Impact Of Butylphthalide On Cognitive Impairment And Calcium/calmodulin-dependent Protein Kinase-â…¡ In Hippocampus Of STZ Diabetic Rats

Objective: Diabetes could cause the central nervous system damage andform diabetic ncephalopathy. The diabetes encephalopathy was clinicallycharacterized by cognitive dysfunction. Studies have shown that changes inhippocampal synaptic plasticity were associated with diabetic cognitivedysfunction. The calcium/calmodulin dependent protein kinaseII(calcium/calmodulin-dependent protein kinase II, CaMKII) was an importantprotein in regulating learning memory and synaptic plasticity. The content ofCaMKII reflectd the ability of learning and memory indirectly. The presentstudy was performed to observe the changes of hippocampal CaMKII ofStreptozotocin-induced diabetic rats with cognitive disfunction and whetherbutylphthalide could protect diabetic cognitive function.Methods: Fifty-five healthy male SD rats(weight140-160g) wererandomly divided into the normal controlling group (NC group, n=10) andthe diabetes group (n=45). The diabetic rat models were induced byintraperitoneal injection of STZ (60mg／kg) after abrosia for12hours, whilethe rats of NC group received equal volume of citrate buffer solution byintrapertoneal injection.Random blood glucose monitored by tail-veinsampling≥16.7mmol／L was our criterion for experimental diabetes. Thenthe rats of the diabetes group were randomly divided into four groups:diabeticcontrol group (DC group,n=15), diabetic group treated with low dosebutylphthalide (DL group,n=15), and diabetic group treated with high dosebutylphthalide (DH group,n=15). The rats of DL and DH group were given agavage of butylphthalide (60mg／kg) and butylphthalide (120mg／kg) oncedaily for12weeks respectively. The rats of NC and DC roup were intragastricadministration with equal volume corn germ oil one time every day for12 weeks. In the12weeks, Mental status and general conditions such as eating,drinking and urine volume of the rats were observed and recorded. The bloodglucose and weight of the rats were detected and recorded. All rats acceptedthe Morris water maze test at the12w point and then were sacrificed bydecapitation, the whole hippocampus were took out quickly. HE staining wasperformed to observe the changes of microstructure in the hippocampus ofrats. The expression of CaMKII in hippocampal slices by SABCimmunohistochemical method. The mRNA level and protein level of CaMKIIin hippocampus were detected by Real-time polymerase chain reaction(R-TPCR) and Western blotting.All the Datas were dealed with statistical software packages SPSS13.0，the Datas were analyzed using Two independent sample t test and, OnewayANOVA of completely random design, one-way repeated-measures ANOVAand Rank sum test the results were expressed as the mean±standard deviation。Statistical significance was set at P＜0.05.Results:1Generally observationThe rats of DC group appeared polyuria、polydipsia、polyphagia after thediabetic model was established, the blood glucoses of diabetic rats maintainedat high levels (＞20.0mmol/l).Followed with the progression of experiment,the diabetic rats gradually appeared gloomy sparse fur and dull、weight lose、slow in response and so on. While the NC group rats were in good shape withpure white and glossy fur and gain syeady growth.After intervention,thegeneral conditions of DH group were better than that of DL group.2Results of Morris water mazeBasic swimming speed: There was no statistical significance inswimming speed to each group.Navigation test: The escape latency of DC group was prolonged than NCgroup (P <0.05).The escape latency of DL group and DH group was shorterthan DC group (P <0.05). In1day and2day,there was no significantdifferences in escape latency between DL droup and DH group, but the escape latency of DH group was prolonged than DL group in3to5day (P <0.05).There were no significant differences in escape latency among differentdays (P <0.05). There was no interaction between days and groups.Probe test: The number in60s of crossing platform of DC group,DLgroup and DH group were less than NC group (P <0.05). The number in60sof crossing platform of DL group and DH group were more than DC group (P<0.05). The number in60s of crossing platform of DH group was more thanDL group (P <0.05).3Results of HE stainingA series of histopathologic alterations were observed in hippocampusneurons of DC group，and the number of normal neurons was decreasedsignificantly.In contrast, the majority of the CAl neurons in DL and DH groupremained normal morphology，and neurons showed integrated structures andlined up inorder, moreover there were also less neurons depletion．4Results of Immunohistochemistey stainingThe expression of CaMKII protein in hippocampus of rats in group DCwas lower than that in the NC group(P＜0.01).And the CaMKII proteinexpression of DL group and DH group were increased compared with DCgroup(P＜0.01). Compared with the DL group, the expression of hippocampalCaMKII protein in the rats of group DH was incresed(P＜0.01).5Results of Rt-PCRCompared with the NC group, the expression of hippocampalCaMKIImRNA in the rats of group DC was decresed(P＜0.05). TheCaMKIImRNA expression of DL group and DH group were increasedcompared with DC group(P＜0.01,P<0.05). The expression of hippocampalCaMKIImRNA in the rats of DH group was increased compared with DLgroup (P＜0.05).6Results of Western blotThe expression of CaMKII protein in hippocampus of rats in group DCwas lower than that in the NC group(P＜0.01). And the CaMKII proteinexpression of DL group and DH group were increased compared with DC group(P＜0.01). Compared with the DL group，the expression of hippocampalCaMKII protein in the rats of group DH was incresed(P＜0.01).Conclusions:1STZ-induced diabetic rats developed significant decilne in learning andmemory function at12-week point. Meanwhile the expression of CaMKII wasdecreased.2Butylphthalide could upregulate CaMKII expression,and improve cognitiveimpairment in diabetic rats, in order to avoid diabetic encephalopathy. And thehigh dose was better than the low dose.