Yang Yongli, Xiang Hui, Xiao Zhiying
Department of Critical Care Medicine, Hubei NO.3 People's Hospital of Jianghan University, Wuhan 430033, Hubei, China.
Department of Critical Care Medicine, Zhongnan Hospital of Wuhan University, Wuhan 430071, Hubei, China. Corresponding author: Xiao Zhiying, Email:
Zhonghua Wei Zhong Bing Ji Jiu Yi Xue. 2021 Feb;33(2):203-206. doi: 10.3760/cma.j.cn121430-20201127-00733.
To investigate the protective effect and potential mechanism of cordycepin on renal proximal tubular cells injury induced by lipopolysaccharide (LPS).
Renal proximal tubular cells NRK-52E were incubated on a cell culture plated at a density of 1×10/mL for experiment, then divided into control group (Ctrl group), LPS group (cells were stimulated with 1 mg/L LPS), 10 μmol/L or 20 μmol/L cordycep in intervention groups (LPS+C 10 group and LPS+C 20 group). Cell viability was measured using cell counting kit-8 (CCK-8) reagent. The level of intracellular reactive oxygen species (ROS) was detected by 2',7'-dichlorofluorescin diacetate (DCFH-DA) staining. The protein expressions of inflammatory factors intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), interleukin-1β (IL-1β), and nuclear factor-κB (NF-κB) were detected by Western blotting.
Compared with the Ctrl group, LPS significantly inhibited NRK-52E cell viability, increased intracellular ROS, and up-regulated the expressions of ICAM-1, VCAM-1, IL-1β and NF-κB. Compared with LPS group, after treated with 10 μmol/L or 20 μmol/L cordycepin, NRK-52E cell viability was significantly increased (Ctrl group as 1: 0.717±0.017, 0.916±0.036 vs. 0.554±0.046) and intracellular ROS level was significantly decreased (Ctrl group as 1: 1.527±0.165, 1.098±0.168 vs. 2.543±0.127), meanwhile the expressions of ICAM-1, VCAM-1, IL-1β and NF-κB were significantly down-regulated [Ctrl group as 1, ICAM-1/GAPDH: 2.364±0.097, 1.561±0.074 vs. 3.101±0.121; VCAM-1/GAPDH: 2.866±0.135, 1.920±0.098 vs. 4.170±0.119; IL-1β/GAPDH: 2.358±0.107, 1.563±0.179 vs. 3.301±0.210; phosphorylation NF-κB p65 (NF-κB p-p65)/GAPDH: 2.559±0.166, 1.596±0.148 vs. 3.183±0.098], the differences were statistically significant (all P < 0.05). Compared with the LPS+C 10 group, the cell activity of LPS+C 20 group was more significant (0.916±0.036 vs. 0.717±0.017, P < 0.01), and the expressions of ICAM-1, VCAM-1, IL-1β, NF-κB were down-regulated more significantly (ICAM-1/GAPDH: 1.561±0.074 vs. 2.364±0.097, VCAM-1/GAPDH: 1.920±0.098 vs. 2.866±0.135, IL-1β/GAPDH: 1.563±0.179 vs. 2.358±0.107, NF-κB p-p65/GAPDH: 1.596±0.148 vs. 2.559±0.166, all P < 0.05).
Cordycepin could significantly increase the survival rate of NRK-52E cells, reduce intracellular ROS level, and inhibit inflammation, and the anti-inflammation effect can be related with NF-κB pathway.
探讨虫草素对脂多糖（LPS）诱导的肾近端小管细胞损伤的保护作用及潜在机制。
将肾近端小管细胞NRK-52E以1×10⁶/mL的密度接种于细胞培养板进行实验，然后分为对照组（Ctrl组）、LPS组（细胞用1 mg/L LPS刺激）、10 μmol/L或20 μmol/L虫草素干预组（LPS+C 10组和LPS+C 20组）。使用细胞计数试剂盒-8（CCK-8）试剂检测细胞活力。用2',7'-二氯荧光素二乙酸酯（DCFH-DA）染色检测细胞内活性氧（ROS）水平。通过蛋白质印迹法检测炎症因子细胞间黏附分子-1（ICAM-1）、血管细胞黏附分子-1（VCAM-1）、白细胞介素-1β（IL-1β）和核因子-κB（NF-κB）的蛋白表达。
与Ctrl组相比，LPS显著抑制NRK-52E细胞活力，增加细胞内ROS，并上调ICAM-1、VCAM-1、IL-1β和NF-κB的表达。与LPS组相比，用10 μmol/L或20 μmol/L虫草素处理后，NRK-52E细胞活力显著增加（Ctrl组为1：0.717±0.017，0.916±0.036 vs. 0.554±0.046），细胞内ROS水平显著降低（Ctrl组为1：1.527±0.165，1.098±0.168 vs. 2.543±0.127），同时ICAM-1、VCAM-1、IL-1β和NF-κB的表达显著下调[Ctrl组为1，ICAM-1/GAPDH：2.364±0.097，1.561±0.074 vs. 3.101±0.121；VCAM-1/GAPDH：2.866±0.135，1.920±0.098 vs. 4.170±0.119；IL-1β/GAPDH：2.358±0.107，1.563±0.179 vs. 3.301±0.210；磷酸化NF-κB p65（NF-κB p-p65）/GAPDH：2.559±0.166，1.596±0.148 vs. 3.183±0.098]，差异有统计学意义（均P < 0.05）。与LPS+C 10组相比，LPS+C 20组的细胞活性更显著（0.916±0.036 vs. 0.717±0.017，P < 0.01），ICAM-1、VCAM-1、IL-1β、NF-κB的表达下调更显著（ICAM-1/GAPDH：1.561±0.074 vs. 2.364±0.097，VCAM-1/GAPDH：1.920±0.098 vs. 2.866±0.135，IL-1β/GAPDH：1.563±0.179 vs. 2.358±0.107，NF-κB p-p65/GAPDH：1.596±0.148 vs. 2.559±0.166，均P < 0.05）。
虫草素可显著提高NRK-52E细胞的存活率，降低细胞内ROS水平，并抑制炎症，其抗炎作用可能与NF-κB通路有关。
