| Objective: To evaluate the effects of artesunate and ceramide on the primary activated HSCs proliferation and extracellular matrix production. To explore the possible mechanisms of artesunate through ceramide actions.Methods : The liver of healthy Wistar male rats was perfused by Ca2+-free perfusion in site perfusion to wash the blood away from liver. Then the liver was removed and perfused with 0.8 mg/mL pronase E and 0.5 mg/mL collagenase, the cell suspension was centrifuged by 8%, 12% and 18% Nycodenz. HSCs were observed at 8% and 12% of the gradient interface, cell density was adjusted and culture the cells in the culture bottle. Morphology and growth characteristics of freshly isolated cells were observed with inverted phase contrast microscope. HSCs were isolated and cultured in the flask for 10 days to make cells activated. HSCs were divided into two groups:control groups and experimental groups. Experimental groups were incubated with various concentrations of artesunate, ceramide for 24, 48 and 72 hours. MTT assay was used to detect rate of cellular proliferation; digestive method was used to determinate concentrations of hydroxyproline in supernatant; the level/activity of MMP-2 and MMP-9 of artesunate and ceramide were analyzed with gelatin zymography; the expression of MMP-13 in HSCs was analyzed with Western Blot;HPLC-FLD was used to analysis and determination of ceramide in biological samples.Results:1. Hepatic stellate cells isolation and culture: freshly isolated HSCs observed by phase contrast microscope were spherical, had strong refraction, and suspended in culture medium. After culturing for 10 days, HSCs had been fully developed,shown stellate or polygonal form, and granules cells were significantly reduced.HSCs growth curve showed that the cultured HSCs were proliferated after 72 h,and entered into the logarithmic growth phase on the 4th-5th day, entered into the plateau phase on the 6th-7th day.2. Effects of artesunate and ceramide on cell proliferation using MTT method: After 125, 150, 175, 200, 225 μmol/L artesunate and 10 μmol/L ceramide stimulated HSCs for 24, 48, 72 h, results of MTT colorimetric showed that inhibition of HSCs, in the artesunate group, ceramide group and combination group,compared with the control group, the difference was statistically significant(P<0.05), the difference of all the concentration of artesunate was statistically significant(F=9.32, P < 0.05), suggesting that inhibition of artesunate on proliferation was in a concentration-dependent and time-dependent manner. In the group 10 μmol/L ceramide, the action of inhibition was also increasing with increasing concentration (F=108.79, P<0.05).3. Hydroxyproline test in the cell culture supernatant: after treated with artesunate for 24 h, secretion of hydroxyproline decreased in all treatment groups, compared with control group, the difference was statistically significant (P<0.05); compared the corresponding dose artesunate or ceramide group, the concentration of the combination group hydroxyproline is increasing, but the difference was not statistically significant.4. Gelatin zymography detection of MMP-2 and MMP-9 levels and activities:Above-mentioned different concentration stimulated HSCs for 24 h,gelatin zymography was detected the MMP-2 and MMP-9 expression. Compared the control group, the difference of MMP-2 expression was statistically significant(P<0.05); compared the corresponding dose artesunate group, the difference of the combination group MMP-2 expression was statistically significant (P<0.05).There is the MMP-9 expression and the difference was not statistically significant.5. Western blot detection of MMP-13 expression: Above-mentioned different concentration stimulated HSCs for 24 h, western blot was used to detected the MMP-13 expression, compared the control group, the difference was statistically significant(P<0.05). In the combination group, compared the artesunate or ceramide group, the difference was not statistically significant.6. HPLC-FLD analysis and determination of ceramide in supernatant: Establish an HPLC method for determination of ceramide in biological samples successfully.Drawing standard curve: y=1.3306×105x+2592.3, r=0.9999. In the group 200、300、400、500 μmol/L artesunate, compared the control group, the difference of ceramide concentration was statistically significant(P < 0.05). With the increase of artesunate, the ceramide concentration is increasing(F=76.64, P < 0.05). In the group 1、1.5、2 μmol/L N-oleoylethanolamide, compared the ethanol control group, the difference of ceramide concentration was statistically significant(P <0.05), the ceramide concentration is increasing(F-36.02, P < 0.05). In conbination group of artesunate and N-oleoylethanolamide, compared the ethanol control group, the difference of ceramide concentration was statistically significant(P < 0.05). In combination group of 100 μmol/L artesunate and 2μmol/L N-oleoylethanolamide, 200 μmol/L and 2 μmol/L N-oleoylethanolamide,compared the corresponding artesunate concentration group, the difference of ceramide concentration was statistically significant(P < 0.05). In all the combination groups, compared the 2 μmol/L N-oleoylethanolamide group, the difference of ceramide concentration was not statistically significant.Conclusion:1. The isolation and culture of rat primary HSCs was successful.2. Establish an HPLC method for determination of ceramide in biological samples.3. The antifibrosis mechanisms of artesunate is through up-regulating ceremide,inhibit the proliferation, decrease the level/activity of MMP-2, increase MMP-13 expression, reduce the concentration of hydroxyproline in the cell culture supernatant, inhibit the ECM whose major constituent is collagen,so artesunate play a role on anti-fibrosis. |
