周络通提取物 Z-6对高糖所致 Schwann 细胞损伤和PI3 K／Akt／nNOS 通路的影响

摘要

目的：探讨磷酯酰肌醇3-激酶/蛋白激酶B/神经元型一氧化氮合酶（PI3K/Akt/nNOS）信号通路在周络通提取物抗糖尿病周围神经病变中的作用。方法：将体外培养的雪旺（ Schwann）细胞分为正常组（ D-葡萄糖25 mmol/L）、高糖模型组（D-葡萄糖100 mmol/L）、周络通提取物活性部位5（Z-5）＋高糖组、周络通提取物活性部位6（Z-6）＋高糖组、周络通＋高糖组和甲钴胺＋高糖组。采用CCK-8试剂盒检测各组细胞的生存活性，相关试剂盒分析培养上清中NO含量和细胞内Ca2＋-ATPase活性的变化，流式细胞仪分析细胞的凋亡情况， Western blotting方法检测Bcl-2、Bcl-xL、Bax、Bak和caspase-3蛋白表达及nNOS和Akt的磷酸化情况。利用PI3K和Akt的显性负性突变体瞬时转染Schwann细胞，探讨PI3K/Akt信号通路在周络通提取物抗糖尿病周围神经病变中的作用。结果：与模型组比较，周络通提取物Z-6能显著提高高糖损伤后Schwann细胞的存活率，提高NO分泌量并明显上调Bcl-2、Bcl-xL蛋白的表达及Akt、nNOS的磷酸化水平，同时显著降低细胞凋亡率及Bax、 Bak、 caspase-3蛋白的水平，利用PI3K的显性负性突变体（δp85）阻断nNOS的上游信号通路PI3K/Akt后，NO分泌量减少，但未对Z-6上调nNOS和Akt磷酸化的作用产生明显影响。结论：在高糖损伤条件下，周络通提取物Z-6上调nNOS蛋白磷酸化水平，促进抗凋亡因子的表达，抑制促凋亡因子及caspase-3关键蛋白酶的表达，降低Schwann细胞凋亡率，从而提高高糖损伤细胞的存活率，这些作用与PI3 K/Akt/nNOS通路的关系有待进一步研究。%AIM: To explore the role of PI3K/Akt/nNOS in Zhouluotong extract resisting diabetic peripheral neuropathy.METHODS:The Schwann cells were divided into normal group ( D-glucose 25 mmol/L) , model group ( D-glucose 100 mmol/L) , Zhouluotong extract Z-6 +high glucose group, Zhouluotong +high glucose group, mecobalamine+high glucose group.The viability, nitric oxide content and the Ca2+-ATPase activity in Schwann cells were determined by Cell Counting Kit-8 , nitric oxide assay kit and Ca2+-ATPase assay kit, respectively.The apoptosis of Schwann cells were analyzed by flow cytometry.The expression of Bcl-2, Bcl-xL, Bax, Bak and caspase-3, and the phosphorylation levels of nNOS and Akt were determined by Western blotting.The signal pathway of PI3K/Akt was explored by dominant negative PI3K and Akt (δp85 and DN-Akt) transient transfection assay.RESULTS:Under high-glucose culture, the cell viability, nitric oxide content in culture supernatant, the expression of Bcl-2 and Bcl-xL, and the phosphorylation levels of Akt and nNOS in the Schwann cells were significantly increased.The cell apoptosis, the expression of Bax, Bak and caspase 3 in the Schwann cells were significantly decreased by Zhouluotong extract Z-6, compared with model group.In-creased nitric oxide content and the up-regulation of nNOS were observed.However, the effects of blocking PI3K/Akt, the upstream pathway of nNOS , by transfection with DN-δp85 on Akt phosphorylation in the Schwann cells was still unclear. CONCLUSION:Zhouluotong extract Z-6 changes the phosphorylation of nNOS, and the expression of anti-apoptotic fac-tors , caspase-3 and pro-apoptotic factors in Schwann cells under high-glucose culture, thus reducing apoptosis and elevating viability.The relationship to PI3K/Akt/nNOS pathway needs further investigation.