Effects of ursolic acid on the signal pathway in activated hepatic stellate cells

Abstract

Objective To observe the effects of ursolic acid (UA) on the activation of nicotinamide adenine dinucleotide phosphate-oxidase (NOX) and the downstream signaling pathways in platelet derived growth factor (PDGF) activated rat hepatic stellate cell (HSC-T6). Methods Rat HSC-T6 cells were divided into blank control group (no treatment), UA control group (50 μmol/L UA), PDGF group (10 μg/L PDGF), UA intervention group (50 μmol/L UA+ 10 μg/L PDGF), diphenyleneiodonium intervention (DPI) group (20 μmol/L DPI+ 10 μg/L PDGF), SB203580 (p38 mitogen-activated protein kirase (p38MAPK) inhibitor) intervention group (10 μmol/L SB203580+ 10 μg/LPDGF), LY294002 (phosphatidylinositop 3 kinase (PI3K) inhibitor) intervention group (10 μmol/L LY294002+ 10 μg/L PDGF) and rosup positive control group (5 μg/mL rosup). Except rosup positive control group, the expression of typeⅠcollagen at mRNA level of each group was detected by fluorescence quantitave-polymerase chain reaction (RT-PCR). The expression of membrane protein p47phox (except rosup positive control group), PI3K (except rosup positive control group and SB203580 intervention group), p-protein kinase B (p-AKT, except rosup positive control group and SB203580 intervention group) and phosphorylated p38 mitogen-activated protein kinase (p-p38MAPK, except rosup positive control group and LY294002 intervention group) were tested by Western blot. Except SB203580 intervention group and LY294002 intervention group, the fluorescence intensity in the cells of each group was analyzed with active oxygen detection kit and fluorescence microplate reader. Single factor analysis of variance and LSD test were performed for comparison between groups. Results Type I collagen at the mRNA level of PDGF group (3.74±0.32) was higher than that of blank control group (1.00±0.00); Type Ⅰ collagen at the mRNA level of UA group (0.21±0.02) was lower than that of blank control group, UA intervention group (1.02±0.12), DPI intervention group (1.09±0.21), SB203580 intervention group (1.18±0.27), and LY294002 intervention group (1.15±0.26) were all lower than PDGF group, and the differences were statistically significant (t=15.667, -4.501, -15.553, -15.154, -14.642 and -14.813, all P<0.05). p47phox at the protein expression level of PDGF group (1.98±0.53) was higher than that of blank control group (1.00±0.00); that of UA group (0.48±0.10) was lower than blank control group; those of UA intervention group (0.95±0.26), DPI intervention group (0.99±0.28), SB203580 intervention group (0.93±0.31), and LY294002 intervention group (1.07±0.19) were all lower than PDGF group (t=4.209, -2.234, -4.424, -4.252, -4.510 and -3.909, all P<0.05). The protein expression level of PI3K of PDGF group (2.27±0.46) was higher than that of blank control group (1.00±0.00); that of UA intervention group (0.14±0.07) was lower than PDGF group and blank control group; that of UA group (0.14±0.07) was lower than blank control group; those of DPI intervention group (0.53±0.25) and LY294002 intervention group (0.35±0.14) were all lower than PDGF group (t=6.205, -8.208, -2.003, -4.202, -8.502 and -9.831, all P<0.05). The protein expression level of p-Akt of PDGF group (2.54±0.49) was higher than that of blank control group (1.00±0.00); those of UA intervention group (0.74±0.20), DPI intervention group (0.94±0.37) and LY294002 intervention group (1.17±0.41) were all lower than PDGF group; that of UA group (0.59±0.15) was lower than blank control group (t=5.927, -6.928, -6.158, -5.273 and -1.578, all P<0.05). The protein expression level of p-p38MAPK of PDGF group (1.98±0.35) was higher than that of blank control group (1.00±0.00); those of UA intervention group (0.68±0.28), DPI intervention group (0.63±0.27) and SB203580 intervention group (0.67±0.29) was all lower than PDGF group; that of UA group (0.28±0.13) was lower than blank control group (t=4.897, -6.479, -6.727, -6.529 and -3.561, all P<0.05). The level of active oxygen of PDGF group (105.57±7.51) was higher than that of blank control group (69.60±8.63); those of UA intervention group (64.56±9.11), DPI intervention group (65.75±6.62) was lower than PDGF group, UA group (29.84±3.19) was lower than blank control group (t=6.368, -7.288, -7.071 and -7.255, all P<0.05). Conclusion UA could inhibit membrane displacement of NOX subunit p47phox and reduce active oxygen production in PDGF induced rat HSC-T6 cells, and then block phosphorylation of PI3K-Akt, p 38MAPK signal pathways and inhibited the expression of type Ⅰ collagen at mRNA level. Key words: Hepatic stellate cell; Ursolic acid; Platelet-derived growth factor; NADPH oxidase