| Objective: Succinate dehydrogenase(SDH),also known as succinic ubiquinone oxidoreductase,or mitochondrial complex II,is the only one of the tricarboxylic acid cycle(TCA)integrated into the mitochondrial inner membrane as the multi-subunitase,which play an important role in TCA and aerobic respiratory chain.Succinate dehydrogenase-flavin(SDHA)is the only active group in the SDH structure,which is a key factor in the mitochondrial electron transport and respiratory function.Therefore,this experiment is intended to observe the process of Ischemia reperfusion(IR)in SDHA in normal and diabetic mellitus rats(DM)during cardiopulmonary bypass(CPB).Clarifing the relationship between ischemic postconditioning(IPO)and SDHA in DM to alleviate myocardial ischemia reperfusion injury(MIRI),as well as its possible role and mechanism.Methods: Ninty adult male SD rats,SPF grade with weighing 300 g approximately.Normal rats were fed with normal diet before CPB model was established.DM rats were fed with high-fat and high-sugar diet for four weeks as well,and then injected with Streptozotocin solution.Seventy-two hours later,if the blood glucose level was more than sixteen mmol/L,we think the diabetic model was conducted successfully.Subsequently,CPB model was created in normal rats and DM rats.Ninty rats were randomly divided into nine groups,ten rats in each group: normal group(N),ischemiareperfusion group(IR),ischemia-postconditioning group(IPO),diabetic group(DM),diabetes+ischemia-reperfusion group(DM+IR),diabetes+ischemia-postconditioning group(DM+IPO),diabetes+inhibitor-dimethyl malonate(dme)group(DM+dme),diabetes+ischemia-reperfusion+dme group(DM+IR+dme)and diabetes ischemiareperfusion postconditioning group(DM+IPO+dme).The rats in the N group were perfused for 100 minutes till the end.After ten minutes of perfused in the IR group,the whole heart was ischemia for 30 minutes and the experiment was end after 60 minutes of reperfusion.The IPO group was treated with three cycles of 30 s ischemia and 30 s reperfusion,henceforth,fifth-seven minutes of reperfusion were continued.The DM group was terminated by DM rat transfer for 100 min;DM+IR group was treated with DM rats,after 10 minutes of transfer,the whole heart was ischemia for 30 minutes,and the experiment was terminated after 60 minutes of reperfusion.As for the DM+IPO group was treated with a total of 3 cycles of 30 s ischemia and 30 s reperfusion,terminated the experiment after 57 minutes of reperfusion under the DM rats.Importantly,in the DM+dme group,DM+IR+dme group,DM+IPO+dme group were all pumped into vein with inhibitor-dme during the ischemia of whole heart,the remaining steps are the same as the previous experimental group.Mean arterial pressue(MAP),Heart Rate(HR),and Pulse Pressure(PP)were monitored during the operation.The detection indexes are as follows:(1)Both DM rats and normal rats were subjected to oral glucose tolerance test(OGTT)and intraperitoneal glucose tolerance test(IPGTT).(2)MAP,HR,and PP were recorded at 5 mins before CPB(T0),at the beginning of CPB(T1),at the beginning of ischemia(T2),at the end of ischemia(T3),5 mins after reperfusion(T4)and at the end of reperfusion(T5).At the corresponding time,arterial blood was collected for the Blood-Gas analysis in the N group.(3)At the T5,the area of myocardial infarction was detected by TTC method.(4)At the T5,1 ml of plasma was taken to detect the contents of CK-MB and CTnI,TNF-? and IL-1?.(6)Left ventricular tissues were taken at the T5,the mRNA and protein relative expression of SDHA were detected by RT-qPCR and Western blotting,respectively,as well as the SDH enzyme activity.Results:(1)In the IPGTT and OGTT experiments,DM rats were at a high blood glucose level of twenty to twenty-five mmol/L after the peak of blood glucose,but the normal rats decreased slowly after the peak,which fluctuated from five to ten mmol/L.(2)At T5,except for the N,DM and DM+dme groups,the other groups were significantly disordered compared with the T0.As for the arterial Blood-Gas analysis showed that compared with T0,Hct was significantly decreased at each time point of T1 to T5(P<0.05).(3)At T5,the change of myocardial infarction area was detected by TTC method: The area of myocardial infarction in the IR group was about 41.42±6.20%,which was significantly higher than that in the N group(P<0.05).The area of myocardial infarction in the IPO group was about 29.44±10.82%,which was significantly lower than that in the IR group(P<0.05).The area of myocardial infarction in the DM+IR group was about 60.26±7.9%,and the area of myocardial infarction in the DM+IPO group was about 57.86±5.65%,both of them were significantly higher than those in the DM group(P<0.05),but there was no statistical difference between them.The myocardial infarction area of DM+IR+dme group was about 59.55±10.2%,which was higher than that of the DM+dme group(P<0.05).and the myocardial infarction area of the DM+IPO+dme group was approximately 36.69±16.2%,compared with the DM+IR+dme group,it was lower(P<0.05).(4)Changes in the serum CKMB&CTnI,TNF-?&IL-1? at T5: IR group was higher than the N group(P<0.05),IPO group was lower than the IR group(P<0.05).The DM+IR group was higher than the DM group and the IR group individually(P<0.05),but there was no significantly difference between the DM+IR group and the DM+IPO group.The DM+IR+dme group was higher than the DM+dme group(P<0.05),and the DM+IPO+dme group was not only lower than the DM+IR+dme(P<0.05),but also the DM+IPO group(P<0.05).(5)Changes in the relative expression of SDHA mRNA and protein: IR group was higher than the N group(P<0.05),IPO group was lower than the IR group(P<0.05).But there was no significantly difference between the DM+IR group and DM+IPO group,although they were all higher than the DM group,and the DM+IR group was higher than the the IR group(P<0.05).The DM+IR+dme group was higher than the DM+dme group(P<0.05),the DM+IPO+dme group was lower than the DM+IR+dme group(P<0.05),and the DM+IPO+dme group was lower than the DM+IPO group(P<0.05).(6)Changes in the SDH enzyme activity: compared with the N group,IR group was significantly increased(P<0.05),compared with the IR group,IPO group was decreased(P<0.05).Compared with the DM group,the DM+IR group and the DM+IPO group were both elevated,and there was no significantly difference between the two groups(P<0.05).Compared with the IR group,the DM+IR group was elevated(P<0.05).The DM+IR+dme group was higher than the DM+dme group(P<0.05),meanwhile,the DM+IPO+dme group was lower than the DM+IR+dme group(P<0.05),and the DM+IPO+dme group was lower than the DM+IPO group as well(P<0.05).Conclusions: 1.Under CPB,IR promotes SDHA expression,enchances its activity,and triggers MIRI.However,IPO can inhibit SDHA expression,weaken enzyme acitivity and exert myocardial protection.2.In the DM state,IR promotes SDHA' gene and protein expression,enhance its activity and triggers MIRI,but IPO cannot inhibit its expression and the myocardial protection effect disappears.3.IPO joint inhibitor dme can reduce the levels of inflammatory factors and myocardial enzymes in serum,reduce the expression of SDHA gene and protein,weaken the enzyme activity,as well as restore the myocardial protection of IPO in the DM state finally. |
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