| BackgroundPost-ischemic reperfusion is a "double-edged sword",which not only restores blood flow to save the dying myocardium,but also leads to further damage of myocardial structure and function.This phenomenon is called ischemia/reperfusion injury(IRI).Diabetes mellitus is an independent risk factor for ischemic heart disease.It could aggravate myocardial ischemia/reperfusion injury(MIRI),and weaken the protective effect of most interventions on MIRI.However,the specific mechanism is still unclear.Thus finding interventions to protect diabetic MIRI is the focus of researchers.Dexmedetomidine(DEX)is a novel highly selective ?2 adrenergic receptor agonist with sedative,analgesic and sympathetic nerve inhibition effects.It is widely used in clinical anesthesia and intensive care units.The effects of DEX on cardiovascular system include reducing heart rate and stabilizing hemodynamics.In the basic research of cardioprotection of DEX on MIRI,DEX plays an anti-inflammatory and anti-apoptotic role by activating signal transduction pathway to reduce the infarct size.However,the role of DEX in diabetic MIRI and its underlying mechanisms have not been studied.It has been found that many mechanisms are involved in the occurrence of MIRI,in which apoptosis and oxidative stress play important roles.Clinical and animal studies have confirmed that reperfusion injury can initiate or accelerate the process of cardiomyocyte apoptosis,resulting in the reduction of cardiomyocyte number and cardiac dysfunction.In addition,diabetes also causes increased apoptosis of cardiomyocytes.Therefore,apoptosis is an important cause of diabetic cardiomyopathy.The process of ischemia-reperfusion can aggravate the production of reactive oxygen species(ROS).Excessive ROS can not only directly act on myocardial cells,causing lipid peroxidation of cell membrane,DNA damage and protein degeneration in cells,but also indirectly act as intracellular messenger and result in tissue injury by activating various signaling pathways.When oxidative stress occurs in myocardial tissue,the Keapl-Nrf2/ARE signal transduction pathway is activated.It drives the antioxidant defense mechanism,regulates the expression of many target genes,and induces the production of endogenous antioxidant enzymes,thus affecting the level of oxidative stress in the body,which is very important in improving cardiac remodeling and cardiac dysfunction.In addition,Keapl-Nrf2/ARE signal transduction pathway plays an important role in avoiding myocardial injury by resisting oxidative stress in diabetic patients.The protective effect of DEX is also closely related to antioxidant stress,which can alleviate IRI in brain,lung and kidney by inhibiting oxidative stress.Therefore,there is a close relationship between diabetic MIRI,Keapl-Nrf2/ARE signal transduction pathway and DEX intervention.However the specific mechanism remains to be further studied by researchers.Based on the above analysis,this experiment was designed to explore the effect of DEX preconditioning on normal and diabetic MIRI,and to explore the roles of anti-apoptotic and anti-oxidative stress in it.This is helpful to further elucidate the cardioprotective mechanism of DEX preconditioning and provide ideas for the treatment of MIRI.This study was divided into four parts:Part 1:Establishment of diabetic rat modelThe aim of this part experiment was to build an appropriate and effective diabetic model for studying the effects of DEX preconditioning on normal and diabetic MIRI.Three hundred and six 8-week-old male SD rats were randomly divided into normal group(N group,138 rats)and diabetic group(D group,168 rats).Rats in the N group were fed with normal diet for 8 weeks.Rats in the D group were intraperitoneal injected of streptozotocin(STZ)50 mg/kg combined with fed with high-fat and high-sugar diet for 8 weeks,during which the blood glucose level continued to exceed 16.65 mmol/L.During the feeding process,the weight and blood glucose level of rats were observed for 8 weeks.After feeding,the mortality rate in each group and the success rate of establishing diabetic model were counted.The results showed that the mortality rates of rats in the N group and D