目的:慢性糖尿病并发症,包括糖尿病肾病(DN),常导致终末期肾衰竭。这项研究调查了依帕列净(SGLT2i)对胶原蛋白合成的影响,氧化应激,细胞存活,和DN的LLC-PK1模型中的蛋白质表达。
方法:高葡萄糖(HG)和增加依帕列净浓度(100nM和500nM)的组合,以及HG的组合,H2O2和依帕列净,用于细胞培养处理。细胞活力,谷胱甘肽(tGSH),ECM表达,测定TGF-β1浓度。此外,Akt的蛋白表达,pAkt,测定GSK3、pGSK3、pSTAT3和SMAD7。
结果:向先前暴露于葡萄糖和氧化应激的细胞中添加两种浓度的依帕列净通常会改善细胞活力并增加GSH水平(p<0.001,p<0.05)。在HG30/H2O2/Empa500处理的细胞中,pSTAT3、pGSK3β显著增加,GSK3β,除AKT外,观察到SMAD7和pAKT水平(p&lt;0.001,p&lt;0.001,p&lt;0.05)。较低的药物浓度不影响蛋白质表达水平。此外,empagliflozin处理(100nM和500nM)HG30/H2O2损伤的细胞导致TGF-β1水平降低(p<0.001)。在暴露于氧化应激和高血糖的细胞中,胶原蛋白生产保持不变。
结论:依帕列净的肾保护作用,在DN的LLC-PK1细胞模型中,是通过激活Akt/GSK-3信号通路介导的,从而减少氧化应激引起的损伤,以及增强SMAD7表达导致TGF-β1下调,TGF-β1是炎症和纤维化的关键介质之一。
Aims: Chronic diabetes complications, including diabetic nephropathy (DN), frequently result in end-stage renal failure. This study investigated empagliflozin (SGLT2i) effects on collagen synthesis, oxidative stress, cell survival, and protein expression in an LLC-PK1 model of DN. Methods: Combinations of high glucose (HG) and increasing empagliflozin concentrations (100 nM and 500 nM), as well as combinations of HG, H2O2, and empagliflozin, were used for cell culture treatment. The cell viability, glutathione (tGSH), ECM expression, and TGF-β1 concentration were measured. In addition, the protein expression of Akt, pAkt, GSK3, pGSK3, pSTAT3, and SMAD7 was determined. Results: The addition of both concentrations of empagliflozin to cells previously exposed to glucose and oxidative stress generally improved cell viability and increased GSH levels (p < 0.001, p < 0.05). In HG30/H2O2/Empa500-treated cells, significant increase in pSTAT3, pGSK3β, GSK3β, SMAD7, and pAKT levels (p < 0.001, p < 0.001, p < 0.05) was observed except for AKT. Lower drug concentrations did not affect the protein expression levels. Furthermore, empagliflozin treatment (100 nM and 500 nM) of HG30/H2O2-injured cells led to a decrease in TGF-β1 levels (p < 0.001). In cells exposed to oxidative stress and hyperglycemia, collagen production remained unchanged. Conclusion: Renoprotective effects of empagliflozin, in this LLC-PK1 cell model of DN, are mediated via activation of the Akt/GSK-3 signalling pathway, thus reducing oxidative stress-induced damage, as well as enhanced SMAD7 expression leading to downregulation of TGF-β1, one of the key mediators of inflammation and fibrosis.