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 type Ⅰ collagen at mRNA level of each group was detected by fluorescence quantitavepolymerase 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 PDGFgroup (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 UAgroup (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.
ABSTRACT
Objective To observe the anti-tumor effects of Agrimonia Pilosa Ledeb(APL) ,a Chinese herbal medicine ,on hepato-cellular carcinoma cells in vitro and investigate the underlined mechanisms preliminarily .Methods APL water extracts were pre-pared .SMMC-7721 cells were cultured with the medium containing different concentrations of APL water extracts ,and at different time points ,cell viabilities were measured by the MTT assay and inhibitory rates (IR) were calculated ;cell morphologic changes were observed under a light microscope ;apoptotic ratios were measured by flow cytometry ;and the expressions of Bcl-2 and P53 proteins were examined by immunocytochemistry .Results After the cells were cultured with the medium containing APL water ex-tracts for 24 h ,48 h and 72 h ,no obvious effects were found on the cell proliferation in 5 mg/mL group and 10 mg/mL group ,but IR were 0 .5% ,23 .9% and 27 .5% in 20 mg/mL group and 23 .3% ,51 .7% and 71 .6% in the 40 mg/mL group ,respectively .In the groups with effects on the cells proliferation ,morphological characteristics of apoptosis were obvious ,and the cell apoptotic ratios were 19 .5% and 23 .0% in 20 mg/mL group and 33 .4% and 42 .7% in 40 mg/mL group at 48 h and 72 h .The expressions of Bcl-2 protein were 71 .9% and 58 .5% in 20 mg/mL group and 47 .9% and 26 .5% in 40 mg/mL group at 48 h and 72 h ,and the ex-pressions of P53 protein were 22 .9% and 50 .6% in 20 mg/mL group and 48 .7% and 83 .7% in 40 mg/mL group at 48 h and 72 h . Conclusion The water extracts of APL are able to inhibit proliferation and induce apoptosis of SMMC-7721 cells dose-time depend-ently in vitro ,which might be associated with the expression changes of Bcl-2 and P53 protein .