周络通提取物Z-6对高糖所致Schwann细胞损伤和PI3K/Akt/nNOS通路的影响  

Protective effects of Zhouluotong extract Z-6 on Schwann cells damaged by high-glucose and PI3K /Akt /nNOS pathway

作　　者：梁俊清 徐海波[2] 陈檬 王志鑫 徐明远 姚兵 李文燕 李辉欣 侯斌 宋燕飞 王娜 庞洁 

机构地区：[1]北京以岭药业有限公司,北京102600 [2]承德医学院附属医院,河北承德067000

出　　处：《中国病理生理杂志》2014年第10期1778-1783,共6页Chinese Journal of Pathophysiology

基　　金：国家重点基础研究发展计划(国家973计划)项目(No.2012CB518606);十二五科技重大专项重大新药创制课题(No.2011ZX0940-020)

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PI3K/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（ Dglucose 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 Ca^2＋-ATPase activity in Schwann cells were determined by Cell Counting Kit-8,nitric oxide assay kit and Ca^2＋-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 PI3 K /Akt was explored by dominant negative PI3 K 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. Increased nitric oxide content and the up-regulation of nNOS were observed. However,the effects of blocking PI3 K /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 factors,caspase-3 and pro-apoptotic factors in Schwann cells under high-glucose culture,thus reducing apoptosis and elevating viability. The relationship to PI3 K /Akt /nNOS pathway needs further investigation.

关 键 词：周络通提取物 PI3K/Akt/nNOS信号通路 雪旺细胞 糖尿病周围神经病变 

分 类 号：R363[医药卫生—病理学]