益肺温阳化浊汤调控PI3K/Akt-mTOR信号通路保护AD大鼠神经细胞的作用机制研究

目的探讨中药复方益肺温阳化浊汤对阿尔茨海默病(AD)大鼠模型调控PI3K/Akt-mTOR信号通路、保护AD大鼠神经元的作用机制。方法选取SD大鼠120只,随机分为假手术组、模型组、对照组、中药低剂量组、中药中剂量组和中药高剂量组,每组大鼠20只。采用β-淀粉样蛋白1-40(Aβ均1-40)制备AD大鼠模型。采用Real-timePCR、Western Blot等方法检测PI3K/Akt-mTOR通路及其下游自噬与凋亡相关因子的mRNA和蛋白表达水平,观察益肺温阳化浊汤灌胃对AD模型大鼠海马区PI3K/Akt-mTOR信号通路相关因子及Aβ含量的影响;探讨益肺温阳化浊汤治疗AD的作用机制。结果对照组、低剂量组、中剂量组、高剂量组大鼠定位航行试验逃避潜伏期、平台象限游泳距离、非平台象限游泳距离、平台象限游泳时间、非平台象限游泳时间、跨越原平台次数均显著优于模型组,差异均有统计学意义(P<0.05)。假手术组大鼠的海马组织神经细胞凋亡率及Aβ含量均低于其他5组,模型组高于其他用药4组,差异有统计学意义(P<0.05)。六组大鼠海马组织的神经细胞凋亡率表现为模型组>低剂量组>对照组>中剂量组>高剂量组>假手术组,海马组织Aβ含量表现为模型组>低剂量组>中剂量组>对照组>高剂量组>假手术组。大鼠脑组织中PI3K、Akt、Beclin1、LC3蛋白表达水平均表现为模型组>对照组>低剂量组>中剂量组>高剂量组,mTOR水平表现为模型组<对照组<低剂量组<中剂量组<高剂量组<假手术组。与假手术组大鼠比较,模型组大鼠脑组织中PI3K、Akt、Beclin1的mRNA表达水平显著升高,mTOR的mRNA水平显著降低,差异有统计学意义(P<0.05);与模型组大鼠比较,低剂量组、中剂量组、高剂量组及对照组大鼠脑组织的PI3K、Akt的mRNA表达水平均明显降低,mTOR的mRNA表达水平均明显升高;低剂量组、中剂量组、高剂量组大鼠脑组织的Akt的mRNA水平显著低于模型组,差异均有统计学意义(P<0.05)。结论益肺温阳化浊汤灌胃可通过调控PI3K/Akt-mTOR信号通路,调节AD模型大鼠自噬系统,清除神经细胞Aβ,抑制神经细胞凋亡,发挥保护神经细胞的作用。

Mechanism research of Yifei Wenyang Huazhuo decoction regulating PI3K/Akt-mTOR signal pathway in protecting nerve cells of AD rat

Objective To explore the mechanism of traditional Chinese medicine-compound Yifei Wenyang Huazhuo decoction in regulating the PI3K/Akt-mTOR signal pathway,and protecting nerve cells in rat model with Alzheimer′s disease (AD).Methods 120 SD rats were selected and randomly divided into sham-operative group,model group,control group,low-dose traditional Chinese medicine group,middle-dose traditional Chinese medicine group and high-dose traditional Chinese medicine group,with 20 rats in each group.AD rat model was prepared by β-amyloid 1-40 (Aβ).The mRNA and protein expression levels of PI3K/Akt-mTOR signal pathway and its downstream autophagy and apoptosis-related factors were detected by Real-time PCR and Western Blot.The effects of using Yifei Wenyang Huazhuo decoction to administered intragastrically on related factors of PI3K/Akt-mTOR signal pathway and Aβ content in hippocampus of AD model rats was observed,and the mechanism of Yifei Wenyang Huazhuo decoction treating AD was explored.Results The evasive incubation of navigation trial,swimming distance of platform quadrant,swimming distance of non-platform quadrant,swimming time of platform quadrant,swimming time of non-platform quadrant,and the times across the original platform in the control group,low-dose group,middle-dose group,and high-dose group were significantly better than those in the model group,with statistically significant difference ( P <0.05).The apoptosis rate of nerve cells and Aβ content in hippocampus tissue of rats in the sham-operative group were lower than the other 5 groups,and the above mentioned rate and content in the model group were higher than the remaining 4 groups,with statistically significant difference ( P <0.05).The apoptosis rate of nerve cells in hippocampus tissue of rats in the 6 groups exhibited in a descending order as follows: the model group,low-dose group,control group,middle-dose group,high-dose group,and sham-operative group.The Aβ content in hippocampus tissue exhibited in a descending order as follows: the model group,low-dose group,middle-dose group,control group,high-dose group,and sham-operative group,with no statistically significant difference ( P >0.05).The protein expression levels of PI3K,Akt,Beclin1 and LC3 of the rat brain tissue exhibited in a descending order as follows : the model group,control group,low-dose group,middle-dose group,high-dose group.The level of mTOR exhibited in a descending order as follows: the sham-operative group,high-dose group,middle-dose group,low-dose group,control group,and model group.Compared to the sham-operative group,the mRNA expression levels of PI3K,Akt and Beclin1 of the rat brain tissue in the model group were significantly increased,whereas the mRNA level of mTOR was significantly decreased,with statistically significant difference ( P <0.05).Compared to the model group,the mRNA expression levels of PI3K and Akt of the rat brain tissue in low-dose group,middle-dose group,high-dose group and comtrol group were significantly decreased,whereas the mRNA expression level of mTOR was significantly increased.The mRNA levels of Akt of the rat brain tissue in the low-dose group,middle-dose group,and high-dose group were significantly lower than the model group,with statistically significant difference ( P <0.05).Conclusion Using Yifei Wenyang Huazhuo decoction to administered intragastrically can significantly regulate the autophagy in AD model rats,eliminate Aβ of nerve cells,inhibit the apoptosis of nerve cells,and protect nerve cells by regulating PI3K/Akt-mTOR signal pathway.