Yin Caocao, Wang Yue, Peng Yue, Zhang Rongqiang, Sun Na, Shi Chuandao, Liu Qiling
Department of Public Health, Shaanxi University of Chinese Medicine, Xi'an 712046, China.
Department of Public Health, Shaanxi University of Chinese Medicine, Xi'an 712046, China. *Corresponding authors, E-mail:
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2021 Nov;37(11):973-980.
Objective To investigate the effect and mechanism of urolithin A (UA) on the inflammation and lipid accumulation induced by hyperlipidemia in L02 hepatocytes. Methods Nuclear erythroid 2-related factor 2 (Nrf2) short hairpin RNA (shRNA) lentivirus was used to establish a stable Nrf2 knockdown cell line in L02 cells. Empty vector control cells and Nrf2 knockdown cells were treated with free fatty acids (FFAs) or bovine serum albumin (BSA) to establish the hyperlipidemic cell model, and Urolithin A was treated on this basis. Specifically, they were divided into control group (BSA treatment), FFA treatment group (0.6 mmol/L), FFA (0.6 mmol/L) combined with UA low-dose group (10 μmol/L) and FFA (0.6 mmol/L) combined with UA high-dose group (20 μmol/L). All of these groups were treated for 48 h. The dye of BODIPY493/503 was used to detect the accumulation of lipid droplets in the cell. The levels of triglyceride (TG) was detected by TG assay kit. TNF-α and IL-6 in the supernatant of the cells were detected by ELISA. The level of cellular reactive oxygen species (ROS) was detected by flow cytometry combined with DCFH-DA. Malondialdehyde (MDA) kit was used to test the level of MDA. Total superoxide dismutase (SOD) kit and catalase (CAT) kit were used to detect the activities of total SOD and CAT, respectively. The mRNA levels of SOD2 and CAT were detected by real-time quantitative PCR. The protein levels of SOD2, CAT, Nrf2 as well as P62, LC3 were detected by Western blot analysis. The adenovirus of RFP-GFP-LC3 was used to measure the autophagy flux in the cells. Results FFA increased the levels of TNF-α, IL-6 and TG as well as the positive rate of BODIPY493/503 staining in L02 cells. The levels of MDA and ROS increased, while the mRNA and protein expressions of SOD2, CAT and Nrf2 decreased when treated with FFA. FFA treatment also suppressed the levels of autophagy markers LC33-II and promoted the level of P62, and blocked autophagy flux. UA treatment could reverse the above effects of FFA, with significant difference. When Nrf2 was knocked down, the above effects of UA disappeared. Conclusion Through activating autophagy and antioxidative pathways which are mediated by Nrf2 pathway, urolithin A alleviates inflammation and oxidative stress induced by high lipid in L02 hepatocytes.
目的 探讨尿石素A（UA）对高脂血症诱导的L02肝细胞炎症和脂质蓄积的影响及其机制。方法 利用核因子E2相关因子2（Nrf2）短发夹RNA（shRNA）慢病毒在L02细胞中建立稳定的Nrf2敲低细胞系。用游离脂肪酸（FFA）或牛血清白蛋白（BSA）处理空载体对照细胞和Nrf2敲低细胞，建立高脂血症细胞模型，并在此基础上给予尿石素A处理。具体分为对照组（BSA处理）、FFA处理组（0.6 mmol/L）、FFA（0.6 mmol/L）联合UA低剂量组（10 μmol/L）和FFA（0.6 mmol/L）联合UA高剂量组（20 μmol/L）。所有组均处理48 h。用BODIPY493/503染料检测细胞内脂滴的蓄积情况。用甘油三酯（TG）检测试剂盒检测TG水平。用酶联免疫吸附测定（ELISA）法检测细胞上清液中肿瘤坏死因子-α（TNF-α）和白细胞介素-6（IL-6）水平。采用流式细胞术结合2′,7′-二氯二氢荧光素二乙酸酯（DCFH-DA）检测细胞内活性氧（ROS）水平。用丙二醛（MDA）试剂盒检测MDA水平。分别用总超氧化物歧化酶（SOD）试剂盒和过氧化氢酶（CAT）试剂盒检测总SOD和CAT的活性。用实时定量聚合酶链反应（PCR）检测SOD2和CAT的mRNA水平。用蛋白质免疫印迹分析检测SOD2、CAT、Nrf2以及P62、微管相关蛋白轻链3（LC3）的蛋白水平。用红色荧光蛋白-绿色荧光蛋白-LC3腺病毒检测细胞中的自噬通量。结果 FFA增加了L02细胞中TNF-α、IL-6和TG水平以及BODIPY493/503染色阳性率。FFA处理后，MDA和ROS水平升高，而SOD2、CAT和Nrf2的mRNA和蛋白表达降低。FFA处理还抑制了自噬标志物LC3-II水平，促进了P62水平，并阻断了自噬通量。UA处理可逆转FFA的上述作用，差异有统计学意义。当Nrf2被敲低时，UA的上述作用消失。结论 尿石素A通过激活由Nrf2途径介导的自噬和抗氧化途径，减轻L02肝细胞中高脂诱导的炎症和氧化应激。
