羟基红花黄色素A对肾小管上皮细胞冷缺氧/复氧损伤的保护作用

Protective Effect of Hydroxysafflor Yellow A on Cold Hypoxia/Reoxygenation Injury in Renal Tubular Epithelial Cells

目的观察羟基红花黄色素A(HSYA)对人肾小管上皮细胞(HK-2细胞)冷缺氧/复氧(H/R)损伤的作用。方法HK-2细胞常规培养后分3组:对照组(sham组)、冷H/R组即细胞冷缺氧4 h后复氧4 h、HSYA预处理组即缺氧前0.5 h给予不同剂量(15、25、35、45、55、65、75μmol·L^(-1))的HSYA处理,其余同冷H/R组。倒置相差显微镜观察各组细胞形态;CCK-8法检测各组细胞存活率;电镜观察各组细胞超微结构。结果与sham组比较,冷H/R组细胞变圆,贴壁细胞数量减少,但经HSYA(25μmol·L^(-1))处理后细胞形态有所恢复,贴壁细胞数量增多;与sham组比较,冷H/R组细胞存活率明显下降[(100.00±0.09)%比(59.00±3.80)%,P<0.05];与冷H/R组比较,HSYA(25μmol·L^(-1))处理组细胞存活率显著增加[(59.00±3.80)%比(74.00±5.50)%,P<0.05];sham组见细胞质内线粒体、糖原较丰富,冷H/R组细胞质内见线粒体肿胀或固缩,次级溶酶体中含残存细胞器,糖原散在、减少,明显扩张的内质网;而HSYA预处理(25、35、55μmol·L^(-1)亚组)后细胞质内见自噬小体增多,其他4个亚组(15、45、65、75μmol·L^(-1)组)见糖原数量有所恢复且部分聚集。结论HSYA预处理对人肾小管上皮细胞冷H/R损伤具有保护作用,其最佳预处理浓度为25μmol·L^(-1),其作用机制可能与调节细胞自噬、线粒体功能和直接抗氧化有关。

Objective To investigate the protective effect of hydroxysafflor yellow A(HSYA)on cold hypoxia/reoxygenation(cold H/R)injury in human renal tubular epithelial HK-2 cells.Methods The cultured HK-2 cells were divided into sham group,cold H/R group and HSYA group.In cold H/R group,cells were reoxygenated for 4 h after cold hypoxia for 4 h.In HSYA group,cells were treated with different doses of HSYA(15,25,35,45,55,65 and 75μmol·L^(-1))0.5 h before hypoxia,followed by 4 h of cold hypoxia and 4 h of reoxygenation.Cell morphology was evaluated by an inverted phase contrast microscope.The cell viability was measured by CCK-8 assay.The ultrastructure of cells was observed by transmission electron microscope.ResultsCompared with the sham group,the cells became round in shape and the number of adherent cells decreased in the cold H/R group.However,pretreatment with HSYA(25μmol·L^(-1))recovered cell morphology and increased the number of adherent cells when compared to the cold H/R group.The survival rate of cells in the cold H/R group was lower than that in the sham group((59.00±3.80)%vs(100.00±0.09)%,P<0.05),while that in 25μmol/L HSYA group was higher than that in the cold H/R group((74.00±5.50)%vs(59.00±3.80)%,P<0.05).In the sham group,abundant mitochondria and glycogen were observed in the cytoplasm.In the cold H/R group,cells showed mitochondrial swelling or pyknosis along with residual organelles in secondary lysosomes,scattered and reduced glycogen and dilated endoplasmic reticulum.However,pretreatment with HSYA at doses of 25,35 and 55μmol/L increased autophagosomes in the cytoplasm.Furthermore,glycogen restoration and aggregation were achieved by pretreatment with HSYA at concertrations of 15,45,65 and 75μmol·L^(-1).Conclusion HSYA pretreatment has a protective effect on cold H/R injury in human renal tubular epithelial cells with the optimal concentration at 25μmol·L-1.The mechanisms of HSYA action may be related to the regulation of autophagy,mitochondrial function and antioxidation.