| Objectives To investigate the effect of Nrf2 gene knockout on cognitive impairment in type 2 diabetic mice and its related mechanisms.Methods SPF classic ICR male wild-type(WT)and Nrf2 gene knockout(KO)mice,40 each,8 weeks old,25 to 30 g.Mise were randomly divided into 4 groups:(20 in each group): the wild type negative control group(WT-NC),type 2 diabetes model group(WT-T2DM),Nrf2 gene knock out-negative control group(KO-NC)and Nrf2 knockout type 2 diabetic model group(KO-T2DM).Mice of diabetes group were fed high fathigh sucrose diet(HFD),a week after intraperitoneal injection of treptozotocin(STZ)50 mg/kg.Mice of the corresponding control groups were fed with normal diet,a week after intraperitoneal injection of citrate buffer solution.The weight and blood glucose indexes of each group were measured to determine whether the model was established;The abilitis of learning and spatial memory were evaluated by the water maze experiment;Ninig staining was used to observe the effect of Nrf2 gene knockout on hippocampal neurons in T2 DM mice;the expression level of tumor necrosis factor-?(TNF-?)and interleukin-1?(IL-1?)were evaluated using Western blot;The oxidative stress index of MDA and SOD was measured using ELISA;qRT-PCR wad used to examine the effect of Nrf2 gene on heme oxygenase-1(HO-1)and quinone oxidoreductase(NQO1)in T2 DM mice.Results 1 Both the KO-NC group and the KO-T2 DM group lost weight compared with the corresponding wild-type mice,and the difference was statistically significant(P<0.05).2 Compared with the WT-NC group,blood glucose in the KO-NC group was significantly increased(P<0.05),but the blood glucose did not meet the criteria for diabetes.Both the WT-T2 DM group and the KO-T2 DM group reached the criteria for diabetes,and the blood glucose in the KO-T2 DM group was higher than that in the WT-T2 DM group,with statistically significant differences(P<0.05).3 The results of the navigation experiment in water maze showed that the KO-NC group evaded the latent time longer than the WT-NC group,with statistical significance(P<0.05).Compared with the WT-T2 DM group,the KO-T2 DM group had longer evasive latency,which was statistically significant(P<0.05).4.The results of space exploration experiments showed that the time percentages of mice in the KO-NC group and KO-T2 DM group in the target quadrant were shorter than those in the corresponding WT-NC group and WT-T2 DM group,suggesting that Nrf2 gene has a protective effect on cognitive impairment in type 2 diabetes.5 Nissl's staining results showed that the hippocampal neurons in the WT-NC group were in normal shape,orderly arranged,round and clear,with obvious nucleo.In the WT-T2 DM group,the nerve cells were denatured and necrotic,the cells were shrivelled and hyperchromatic,the nuclei became smaller and even disappeared,and the intercellular space became larger.The atrophy and nuclear lysis of neurons in the KO-T2 DM group was more severe than that in the WT-T2 DM group.The microscopic counting results showed that compared with the WT-NC group,the loss of neurons in the WT-T2 DM group was significant(P<0.05),while the loss of cells in the KO-T2 DM group was more significant than that in the WT-T2 DM group,and the difference was statistically significant(P<0.05).6 Western blot analysis results showed that the expression levels of TNF-? and IL-?1 protein in the hippocampus of the WT-T2 DM group were significantly higher than that of the WT-NC group(P<0.05).The expression levels of TNF-? and IL-?1 protein were significantly higher in the KO-T2 DM group than in the WT-T2 DM group(P<0.05).However,there was no statistically significant difference in the expression levels of TNF-and IL-1 beta protein between the KO-T2 DM group and the WT-T2 DM group(P>0.05).7 ELISA results showed that the content of MDA in the hippocampus of WT-T2 DM group was higher than that of WT-NC group.MDA content in KO-T2 DM group were more than in WT-T2 DM group,the differences were statistically significant(P<0.05),however,the content of MDA between KO-T2 DM group and WT-T2 DM group was no statistical difference(P>0.05),in addition,the SOD activity test results showed that compared with WT-NC group,SOD activity in mice hippocampal in WT-T2 DM group,the KO-NC group and KO-T2 DM group were significantly down-regulated(P<0.05);Moreover,SOD activity in the KO-T2 DM group was significantly lower than that in the WT-T2 DM group,with statistically significant differences(P<0.05).8.The results of qRT-PCR showed that the expression level of Nrf2 mRNA in hippocampus of WT-T2 DM group was significantly lower than that of WT-NC group(P<0.05).There was no expression of Nrf2 mRNA in KO-NC group and KO-T2 DM group.Compared with the WT-NC group,the expressions of NQO1 and HO-1 mRNA in the hippocampus of mice in the WT-T2 DM group,KO-NC group and KO-T2 DM group were all down-regulated(P<0.05),and the expressions of NQO1 mRNA and HO-1 mRNA in the KO-NC group and KO-T2 DM group were significantly lower than those in the WT group and WT-T2 DM group(P<0.05).Conclusions 1 Nrf2 gene knockout can cause cognitive impairment in type 2 diabetic mice,and Nrf2 gene has a protective effect on cognitive impairment in type 2 diabetes.2 Type 2 diabetes mellitus will lead to increased oxidative stress and inflammatory response in the hippocampus,decreased antioxidant defense ability,and then cognitive function damage followed with abnormal neuronal structure and morphology.The deletion of Nrf2 gene will lead to more enhancement of oxidative stress and inflammatory response in the hippocampus of type 2 diabetic mice,further decrease in antioxidant defense ability,and more serious cognitive function damage,neuronal structural and morphological abnormalities.Figure9;Table0;Reference 93... |
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