The molecular mechanism of angiotensin-converting enzyme 2 alleviating hepatocyte inflammation

Objective To observe the protective mechanism of angiotensin-converting enzyme (ACE) 2 on lipopolysaccharide (LPS) -induced hepatocyte inflammation by inhibiting P38 mitogen activated protein kinase (MAPK)/ activator protein (AP)-1 pathway. Methods Rat liver BRL cells after immortalized culture were randomly divided into five kinds of groups: control group, LPS (10 μg/mL) group, LPS + recombinant(r) ACE2 (5, 10, 20 ng/mL rACE2 for 30 min before cells stimulated with LPS) groups, LPS+ACE2 inhibitor MLN-4760 (10-7, 10-6, 10-5 mmol/L MLN-4760 for 30 min before cells stimulated with LPS) groups, LPS + rACE2 (20 ng/mL rACE2 for 30 min before cells stimulated with LPS) + P38MAPK inhibitor SB203580 (10-5 mmol/L SB203580 for 30 min before cells stimulated with LPS) groups. The changes in protein levels of ACE2, P38MAPK, p-P38MAPK and AP-1 were detected by western blot after LPS exposure for 6, 12 and 24 hours, and the mRNA expressions of P38MAPK, AP-1 and tumor necrosis factor-α were quantified by real-time RT-PCR. Results Compared with control group, the protein levels of ACE2, P38MAPK and AP-1 were up-regulated in LPS-induced hepatic cells in a time-dependent manner, peaking at 12 h after LPS stimulation (all P<0.05). Compared with LPS group, the mRNA expressions of AP-1, P38MAPK, p-P38MAPK and tumor necrosis factor-α decreased significantly in rACE2 group (all P<0.05). The dose of 20 ng/mL rACE2 had the best inhibitory effects on the mRNA expression of AP-1 (0.12±0.002 vs. 0.04±0.005, P<0.01), P38MAPK (0.17±0.02 vs. 0.02±0.002, P<0.01) and p-P38MAPK(0.29±0.01 vs. 0.02±0.01, P<0.01)compared with LPS group. The mRNA expressions of AP-1, P38MAPK and p-P38MAPK increased in MLN-4760 group in a concentration dependent manner (all P<0.05). Furthermore, the inhibitory effects of rACE2 on AP-1 and tumor necrosis factor-α levels were cancelled by SB203580. Conclusion The rACE2 can alleviate the LPS-induced hepatocyte injury by down regulating the P38MAPK/AP-1 signaling pathway. Key words: Angiotensin-converting enzyme 2; p38 mitogen activated protein kinase; Lipopolysaccharide; Hepatocyte inflammation; Molecular mechanism