To explore whether epigallocatechin-3-gallate (EGCG) improves renal damage in diabetic db/db mice and high-glucose- (HG-) induced injury in HK-2 cells by regulating the level of Klotho gene promoter methylation. Western blotting was used to detect the protein expression levels of DNA methyltransferase 1 (DNMT1), DNMT3a, DNMT3b, transforming growth factor-β1 (TGF-β1), α-smooth muscle actin (α-SMA), and Klotho. The methylation level of the Klotho gene promoter was detected by pyrosequencing. Chromatin immunoprecipitation was used to detect the binding of the Klotho gene promoter to DNMT1 and DNMT3a. The expression of oxidative stress markers (reactive oxygen species (ROS), superoxide dismutase (SOD), malondialdehyde (MDA), catalase (CAT), and 8-hydroxy-2-deoxyguanosine (8-OHdG)) and inflammatory cytokines (interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α)) in kidney homogenates was also measured using ELISA. Klotho and DNMT3b protein expression was upregulated, while DNMT1, DNMT3a, TGF-β1, and α-SMA protein expression was downregulated after EGCG treatment. EGCG treatment also reduced the methylation level of the Klotho gene promoter as well as the binding of DNMT3a to the Klotho gene promoter. In addition, EGCG treatment significantly decreased the levels of ROS, MDA, 8-OHdG, IL-1β, IL-6, and TNF-α and increased the levels of CAT and SOD. Under HG conditions, EGCG regulated Klotho gene promoter methylation via DNMT3a and decreased the methylation level of the Klotho gene promoter, thereby upregulating the expression of the Klotho protein to exert its protective effect.

中文翻译：

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EGCG通过逆转糖尿病db / db小鼠和HK-2细胞的Klotho高甲基化来减轻肾脏损害。

探讨表没食子儿茶素-3-没食子酸酯（EGCG）是否通过调节Klotho基因启动子甲基化水平来改善糖尿病db / db小鼠的肾脏损伤和高糖（HG-）诱导的HK-2细胞损伤。Western印迹法检测蛋白质的DNA的表达水平甲基1（DNMT1），DNMT3A，DNMT3B，转化生长因子β 1（TGF- β 1），α平滑肌肌动蛋白（α-SMA）和Klotho。通过焦磷酸测序检测Klotho基因启动子的甲基化水平。染色质免疫沉淀用于检测Klotho基因启动子与DNMT1和DNMT3a的结合。的氧化应激标志物的表达（活性氧物质（ROS），超氧化物歧化酶（SOD），丙二醛（MDA），过氧化氢酶（CAT），和8-羟基-2- -脱氧鸟苷（8-OHdG水平））和炎性细胞因子（白介素1个β（IL-1 β），IL-6和肿瘤坏死因子α（TNF- α））在肾匀浆用ELISA还测量。Klotho和DNMT3b蛋白表达上调，而DNMT1，DNMT3a，TGF- β1和αEGCG处理后-SMA蛋白表达下调。EGCG处理还降低了Klotho基因启动子的甲基化水平以及DNMT3a与Klotho基因启动子的结合。另外，EGCG治疗显着降低了ROS，MDA，8-OHdG，IL- 1β，IL-6和TNF- α的水平，并增加了CAT和SOD的水平。在HG条件下，EGCG通过DNMT3a调节Klotho基因启动子的甲基化并降低Klotho基因启动子的甲基化水平，从而上调Klotho蛋白的表达以发挥其保护作用。