| Objective:To estimate expression of p53in the effects of Artesunate on proliferation, expression of p53, cell cycle and apoptosis of primany hepatic stellate cells (HSCs). To provide experimental evidences for clinical application of Aartesunate to treat hepatic fibrosis.Methods:Healthy Wistar rats were disinfected after intraperitoneal injection of25%urethane anesthesia,0.8mg/mL protease E50ml and50ml0.5mg/mL collagenase digested liver in situ, the cell suspension was centrifuged by8%,12%and18%Nycodenz. HSCs were collected at8%and12%of the gradient interface, cell density was adjusted to5×105/mL. Then HSCs were seeded in culture flasks and cultured in the DMEM containing20%FBS. Morphology and growth characteristics of freshly isolated cells were observed with inverted phase contrast microscope, cell purity was detected by flow cytometry. Preparation of passage cells climbing films, staining desmin, glial fibrillary acidic protein and nuclei by anti-desmin/FITC, anti-GFAP/TRITC, DAPI to identify the cells whether they were HSCs. Rat primary HSCs were isolated and cultured in the flask for10days to make cells activated. HSCs were divided into two groups:control groups and experimental groups. Experimental groups were incubated with various concentrations of Artesunate (125,150,175,200,225μmol/L) for24,48and72hours. MTT assay was used to detect rate of cellular proliferation; digestive method was used to determinate concentrations of hydroxyproline in supernatant; microplate method was used to detect activity of lactate dehydrogenase (LDH) in cell culture supernatant; flow cytometry was used to detect cell cycle; flow cytometry with Annexin V-FITC/PI double staining and Hoechst33258dye staining were used to detect apoptosis; western blotting and PT-PCR were used to observe expression of p53.Results:(1) Hepatic stellate cells isolation, culture and identification:freshly isolated HSCs observed by phase contrast microscope were spherical, had strong refraction, and suspended in culture medium. After Culturing for10days, HSCs had been fully developed, shown stellate or polygonal form, and granules cells were significantly reduced. Newly isolated HSCs binded with anti-desmin. Cell purity was detected by flow cytometry, result showed that the purity was>95%. The secondary HSCs were anti-desmin/FITC, anti-GFAP/TRITC staining positive. The number of positive staining cells>99%. HSCs growth curve showed that the cultured HSCs were proliferated after72h, and entered into the logarithmic growth phase on the4th-5th day, entered into the plateau phase on the6th-7th day.(2) Effects of Artesunate on cell proliferation:Results of MTT colorimetric showed that inhibition of HSCs which stimulated with different concentrations of Artesunate for24,48,72h, significantly increased in groups that stimulated with150,175,200,225μmol/L, compared with the control group, the difference was statistically significant (P<0.01). In the treatment groups, the inhibitory effect increased with the concentration when stimulated for24h and48h (P<0.01), prompting that inhibition of Artesunate on proliferation of HSCs was in a dose-dependent manner. In group175μmol/L, the action of inhibition was with increasing time (P<0.01), at72h, the inhibition rate reached92.6%in group150μmol/L, suggesting that inhibition of Artesunate on proliferation was in a time-dependent manner.(3) Hydroxyproline test in the cell culture supernatant:after treated with Artesunate for24h, secretion of hydroxyproline decreased in all treatment groups. Compared with control group, the difference was statistically significant (P <0.01).(4) LDH test in the cell culture supernatant:after stimulated with Artesunate for24h, activity of LDH in supernatant was low in all treatment groups. Compared with control group, the difference had no statistically significance (P>0.05).(5) Effects of Artesunate on cell cycle of HSCs:in groups175,200μmol/L, the percentage of HSCs at G0/G1phase increased significantly (P<0.01), compared with the control group, the percentage of S phase was significantly decreased (P <0.05), G2/M phase cells had no obvious change.(6) Effects of Artesunate on apoptosis of HSCs:flow cytometry results showed that as the concentration of Artesunate increased, the number of apoptosis HSCs were on the rise. Rate of apoptosis in control group was (40.73±0.81)%, rate of treatment groups (Artesunate concentration was150,175,200μmol/L) were (52.63±0.84)%,(63.97±0.50)%and (63.97±0.50)%. Compared with control group, the difference was statistically significant (P<0.01). With the concentrations of Artesunate increasing, apoptotic index increased. With no Artesunate, HSCs apoptosis progressed spontaneously. Hoechst33258dye staining showed that the nucleus of Artesunate group showed typical morphological changes of apoptosis. Nucleus of the control group without morphological changes of apoptosis.(7) Western blotting and RT-PCR detection of p53expression:after acted with Artesunate150μmol/L,175μmol/L,200μmol/L, respectively, for24h, Western blotting and RT-PCR showed that the expression of p53was significantly increased in dose-effect relationship (P<0.01).Conclusion:(1) The isolation, culture and identification of rat primary HSCs was successful.(2) Artesunate could inhibit HSCs proliferation in dose-dependent and time-dependent manners, accordingly decrease production/secretion of collagen.(3) One of the mechanisms that Artesunate inhibited proliferation of HSCs was to increase the expression of p53, so that cell cycle arrested in G1phase, and also Artesunate could induce apoptosis of HSCs, and the latter accounted for the main effect. |
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