胰岛素样生长因子结合蛋白2对高血糖环境诱导人足细胞凋亡的影响及机制研究

王晓晨; 迟坤; 杜军霞; 宋晨雯; 丁潇楠; 冀雨薇; 张可颖; 张益帆; 韩秋霞; 傅博; 洪权; 朱晗玉

doi:10.12435/j.issn.2095-5227.2024.084

胰岛素样生长因子结合蛋白2对高血糖环境诱导人足细胞凋亡的影响及机制研究

Effect and mechanism of insulin-like growth factor binding protein 2 on hyperglycemia-induced apoptosis in human podocyte

摘要

摘要:
背景糖尿病肾病患者日益增加，仍有患者在现有治疗中进展为终末期肾病，因此迫切需要新型治疗靶点。
目的探讨胰岛素样生长因子结合蛋白2(insulin like growth factor binding protein 2，IGFBP2)对高血糖环境诱导人足细胞凋亡的影响及机制。
方法体外培养的人足细胞随机分为正常血糖(normal glucose，NG)组(5 mmol/L)以及高血糖(high glucose，HG) 24 h组、HG 48 h组、HG 72 h组(30 mmol/L)。采用RT-qPCR法检测IGFBP2、肿瘤坏死因子α(tumor necrosis factor，TNF-α)和细胞间黏附分子-1(intercellular cell adhesion molecule-1，ICAM-1) mRNA表达水平，Western blot检测IGFBP2和Cleaved Caspase 3蛋白表达水平，以此确定后续实验HG处理的最佳时间点。将IGFBP2小干扰RNA转染进入足细胞并分为NG组、阴性对照干预组(NG-NC-siRNA)、IGFBP2敲低siRNA干预组(NG-IGFBP2-siRNA1、NG-IGFBP2-siRNA2、NG-IGFBP2-siRNA3)，RT-qPCR检测IGFBP2 mRNA表达水平，选择敲低效率最高的IGFBP2-siRNA用于后续实验。根据实验内容将足细胞随机分为：(1) NG组和HG组；(2) NG组和NG + 125 ng/mL rhIGFBP2组；(3) HG组和HG-IGFBP2-siRNA组。(1)(2)(3)均通过RT-qPCR检测TNF-α和ICAM-1 mRNA表达水平，JC-1染色法检测线粒体膜电位，共聚焦显微镜检测线粒体超氧化物和活性氧荧光强度，流式细胞术检测细胞凋亡率。
结果随HG处理时间增加，RT-qPCR结果显示IGFBP2、TNF-α和ICAM-1 mRNA水平随时间升高，Western blot结果显示IGFBP2和Cleaved Caspase 3蛋白水平随时间升高。与NG组比较，RT-qPCR结果显示IGFBP2、TNF-α和ICAM-1均在HG 72 h时mRNA水平最高(P＜0.05)，Western blot结果显示IGFBP2在HG 72 h时和Cleaved Caspase 3在HG 48 h时蛋白水平最高(P＜0.05)，据此选72 h为后续实验诱导时间点。RT-qPCR检测结果显示，与NG组相比，阴性对照干预组mRNA表达无统计学差异(P＞0.05)，NG-IGFBP2-siRNA2组IGFBP2 mRNA表达水平最低(P＜0.05)，敲除效率最高，因此选择IGFBP2-siRNA2进行后续实验。与NG组相比较，HG组线粒体膜电位绿/红色荧光强度比值、线粒体超氧化物和活性氧荧光强度以及细胞凋亡率均增强(P＜0.05)。与NG组相比较，NG + 125 ng/mL rhIGFBP2组的TNF-α和ICAM-1 mRNA水平、线粒体膜电位绿/红色荧光强度比值、线粒体超氧化物和活性氧荧光强度以及细胞凋亡率均升高(P＜0.05)。与HG组相比较，HG-IGFBP2-siRNA组的TNF-α和ICAM-1 mRNA表达水平、线粒体膜电位绿/红色荧光强度比值、线粒体超氧化物和活性氧荧光强度以及细胞凋亡率均降低(P＜0.05)。
结论敲除IGFBP2后通过减弱高血糖处理下的线粒体功能紊乱和氧化应激降低人足细胞凋亡，因此抑制IGFBP2的表达有望成为糖尿病肾病的潜在治疗策略。

