Effect Of L-3-n-butyphlthalide On Expression Of Inflammatory Factor In Hippocampus Of Db/db Mice

Objective:With the progressive aging of the population in developing counties,the central nervous system disorders induced by diabetes,e.i.,diabetic cognitive dysfunction come to be called attention by human beings.Diabetic cognitive dysfunction is characterized by electrophysiological,neurochemical,structural,and degenerative neuronal changes that gradually lead to cognitive functioning limitations.Type 2 diabetes is considered to be one of the most relevant risk factor for cognitive dysfunction,increased incidence of dementia,consequently Alzhermer's disease(AD),referred to as“type 3 diabetes”.L-3-n-Butylphthalide(L-NBP)is a successfully synthesized and stable chemical drug used for the treatment of ischemic stroke that has Chinese independent intellectual property rights.Studies showed L-NBP had potent neuroprotective effects by improving mitochondrial function,mitigating oxidative damage,decreasing neuronal apoptosis,reducing tau phosphorylation,inhibiting inflammatory responses in middle cerebral artery occlusion rat models.L-NBP has been proved to improve learning and memory by improving microcirculation,protecting mitochondria in vascular dementia animal models,but its specific pathogenesis is not yet clear.In the present study,we aim to investigate L-NBP on expression of nuclear factor-?B and inflammatory factor(NF-?B),interleukin-6(IL-6)interleukin-1(IL-1?),the proinflammatorycytokinetumornecrosisfactor-alpha(TNF-?),in hippocampus of db/db mice.Method:1.AnimalsSixteen healthy male db/db mice used as type 2 diabetic animal models(6weeks of age)and eight same age male lean nondiabetic db/m mice(17~19g)used as control group.2.Drug interventionsDM-NBP group was given L-NBP dissolved in vegetable oil by oral gavage(at a dose of 120mg/kg).NC group and DM group were given vegetable oil only and continued for 6 weeks.3.General observationsThe metal state,fur color,urine volume,water and food intake of mice were observed and recorded.Meanwhile,the weight and blood glucose of mice were detected weekly.4.Cognitive behavior testsMorris water maze(MWM)consists of place navigation test and space probe test.The experiment of place navigation lasted for five days to record the escape latency.At the sixth day,the space probe test was performed to record the number of crossing the platform during the free swimming.5.The expression of NF-?B,IL-6,IL-1?,TNF-?After the behavior experiment,RT-PCR Analysis was used to detect the expression of NF-?B,IL-6,IL-1?,TNF-?mRNA in mice hippocampus.Western blotting was used to detect the expression of NF-?B,IL-6,IL-1?,TNF-?protein in mice hippocampus.6.Statistical methodAll statistical analyses were processed by SPSS 21.0.The measurement data was expressed as the mean±standard deviation.In the results of escape latency,we used repeated measure analysis of variance.The date of space probe test was compared with one-way analysis of variance.P<0.05 was considered as statistically significant difference.Result:1.General conditionThe mice of NC group appeared good mental state,sensitive reaction and bright fur.The drinking,eating and urine were normal.Nevertheless,the mice of DM group and DM-NBP group gradually showed unresponsive,sparse fur,polydipsia and polyphagia.After 6 weeks of drug treatment,the blood glucose level in DM and DM-NBP group were increased obviously that compared to NC group.The difference was statistically significant(P<0.05).The weight of DM and DM-NBP group gradually increased,which was higher obviously than the NC group.The difference was statistically significant(P<0.05).2.Cognitive behavior tests2.1 Basic swimming speedThe result showed that DM and DM-NBP group had no statistical significance(P>0.05).2.2 Space probe testCompared to NC group,the time of crossing the platform in DM group was decreased(P<0.05);However,the DM-NBP group could reverse the situation that the time was increased that comparing to DM group.The difference was statistically significant(P<0.05).2.3 Place navigation testAt the first day of this test,there was no difference between each groups(P>0.05).During 2~thh day to 5~thh day,the escape latency of DM group was markedly prolonged compared to NC group(P<0.05).The DM-NBP group had reduced the prolonged escape latency effectively,but it was shorter than DM group(P<0.05).3.RT-PCR AnalysisCompared with the NC group,the expression of hippocampal NF-?B,IL-6,IL-1?,TNF-?mRNA in DM and DM-NBP group were higher(each P<0.05).Compared with the DM group,the expression of hippocampal NF-?B,IL-6,IL-1?,TNF-?mRNA in DM-NBP were decreased,but higher than NC group(each P<0.05).4.Western blotCompared with the NC group,the expression of hippocampal NF-?B,IL-6,IL-1?,TNF-?protein in DM and DM-NBP group were higher(each P<0.05).Compared with the DM group,the expression of hippocampal NF-?B,IL-6,IL-1?,TNF-?protein in DM-NBP were decreased,but higher than NC group(each P<0.05).Conclusion:1.db/db mouse could develop learning and memory impairment at 13weeks,and the expression of NF-?B,IL-6,IL-1?,TNF-?mRNA and protein in hippocampus of db/db mice were increased.2.L-NBP might prevent the mouse from getting cognitive impairment by down-regulating NF-?B,IL-6,IL-1?,TNF-?expression in hippocampus of diabetic mouse.