开心散对Aβ1-42诱导Alzheimer病大鼠模型Keap-1/Nrf2/MnSOD信号通路的作用

目的:基于Kelch样环氧氯丙烷相关蛋白-1(Keap-1)/核转录因子E2相关因子2(Nrf2)/锰超氧化物歧化酶(Mn SOD)信号通路探讨开心散改善阿尔茨海默病(AD)模型大鼠认知障碍的可能机制。方法:通过侧脑室注射β淀粉样蛋白1-42(Aβ1-42,5μL)建立AD模型。造模后将大鼠随机分为模型组,多奈哌齐组和开心散低、中、高剂量组,并另设正常组。多奈哌齐组给予多奈哌齐片(1.8 g·kg^-1·d^-1),开心散低、中、高剂量组给予开心散制成的药液(10,20,40 g·kg^-1·d^-1),正常组和模型组给予等体积的纯水。采用Morris水迷宫检测大鼠的学习记忆力。采用比色法检测血清中髓过氧化酶(MPO),诱导型一氧化氮合酶(i NOS),超氧化物歧化酶(SOD)的表达水平。采用尼氏染色观察海马CA3区病理形态。采用免疫组化(IHC)和蛋白免疫印迹法(Western bolt)检测海马中Keap-1,Nrf2,Mn SOD的蛋白表达水平。结果:与正常组比较,模型组大鼠平台潜伏期、游泳总路程和第1次抵原平台时间显著增加(P<0.01),穿越平台次数和目标象限时间显著降低(P<0.01);与模型组比较,多奈哌齐组和开心散低、中、高组大鼠平台潜伏期、游泳总路程和第1次抵原平台时间明显减少(P<0.05,P<0.01),穿越平台次数和目标象限时间明显增加(P<0.05,P<0.01)。与正常组比较,模型组大鼠血清中MPO,i NOS表达水平显著升高(P<0.01),而SOD表达水平降低(P<0.01)。与模型组比较,多奈哌齐组和开心散低、中、高组大鼠血清中MPO,i NOS表达水平明显降低(P<0.05,P<0.01),而SOD表达水平明显升高(P<0.05,P<0.01)。正常组大鼠海马CA3区神经元排列整齐,未有尼氏体固缩。模型组大鼠海马CA3区神经元排列欠整齐,有明显的神经元丢失和尼氏体固缩。多奈哌齐组和开心散低、中、高组大鼠海马CA3区神经元排列较整齐,神经元数量增多,尼氏体固缩减少。与正常组比较,模型组大鼠海马中Keap-1的积分吸光度IA和蛋白水平降低(P<0.01),而Nrf2,Mn SOD的IA和蛋白水平显著升高(P<0.01)。与模型组比较,多奈哌齐组和开心散低、中、高组大鼠海马中Keap-1和Mn SOD的IA和蛋白水平明显升高(P<0.05,P<0.01),而Nrf2的IA和蛋白水平降低(P<0.05,P<0.01)。结论:开心散可通过调节Keap-1/Nrf2/Mn SOD信号通路缓解AD模型大鼠的认知障碍。

Effect of Kaixinsan on Keap-1/Nrf2/MnSOD Signaling Pathway in Rats with Alzheimer’s Disease Induced by Aβ

Objective:To explore the possible mechanism of Kaixinsan in improving cognitive impairment in Alzheimer’s disease(AD)model rats based on the epichlorohydrin associated protein-1(Keap-1)/nuclear factor E2 related factor (Nrf2)/manganese superoxide dismutase (MnSOD) signaling pathway.Method:The AD model was established by injecting Amyloidβ1-42(Aβ1-42,5μL)into the lateral ventricle.After modeling,the experimental rats were randomly divided into model group,donepezil group,and Kaixinsan low dose,medium dose and high dose groups.Another normal control group was also established.The donepezil group received donepezil tablets(1.8 g·kg^-1·d^-1),Kaixinsan low dose,medium dose and high dose groups received corresponding doses of Kaixinsan(10,20,40 g·kg^-1·d^-1,respectively),and the normal control group and model group were given with equal volume of pure water.Morris water maze was used to test the learning and memory ability of rats.The pathological morphology of hippocampal CA3 area was observed by Nissl staining.The expression levels of myeloperoxidase(MPO),inducible nitric oxide synthase(i NOS) and superoxide dismutase(SOD)in serum were detected by colorimetry,and the protein expression levels of Keap-1,Nrf2 and Mn SOD in hippocampus were detected by immunohistochemistry(IHC)and Western bolt.Result:Compared with the normal control group,the escape latency,total swimming distance and first arrival time of the plateau in the model group increased(P<0.01),while the times of crossing the plateau and the time in target quadrant decreased(P<0.01).Compared with the model group,the rats in donepezil group and Kaixinsan groups showed less latency,lower total swimming distance and first arrival time on the platform(P<0.05,P<0.01),while the times of crossing the platform and time in target quadrant increased(P<0.05,P<0.01).Compared with the normal control group,the expression levels of MPO and i NOS in serum of the model group increased(P<0.01),while the expression levels of SOD decreased(P<0.01).Compared with model group,the expression of MPO and i NOS in serum of donepezil group and Kaixinsan groups decreased(P<0.05,P<0.01),while the expression of SOD increased(P<0.05,P<0.01).In the normal control group,the neurons in the hippocampal CA3 of the rats were arranged neatly,without obvious Nissl body shrinkage.The neurons in the CA3 of the hippocampus of the model group were not arranged neatly,with obvious neuron loss and pyknosis of Nissl body.The neurons in the CA3 of the hippocampus of the rats in the donepezil group and Kaixinsan groups were arranged neatly,with increased number of neurons and decreased Nissl body shrinkage.Compared with the normal control group,the integrated optical density(IA)and protein level of Keap-1 in the hippocampus of the model group decreased(P<0.01),while the IA and protein level of Nrf2 and Mn SOD increased(P<0.01).Compared with model group,IA and protein levels of Keap-1 and Mn SOD in hippocampus of rats in donepezil group and Kaixinsan groups increased(P<0.05,P<0.01),while IA and protein levels of Nrf2 decreased(P<0.05,P<0.01).Conclusion:Kaixinsan could alleviate memory impairment in AD rats,and its mechanism may be related to its regulation of Keap-1/Nrf2/Mn SOD signaling pathway.