Urolithin A inhibits inflammation and oxidative stress induced by high lipid in hepatocytes via activating Nrf2 pathway and autophagy

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.