健脾消渴方通过AMPK/mTORC1/SAD-A信号通路改善胰岛β细胞功能的机制探讨

Islet β-cell dysfunction is a key factor in the pathogenesis of diabetes mellitus (DM). The protection of islet βcells has been a hot and difficult field of endocrine. The invigorating spleen and resolving turbidity is the main method of treatment of diabetes. Our previous study revealed that the effect of Jianpixiaoke recipe improved islet β cell function associated with AMPK/mTORC1/SAD-A signaling pathway. To further explore the mechanism of Jianpixiaoke recipe improving the function of islet β cells by the signaling pathway, this study will apply the combination vivo with vitro methods. Because mTORC1 as the key target in the signal pathway impacts the activity of AMPK and SAD-A through a positive and negative regulator. In vivo and vitro study, activating or blocking the key target mTORC1, point to point analysis the targets for jianpixiaoke recipe. In vivo experiments the T2DM model rats were established, then injected Activated mTORC1 adenovirus via tail veins. After intervention of Jianpixiaoke recipe, methods of enzyme-linked immunosorbent assay, Western Blot and RT-PCR were applied to observe the influence of Jianpixiaoke recipe on islet βcell function, and phosphorylation and non-phosphorylated cytokine protein, the expression of mRNA in the AMPK/mTORC1/SAD-A signaling pathway. In vitro experiment analyzes molecular mechanism of Jianpixiaoke recipe by activating and blocking mTORC1, observing the effect of Jianpixiaoke recipe on islet β cell with high sugar irritation, and analyzing the mechanism of changing of differences in protein mass spectrometry. The study illustrates the influence of Jianpixiaoke recipe on the improvement of islet β cell by AMPK/mTORC1/SAD-A signaling pathway, and the mechanism of molecular and the effect of target of Jianpixiaoke recipe.

胰岛β细胞功能受损是糖尿病(DM)发病的关键环节，保护胰岛β细胞是研究的热点和难点。健脾清热法是DM的主要治法。前期研究发现健脾消渴方改善胰岛β细胞功能与AMPK/mTORC1/SAD-A 信号通路有关。为进一步探讨健脾消渴方通过该信号通路改善胰岛β细胞的机制，体内和体外实验相结合方法，mTORC1双向调节AMPK和SAD-A活性，是信号通路的关键靶位，故体内和体外均激活或阻断mTORC1。体内实验建立T2DM大鼠模型，导入激活mTORC1重组腺病毒，健脾消渴方干预后，应用ELISA、Western Blot和RT-PCR等方法，观察该通路磷酸化及非磷酸化细胞因子蛋白量和mRNA表达的改变，及对胰岛β细胞的影响。体外实验激活和阻断mTORC1，观察健脾消渴方含药血清对高糖刺激胰岛β细胞及信号通路细胞因子蛋白量和mRNA表达的影响，明确健脾消渴方改善DM胰岛β细胞功能的作用靶点和分子机制。

糖尿病(diabetes mellitus, DM)是严重危害人类健康的疾病之一，发病率及并发症逐年增高。DM终生治疗及后期严重的并发症严重影响着人们的生活质量，也带来了巨大的经济负担。保护胰岛β细胞数量及功能是防治DM的重要研究方向，健脾消渴方已经多项临床研究证实其疗效。为进一步研究健脾消渴方防治T2DM的分子机制，本研究通过体内和体外研究结合的方式研究了健脾消渴方对胰岛β细胞的保护作用，体内研究通过构建大鼠T2DM模型，尾静脉导入mTORC1干扰腺病毒mTORC1，观察健脾消渴方对AMPK/mTORC1/SAD-A信号通路各细胞因子的改变及对胰岛β细胞功能的影响。体外研究通过制备大鼠健脾消渴方含药血清，激活及阻断AMPK/mTORC1/S AD-A通路关键靶位mTORC1，采用健脾消渴方对体外高糖环境下培养的min6细胞株进行干预，观察min6细胞功能及AMPK/mTORC1/SAD-A信号通路中各细胞因子的改变，进一步明确健脾消渴方保护胰岛β细胞的作用靶点和分子机制。体内研究发现：健脾消渴方可改善T2DM模型大鼠一般状态，降低血糖及糖化血红蛋白，改善胰岛素抵抗，增强胰岛素敏感性，提升大鼠胰岛对葡萄糖刺激的响应性，保护胰岛的组织形态，调整胰岛α/β细胞的定位和分布，纠正高糖对自噬的抑制作用，其发挥作用的机制之一是纠正高糖对AMPK的抑制，抑制mTOR表达和SAD-A实现的。体外研究发现：高糖损伤胰岛β细胞细胞活力，健脾消渴方可改善高糖导致的细胞活力降低；高糖导致胰岛β细胞对葡萄糖刺激的胰岛素分泌响应性降低，健脾消渴方可改善高糖环境下胰岛β细胞对葡萄糖刺激的响应性，促进胰岛素分泌；高糖抑制AMPK表达，促进mTOR、SAD-A表达，健脾消渴方可促进高糖环境下AMPK表达，抑制mTOR和SAD-A的表达，但对应用雷帕霉素阻断mTOR后的mTOR及SAD-A表达无明显作用，提示健脾消渴方保护胰岛β细胞数量及功能的作用机制之一是通过调节AMPK/mTOR/SAD-A信号通路实现的。证实了本研究提出的假说，验证了健脾消渴方防治T2DM的分子机制，阐明了健脾消渴方对胰岛β细胞发挥作用的分子基础，为健脾清热法防治糖尿病提供了分子细胞学的证据，拓展了中医学理论的内涵和外延。

内容获取失败，请点击重试