丹酚酸B对高糖诱导的视网膜Müller细胞凋亡、自噬及AMPK信号通路的影响

目的　探讨丹酚酸B对高糖诱导的视网膜Müller细胞凋亡、自噬及AMPK信号通路的影响。方法　提取大鼠视网膜原代Müller细胞，随机分为对照组、高糖组、高糖+丹酚酸B组、高糖+ AMPK抑制剂Dorsomorphin（DS）组、高糖+DS+丹酚酸B组、高糖+AICAR组和高糖+AICAR+丹酚酸B组。其中，对照组细胞给予5 mmol·L^-1葡萄糖处理，高糖为35 mmol·L^-1高浓度葡萄糖，丹酚酸B浓度为40 μmol·L^-1，DS浓度为10 μmol·L^-1，AICAR浓度为1 mmol·L^-1 。MTT法检测各组细胞活力，Hoechst 33258染色观察各组细胞凋亡情况，qRT-PCR检测各组细胞自噬相关基因Beclin1和LC3-II mRNA的表达，Western blot检测各组细胞Beclin1、LC3-I、LC3-II、p-AMPK、AMPK、Bax和Bcl-2蛋白表达情况。结果　与对照组比较，高糖组细胞Beclin1、LC3-II、p-AMPK、Bcl-2蛋白及Beclin1、LC3-II mRNA表达均显著减少（均为P<0.01)，Bax蛋白表达增加（P<0.01)，细胞活力降低(P<0.01)，细胞核浓缩和DNA片段化加剧，凋亡亢进。与高糖组比较，高糖+丹酚酸B组和高糖+AICAR+丹酚酸B组细胞Beclin1、LC3-II、p-AMPK、Bcl-2蛋白及Beclin1、LC3-II mRNA表达均显著增加（均为P<0.05)，Bax蛋白表达均显著减少（均为P<0.01)，细胞活力增加，细胞核浓缩和DNA片段化改善，凋亡被抑制；高糖+DS+丹酚酸B组细胞Beclin1、LC3-II、p-AMPK、Bcl-2、Bax蛋白表达及细胞活力均无显著变化（均为P>0.05)，细胞核浓缩和DNA片段化无显著变化。各组细胞间LC3-I和AMPK蛋白表达差异均无统计学意义（均为P>0.05)。结论　丹酚酸B可通过激活AMPK信号显著增强细胞自噬而抑制Müller细胞凋亡，从而对高糖诱导的Müller细胞起到保护作用。

Effects of salvianolic acid B on high glucose-induced apoptosis and autophagy of retinal Müller cells and AMPK signaling pathway

Objective To investigate the effects of salvianolic acid B(Sal B)on high glucose(HG)induced apoptosis and autophagy of retinal Müller cells and AMPK signaling pathway.Methods Primary retinal Müller cells extracted from rats were randomly divided into the control group,HG group,HG+Sal B group,HG+dorsomorphin(DS,an AMPK inhibitor)group,HG+DS+Sal B group,HG+5-Aminoimidazole-4-carboxamide1-β-D-ribofuranoside(AICAR)group,and HG+AICAR+Sal B group.Among them,cells in the control group were treated with 5 mmol·L^-1 glucose,while cells in the HG group were treated with 35 mmol·L^-1 glucose.The concentration of Sal B,DS,and AICAR used for treatment was 40μmol·L^-1,10μmol·L^-1,and 1 mmol·L^-1,respectively.Cell viability was detected by MTT assay,cell apoptosis was assessed by Hoechst 33258 staining,mRNA expression levels of autophagy-related genes Beclin1 and LC3-II were measured by quantitative real-time polymerase chain reaction,and protein expression levels of Beclin1,LC3-I,LC3-II,p-AMPK,AMPK,Bax,and Bcl-2 were detected by Western blot.Results Compared with the control group,the HG group showed significantly decreased Beclin1,LC3-II,p-AMPK and Bcl-2 protein expression,and Beclin1 and LC3-II mRNA expression(all P<0.01),increased Bax protein expression(P<0.01),decreased cell viability(P<0.01),increased nuclear condensation and DNA fragmentation,and hyper-apoptosis.Compared with the HG group,in the HG+Sal B group and HG+AICAR+Sal B group,Beclin1,LC3-II,p-AMPK and Bcl-2 protein expression,and Beclin1 and LC3-II mRNA expression were significantly increased(all P<0.05),Bax protein expression was significantly decreased(both P<0.01),cell viability increased,nuclear condensation and DNA fragmentation were alleviated,and apoptosis was suppressed;on the other hand,in the HG+DS+Sal B group,Beclin1,LC3-II,p-AMPK,Bcl-2 and Bax protein expression and cell viability were not significantly different(all P>0.05),and nuclear condensation and DNA fragmentation were also not significantly different.Differences in the expression levels of LC3-I and AMPK proteins among different groups were not statistically significant(all P>0.05).Conclusion Sal B inhibits Müller cell apoptosis by activating AMPK signaling and reinforcing autophagy,thus protecting HG-challenged Müller cells.