黄芪甲苷抑制高糖腹透液诱导HMrSV5氧化应激与EMT的实验研究

The Effect of Astragaloside-Ⅳ on Oxygen Stress and EMT of HMrSV5 Induced by High-glucose Peritoneal Dialysate

目的观察黄芪甲苷(AstragalosideⅣ,AS-Ⅳ)对高糖腹透液诱导人腹膜间皮细胞HMrSV5氧化应激及EMT的影响,探讨ROS在EMT中的作用及AS-Ⅳ干预机制。方法采用葡萄糖腹透液诱导建立HMrSV5氧化应激模型,相差显微镜观察细胞形态变化,MTT检测AS-Ⅳ及葡萄糖腹透液对细胞活力的影响;予40μg/mL AS-Ⅳ干预HMrSV5氧化应激,DHE荧光探针检测药物作用前后ROS生成水平变化,Western blot检测EMT相关蛋白变化;与ROS清除剂NAC作对照,观察AS-Ⅳ干预HMrSV5氧化应激EMT指标及Smads蛋白的变化。结果 140、50μg/mL AS-Ⅳ干预后细胞活力稍有上升(P<0.05);随腹透液作用时间的延长及葡萄糖浓度提高,细胞活力明显抑制,以4.25%高糖腹透液干预48h最明显(P<0.05),细胞拉伸变长;240μg/mL AS-Ⅳ作用细胞能抑制形态改变,降低模型组ROS的生成(P<0.05),上调E-cadherin、ZO-1表达,下调α-SMA表达,并抑制Smad2/3磷酸化;35mmol/L NAC产生类似调控作用。结论高糖腹透液可抑制PMCs细胞活性,增加ROS生成,导致PMCs EMT,其分子机制可能与ROS参与调控的Smads通路激活有关,而AS-Ⅳ可能通过减少ROS生成,抑制Smad2/3磷酸化,阻抑PMCs EMT。

OBJECTIVE To observe the effect of Astragaloside- Ⅳ（AS- Ⅳ）on oxygen stress and EMT of peritoneal mesothelial cells（PMCs）HMrSV5,explore the function of ROS in EMT and regulatory mechanism of AS-Ⅳ.METHODSWe used peritoneal dialysis solutions（PDS）with high glucose concentration to establish oxygen stress cell model of HMrSV5.Morphologic alterations of cells were observed and MTT assay was used to detect cell viability.DHE fluorescent probes were loaded to label secreted ROS.EMT-associated proteins were expressed by western blotting analysis.RESULTS 140 or 50μg/mL AS-Ⅳincreased the viability of HMrSV5（P〈0.05）.Cell viability was supressed due to the longstanding effect of PDS and high glucose concentration,notably in intervention of PDS containing 4.25% glucose for 48 h.Phenotypic changes of HMrSV5 characterized by cell elongation and losing cobblestone like feature.240μg/mL AS-Ⅳcould suppress morphologic changes of cells,decrease ROS expression in model group（P〈0.05）,upregulate the protein expression of E-cadherin and ZO-1 while downregulate α-SMA.Furthermore,the phosphorylation of Smad2/3protein was inhibited.3 5mmol/L NAC had the similar effect on EMT of PMCs.CONCLUSION PDS with high glucose concentration can suppress cell viability and induce EMT of PMCs by upregulating ROS expression and activation of Smads pathway.AS-Ⅳ can reduce ROS generation,suppress the phosphorylation of Smad2/3and thus,inhibite the EMT of PMCs.