Effects Of Artesunate On Inhibition Of Hepatic Fibrosis Through Ceramide Synthase-ceramide Pathway

Liver fibrosis is the large deposits of extracellular matrix, which generates more than degradation, especially a large number of type I collagen deposition. Failure to take treatment of liver fibrosis may develop to cirrhosis or liver cancer. In the process of liver fibrosis, HSCs proliferation and activation is the central part. Activation of HSCs proliferation and secretion of collagen significantly increase, extracellular matrix deposition and its degradation out of balance, thus liver function is damaged.In recent years, with the deepening study of the pathogenesis of hepatic fibrosis causes and mechanism, People proposed fibrosis is reversible. There are a lot of medicine to treat liver fibrosis, This study we use artesunate as a tool drug.Objective:To investigate the effect of Artesunate on the ability of hepatic stellate cell to produce ceramide. This study preliminarily reveals the effect of Artesunate on inhibition of hepatic fibrosis through ceramide synthase-ceramide pathway.Methods:1. LX-2 were divided into experimental groups and control group, The experimental groups including Art-treated groups, NOE-treated groups and Co-administration of NOE and Artesunate group. The rate of proliferation of LX-2was measured by MTT assay; Colorimetric method was used to determine the activity of LDH; HPLC-FLD method was adopted to evaluate the content of ceramide; the expressions of PPAR-γ, p53 and Caspase-3 were evaluated by western blotting.Digestive method was adopted to determine the content of hydroxyproline.2. LX-2 were divided into control group and experimental groups. The experimental groups included Fumonisin B1-treated groups, Art-treated groups and Co-administration of Artesunate and Fumonisin B1 groups. The rate of proliferation of LX-2 was measured by MTT assay; The expression of LASS2 was evaluated by western blotting; Colorimetric method was used to determine the activity of LDH;HPLC-FLD method was adopted to evaluate the content of ceramide; The expressions of PPAR-γ, p53 and Caspase-3 were evaluated by western blotting.Digestive method was adopted to determine the content of hydroxyproline.Results:1. Compared with control group, The proliferation of LX-2 was inhibited significantly by Artesunate(P<0.05), with dose-dependent and time-dependent manner; Compared with control group, After Artesunate treated LX-2 24 hours, the activity of LDH had no significant change(P>0.05); Ceramide content was significantly increased by Artesunate in LX-2(P<0.05); Artesunate could increase the expressions of PPAR-γ, p53 and Caspase-3, and the secretion of hydroxyproline was decreased compared with control group(P<0.05). MTT results revealed that N-oleoylethanolamine could inhibit the proliferation of LX-2 on dose-effect relationship; the activity of LDH had no significant change compared with control group(P>0.05); Ceramide content was significantly increased by N-oleoylethanolamine in LX-2 compared with control group(P<0.05);N-oleoylethanolamine could increase the expressions of PPAR-γ, p53 and Caspase-3,and the secretion of hydroxyproline was decreased compared with control group(P<0.05). The efficacy of combination group was better than that of single treated group.2. Compared with control group, Fumonisin B1 6 μmol/L treated LX-2, the proliferation of LX-2 was increased significantly(P>0.05); Fumonisin B1 6 μmol/L combined with Artesunate(250、350、450 μmol/L) significantly decreased the effect of Artesunate on the inhibition of proliferation in LX-2; Fumonisin B1 6 μmol/L treated LX-2, the expression of LASS2 was decreased significantly. Artesunate treated LX-2, the expression of LASS2 was increased significantly, but the combination of two drugs could decrease the effect of Artesunate on the expression of LASS2; Fumonisin B1 could decrease the effect of Artesunate on the increased content of ceramide. Compared with combination groups, the expression of PPAR-γ,p53, Caspase-3 and hydroxyproline level was highest in Art-treated groups and was lowest in Fumonisn B1 groups, the difference had statistical significance(P < 0.05).Conclusions:1. Artesunate and NOE could increase the level of ceramide in LX-2 through different ways, thus increasing the expressions of PPAR-γ, p53 and Caspase-3. The proliferation of LX-2 was inhibited and the apoptosis was accelerated significantly byArtesunate, with dose-dependent and time-dependent manner, then the production of hydroxyproline.2. Artesunate plays a part in the inhibition of hepatic fibrosis through up-regulating ceramide synthase-ceramide pathway, thus increasing the level of ceramide and the expressions of PPAR-γ, p53 and Caspase-3.