Effect And Mechanism Of Incretin Analogues On Oxidative Stress In Diabetic Rats With Cerebral Ischemia-reperfusion Injury

Objective:1.To explore the neuroprotective effects of incretin analogues DA3-CH and liraglutide on diabetic cerebral ischemia-reperfusion injury model rats(neurological deficit score,infarction area percentage and histopathological changes),and compare the effects of the two analogues.2.To explore the effects of DA3-CH and Liraglutide on oxidative stress in diabetic cerebral ischemia-reperfusion injury model rats and its related mechanisms,whether it is related to activation of Nrf2/ARE signaling pathway,and compare the effects of two drugs on this pathway.Methods:72 male SD rats were randomly divided into 4 groups: blank group(Sham group,n=18),Model group(Model group,n=18),DA3-CH treatment group(DA3-CH group,n=18),and Liraglutide treatment group(Liraglutide group,n=18).The Sham group is not given special treatment,and is raised in the same environment as other groups’ rats,the other three groups were all used STZ to model diabetes.After successful diabetes modeling,DA3-CH group and Liraglutide group were respectively intraperitoneally injected with DA3-CH(10nmol/kg,once a day)and liraglutide(10nmol/kg,once a day)for 14 days,and the remaining groups were intraperitoneally injected with the same volume of normal saline for 14 days.After 14 days,the blood glucose in tail vein of rats in each group was measured,and then the Model group,DA3-CH group and Liraglutide group were established with cerebral ischemiareperfusion injury model.After 14 days,the blood glucose in tail vein of rats in each group was measured,and then the Model group,DA3-CH group and Liraglutide group were established with cerebral ischemia-reperfusion injury model.After 2 hours of ischemia and22 hours of reperfusion,the neurological deficit score(m NSS)of each group was measured,after decapitation,the percentage of cerebral infarction area was measured by TTC method,the pathological morphology of ischemic brain tissue was observed by HE staining,SOD activity was measured by WST-1 method,MDA content was measured by TBA method,and the expressions of Nrf2/ARE pathway proteins Nrf2,HO-1 and NQO1 were detected by Western-blot and immunohistochemistry.Results:1.Blood glucose level: Compared with Sham group,the blood glucose levels of the other three groups were significantly higher(P < 0.001).Compared with the Model group,the blood glucose level of DA3-CH group and Liraglutide group decreased(P < 0.01).There was no significant difference in blood glucose level between DA3-CH group and Liraglutide group(P > 0.05).2.Neurological impairment score(m NSS): Compared with Sham group,m NSS scores of the other three groups were significantly higher(P < 0.001).Compared with the Model group,m NSS scores of DA3-CH group and Liraglutide group decreased(DA3-CH,P <0.001;Liraglutide,P<0.01).The m NSS score of DA3-CH group was lower than that of Liraglutide group(p < 0.05).3.Infarct area percentage: Sham group was uniformly stained with red normal brain tissue,while white infarcted brain tissue and red normal brain tissue were found in the other three groups.Compared with Sham group,the infarct size of the other three groups increased significantly(P < 0.001).Compared with the Model group,the infarct size of DA3-CH group and Liraglutide group decreased(P < 0.001).The infarct size of DA3-CH group was lower than that of Liraglutide group(P < 0.01).4.HE staining: HE staining: The morphology and size of brain cells in Sham group were basically the same,with uniform distribution,full cytoplasm and clear nucleoli.Compared with Sham group,Model group,DA3-CH group and Liraglutide group all showed damaged brain tissue cells,relatively sparse distribution,more vacuolar edema,degeneration,nuclear pyknosis and nuclear lysis.Compared with the Model group,the vacuolar edema,degeneration,nuclear pyknosis and nuclear dissolution were reduced in the two groups,especially in the DA3-CH group.5.Oxidative stress indicatorsCompared with Sham group,MDA content in ischemic penumbra of the other three groups increased significantly(P < 0.001).Compared with Model group,MDA content in DA3-CH group and Liraglutide group decreased(P < 0.001),and MDA content in DA3-CH group was lower than that in Liraglutide group(P < 0.001).Compared with Sham group,SOD activity in ischemic penumbra of the other three groups decreased significantly(P < 0.001).Compared with Model group,SOD activity in DA3-CH group and Liraglutide group increased(P < 0.001),and SOD activity in DA3-CH group was higher than that in Liraglutide group(P < 0.001).6.Expression of NRF2/ARE pathway proteinImmunohistochemical results showed that compared with Sham group,the number of Nrf2 positive cells in cerebral cortex of the other three groups increased(P < 0.001).Compared with Model group,the number of Nrf2 positive cells in DA3-CH group and Liraglutide group increased(P < 0.001).Compared with Liraglutide group,the number of Nrf2 positive cells in DA3-CH group was more(P < 0.001).Compared with Sham group,the number of HO-1 and NQO1 positive cells in downstream protein of this pathway increased in the other three groups(P < 0.001).compared with Model group,the number of HO-1 and NQO1 positive cells in DA3-CH group and Liraglutide group increased(P < 0.001)Western blot showed that compared with Sham group,the expression of Nrf2 in cerebral cortex of ischemic penumbra increased in the other three groups(P < 0.001).Compared with Model group,Nrf2 expression in DA3-CH group and Liraglutide group increased(P < 0.001).The expression of Nrf2 in DA3-CH group was higher than that in Liraglutide group(P <0.01).Similarly,compared with the Sham group,the expression of the downstream proteins HO-1 and NQO1 of this pathway increased in the other three groups(P<0.001).Compared with the Model group,the DA3-CH group and the Liraglutide group had HO-1 and NQO1 expression increased(P<0.001).The DA3-CH group had more HO-1 and NQO1 expressions than the Liraglutide group(HO-1,P<0.001;NQO1,P<0.01).Conclusion:1.Both the incretin analogues DA3-CH and liraglutide can reduce the infarct size,reduce the neurological deficit score,alleviate the pathological changes of the ischemic brain tissue,and have definite neuroprotective effect,DA3-CH is superior to liraglutide.2.DA3-CH and liraglutide can alleviate oxidative stress injury in diabetic cerebral ischemia-reperfusion injury model rats by activating Nrf2/ARE pathway,and DA3-CH has stronger effect.