桃叶珊瑚苷通过调控miR-30a-5p表达对高糖诱导的肾小管上皮细胞损伤的影响

Effects of aucubin on renal tubular epithelial cell injury induced by high glucose by regulating expression of miR-30a-5p

目的:探讨桃叶珊瑚苷是否通过调控miR-30a-5p表达,影响高糖诱导的肾小管上皮细胞损伤。方法:肾小管上皮细胞HK-2分为Con组(5.5 mmol/L葡萄糖)、HG组(30 mmol/L葡萄糖)、HG+AU-L组(30 mmol/L葡萄糖+25μmol/L桃叶珊瑚苷)、HG+AU-M组(30 mmol/L葡萄糖+50μmol/L桃叶珊瑚苷)、HG+AU-H组(30 mmol/L葡萄糖+100μmol/L桃叶珊瑚苷)、HG+miR-NC组(30 mmol/L葡萄糖+转染miR-NC)、HG+miR-30a-5p组(30 mmol/L葡萄糖+转染miR-30a-5p)、HG+AU+anti-miR-NC组(30 mmol/L葡萄糖+100μmol/L桃叶珊瑚苷+转染anti-miR-NC)、HG+AU+anti-miR-30a-5p组(30 mmol/L葡萄糖+100μmol/L桃叶珊瑚苷+转染anti-miR-30a-5p)。应用ELISA、Annexin V-FITC/PI双染法、Western blot和逆转录定量聚合酶链反应(RT-qPCR)分别检测IL-1β、TNF-α表达、细胞凋亡、B细胞淋巴瘤/白血病-2(Bcl-2)、Bcl-2相关X蛋白(Bax)蛋白表达和miR-30a-5p表达。结果:与Con组比较,HG组肾小管上皮细胞IL-1β、TNF-α表达、细胞凋亡率、Bax蛋白表达急剧增加,Bcl-2蛋白、miR-30a-5p表达水平降低;相较于HG组,HG+AU-L、HG+AU-M、HG+AU-H组肾小管上皮细胞IL-1β、TNF-α表达、凋亡率、Bax蛋白表达逐渐降低,Bcl-2蛋白、miR-30a-5p表达逐渐升高,并均呈浓度依赖性;HG+miR-30a-5p组肾小管上皮细胞中miR-30a-5p、Bcl-2蛋白表达水平高于HG+miR-NC组,IL-1β、TNF-α表达、凋亡率、Bax蛋白表达水平低于HG+miR-NC组;与HG+AU+anti-miR-NC组比较,HG+AU+anti-miR-30a-5p组肾小管上皮细胞中miR-30a-5p、Bcl-2蛋白表达水平减少,IL-1β、TNF-α表达、细胞凋亡率、Bax蛋白表达水平均升高;上述差异均具有统计学意义(P<0.05)。结论:桃叶珊瑚苷可能通过上调miR-30a-5p的表达,抑制高糖诱导的肾小管上皮细胞炎症因子表达并减轻细胞凋亡。

Objective:To investigate whether aucubin affects the renal tubular epithelial cell damage induced by high glucose by regulating the expression of miR-30 a-5 p.Methods:Renal tubular epithelial cells HK-2 were divided into Con group(5.5 mmol/L glucose),HG group(30 mmol/L glucose),HG+AU-L group(30 mmol/L glucose+25μmol/L aucubin),HG+AU-M group(30 mmol/L glucose+50μmol/L aucubin),HG+AU-H group(30 mmol/L glucose+100μmol/L aucubin),HG+miR-NC group(30 mmol/L glucose+transfected miR-NC),HG+miR-30 a-5 p group(30 mmol/L glucose+transfected miR-30 a-5 p),HG+AU+anti-miR-NC group(30 mmol/L glucose+100μmol/L aucubin+transfection anti-miR-NC),HG+AU+anti-miR-30 a-5 p group(30 mmol/L glucose+100μmol/L aucubin+transfection anti-miR-30 a-5 p).ELISA,Annexin V-FITC/PI double staining,Western blot and RT-qPCR were used to detect IL-1βand TNF-αexpression,apoptosis rate,B-cell lymphoma/leukemia-2(Bcl-2),Bcl-2 related X protein(Bax)protein expression and miR-30 a-5 p expression.Results:Compared with the Con group,the expression of IL-1β,TNF-α,apoptosis rate and Bax protein expression of renal tubular epithelial cells in the HG group increased sharply,while the expression levels of Bcl-2 protein and miR-30 a-5 p decreased.Compared with the HG group,the IL-1β,TNF-αexpression,apoptosis rate and Bax protein expression of renal tubular epithelial cells in the HG+AU-L,HG+AU-M and HG+AU-H groups gradually decreased,and the expression of Bcl-2 protein and miR-30 a-5 p gradually increased,and all in a concentration-dependent;the expression levels of miR-30 a-5 p and Bcl-2 protein in renal tubular epithelial cells in the HG+miR-30 a-5 p group were higher than those in the HG+miR-NC group,while the IL-1β,TNF-αexpression,apoptosis rate and Bax protein expression levels were lower than those in the HG+miR-NC group.Compared with the HG+AU+anti-miR-NC group,the expression levels of miR-30 a-5 p and Bcl-2 protein in renal tubular epithelial cells of the HG+AU+anti-miR-30 a-5 p group decreased,and the expression of IL-1β,TNF-α,apoptosis rate and Bax protein expression levels increased;the above differences were all statistically significant(P<0.05).Conclusion:Aucubin may up-regulate the expression of miR-30 a-5 p,inhibit the expression of inflammatory factors in renal tubular epithelial cells induced by high glucose and reduce cell apoptosis.