目的:观察绿原酸(CGA)对高糖(HG)诱导的HK-2细胞氧化损伤的缓解作用并探讨其可能机制。
方法:我们培养了人近端肾小管细胞系HK-2,并将其分为对照组和不同浓度的CGA组(0、5、10、25、50、100、200μM)。锥虫蓝染料试验用于检测CGA对HK-2细胞的潜在细胞毒性。然后,我们用HG和CGA处理HK-2;细胞计数试剂盒-8(CCK-8)方法用于检测各组HK-2细胞的细胞活力。采用流式细胞术检测细胞凋亡率。Westernblot检测凋亡蛋白B细胞淋巴瘤-2(BCL-2)的表达,BCL-2相关X蛋白(BAX),半胱氨酰天冬氨酸特异性蛋白酶(CASPASE)-9和CASPASE-3。此外,酶活性,包括超氧化物歧化酶(SOD),谷胱甘肽过氧化物酶(GSH-Px),过氧化氢酶(CAT),和脂质过氧化物(LPO),用相应的检测试剂盒测量。2\',进行7'-二氯二氢荧光素二乙酸酯(DCFH-DA)测定和流式细胞术以检测活性氧(ROS)的产生。通过Westernblot分析和逆转录聚合酶链反应(RT-PCR)评估Kelch样ECH相关蛋白1(KEAP1)/核因子红细胞2相关因子2(NRF2)/抗氧化反应元件(ARE)信号通路的蛋白和mRNA表达。
结果:结果显示,以剂量依赖的方式,CGA显著提高了HG诱导的HK-2的活力。此外,CGA显著降低HG刺激的HK-2细胞凋亡,这可能与促进BCL-2和抑制BAX有关,切割的CASPASE-3和切割的CASPASE-9表达。在HK-2细胞中,CGA减少了HG水平产生的ROS的形成,并显着增强了抗氧化酶SOD的活性,GSH-Px,和CAT。此外,与HG组相比,CGA显著上调NRF2核表达,下调NRF2胞浆表达,上调NRF2及其靶基因的mRNA表达,血红素加氧酶-1(HO-1),KEAP1和NAD(P)H脱氢酶醌1(NQO1)。
结论:这些结果表明,CGA可能有助于控制HG诱导的HK-2细胞的氧化损伤。
OBJECTIVE: To investigate the alleviating effect of chlorogenic acid (CGA) on oxidative damage in high glucose (HG)-induced HK-2 cells and to explore its potential mechanisms.
METHODS: We cultured the human proximal tubular cell line HK-2 and divided them into the control group and different concentrations of CGA groups (0, 5, 10, 25, 50, 100, 200 μM). The trypan blue dye test was used to detect CGA\'s potential cytotoxicity on HK-2 cells. Then, we treated HK-2 with HG and CGA; the Cell Counting Kit-8 (CCK-8) method was used to detect the cell viability of HK-2 cells in each group. Flow cytometry was employed to measure the apoptosis rate of cells. Western blot was performed to detect the expression of apoptosis proteins B-cell lymphoma-2 (BCL-2), BCL-2-associated X protein (BAX), cysteinyl aspartate specific proteinase (CASPASE)-9, and CASPASE-3. In addition, enzymatic activities, including superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), and lipid peroxide (LPO), were measured with the corresponding detection kits. 2\',7\'-Dichlorodihydrofluorescein diacetate (DCFH-DA) assay and flow cytometry were performed to detect reactive oxygen species (ROS) production. Western blot analysis and Reverse Transcription-Polymerase Chain Reaction (RT-PCR) were conducted to evaluate protein and mRNA expressions of the Kelch-like ECH-associated protein-1 (KEAP1)/Nuclear factor erythroid 2-related factor 2 (NRF2)/Antioxidant Response Elements (ARE) signaling pathway.
RESULTS: The outcomes showed that, in a dose-dependent way, CGA dramatically increased the vitality of HK-2 induced by HG. Furthermore, CGA significantly reduced the HG-stimulated HK-2 cell apoptosis, which may be linked to the promotion of BCL-2 and the suppression of BAX, cleaved-CASPASE-3, and cleaved-CASPASE-9 expression. In HK-2 cells, CGA reduced the formation of ROS generated by HG levels and markedly boosted the activity of the antioxidant enzymes SOD, GSH-Px, and CAT. Furthermore, compared with the HG group, CGA significantly raised NRF2 nuclear expression and downregulated NRF2 cytosolic expression and increased the mRNA expression of NRF2 and its target genes, heme oxygenase-1 (HO-1), KEAP1, and NAD(P)H dehydrogenase quinone 1 (NQO1).
CONCLUSIONS: These results show that CGA might be useful in managing oxidative damage in HG-induced HK-2 cells.