Abstract:
Background The number of diabetic nephropathy patients is increasing, and there are still patients who progress to end-stage renal disease with existing treatments. Thus, new effective therapeutic targets are urgent to be discovered.
Objective To investigate the effect and mechanism of insulin-like growth factor binding protein 2 (IGFBP2) on apoptosis of human podocyte under hyperglycemia stimulation.
Methods The in vitro cultured human podocyte were randomly divided into four groups: the normal glucose group (NG=5 mM) and the high glucose groups (HG=30 mM) treated with different durations (24, 48, 72 h). IGFBP2, TNFα and ICAM-1 mRNA were detected by RT-qPCR, as well as IGFBP2 and cleaved caspase3 protein levels were detected by Western blotting. The best HG treatment time was determined to use in subsequent research. Podocyte transfected with IGFBP2 small interfering RNA (IGFBP2-siRNA) was divided into NG group, negative control intervention group and IGFBP2 knockdown siRNA intervention group (NG-IGFBP2-siRNA1, NG-IGFBP2-siRNA2, NG-IGFBP2-siRNA3). RT-qPCR was used to detect IGFBP2 mRNA expression level, and the group with the highest IGFBP2-siRNA knockdown efficiency was selected for subsequent transfection experiment. Podocyte were randomly divided into NG and HG group, NG and NG + 125 ng/mL rhIGFBP2 group, HG and HG-IGFBP2-siRNA group. In the three groups, RT-qPCR was used to detect TNFα and ICAM-1 mRNA levels, JC-1 staining was used to detect mitochondrial membrane potential, confocal microscopy to detect fluorescence intensity of mitochondrial superoxide (Mito SOX) and reactive oxygen species (ROS), and flow cytometry to detect the apoptosis rate.
Results RT-qPCR showed IGFBP2, TNFα and ICAM-1 mRNA levels, as well as Western blotting results showed IGFBP2 and cleaved caspase3 protein levels increased with HG treatment time. Compared with NG group, RT-qPCR showed that IGFBP2, TNFα and ICAM-1 mRNA levels in human podocyte all peaked at HG 72 h (P＜0.05), while Western blotting results showed that IGFBP2 level peaked at HG 72 h (P ＜ 0.05) and cleaved caspase3 protein level peaked at HG 48 h (P ＜ 0.05). Therefore, 72 h was selected as the time point to induce in further studies. RT-qPCR result showed that compared with NG group, the mRNA expression of negative control intervention group had no significant difference (P ＞ 0.05). The mRNA expression level of NG-IGFBP2-siRNA2 group was lowest with the highest knockdown efficiency (P ＜ 0.05). Therefore, IGFBP2-siRNA2 was selected for follow-up experiments. Compared with NG group, green/red fluorescence intensity ratio of mitochondrial membrane potential, Mito SOX and ROS, and apoptosis rate in HG group were all increased (P＜0.05). Compared with NG group, TNFα and ICAM-1 mRNA levels, green/red fluorescence intensity ratio of mitochondrial membrane potential, Mito SOX and ROS, and apoptosis rate in NG + 125 ng/mL rhIGFBP2 group were also all increased (P＜0.05). Compared with HG group, TNFα and ICAM-1 mRNA levels, green/red fluorescence intensity ratio of mitochondrial membrane potential, Mito SOX and ROS, apoptosis rate in HG-IGFBP2-siRNA group were all decreased (P＜0.05).
Conclusion IGFBP2 knockdown decrease human podocyte apoptosis by alleviating mitochondrial dysfunction and oxidative stress under hyperglycemia. Therefore, inhibition of IGFBP2 may be a potential therapeutic target for diabetic nephropathy.

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