糖络宁含药血清对高糖环境下RSC96细胞Keap1/Nrf2/Bcl-2途径的作用机制研究

Effect of Tangluoning incubated serum on Keap1/Nrf2/ Bcl-2 pathway of RSC96 cells in high glucose environment

目的针对糖尿病周围神经病变氧化应激的核心发病机制,以Keap1/Nrf2/Bcl-2途径为切入点,观察糖络宁对高糖环境下雪旺细胞的氧化应激和细胞凋亡的影响,探讨糖络宁防治DPN的可能作用机制。方法采用150 mmol/L葡萄糖培养雪旺细胞,随机分为空白对照组(Control)、高糖组(High Glucose)、α-硫辛酸含药血清组(ALA)、糖络宁含药血清组(TLN)。干预36 h后,应用流式细胞仪检测高糖环境下雪旺细胞超氧自由基和细胞凋亡,应用JC-1探针观察高糖环境下雪旺细胞线粒体膜电势(MMP),应用高内涵分析(HCA)法和Western Blot分析高糖环境下雪旺细胞Keap1/Nrf2通路中Keap1、Nrf2、HO-1和γGCS蛋白表达的变化及细胞凋亡调节蛋白Bcl-2、Bax、Cyto C和Caspase-3的蛋白表达的变化。结果高糖干预36 h后,与空白对照组比较,高糖组细胞凋亡、ROS含量增加,MMP降低(P<0.01);与高糖组比较,糖络宁组能够降低细胞凋亡和ROS含量,增加MMP(P<0.05或P<0.01)。HCA和Western Blot分析结果显示,与空白对照组比较,高糖组Keap1、Nrf2、HO-1和γGCS增加(P<0.01或P<0.05);与高糖组比较,糖络宁组能进一步增加Keap1、Nrf2、HO-1和γGCS蛋白表达(P<0.01或P<0.05)。与空白组比较,高糖组Bcl-2、Bax、Cyto C和Caspase-3蛋白表达增加(P<0.01或P<0.05);与高糖组比较,糖络宁组能够增加Bcl-2蛋白表达,降低Bax、Cyto C和Caspase-3蛋白表达(P<0.01或P<0.05)。结论糖络宁能够抑制高糖环境下雪旺细胞的氧化应激和细胞凋亡,与上调Keap1/Nrf2通路的蛋白表达对抗氧化应激,调控Bcl-2相关细胞凋亡通路的蛋白表达从而抑制细胞凋亡有关。

Objective To observe the effect of Tangluoning ( TLN ) on oxidative stress and apoptosis in high glucose incubated Schwann cells with Keap 1 /Nrf2/Bel-2signal pathway as breakthrough point according to the key pathogenesis of oxidative stress in the prevention and treatment of diabetic peripheral neuropathy ( DPN). Methods 150 mmol/L glucose incubated Schwann cells ( SCs) were used as cell models followed by setting control group, high glucose group, ALA serum group and TLN serum group, and after intervention for 36 hour( h), flow cytometry were used to analyze superoxide radical ( ROS) and apoptosis. JC-1 staining was used to observe mitochondrial membrane potential ( MMP). High content analysis ( HCA) and Western blot analysis were used to assess the changes of the expression of Keapl , Nr￡2, HO-1 and "yGCS in Keapl/Nrf2 pathway and Bcl-2 mediated apoptosis related protein including Bcl-2, Bax, Cyto C and Caspase-3. Results After incubated with high glucose for 36h, apoptosis and ROS levels were increased but MMP decreased in high glucose group compared with the control group ( P < 0. 01 ). Apoptosis and ROS levels were decreased and MMP increased in TLN serum group compared with high glucose group(P <0. 05 or P < 0. 01 ). The results of HCA and Western bolt analysis showed that Keapl , Nr￡2, HO-1 and "yGCS were increased in high glucose group compared with the control group( P <0. 01 or P < 0. 05) and Keapl , Nrf2, HO-1 and -yGCS further increased in TLN group compared with high glucose group( P <0. 01 or P <0. 05). Bcl-2, Bax, Cyto C and Caspase-3 were increased in high glucose group compared with control group(P <0. 01 or P < 0. 05 ) and Bcl-2 expression further increased but Bax, Cyto C and Caspase-3 decreased in the TLN group compared with the high glucose group(P <0. 01 or P < 0. 05 ). Conclusion TLN can inhibit the oxidative stress and apoptosis of Schwann cells in high glucose environment, and it is related with the up-regulation of protein expression in Keapl/Nrf2 pathway against antioxidative stress, and regulation of the expression of Bel-2-related apoptotic pathway protein to inhibit cell apoptosis.