group were 2.2%and 4.8%respectively.There was no significant difference between N group and D group(P>0.05).The success rate of diabetes model was 90%.There was no significant difference in body weight between N group and D group before and 2 days after injection(P>0.05).At 1,2,4,6 and 8 weeks after injection,body weight in the D group was significantly higher than that in the N group(P<0.05).Before injection,there was no significant difference in blood glucose level between N group and D group(P>0.05).At 2 days,1,2,4,6 and 8 weeks after injection,the blood glucose levels were significantly higher in the D group than those in the N group(P<0.05).Part 2:Experimental study on the protecton of DEX preconditioning against diabetic myocardial ischemia/reperfusion injuryThe aim of this part experiment was to evaluate the effect of diabetes mellitus on cardioprotection of DEX preconditioning by comparing the protective effects of DEX preconditioning on normal and diabetic MIRI.Thirty-two normal and thirty-two diabetic male SD rats were randomly divided equally into 8 groups(n=8 in each group):Normal blank control group(S group);Normal IRI group(C group);Normal IPC group(IPC group);Normal DEX preconditioning group(DEX group);Diabetic blank control group(DS group);Diabetic IRI group(DC group);Diabetic IPC group(D+IPC group);Diabetic DEX preconditioning group(D+DEX group).Throughout the experiment,hemodynamics and arrhythmia were continuously monitored.After reperfusion,blood samples were collected to measure serum cardiac troponin I(cTnI)and creatine kinase-MB(CK-MB)levels.IS was assessed by Evan's blue and TTC staining.The results showed that the blood glucose level of diabetic rats was significantly higher than that of normal rats(P<0.05).There were no significant differences in rat's body weight,body temperature and baselines of HR,MAP and RPP among eight groups(P>0.05).Compared with C group,arrhythmia scores during ischemia and reperfusion period were significantly decreased in the IPC group(P<0.05).Compared with the IPC group,arrhythmia score during reperfusion period was significantly increased in the DEX group(P<0.05).Compared with the C group,the IS,serum CK-MB and cTnI levels decreased significantly(P<0.05)in the IPC and DEX groups.Compared with the IPC group,serum CK-MB and cTnI levels increased significantly(P<0.05)but IS was not significantly different(P>0.05)in the DEX group.Compared with the DC group,serum CK-MB and cTnI levels decreased significantly(P<0.05)but IS was not significantly different(P>0.05)in the D+IPC and D+DEX groups.Compared with the D+IPC group,serum CK-MB level increased significantly(P<0.05),but serum cTnI level and IS were not significantly different(P>0.05)in the D+DEX group.Compared with the C and DEX groups in the normal rats,serum CK-MB and cTnI levels increased significantly(P<0.05),but IS was not significantly different(P>0.05)in the DC and D+DEX groups in the diabetic rats.Compared with the IPC group in the normal rats,IS,serum CK-MB and cTnI levels increased significantly(P<0.05)in the D+IPC group in the diabetic rats.Part 3:Experimental study on role of apoptosis in protection of DEX preconditioning against diabetic myocardial ischemia/reperfusion injuryThe aim of this part experiment was to explore the mechanism of apoptosis in cardioprotection of DEX preconditioning against diabetic MIRI by comparing the effects of DEX preconditioning on apoptosis in normal and diabetic MIRI.Thirty normal and thirty diabetic male SD rats were randomly divided equally into 6 groups(n=10 in each group):Normal blank control group(S group);Normal IRI group(C group);Normal DEX preconditioning group(DEX group);Diabetic blank control group(DS group);Diabetic IRI group(DC group);Diabetic DEX preconditioning group(D+DEX group).The apoptosis in the myocardial ischemic area were assessed through Tunel assay and Western-blot for expressions of Bax and Bcl-2 proteins.Optical microscope and electron microscope were used to determine the cardiomyocyte morphology.The results showed that compared with the S group,AI and the myocardial expression of Bax increased significantly(P<0.05),the myocardial expression of Bcl-2 and the ratio of Bcl-2/Bax