Zonisamide Ameliorates Cognitive Impairment By Inhibiting ER Stress In Type 2 Diabetic Mice

BackgroundType 2 diabetes mellitus(T2DM)is one of the most common diseases whose prevalence is on the rise.The deleterious effects of diabetes mellitus on the retinal,renal,cardiovascular,and peripheral nervous systems are widely acknowledged.Similar to Alzheimer’s disease,patients with type 2 diabetes show hyperphosphorylation of Tau protein,impaired synapses,and cognitive impairment.Hyperglycemia,vascular disease,and insulin resistance in type 2 diabetes increase the risk of dementia.In addition,endoplasmic reticulum stress is involved in neuronal apoptosis and cognitive decline in diabetic mice.Therefore,reducing endoplasmic reticulum stress may be a potential target for the prevention and treatment of diabetic encephalopathy.Zonisamide,an antiepileptic drug,was found to suppress ER stress–induced neuronal cell damage in the experimental models of Parkinson’s disease.However,the protective effect of Zonisamide in the treatment of diabetes-related dementia is not determined.In the study,we intend to construct a mice model of type 2 diabetes induced by high-fat diet and low-dose STZ injection,to investigate the therapeutic effect of Zonisamide on diabetes-related dementia,and to explore the possible mechanism.Thus the study will provide a new evidence for clinical drug treatment of diabetes-related dementia.Methods1.Construction of type 2 diabetes mice model model Mice were given a high-fat diet combined with a single injection of streptozotocin to develop type 2 diabetes.Only the mice with hyperglycemia(fasting blood glucose levels > 11.6 mmol/L)were considered as the diabetic mice and were selected for studies.Mice of the same age received a normal diet as a control group.Throughout the whole experiment,the control mice were given a normal diet,while the diabetic mice were given the HFD.4-week-old male C57BL/6J mice(SPF grade)were randomly divided into control group(Con),type2 diabetes group(T2DM)and Zonisamide treatment group(ZNS)(10 animals per group).Administration: At the 9th week of high-fat diet feeding,mice were administered Zonisamide(40 mg/kg)orally by gavage at 9: 00 daily for 16 weeks.Fasting blood glucose and body weight of mice were regularly measured every weeks.2.At the 16 th week of the Zonisamide administration,Morris water maze test(MWM)was performed to evaluate the learning and spatial memory abilities of the mice.3.Oral glucose tolerance test(OGTT)was used to to test insulin sensitivity in mice after behavioral experiments.4.Western blot was used to determine the protein expression levels in the hippocampus and cortex,including the critical synaptic-related proteins involved in learning and memory(PKA-C?,PKA-RI,CREB,PSD95 and synaptophysin),Tau phosphorylation,Tau kinase(PHF1,Tau-5,JNK and ERK1/2)and ER stress markers(XBP-1s,PERK,ATF4,ATF6,GRP78 and Hrd1).5.Immunofluorescence of Neu N and Nissl’s staining were used to detect neuronal changes.Immunofluorescence of Aβ was used to detect the Aβ load and distribution in the superior and lateral cortex of mice.6.Statistical analysis: Data are expressed as mean ± standard error(Mean ± SEM).Prism 7.0 software was used for data analysis and processing.We used One-way ANOVA and Two-way ANOVA followed by Tukey tests to analyse the data.Bilateral P <0.05 was considered statistical significance in all experiments.Results1.After the high-fat diet and the injection of streptozotocin,Compared with the normal group(Con)mice,the fasting blood glucose in the T2 DM group increased significantly(P < 0.05)while the body weight decreased significantly(P <0.01).After Zonisamide treatment,in the 25 th week the fasting blood glucose of mice was reduced(P <0.05)and the body weight of the mice increased to a normal level(P<0.05).2.The results of OGTT showed that the blood glucose levels of the T2 DM group were higher than that of normal group at all-time points(P <0.01),area under the concentration curve(AUC)was increased(P <0.01),suggesting that the model of T2 DM was successfully constructed.After Zonisamide treatment,the glucose levels both at 90 and 120 minutes were declined by Zonisamide treatment(P <0.05),area under the concentration curve(AUC)was reduced(P <0.05).3.The results of the MWM showed that compared with normal group,the swimming distance to platform,latency to targets,and the time to first target crossing in the T2 DM group were significantly increased(P <0.05),while the values of total distance,latency and time to first target crossing of mice treated with Zonisamide were significantly decreased(P<0.05).There was no significant difference in the swimming speed of mice in each group(P> 0.05).4.Western blotting results(1)Results of synaptic-related proteins involved in learning and memory showed that the p-CREB/CREB ratios was significantly reduced(P<0.05),and protein expression of PSD95 and Synaptophysin were significantly down-regulated in the T2 DM mice compared with the normal group(P<0.05).The p CREB/CREB ratio and the protein expression of PSD95 and Synaptophysin were significant increased both in hippocumpus and cortex of T2 DM mice after Zonisamide treatment(P<0.05).(2)In the results of Tau phosphorylation and Tau kinase protein of hippocampus and cortex,the ratios of PHF1/Tau-5,p-ERK/ERK,and p-JNK/JNK in the T2 DM group increased significantly compared with Con mice(P <0.05),and the ratios of PHF1/Tau-5,p-ERK/ERK,p-JNK/JNK in T2 DM mice were significantly decreased after Zonisamide treatment(P <0.05).(3)In the results of ER Stress marker proteins in hippocampus and cortex,the p-PERK/PERK ratio of the T2 DM group significantly increased compared with the Con group(P <0.05),and XBP-1s,ATF4,ATF6,and GRP78 protein expressions were significantly up-regulated(P <0.05),while the p-PERK/PERK ratio,XBP-1s,ATF4,ATF6,GRP78 protein and Hrd1 expression decreased significantly in T2 DM mice after Zonisamide treatment(P <0.05).5.Immunofluorescence results showed that the staining of Neu N was reduced among the regions of CA1,CA3 and DG of hippocampus in T2 DM mice compared with control mice,while Zonisamide treatment increased the Neu N positiv staining,which suggested that Zonisamide attenuated the neuronal loss induced by T2DM;And the results of Nissl’s staining were consistent with the Neu N staining.Positive puncta Aβfluorescence staining was more and stronger both in the superior and lateral cortex of the T2 DM mice than in the Con mice,while the positive fluorescence density was significantly decreased(P <0.05)after Zonisamide treatment(P <0.05).ConclusionZonisamide improved peripheral blood glucose levels in the mice of type 2diabetes,attenuated the neuronal loss in hippocampus,relieved learning and memory impairment in T2 DM mice.Zonisamide may ameliorate the cognitive impairment through increasing the PSD95 and CREB in T2 DM mice.And Zonisamide also decreased the Aβ load,and rescued the tau hyperphosphorylation at Ser396/404 through inhibiting the activity of JNK.The above results may be related to the regulation of ER stress through the elevation of Hrd1 by Zonisamide.