions of NLRP3 were significantly decreased(P<0.05).Compared with the NS-I/R group,the protein expressions of BMAL1 and SIRT3 in DMS-I/R group were down-regulated,while the protein expressions of NLRP3 and BNIP3 were up-regulated(P<0.05).Conclusion:1.Ischemia-reperfusion injury can induce the down-regulation of Bmal1 expression in the myocardium of non-diabetic rats,and diabetic factors further down-regulate the expression of Bmal1 in rat myocardial cells.2.Bmal1 activates the mitochondrial Bnip3 autophagy pathway induced by sirt3 and inhibits the Nlrp3 inflammasome,thereby protecting diabetic myocardial ischemia and reperfusion. |
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