黄芪甲苷对高糖环境下脂肪干细胞衰老的延缓作用

Effect of astragaloside Ⅳ alleviating senescence of adipose-derived stem cells under high-glucose environment

目的探讨黄芪甲苷(astragalosideⅣ,Ast)减缓高糖环境下人脂肪间充质干细胞(human adipose-derived stem cells,hADSCs)衰老的作用。方法通过用含不同浓度葡萄糖的培养基培养hADSCs,筛选出最适高葡萄糖浓度;用不同浓度的Ast干预高糖环境下的hADSCs,筛选出最适Ast干预浓度。将hADSCs分为4组:对照组用含5.5 mmol/L葡萄糖的培养基培养;甘露醇组用含5.5 mmol/L葡萄糖和19.5 mmol/L甘露醇的培养基培养;高糖组用含最适高浓度葡萄糖的培养基培养;Ast组用含最适高浓度葡萄糖和最适浓度Ast的培养基培养。4组均培养72 h,通过SA-β-Gal染色观察细胞衰老,Annexin Ⅴ-FITC/PI检测凋亡;qRT-PCR及Western印迹检测hADSCs中p16、Pink1、Parkin、LC3(LC3Ⅱ/Ⅰ)及p62的表达情况;透射电镜观察线粒体及自噬小体情况。结果最适高葡萄糖浓度为25 mmol/L,最适Ast干预浓度为20 mg/L。Annexin Ⅴ-FITC/PI检测显示,与对照组比较,高糖组hADSCs凋亡增加(P<0.001);与高糖组比较,Ast组hADSCs凋亡减少(P<0.001)。SA-β-Gal染色显示,与对照组比较,高糖组衰老细胞增加(P<0.001);与高糖组比较,Ast组衰老细胞减少(P<0.001)。qRT-PCR及Western印迹显示:与对照组比较,高糖组hADSCs中p16及p62表达增加(P<0.001),LC3、Pink1及Parkin表达减少(P<0.001);与高糖组比较,Ast组hADSCs中p16及p62表达减少(P<0.001),LC3、Pink1及Parkin表达增加(P<0.01)。透射电镜下发现,高糖组线粒体肿胀变形、体积较大,自噬小体较少,Ast组线粒体肿胀变形减轻、体积较小,可见大量自噬小体。结论Ast可减缓高糖引起的hADSCs衰老,可能与其上调线粒体自噬有关。

Objective To investigate the effect of astragalosideⅣ(Ast)alleviating human adipose-derived stem cells(hADSCs)senescence induced by high glucose.Methods The high glucose concentration was screened by culturing hADSCs with glucose at different concentrations.The optimal Ast concentration was screened by culturing hADSCs with Ast at different concentrations.hADSCs were divided into four groups:the control group was treated with medium containing 5.5 mmol/L glucose;the mannitol group was treated with medium containing 5.5 mmol/L glucose and 19.5 mmol/L mannitol;the high glucose group was treated with medium containing glucose at optimal high-concentration;and the Ast group was treated with medium containing Ast at optimal concentration and glucose at optimal high-concentration.After 72 hours of culture,SA-β-Gal staining was used to observe cell senescence;Annexin Ⅴ-FITC/PI was used to detect apoptosis;qRT-PCR and Western blotting were used to detect the expressions of p16,Pink1,Parkin,LC3(LC3Ⅱ/Ⅰ),and p62 in hADSCs;and transmission electron microscopy was used to observe mitochondria and autophagosomes.Results The optimal high glucose concentration was 25 mmol/L and the optimal Ast concentration was 20 mg/L.Annexin Ⅴ-FITC/PI showed that the apoptosis of hADSCs in the high glucose group was increased compared with the control group(P<0.001),and the apoptosis of hADSCs in the Ast group was decreased compared with the high glucose group(P<0.001).SA-β-Gal staining showed that the senescent cells increased in the high glucose group compared with the control group(P<0.001),and the senescent cells decreased in the Ast group compared with the high glucose group(P<0.001).qRTPCR and Western blotting showed that compared with the control group,the expression levels of p16 and p62 in hADSCs in the high glucose group were higher(P<0.001),and the expression levels of LC3,Pink1,and Parkin were lower(P<0.001);compared with the high glucose group,the expression levels of p16 and p62 in hADSCs in Ast group were lower(P<0.001),and the expression levels of LC3,Pink1,and Parkin were higher(P<0.01).Transmission electron microscopy showed that the mitochondria in the high glucose group were swollen and deformed,with a large volume and a small number of autophagosomes;after Ast intervention,a small number of swollen and deformed mitochondria were observed,with a large number of autophagosomes.Conclusions Ast could alleviate hADSCs senescence induced by high glucose,and the mechanism may be related to the up-regulation of mitophagy.