decreased significantly(P<0.05)in the C and DEX groups.Compared with the C group,AI decreased significantly(P<0.05),and the myocardial expression of Bcl-2 and the ratio of Bcl-2/Bax increased significantly(P<0.05)in the DEX group.Compared with the DS group,AI and the myocardial expression of Bax increased significantly(P<0.05),the myocardial expression of Bcl-2 and the ratio of Bcl-2/Bax decreased significantly(P<0.05)in the DC group,while AI increased significantly(P<0.05),and the myocardial expressions of Bax and Bcl-2 and the ratio of Bcl-2/Bax were not significantly different(P>0.05)in the D+DEX group.Compared with the DC group,AI and the myocardial expression of Bax protein decreased significantly(P<0.05)and the ratio of Bcl-2/Bax increased significantly(P<0.05)in the D+DEX group.Compared with the S group in the normal rats,AI and myocardial expression of Bax increased significantly(P<0.05)and ratio of Bcl-2/Bax decreased significantly(P<0.05)in the DS group in the diabetic rats.Compared with the C group in the normal rats,AI and myocardial expression of Bax increased significantly(P<0.05)and ratio of Bcl-2/Bax was not significantly different in the DC group in the diabetic rats(P>0.05).Compared with the DEX group in the normal rats,AI increased significantly(P<0.05),and myocardial expressions of Bax and Bcl-2 and ratio of Bcl-2/Bax were not significantly different(P>0.05)in the D+DEX group in the diabetic rats.The optical microscopic and electron microscopic examinations showed that the cardiomyocytes in the DEX group were slightly swollen,had a comparative normal shape,mitochondria cristae was lucid,and the injury was less than that in the C group.The cardiomyocytes in the D+DEX group were swollen,the arrangement of myocardial cells was irregular,mitochondria were swollen or even rupture,and the injury was less than that in the DC group.Part 4:Experimental study on role of Keap1-Nrf2/ARE signaling transduction pathway in protection of DEX preconditioning against diabetic myocardial ischemia/reperfusion injuryThe aim of this part study was to explore the role and mechanism of Keap1-Nrf2/ARE signal transduction pathway in protection of DEX preconditioning on normal and diabetic MIRI.Seventy normal and seventy diabetic male SD rats were randomly divided equally into 14 groups(n=10 in each group):Normal blank control group(S group);Normal IRI group(C group);Normal DEX preconditioning group(DEX group);Normal tBHQ control group(tBHQ group);Normal combined tBHQ and DEX preconditioning group(tBHQ+DEX group);Normal ATRA control group(ATRA group);Normal combined ATRA and DEX preconditioning group(ATRA+DEX group);Diabetic blank control group(DS group);Diabetic IRI group(DC group);Diabetic DEX preconditioning group(D+DEX group);Diabetic tBHQ control group(D+tBHQ group);Diabetic combined tBHQ and DEX preconditioning group(D+tBHQ+DEX group);Diabetic ATRA control group(D+ATRA group);Diabetic combined ATRA and DEX preconditioning group(D+ATRA+DEX group).After reperfusion,blood samples were collected to measure cTnI and CK-MB levels,and IS was assessed by Evan's blue and TTC staining.The oxidative stress in the myocardial ischemic area was assessed through Western-blot for expressions of Nrf2,HO-1 and NOQ1 proteins and the expressions of SOD,MDA and GSH-Px levels.Compared with the C group,the IS,serum CK-MB and cTnI levels decreased significantly(P<0.05)in the DEX,tBHQ and tBHQ+DEX groups.Compared with the DEX group,the IS,serum CK-MB and cTnI levels decreased significantly(P<0.05)in the tBHQ+DEX group,while increased significantly(P<0.05)in the ATRA+DEX group.Compared with the tBHQ group,the IS,serum CK-MB and cTnI levels decreased significantly(P<0.05)in the tBHQ+DEX group.Compared with the ATRA group,the IS decreased significantly(P<0.05)and serum CK-MB and cTnI levels were not significantly different(P>0.05)in the ATRA+DEX group.Compared with the DC group,the IS,serum CK-MB and cTnI levels decreased significantly in(P<0.05)the D+ATRA group.Compared with the D+DEX group,the IS and serum CK-MB level increased significantly(P<0.05)in the D+tBHQ+DEX group,while the IS,serum CK-MB and cTnI levels were not significantly different(P>0.05)in the D+ATRA+DEX