恩格列净改善高糖诱导HK-2细胞凋亡的机制探讨

王美君?梁日英?徐芬?梁小奇?李绮芊?蔡梦茵

【摘要】目的 探討恩格列净改善人肾皮质近曲小管上皮细胞（HK-2细胞）凋亡的机制。方法 将HK-2细胞置于正常葡萄糖、高葡萄糖、高葡萄糖+不同浓度的恩格列净共干预环境下培养48 h。采用蛋白免疫印迹法检测肾脏沉默信息调节因子2相关酶1（SIRT1）、硫氧还蛋白互作蛋白（TXNIP）以及裂解的半胱天冬酶-3（cleaved Caspase-3）的表达水平。采用TUNEL染色评估HK-2细胞凋亡情况。结果 蛋白免疫印迹结果显示，高葡萄糖培养48 h后HK-2细胞的TXNIP和cleaved Caspase-3表达水平均高于正常葡萄糖组，而SIRT1表达水平低于正常葡萄糖组（P均< 0.05）。1000 nmol/L恩格列净和30 mmol/L葡萄糖共干预48 h后，HK-2细胞的TXNIP和cleaved Caspase-3的蛋白表达水平降低，SIRT1表达水平升高（P均< 0.001）。TUNEL染色结果显示恩格列净和30 mmol/L葡萄糖共干预组较30 mmol/L葡萄糖组TUNEL阳性细胞数少，且1000 nmol/L恩格列净和30 mmol/L葡萄糖共干预组凋亡改善更明显。结论 恩格列净可能通过上调SIRT1的表达、抑制TXNIP表达，改善高糖诱导HK-2细胞凋亡。

【关键词】恩格列净;沉默信息调节因子2相关酶1;硫氧还蛋白互作蛋白;

糖尿病肾脏疾病;人肾皮质近曲小管上皮细胞

Mechanism of empagliflozin in mitigating HK-2 cells apoptosis induced by high glucose Wang Meijun， Liang Riying， Xu Fen， Liang Xiaoqi， Li Qiqian， Cai Mengyin. Department of Endocrinology， the Third Affiliated Hospital of Sun Yat-sen University， Guangzhou 510630， China

Corresponding author， Cai Mengyin， E-mail： caimengyin@ mail. sysu. edu. cn

【Abstract】Objective To investigate the mechanism of empagliflozin in mitigating the apoptosis of human renal proximal tubule epithelial cells （HK-2 cells）. Methods HK-2 cells were incubated in normal glucose （NG） or high glucose （HG） combined without or with empagliflozin at different concentrations for 48 h. The expression levels of silent mating type information regulation 2 homolog 1（SIRT1）， thioredoxin-interacting protein （TXNIP） and cleaved Caspase-3 were determined by Western blot. The apoptosis of HK-2 cells was determined by TUNEL staining. Results Western blot demonstrated that the expression levels of TXNIP and cleaved Caspase-3 at 48 h in the HG group were significantly higher， whereas that of SIRT1 was remarkably lower compared with those in the NG group （all P < 0.05）. After combined interventions of 1000 nmol/L empagliflozin and 30 mmol/L glucose for 48 h， the expression levels of TXNIP and cleaved Caspase-3 were significantly down-regulated， whereas that of SIRT1 was considerably up-regulated （all P < 0.001）.TUNEL staining demonstrated that the number of TUNEL-positive cells in the empagliflozin combined with 30 mmol/L

glucose group was significantly less than that in the 30 mmol/L glucose group. In the 1000 nmol/L combined with 30 mmol/L glucose group， the cell apoptosis was more evident. Conclusion Empagliflozin can protect HK-2 cells from HG-mediated apoptosis probably by up-regulating the expression level of SIRT1 protein and down-regulating that of TXNIP protein.

【Key words】Empagliflozin;SIRT1;TXNIP;Diabetic kidney disease;

Human renal proximal tubule epithelial cells

中国 2型糖尿病（T2DM）患者中肾病患病率高达 30% ～ 50%，白蛋白尿和肾小球滤过率受损的患者10 年累积病死率达47%，给社会带来极大的卫生经济负担[1]。强化降糖、降压、ARB/ACEI虽可降低蛋白尿风险，但仍缺乏肾脏硬终点获益的证据。恩格列净心血管结局事件试验（EMPA-REG OUTCOME）研究显示恩格列净治疗组血清肌酐水平增加1倍的相对风险显著降低44%、采用肾脏替代疗法较安慰剂组的相对风险降低了55%[2-3]。钠-葡萄糖共转运蛋白2（SGLT2）抑制剂可改善肾小球血流动力学、肾小管能量代谢和肾脏疾病高危因素等，延缓糖尿病肾病的进展，但目前还不能完全解释其肾脏保护机制。

2014年美国糖尿病协会（ADA）弃用糖尿病肾病名称，改为糖尿病肾脏疾病（DKD），说明糖尿病肾功能损伤不仅仅与肾小球病变相关。……