Effects of ursolic acid on NADPH oxidase subunit p47Phoxexpression and ERKL/2 pathway activation in rat hepatic stellate cells / 第二军医大学学报

ABSTRACT

Objective To investigate the effects of ursolic acid (UA) on leptin-induced NADPH oxidase (NOX) subunits p47Phox expression and ERKl/2 pathway activation of rat hepatic stellate cells (HSC-T6), and to observe the cells proliferation and collagen I synthesis. Methods Culture-activated HSC-T6 cells were divided into six groups normal control group received no treatment; leptin group received recombinant rat leptin (100 ng/ml); the four intervention groups were pretreated with UA(50 μmol/L), JAK inhibitor AG490 (50 μmol/L), NOX inhibitor DPI(20 μmol/L), and ERK inhibitor PD98059 (30 μmol/L) for 30 min, and then stimulated with leptin for different time periods. Western blotting analysis was used to examined the expression of p47Phox protein translocation to the cell membrane, total cellular p47Phox protein and phosphorylated ERKl/2 (p-ERKl/2) protein. Collagen I mRNA expression was detected by RT-PCR and cell proliferation was examined by MTT assay. Results Expression of p47Phox membrane protein was significantly increased compared with the normal control 30 min after leptin stumulation (P<0. 01), and p-ERKl/2 protein expression was also significantly increased correspondingly (P<0. 05). UA, AG490, DPI and PD98059 inhibited the p47Phox protein translocation to membrane and ERKl/2 protein phosphorylation in HSC-T6 cells. Compared with normal control group, leptin stimulation for 12 h significantly up-regulated collagen I mRNA expression in HSC-T6 cells (P<0. 01); UA, AG490, DPI and PD98059 treatment inhibited collagen I mRNA expression in HSC-T6 cells compared with the leptin group (P<0. 01). Proliferation rates of HSC-T6 cells were significantly higher at 12, 24 and 48 h after leptin stimulation compared with the control group (all P<0. 01); UA, AG490, DPI and PD98059 treatment inhibited leptin-induced cell proliferation at different time points (all P<0. 01), with the inhibitory effect of UA being significantly weaker than that of DPI (P<0. 01). Conclusion UA can inhibit leptin-induced proliferation of HSC-T6 cells and collagen I expression, which might be associated with the inhibition of NOX subunit p47Phox translocation to the cell membrane and the ERK1/2 pathway activation.