group.Compared with the D+tBHQ group,the IS,serum CK-MB and cTnI levels were not significantly different(P>0.05)in the D+tBHQ+DEX group.Compared with the D+ATRA group,the IS,serum CK-MB and cTnI levels increased significantly(P<0.05)in the D+ATRA+DEX group.Compared with the C and DEX groups in the normal rats,the IS,serum CK-MB and cTnI levels increased significantly(P<0.05)in the DC and D+DEX groups in the diabetic rats.Compared with the C group,the myocardial expressions of Nrf2,HO-1 and NOQ1 increased significantly(P<0.05)in the DEX,tBHQ and tBHQ+DEX groups,while the myocardial expressions of Nrf2,HO-1 and NOQ1 were not significantly different(P>0.05)in the ATRA and ATRA+DEX groups.Compared with the DEX group,the myocardial expressions of Nrf2,HO-1 and NOQ1 increased significantly(P<0.05)in the tBHQ and tBHQ+DEX groups,decreased significantly(P<0.05)in the ATRA and the ATRA+DEX groups.Compared with the DC group,the myocardial expressions of Nrf2,HO-1 and NOQ1 increased significantly(P<0.05)in the D+DEX,D+tBHQ and D+tBHQ+DEX groups,while the myocardial expressions of Nrf2 and HO-1 decreased significantly(P<0.05)in the D+ATRA and D+ATRA+DEX groups.Compared with D+DEX group,the myocardial expressions of HO-1 and NOQ1 increased significantly(P<0.05)and the myocardial expression of Nrf2 was not significantly different(P>0.05)in the D+tBHQ and D+tBHQ+DEX groups,while the myocardial expressions of Nrf2 and HO-1 decreased significantly(P<0.05)in the D+ATRA and D+ATRA+DEX groups.Compared with the C and DEX groups in the normal rats,the myocardial expressions of Nrf2 and HO-1 increased significantly(P<0.05)and NOQ1 decreased significantly(P<0.05)in the DC and D+DEX groups in the diabetic rats.Compared with the C group,the GSH-Px level in the ischemic myocardium increased significantly(P<0.05),the MDA level in the ischemic myocardium decreased significantly(P<0.05)in the DEX,tBHQ and tBHQ+DEX groups,and the GSH-Px and the MDA levels in the ischemic myocardium were not significantly different(P>0.05)in the ATRA and ATRA+DEX groups.Compared with the DEX group,the SOD and GSH-Px levels in the ischemic myocardium increased significantly(P<0.05)and MDA level in the ischemic myocardium decreased significantly(P<0.05)in the tBHQ+DEX group.Compared with the DC group,the SOD and GSH-Px levels in the ischemic myocardium increased significantly(P<0.05)and MDA level in the ischemic myocardium decreased significantly(P<0.05)in the D+DEX,D+tBHQ and D+tBHQ+DEX groups.Compared with the D+DEX group,the GSH-Px level in the ischemic myocardium decreased significantly(P<0.05)and the MDA level in the ischemic myocardium increased significantly(P<0.05)in the D+ATRA and D+ATRA+DEX groups.Compared with the C and DEX groups in the normal rats,GSH-Px level in the ischemic myocardium decreased significantly(P<0.05)and the MDA level in the ischemic myocardium increased significantly(P<0.05)in the DC and D+DEX groups in the diabetic rats.ConclusionsBased on the results of all experiments,the following conclusions can be drawn:1.Both IPC and DEX preconditioning have protective effect on MIRI in normal rats,but the protective effect is significantly weakened in diabetic rats.2.The apoptosis induced by ischemia/reperfusion is significantly stronger in diabetic myocardium than in normal myocardium.3.DEX preconditioning can significantly inhibit cardiomyocyte apoptosis and improve cardiomyocyte morphology after ischemia/reperfusion in normal and diabetic myocardium,suggesting that inhibition of apoptosis may be one of the cardioprotective mechanisms of DEX preconditioning.However,diabetes mellitus can significantly attenuate the anti-apoptotic effect of DEX preconditioning on cardiomyocytes after ischemia/reperfusion.4.DEX preconditioning protects MIRI by exerting antioxidant stress through activating Keapl-Nrf2/ARE signal transduction pathway in normal rats;but the protective effect is significantly weakened in diabetic rats.5.The weakened protection of DEX preconditioning against diabetic MIRI may be attributable to the enhancement of apoptosis and disorder of Keapl-Nrf2/ARE signal transduction pathway in diabetic myocardium. |
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