沉默信息调节因子3在高糖诱导的肾小管上皮细胞铁死亡中的作用

Role of silent information regulator 3 in high glucose-induced ferroptosis of renal tubular epithelial cells

目的初步探讨沉默信息调节因子3(silent information regulator 3,SIRT3)在高糖诱导的肾小管上皮细胞铁死亡中的作用,以期为糖尿病肾脏疾病患者肾小管损伤提供新的理论依据和治疗思路。方法通过"Tabula-muris"单细胞转录组数据库分析肾组织各细胞亚群SIRT3基因的表达。体外培养人永生化肾小管上皮细胞(HK-2细胞)进行以下分组:(1)对照组、甘露醇组及高糖组。(2)对照组、阴性对照组、过表达SIRT3组、高糖组及过表达SIRT3+高糖组。(3)对照组、阴性对照组、敲低SIRT3组、高糖组及敲低SIRT3+高糖组。(4)对照组、Erastin干预组及过表达SIRT3+Erastin干预组。正常葡萄糖5.5 mmol/L,高糖30 mmol/L,甘露醇24.5 mmol/L,Erastin 10μmol/L,干预时间为48 h。细胞增殖与毒性检测试剂盒实验检测细胞活力。实时荧光定量PCR和Western印迹法分别检测SIRT3、肾损伤分子1及铁死亡相关蛋白酰基辅酶A合成酶长链家族成员4(acyl-CoA synthetase long chain family member 4,ACSL4)、谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)mRNA和蛋白水平的表达。通过检测丙二醛、谷胱甘肽、铁含量评估细胞铁死亡程度。DCFH-DA法检测细胞内活性氧水平。JC-1染色法检测细胞线粒体膜电位改变。结果(1)单细胞转录组数据库分析结果显示,SIRT3基因在肾组织近端肾小管上皮细胞亚群中表达最高。(2)与对照组比较,高糖组HK-2细胞肾损伤分子1、ACSL4表达均较高,SIRT3、GPX4表达及细胞活力均较低(均P<0.05),甘露醇组与对照组上述指标的差异均无统计学意义(均P>0.05)。(3)与高糖组比较,过表达SIRT3+高糖组HK-2细胞活力、GPX4表达及细胞内谷胱甘肽均较高,ACSL4表达、细胞内铁、丙二醛及活性氧均较低,线粒体膜电位部分恢复(均P<0.05);与高糖组比较,敲低SIRT3+高糖组HK-2细胞活力、GPX4表达均较低,ACSL4表达较高(均P<0.05),细胞内铁、丙二醛、谷胱甘肽的差异均无统计学意义(均P>0.05)。(4)与对照组比较,Erastin干预组HK-2细胞ACSL4表达较高,GPX4表达较低(均P<0.05);与Erastin干预组比较,过表达SIRT3+Erastin干预组ACSL4表达较低,GPX4表达较高(均P<0.05)。结论高糖可诱导HK-2细胞SIRT3表达下调、线粒体膜电位降低以及氧化应激和铁死亡增加,过表达SIRT3可能通过减少氧化应激及缓解线粒体功能障碍减轻高糖诱导的HK-2细胞铁死亡。

Objective To preliminarily explore the role of silent information regulator 3(SIRT3)in ferroptosis induced by high glucose in renal tubular epithelial cells,and to provide a new theoretical basis and treatment ideas for renal tubular injury in diabetic kidney disease patients.Methods The single-cell transcriptomic analysis from"Tabula-muris"database was used to evaluate the expression of SIRT3 gene in different cellular subtypes of kidney tissues.HK-2 cells,a human immortalized proximal tubule epithelial cell line,were cultured in vitro and divided into following groups:(1)control group,mannitol group and high glucose group;(2)control group,negative control group,SIRT3 overexpression group,high glucose group and SIRT3 overexpression+high glucose group;(3)control group,negative control group,SIRT3 knockdown group,high glucose group and SIRT3 knockdown+high glucose group;(4)control group,Erastin intervention group and SIRT3 overexpression+Erastin intervention group.Normal glucose was 5.5 mmol/L,high glucose was 30 mmol/L,mannitol was 24.5 mmol/L,Erastin was 10μmol/L,and the intervention time was 48 h.Cell counting kit-8 proliferation and cytotoxicity assay was used to determine cell viability.Real-time quantitative PCR and Western blotting were performed to assess the expression of SIRT3,kidney injury molecule-1(KIM-1),and ferroptosis‐related proteins acyl‐CoA synthetase long chain family member 4(ACSL4)and glutathione peroxidase 4(GPX4)at the mRNA and protein levels.The malondialdehyde,glutathione,and iron levels were measured to evaluate the degree of cellular ferroptosis.DCFH-DA was used to analyze the intracellular reactive oxygen species level,while the JC-1 staining method was employed to evaluate alterations of mitochondrial membrane potential in HK-2 cells.Results(1)The results of single-cell transcriptomic database analysis demonstrated that SIRT3 gene was expressed at the highest level in the subtypes of proximal tubule epithelial cells of kidney tissues.(2)Compared with the control group,the expression levels of KIM-1 and ACSL4 were higher,and the expression levels of SIRT3 and GPX4 and cell viability were lower in the high glucose group(all P<0.05),while there was no statistically significant difference of the aforementioned indicators between the mannitol group and the control group(all P>0.05).(3)Compared with the high glucose group,HK-2 cell vitality,GPX4 expression and intracellular glutathione were higher,ACSL4 expression,intracellular iron,malondialdehyde and reactive oxygen species were lower,mitochondrial membrane potential partially recovered in SIRT3 overexpression+high glucose group(all P<0.05).Compared with the high glucose group,HK‐2 cell vitality and GPX4 expression were lower,ACSL4 expression was higher in SIRT3 knockdown+high glucose group(all P<0.05),and there were no statistically significant differences in intracellular iron,malondialdehyde and glutathione(all P>0.05).(4)Compared with the control group,Erastin intervention group had upregulated ACSL4 expression and downregulated GPX4 expression in HK-2 cells(all P<0.05).Compared with the Erastin intervention group,SIRT3 overexpression+Erastin intervention group had upregulated GPX4 expression and downregulated ACSL4 expression(all P<0.05).Conclusions High glucose can decrease SIRT3 expression and mitochondrial membrane potential,and increase oxidative stress and ferroptosis in HK-2 cells.Overexpression of SIRT3 may reduce oxidative stress and alleviate mitochondrial dysfunction,thereby mitigating glucose-induced ferroptosis in HK-2 cells.