Effect Of Artesunate On Anti-fibrogenesis In Hepatic Stellate Cells Via ERK Signaling Pathway

ObjectiveTo investigate the impact of artesunate and ceramide on cell proliferation and collagen generated,and to analyze the underlying molecular mechanisms of anti-fibrogenic effects involving ERK1/2 signaling pathway in hepatic stellate cells.To provide experimental evidences for clinical application of artesunate to treat hepatic fibrosis.MethodsHealthy male Wistar rats were disinfected after intraperitoneal injection of 25%urethane anesthesia,0.8 mg/mL protease E and collagenase IV were used to digest the liver,then the cell suspension was centrifuged by 8%,12%and 18%Nycodenz.HSCs were collected at 8%and 12%of the gradient interface,and cell density was adjusted to 5×105/mL.Then HSCs were seeded in culture flasks and cultured in the DMEM containing 20%FBS.Morphology and growth characteristics of freshly isolated cells were observed with inverted phase contrast microscope.Isolated,cultured,and activated,HSCs were divided into control group and expremental groups.Experimental groups included five artesunate-treated experimental groups with 125，150,175,200 or 225 μmol/L and one ceramide-treated experimental group with 10 μmol/L for 24,48 or 72 hours.The rate of cellular proliferation was measured using the 3-(4,5-dime-thylthiazol-2-yl)-2，5-diphenylterazolium bromide(MTT)assay,and the determination of hydroxyproline(Hyp)was acquired by the enzyme digestion method.Collagen-I,p-ERK1/2 and MAPK1+3 expression was evaluated by Western Blot.Results(1)Hepatic stellate cells isolation,cultured and identification:freshly isolated HSCs observed by inverted phase contrast microscope were spherical,had strong refraction,and presented blue-green fluorescent under 328 nm UV-light.After culturing for 10 days,HSCs had been fully developed,shown stellate or polygonal form,and granules in cytoplasm 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-5h day,then entered into the plateau phase on the 6th-7th day.(2)Effects of artesunate and cerimade on cell proliferation:Results of MTT colorimetric showed that the absorbance value in artesunate and ceramide groups reduced significantly compared with that in the control group at different points(P<0.05).As the concentration increasing and time forwarding,the inhibition of proliferation enhanced.(3)Hydroxyproline test in the cell culture supernatant:After treated with artesunate and cerimade for 24 h,the secretion of hydroxyproline decreased in all treatment groups.Compared with control group,the difference was statistically significant(P<0.05).(4)Western Blot detection of Collagen-Ⅰ:The results of Western Blot indicated that Artesunate in different doses and ceramide could reduce Collagen-Ⅰ expression,compared with the control group,the difference was statistically significant(P<0.05).(5)Western Blot detection of p-ERK1/2:The results of Western Blot indicated that Artesunate,ceramide and PD98059 could reduce p-ERK expression,compared with the control group,the difference was statistically significant(P<0.05).(6)Western Blot detection MAPK1+3:The results of Western Blot indicated that Artesunate in different doses and ceramide could reduce MAPK1+3 expression,compared with the control group,the difference was statistically significant(P<0.05).Conclusion(1)The isolation,culture and identification of rat primary HSCs were successful.(2)Artesunate and cerimade inhibit the proliferation of HSCs in dose-dependent and time-depentent manners.(3)Artesunate and cerimade may decrease the production/secretion of collagen.(4)Artesunate and ceramide show antifibrogenic effects,that may involve ERK1/2 signaling pathway.