College of Pharmacy, Henan University of Chinese Medicine, Zhengzhou 450046, China.
The Engineering and Technology Center for Chinese Medicine Development of Henan Province, Zhengzhou 450046, China.
Molecules. 2021 Oct 11;26(20):6133. doi: 10.3390/molecules26206133.
Lipid deposition in the kidney can cause serious damage to the kidney, and there is an obvious epithelial-mesenchymal transition (EMT) and fibrosis in the late stage. To investigate the interventional effects and mechanisms of phenolic compounds from on the EMT and fibrosis induced by sodium oleate-induced lipid deposition in renal tubular epithelial cells (NRK-52e cells), and the role played by CD36 in the adjustment process, NRK-52e cells induced by 200 μmol/L sodium oleate were given 10 μmoL/L moracin-P-2″--β-d-glucopyranoside (Y-1), moracin-P-3'--β-d-glucopyranoside (Y-2), moracin-P-3'--α-l-arabinopyranoside (Y-3), and moracin-P-3'-O-[β-glucopyranoside-(1→2)arabinopyranoside] (Y-4), and Oil Red O staining was used to detect lipid deposition. A Western blot was used to detect lipid deposition-related protein CD36, inflammation-related protein (p-NF-κB-P65, NF-κB-P65, IL-1β), oxidative stress-related protein (NOX1, Nrf2, Keap1), EMT-related proteins (CD31, α-SMA), and fibrosis-related proteins (TGF-β, ZEB1, Snail1). A qRT-PCR test detected inflammation, EMT, and fibrosis-related gene mRNA levels. The TNF-α levels were detected by ELISA, and the colorimetric method was used to detects SOD and MDA levels. The ROS was measured by flow cytometry. A high-content imaging analysis system was applied to observe EMT and fibrosis-related proteins. At the same time, the experiment silenced CD36 and compared the difference between before and after drug treatment, then used molecular docking technology to predict the potential binding site of the active compounds with CD36. The research results show that sodium oleate can induce lipid deposition, inflammation, oxidative stress, and fibrosis in NRK-52e cells. Y-1 and Y-2 could significantly ameliorate the damage caused by sodium oleate, and Y-2 had a better ameliorating effect, while there was no significant change in Y-3 or Y-4. The amelioration effect of Y-1 and Y-2 disappeared after silencing CD36. Molecular docking technology showed that the Y-1 and Y-2 had hydrogen bonds to CD36 and that, compared with Y-1, Y-2 requires less binding energy. In summary, moracin-P-2″--β-d-glucopyranoside and moracin-P-3'--β-d-glucopyranoside from ameliorated lipid deposition, EMT, and fibrosis induced by sodium oleate in NRK-52e cells through CD36.
脂质在肾脏中的沉积会对肾脏造成严重损害，晚期会出现明显的上皮-间充质转化（EMT）和纤维化。为了研究酚类化合物对油酸钠诱导的肾小管上皮细胞（NRK-52e 细胞）脂质沉积诱导的 EMT 和纤维化的干预作用及其机制，以及 CD36 在调节过程中所起的作用，用 200μmol/L 油酸钠诱导 NRK-52e 细胞，给予 10μmoL/L 麦角甾-4-烯-3β,22α-二醇-3-O-β-D-吡喃葡萄糖苷（Y-1）、麦角甾-4-烯-3β,22α-二醇-3-O-α-L-吡喃阿拉伯糖苷（Y-3）、麦角甾-4-烯-3β,22α-二醇-3-O-[β-D-吡喃葡萄糖基-（1→2）-α-L-吡喃阿拉伯糖苷]（Y-4），油红 O 染色检测脂质沉积。Western blot 检测脂质沉积相关蛋白 CD36、炎症相关蛋白（p-NF-κB-P65、NF-κB-P65、IL-1β）、氧化应激相关蛋白（NOX1、Nrf2、Keap1）、EMT 相关蛋白（CD31、α-SMA）和纤维化相关蛋白（TGF-β、ZEB1、Snail1）。qRT-PCR 检测炎症、EMT 和纤维化相关基因 mRNA 水平。ELISA 检测 TNF-α 水平，比色法检测 SOD 和 MDA 水平。用流式细胞术检测 ROS。高内涵成像分析系统观察 EMT 和纤维化相关蛋白。同时，该实验沉默了 CD36，并比较了药物治疗前后的差异，然后利用分子对接技术预测了活性化合物与 CD36 的潜在结合位点。研究结果表明，油酸钠可诱导 NRK-52e 细胞发生脂质沉积、炎症、氧化应激和纤维化。Y-1 和 Y-2 可显著改善油酸钠引起的损伤，且 Y-2 改善效果更好，而 Y-3 和 Y-4 则无明显变化。沉默 CD36 后，Y-1 和 Y-2 的改善作用消失。分子对接技术显示，Y-1 和 Y-2 与 CD36 形成氢键，与 Y-1 相比，Y-2 需要更少的结合能。综上所述，麦角甾-4-烯-3β,22α-二醇-3-O-β-D-吡喃葡萄糖苷和麦角甾-4-烯-3β,22α-二醇-3-O-β-D-吡喃葡萄糖苷通过 CD36 改善了油酸钠诱导的 NRK-52e 细胞中的脂质沉积、EMT 和纤维化。
