Role Of BMAL1 Mediated Nrf2/HO-1 Signaling Pathway In Myocardial Ischemia/reperfusion Injury In Diabetic Rats

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The involvement of BMAL1 and Nrf2/HO-1 signaling in diabetic rats subjected to myocardial ischemia/reperfusion injury ObjectiveThe changes of myocardial tissue clock gene BMAL1 and Nrf2/HO-1 signaling pathway were detected by establishing a model of myocardial ischemia-reperfusion injury in type(40)diabetic rats,and the mechanisms of BMAL1 and Nrf2/HO-1 signaling pathway in the increase of diabetic myocardial vulnerability were investigated.Methods48 SPF grade male Sprague-Dawley rats were randomly divided into 4 groups(n=12):(1)non-diabetic rat sham operation group(NS group);(2)non-diabetic rats with myocardial ischemia reperfusion group(NI/R group);(3)diabetic rats sham operation group(DS group);(4)diabetic rats myocardial ischemia reperfusion group(DI/R group).All rats were investigated to establish a model of diabetes by intraperitoneal injection of 1% streptozotocin 60 mg/kg.After 72 hours of intraperitoneal injection of streptozotocin,blood glucose was detected in rats tail vein,and rats with blood glucose?16.7 mmol/L were considered as diabetic model.A model of myocardial ischemia-reperfusion injury was established by ligation of the left anterior descending coronary artery(LDA)of the coronary artery: ischemia for 30 min followed by release of the ligature for 120 min.The electrocardiogram was monitored during the procedure using an electrocardiogram II lead and the heart rate,left ventricular systolic pressure,maximum rise of left ventricular pressure,and fall rate of left ventricular pressure were recorded at various time points.The myocardial infarct size of the rats was determined by TTC and Evans blue double staining.The contents of serum CK-MB,myocardial tissue MDA and SOD were determined by ELISA.The expression level of BMAL1 in rat myocardial tissue was detected by immunohistochemistry.The expression levels of BMAL1,Nrf2 and HO-1 proteins in rat myocardial tissue were detected by Western Blot.Results1.Compared with non-diabetic rats,the myocardial oxidative stress level increased in diabetic rats,which showed an increase in MDA levels and a decrease in antioxidant enzyme SOD activity.Further experiments revealed that the expression level of myocardial clock gene BMAL1 was down-regulated,accompanied with the decrease in protein expression of Nrf2 and HO-1;2.After the treatment of myocardial ischemia-reperfusion,the myocardial infarct size and serum myocardial injury marker CK-MB level in diabetic rats were significantly increased compared with non-diabetic group,while the expression levels of BMAL1,Nrf2 and HO-1 in myocardial tissue were further decreased.Conclusions1.Diabetic myocardium is more susceptible to ischemia-reperfusion injury than non-diabetic myocardium,and oxidative stress injury is one of its important mechanisms.2.The decrease of BMAL1 level in diabetic myocardium and the damage of Nrf2/HO-1 signaling pathway may be related to the increased vulnerability of diabetic myocardial ischemia-reperfusion.However,the exact mechanism remains to be further studied.Part ? Role of BMAL1 modulated Nrf2/HO-1 signaling in high glucose and hypoxia/reoxygenation induced injury in H9c2 cellsObjective To establish a model of hyperglycemia and hypoxia-reoxygenation injury in H9c2 cardiomyocytes,and to further investigate the changes of BMAL1 and Nrf2/HO-1 signaling pathway,and to explore whether the increase of vulnerability in cardiomyocytes under high glucose and hypoxia-reoxygenation environment is related to the mechanism of BMAL1 and Nrf2/HO-1 signaling pathway.Methods H9c2 cells were routinely maintained in Dulbecco's modified Eagle medium.In order to study the levels of cell damage,oxidative stress and the protein expression of BMAL1,Nrf2 and HO-1 in high glucose and high glucose hypoxia reoxygenation,the culture time of high glucose condition in this part was determined for 24 h and hypoxia time were determined for 6h.The experiments in this part were divided into the following five groups:(1)low glucose control group(LG);(2)low glucose hypoxia/reoxygenation group(LG+H/R);(3)High glucose control group(HG);(4)High glucose hypoxia/reoxygenation group(HG+H/R).(5)Mannitol control group(Mannitol).In order to study the role of BMAL1 in high glucose and hypoxia-reoxygenation,and the effect of BMAL1 on the oxidative stress level of H9c2 cells under high glucose and hypoxia-reoxygenation condition,and the changes of Nrf2 and HO-1 proteins,this part was divided into the following six groups:(1)HG+ Ctrl group;(2)HG+Lv-BMAL1 group;(3)HG+Lv-BMAL1-sh RNA group;(4)HG+H/R+ Ctrl group;(5)HG+H/R+Lv-BMAL1 group;(6)HG+H/R + Lv-BMAL1-sh RNA group.ROS levels were detected using the DHE method.Immunofluorescence was used to detect the expression of BMAL1 and Nrf2 proteins.CCK-8 was used to detect the survival rate of cells.The level of LDH was measured to determine the degree of cell damage.The levels of MDA and SOD were measured to determine the level of oxidative stress.The protein expression levels of BMAL1,Nrf2 and HO-1 were detected by Western blot.Results 1.The experimental method of hypoxia for 6 h and reoxygenation for 2 h was carried out after incubation for 24 h in high glucose for subsequent experimental studies.The cell viability detected by CCK-8 in H9c2 cells after high glucose hypoxia and reoxygenation was significantly lower than low glucose culture groups,and the level of LDH,which is a marker of myocardial injury,was significantly higher than low glucose culture groups.The oxidative stress injury of H9c2 cells was significantly higher than low glucose culture groups.The MDA level in H9c2 cells was significantly higher than low glucose culture groups,and the SOD level was significantly lower than low glucose culture groups.2.The protein expression of BMAL1,Nrf2 and HO-1 in H9c2 cells of high glucose control group was significantly lower than low glucose control group.The protein expression of BMAL1,Nrf2 and HO-1 in H9c2 cells of high glucose hypoxia-reoxygenation group was significantly lower than high glucose control group.3.Overexpression of BMAL1 increased the activity and SOD level of H9c2 cells in high glucose control group and high glucose hypoxia/reoxygenation group,and decreased the levels of LDH,MDA and ROS in H9c2 cells in high glucose control group and high glucose hypoxia/reoxygenation group.4.Overexpression of BMAL1 increased the expression levels of Nrf2 and HO-1 in high glucose control group and high glucose hypoxia reoxygenation group.Conclusions 1.Important mechanisms of cardiomyocytes susceptible to high glucose and hypoxia-reoxygenation injury,including decreased expression of BMAL1 and inactivation of Nrf2/HO-1 signaling pathway in high glucose condition.2.BMAL1 positively regulates the Nrf2/HO-1 signaling pathway,and measures to restore the level of Nrf2/HO-1 signaling pathway by increasing the expression of BMAL1 could reduce myocardial hyperglycemia and hypoxia-reoxygenation injury.