基于mTOR介导的线粒体自噬探讨左归降糖解郁方对DD海马NVU神经元损伤的保护作用

Protective effect of Zuogui Jiangtang Jieyu Formula against damage of hippocampal neuron of NVU induced by mitophagy via mTOR pathway in diabetes mellitus with depression

目的:基于mTOR介导的线粒体自噬探讨左归降糖解郁方对糖尿病并发抑郁症(DD)神经血管单元(NVU)中海马神经元损伤的保护作用。方法:原代分离、纯化和培养SD大鼠海马神经元(Ne)、星形胶质细胞(As)与脑微血管内皮细胞(Bm),并分别进行细胞鉴定。采用Transwell小室及高糖联合皮质酮干预构建DD海马NVU细胞模型,实验设正常组、模型组、GluR2阻断剂组、mTOR阻断剂、左归降糖解郁方含药血清组、GluR2阻断剂+左归降糖解郁方含药血清组和mTOR阻断剂+左归降糖解郁方含药血清组。采用CCK-8法检测海马神经元细胞存活率;Hoechst染色法检测海马神经元凋亡;免疫荧光检测自噬标志物LC3-Ⅱ、SQSTM1、Parkin及mTOR途径关键蛋白(GluR2、AMPK、mTOR)表达水平。结果:左归降糖解郁方能提高模拟DD环境下NVU中海马神经元细胞存活率(P<0.01),减少海马神经元凋亡,降低自噬标志物LC3-Ⅱ、SQSTM及Parkin水平(P<0.05,P<0.01),并显著抑制mTOR通路中GluR2、mTOR表达下调及AMPK表达上调(P<0.05,P<0.01)。结论:左归降糖解郁方对线粒体自噬介导的DD海马NVU神经元损伤具有明显的保护作用,其提高细胞活力及抗凋亡的机制可能与调控mTOR途径有关。

Objective:To investigate protective effect of Zuogui Jiangtang Jieyu Formula against damage of hippocampal neuron of NVU induced by mitophagy via mTOR pathway in diabetes mellitus with depression.Methods:Hippocampal neurons,astrocytes and brain microvascular endothelial cells from SD rats were primitively isolated,purified and cultured,and immunocytochemistry staining was used to identify primitive cells.The DD model of NVU was established by applying intervention of high glucose and cortisone after hippocampal NVU co-culture system was builded by Transwell in vitro.The cultured cells were randomly divided into normal group,model group,GluR2 blocking group,mTOR blocking group,drugcontaining serum of ZGF group,GluR2 blocking combine with ZGF group,and mTOR blocking combine with ZGF group.Cell survival rate of hippocampal neurons was detected by CCK-8 assay.The apoptosis of hippocampal neurons was detected by Hoechst staining.The expression of autophagy marker LC3-Ⅱ,SQSTM1,Parkin and mTOR pathway key proteins was detected by immunofluorescent staining and high content analysis.Results:Drug-containing serum of ZGF can significantly increase cell survival rate(P<0.01)and reduced apoptosis hippocampal neurons of NVU in mimetic DD condition.The expression of autophagy marker LC3-Ⅱ,SQSTM1,and Parkin was down-regulated in ZGF group(P<0.05,P<0.01).mTOR pathway-related proteins expression of GluR2,mTOR was up-regulated while AMPK down-regulated by Zuogui Jiangtang Jieyu Formula treatment(P<0.05,P<0.01).Conclusion:Zuogui Jiangtang Jieyu Formula have significant protective effects against damage of hippocampal neuron of NVU induced by mitophagy in diabetes mellitus with depression,and its mechanism of improving cell viability and anti-apoptosis may be related to the mTOR pathway.