达格列净通过调节miR-98-5p减轻高糖诱导的肾小管上皮细胞损伤的研究

Dapagliflozin alleviates the damage of renal tubular epithelial cells induced by high glucose by regulating miR-98-5p

目的探讨达格列净介导miR-98-5p对高糖诱导的人肾小管上皮细胞损伤的影响。方法收集糖尿病肾病(DN)患者及健康体检者血液样本,用实时荧光定量聚合酶链反应(RT-qPCR)法检测血清miR-98-5p相对表达水平,用生化免疫分析仪检测肾损伤相关指标水平。体外培养HK-2细胞,并将细胞随机分为对照组(5 mmol·L^(-1)葡萄糖)、高糖组(30 mmol·L^(-1)葡萄糖)、低剂量实验组(30 mmol·L^(-1)葡萄糖+20μmol·L^(-1)达格列净)、高剂量实验组(30 mmol·L^(-1)葡萄糖+40μmol·L^(-1)的达格列净)、高剂量+anti-miR-NC组(转染anti-miR-NC+30 mmol·L^(-1)葡萄糖+40μmol·L^(-1)达格列净)和高剂量+anti-miR-98-5p组(转染anti-miR-98-5p+30 mmol·L^(-1)葡萄糖+40μmol·L^(-1)达格列净)。药物处理24 h后,用细胞计数试剂盒8法检测细胞存活率,用RT-qPCR法检测细胞中miR-98-5p相对表达水平,用流式细胞术检测细胞凋亡率,用酶联免疫吸附试验法检测细胞炎性因子水平。结果DN患者和健康体检者血清中miR-98-5p相对表达水平分别为1.00±0.25和0.39±0.05,在统计学上差异有统计学意义(P<0.05)。对照组、高糖组、高剂量实验组、高剂量+anti-miR-NC组和高剂量+anti-miR-98-5p组的miR-98-5p相对表达水平分别为1.00±0.09、0.31±0.04、0.72±0.06、0.75±0.07和0.22±0.03,细胞存活率分别为(100.00±3.36)%、(51.63±5.89)%、(79.46±9.90)%、(82.88±5.71)%和(59.69±7.43)%,细胞凋亡率分别为(3.52±0.20)%、(35.80±3.67)%、(16.43±1.57)%、(15.71±1.42)%和(29.37±2.18)%,肿瘤坏死因子-α(TNF-α)表达水平分别为(22.46±1.67)、(68.37±6.05)、(34.45±2.47)、(35.11±2.84)和(60.46±3.56)pg·mL^(-1)。高糖组的上述指标与对照组比较,高剂量组的上述指标与高糖组比较,高剂量+anti-miR-98-5p组的上述指标与高剂量+anti-miR-NC组比较,在统计学上差异均有统计学意义(均P<0.05)。结论达格列净可通过上调miR-98-5p的表达来抑制炎性反应和细胞凋亡,减轻高糖诱导的HK-2细胞损伤。

Objective To investigate the effect of dapagliflozin-mediated miR-98-5p on high glucose-induced damage in human renal tubular epithelial cells.Methods Blood samples from patients with diabetic nephropathy(DN)and healthy individuals were collected.The expression of serum miR-98-5p was detected by real-time fluorescence quantitative polymerase chain reaction(RT-qPCR),and kidney injury-related indicators were measured using a biochemical immunoassay analyzer.HK-2 cells were culturedin vitroand randomly divided into control group(5 mmol·L^(-1)glucose),HG group(30 mmol·L^(-1)glucose),experimental-L group(30 mmol·L^(-1)glucose+20μmol·L^(-1)dapagliflozin),experimental-H group(30 mmol·L^(-1)glucose+40μmol·L^(-1)dapagliflozin),anti-miR-NC group(transfected with anti-miR-NC+30 mmol·L^(-1)glucose+40μmol·L^(-1)dapagliflozin),and anti-miR-98-5p group(transfected with anti-miR-98-5p+30 mmol·L^(-1)glucose+40μmol·L^(-1)dapagliflozin).Cell viability was evaluated using the cell counting kit 8(CCK-8)assay 24 hours after drug treatment;miR-98-5p expression in cells was detected by RT-qPCR;cell apoptosis rate was measured by flow cytometry,apoptosis-related protein expression was detected by Western blot;and inflammatory cytokine expression was measured by enzyme-linked immunosorbent assay.Results The expression levels of miR-98-5pin the serum of DN patients and healthy individuals were 1.00±0.25 and 0.39±0.05,respectively,showing a significant difference between the two groups(P<0.05).The expression levels of miR-98-5pin the control group,HG group,experimental-H group,anti-miR-NC group,and anti-miR-98-5p group were 1.00±0.09,0.31±0.04,0.72±0.06,0.75±0.07 and 0.22±0.03;the cell survival rates were(100.00±3.36)%,(51.63±5.89)%,(79.46±9.90)%,(82.88±5.71)%and(59.69±7.43)%;apoptosis rates were(3.52±0.20)%,(35.80±3.67)%,(16.43±1.57)%,(15.71±1.42)%and(29.37±2.18)%;tumornecrosis factor-α(TNF-α)levels were(22.46±1.67),(68.37±6.05),(34.45±2.47),(35.11±2.84)and(60.46±3.56)pg·mL^(-1),respectively.The differences among these indicators were all statistically significant when comparing the HG group to the control group,the experimental-H group to the HG group,and the anti-miR-98-5pgroup to the anti-miR-NC group(allP<0.05).Conclusion Dapagliflozin can alleviate high glucose-induced HK-2 cell damage by upregulating the expression of miR-98-5p,inhibiting inflammation,and reducing cellapoptosis.