鹿茸多肽对轻度认知功能障碍大鼠学习记忆能力的改善作用及其调节Keap1/Nrf2/HO-1信号通路的机制

Improvement effects of pilose antler polypeptide on learning and memory abilities in rats with mild cognitive impairment and their mechanisms of regulating Keap1/Nrf2/HO-1 signaling pathway

目的:观察鹿茸多肽(VAP)对轻度认知功能障碍(MCI)模型大鼠学习和记忆能力及海马组织中KELCH状ECH相关蛋白1(Keap1)/核因子E2相关因子2(Nrf2)/血红素氧合酶1(HO-1)信号通路相关蛋白表达的影响,并探讨其作用机制。方法:50只清洁级Wistar大鼠随机分为正常对照组、MCI组、吡拉西坦组、低剂量VAP组和高剂量VAP组,每组10只,其中吡拉西坦组灌胃500 mg·kg^(-1)吡拉西坦,低剂量VAP组大鼠灌胃200 mg·kg^(-1)VAP,高剂量VAP组大鼠灌胃300 mg·kg^(-1)VAP,灌胃给药共18 d,第18天给药2 h后,采用腹腔中注射东莨菪碱诱导并复制MCI大鼠模型。Morris水迷宫实验观察各组大鼠行为学,HE染色观察活性大鼠海马组织病理形态表现,ELISA法检测各组大鼠血清中超氧化物歧化酶(SOD)活性和丙二醛(MDA)水平,Western blotting法检测各组大鼠海马组织中Keap1、Nrf2和HO-1蛋白表达水平。结果:Morris水迷宫实验,与正常对照组比较,MCI组大鼠逃避潜伏期和行为路径明显延长(P<0.01),穿过平台位置次数和有效区停留时间明显降低(P<0.01);与MCI组比较,吡拉西坦组、低剂量VAP组和高剂量VAP组大鼠逃避潜伏期时间明显降低(P<0.01),吡拉西坦组和高剂量VAP组大鼠行为路径明显缩短(P<0.01),吡拉西坦组、低剂量VAP组和高剂量VAP组大鼠穿过平台位置次数和有效区停留时间明显升高(P<0.05或P<0.01)。HE染色,与正常对照组比较,MCI组大鼠海马组织中细胞数量明显减少,且排列方式由有序变为相对混乱;与MCI组比较,吡拉西坦组和高剂量VAP组大鼠海马组织细胞排列较整齐,形态略规则。ELISA法检测,与正常对照组比较,MCI组大鼠血清中SOD活性明显降低(P<0.01),MDA水平明显升高(P<0.01);与MCI组比较,吡拉西坦组和高剂量VAP组大鼠血清中SOD活性明显升高(P<0.01),MDA水平明显降低(P<0.01)。Western blotting法检测,与正常对照组比较,MCI组大鼠海马组织中Keap1蛋白表达水平明显升高(P<0.01),Nrf2和HO-1蛋白表达水平明显降低(P<0.05或P<0.01);与MCI组比较,吡拉西坦组、低剂量VAP组和高剂量VAP组大鼠海马组织中Keap1蛋白表达水平明显降低(P<0.01),Nrf2和HO-1蛋白表达水平明显升高(P<0.01)。结论:VAP能够改善腹腔注射东莨菪碱诱导并复制的MCI模型大鼠的学习和记忆能力,其机制可能与提高模型大鼠海马组织中Keap1、Nrf2和HO-1蛋白表达水平进而发挥抗氧化应激作用有关。

Objective:To observe the effects of velvet antler polypeptide(VAP)on the learning and memory abilities and the expressions of KELCH-like ECH related protein 1(Keap1),nuclear factor E2 related factor 2(Nrf2)and heme oxygenase-1(HO-1)in hippocampus tissue of the rats with mild cognitive impairment(MCI),and to explore the mechanisms.Methods:Fifty clean Wistar rats were randomly divided into normal control group,MCI group,piracetam group,low dose of VAP group and high dose of VAP group(n=10).Except normal control group,the rats in other groups were intraperitoneally injected scopolamine for 18 d to set up the MCI models.The rats in piracetam group were given piracetam 500 mg·kg^(-1),the rats in low dose of VAP group were given VAP 200 mg·kg^(-1),and the rats in high dose of VAP group waere given VAP 300 mg·kg^(-1).The behavioral changes of rats in various groups were observed by Morris water maze test,the pathomorphology of hippocampus tissue of the rats was observed by HE staining,the activities of superoxide dismutase(SOD)and the levels of malondialdehyde(MDA)in serum of the rats in various groups were detected by ELISA method,and the expression levels of Keap1,Nrf2and HO-1 proteins in hippocampus tissue of the rats in various groups were detected by Western blotting method.Results:The results of Morris water maze test showed that compared with normal control group,the escape latency and behavior path of the rats in MCI group were significantly increased(P<0.01),while the times of crossing the platform and the residence time in the effective area were significantly decreased(P<0.01);compared with MCI group,the escape latencies of piracetam group,low dose of VAP group and high dose of VAP group were decreased significantly(P<0.01),the behavior paths of piracetam group and high dose of VAP group were shortened significantly(P<0.01),and the times of crossing the platform and the residence time in the effective area of the rats in piracetam group,low dose of VAP group and high dose of VAP group were increased significantly(P<0.05 or P<0.01).The HE results showed that compared with normal control group,the number of cells in hippocampus tissue of the rats in MCI group was decreased obviously,and the arrangement mode was changed from orderly to relatively disorder;compared with MCI group,the cells in hippocampus tissue of the rats in piracetam group and high dose of VAP group were arranged more orderly and the shape was slightly regular.Compared with normal control group,the activity of serum SOD of the rats in MCI group was decreased significantly(P<0.01)and the level of MDA was increased significantly(P<0.01);compared with MCI group,the serum SOD activities of the rats in piracetam group and high dose of VAP group were increased significantly(P<0.05 or P<0.01),while the MDA levels were decreased significantly(P<0.05 or P<0.01).The results of Western blotting method showed that compared with normal control group,the expression level of Keap1 protein in hippocampus tissue of the rats in MCI group was significantly increased(P<0.01),while the expression levels of Nrf2 and HO-1 proteins were significantly decreased(P<0.05 or P<0.01);compared with MCI group,the expression levels of Keap1 protein in hippocampus tissue of the rats in piracetam group,low dose of VAP group and high dose of VAP group were decreased significantly(P<0.01),while the expression levels of Nrf2 and HO-1 proteins were increased significantly(P<0.01).Conclusion:VAP can improve the learning and memory abilities of MCI model rats induced by scopolamine,and its mechanism may be related to increasing the expression levels of Keap1,Nrf2 and HO-1 proteins in hippocampus of the rats to play an anti-oxidative stress role.