积雪草苷对癫痫小鼠认知功能、炎症反应、氧化应激及沉默信息调节因子1/内质网应激通路蛋白的影响研究

Effects of Asiaticoside on Cognitive Function,Inflammatory Response, Oxidative Stress and Silent Information Regulator 1/Endoplasmic Reticulum Stress Pathway Protein in Mice with Epileptic

目的:探讨积雪草苷(AS)对癫痫(EP)小鼠认知功能、炎症反应、氧化应激及沉默信息调节因子1(SIRT1)/内质网应激(ER stress)通路蛋白的影响。方法:建立EP小鼠模型,将造模成功的EP小鼠随机分为模型组、AS低剂量组(20 mg/kg)、AS中剂量组(40 mg/kg)、AS高剂量组(80 mg/kg)和阳性对照组(丙戊酸钠0.2 g/kg),每组10只;另取10只健康小鼠作为对照组。除模型组和对照组小鼠灌胃蒸馏水之外,其他各组小鼠给予相应剂量药物干预,1日1次,干预2周。给药结束后,观察小鼠EP持续状态(SE)次数及总持续时间;采用Morris水迷宫实验测定小鼠认知功能,采用酶联免疫吸附试验测定血清中白细胞介素6(IL-6)、肿瘤坏死因子α(TNF-α)水平,脑组织中超氧化物歧化酶(SOD)和丙二醛(MDA)水平,采用蛋白质印迹法检测小鼠脑组织中SIRT1以及ER stress相关蛋白[转录因子C/EBP同源蛋白(CHOP)、免疫球蛋白重链结合蛋白(BIP)、蛋白激酶R样内质网激酶(PERK)、磷酸化PERK(p-PERK)、真核翻译起始因子-2α(eIF2α)和磷酸化eIF2α(p-eIF2α)]表达。结果:与对照组相比,模型组小鼠SE次数增加,总持续时间和逃避潜伏期延长,血清IL-6、TNF-α水平以及脑组织MDA、CHOP、BIP、p-PERK/PERK和p-eIF2α/eIF2α蛋白表达水平升高,游过平台位置的次数减少,脑组织SOD、SIRT1蛋白表达水平降低,差异均有统计学意义(P<0.05)。与模型组相比,AS低、中、高剂量组以及阳性对照组小鼠SE次数减少,总持续时间和逃避潜伏期缩短,血清IL-6、TNF-α水平及脑组织MDA、CHOP、BIP、p-PERK/PERK和p-eIF2α/eIF2α蛋白表达水平降低,游过平台位置的次数增加,脑组织SOD、SIRT1蛋白表达水平升高,差异均有统计学意义(P<0.05),AS低、中及高剂量组呈剂量依赖性(P<0.05);AS高剂量组和阳性对照组上述指标差异无统计学意义(P>0.05)。结论:AS可改善EP小鼠认知功能,减轻炎症反应及氧化应激程度,可能与激活SIRT1蛋白表达,缓解ER stress有关。

OBJECTIVE: To probe into the effects of asiaticoside(AS) on cognitive function, inflammatory response, oxidative stress and silent information regulator 1(SIRT1)/endoplasmic reticulum stress(ER stress) pathway protein in mice with epileptic(EP). METHODS: EP mouse model were established, so that the successfully modeled EP mice were randomly divided into model group, AS low dose group(20 mg/kg AS), AS medium dose group(40 mg/kg AS), AS high dose group(80 mg/kg AS) and positive control group(0.2 g/kg sodium valproate), with 10 mice in each group;and another 10 healthy mice were taken as the control group. Except for the mice in the model group and the control group were given distilled water by gavage, the mice in other groups were given corresponding doses for drug intervention, once a day for two weeks. After administration, the frequency and total duration of status epilepticus(SE) of EP mice were observed;The Morris water maze test was performed to measure the cognitive function of mice;the enzyme-linked immunosorbent assay was used to determine the interleukin-6(IL-6) and tumor necrosis factor-α(TNF-α) levels in serum, superoxide dismutase(SOD) and malondialdehyde(MDA) levels in brain tissue;the Western blot was adopted to detect the expression of SIRT1 and ER stress-related proteins in brain tissue [transcription factor C/EBP homologous protein(CHOP), immunoglobulin heavy chain binding protein(BIP), protein kinase R-like endoplasmic reticulum kinase(PERK), phosphorylated PERK(p-PERK), eukaryotic translation initiation factor-2α(eIF2α) and phosphorylated eIF2α(p-eIF2α)]. RESULTS: Compared with the control group, the frequency of SE was increased, the total duration and escape latency of SE were prolonged, the IL-6 and TNF-α levels in serum, the MDA, CHOP, BIP, p-PERK/PERK and p-eIF2α/eIF2α protein expression levels in brain tissue were increased, the number of times to swim across platform position was decreased, the SOD and SIRT1 protein expression levels in brain tissue were decreased in the model group, with statistically significant differences(P<0.05). Compared with the model group, the frequency of SE was decreased, the total duration and escape latency of SE were shortened, the IL-6 and TNF-α levels in serum, the MDA, CHOP, BIP, p-PERK/PERK and p-eIF2α/eIF2α protein expression levels in brain tissue were decreased, the number of times to swim across platform position was increased, the SOD and SIRT1 protein expression levels in brain tissue were increased in the AS low, medium and high dose groups and the positive control group, with statistically significant differences(P<0.05), there were dose-dependent in the AS low, medium and high dose groups(P<0.05);there was no statistical significance in differences in the above indicators between the AS high dose group and the positive control group(P>0.05). CONCLUSIONS: AS can improve cognitive function, relieve inflammatory response and oxidative stress in EP mice, which may be related to activating the expression of SIRT1 protein and alleviating ER stress.