Effects Of Artesunate On Inhibition Of Hepatic Fibrosis Through Influencing Ceramide De Novo Synthesis Rate-limiting Enzyme SPT

Objective:The present study was designed to investigate the effect of Artesunate on the level of ceramide through affecting the rate-limiting enzyme SPT of ceramides de novo synthesis pathway in hepatic stellate cells, to reveal the mechanism of Artesunate on inhibition of hepatic fibrosis by promoting ceramides de novo synthesis pathway and provide experimental evidence for the prevention and treatment of hepatic fibrosis. Methods:1. Human hepatic stellate cells(HSCs) LX-2 cultured as the experimental subjects were divided into cell control group, Art-treated group, C2-ceramide-treated group and Co-administration of Artesunate and C2-ceramide groups. The proliferation of LX-2 was measured by MTT assay; HPLC-FLD method was used to evaluate the level of ceramide; Western Blot assay the expression of bax, bcl-2 and caspase-3 protein; Colorimetric method was adopted to determine the activity of LDH; the content of hydroxyproline measured by using an enzyme digestion method.2. LX-2 cells were divided into control group, Art-treated groups, myriocin-treated groups and Co-administration of Artesunate and myriocin groups. The expression of SPTLC1 and SPTLC2 was evaluated by Western blot; HPLC-FLD method was used to evaluate the content of ceramide and confirme the effect of artesunate on ceramide de novo synthesis pathway; the rate of proliferation of LX-2, the expression of bcl-2, bax, caspase-3 and the content of hydroxyproline were measured by the method described above. Results:1. Artesunate raised the level of ceramide and improve its effect in LX-2 cells: Compared with control group, artesunate could significantly inhibit LX-2 cells proliferation with serine palmitoyl transferase(P<0.05); Artesunate increased the content of ceramide and the expression of bax and caspase-3, and decreased the expression of bcl-2(P<0.05); the activity of LDH has no significant change; After artesunate treated LX-2 24 hours, the content of hydroxyproline significantly reduced with dose-dependent manner(P<0.05). C2 could inhibite the proliferation of LX-2 on dose-dependent manner(P<0.05); as the concentration of C2 increasd, the expression of bax and caspase-3 increased, the expression of bcl-2 decreased(P<0.05); the activity of LDH has no significant change compared with control group(P>0.05); C2 could significantly reduce the level of hydroxyproline with dose-dependent manner.2. The effect of Artesunate increased the expression of SPTLC1 and SPTLC2 in LX-2 cells: Compared with control group, LX-2 treated with myriocin 15 μmol/L after 24 h, the proliferation was increased significantly(P<0.05); the expression of SPTLC1 and SPTLC2 and the content of ceramide increased in myriocin-treated groups(P<0.05); and myriocin as the specific inhibitor of SPT could reduce the expression of SPTLC1 and SPTLC2 and the level of ceramide in LX-2(P<0.05); Myriocin 15 μmol/L combined with Artesunate(250、350、450 μmol/L) significantly decreased the effect of artesunate on the inhibition of proliferation, the expression of SPTLC1 and SPTLC2 and the level of ceramide in LX-2(P<0.05); myriocin could decrease the effect of artesunate on increased the expression of bax and caspase-3 and increased the expression of bcl-2 and the content of hydroxyproline(P<0.05). Conclusions:1. Artesunate increased the level of ceramide through ceramides de novo synthesis pathway to increase the expression of rate-limiting enzyme SPT in LX-2 cells.2. Artesunate could increase the expression of bax and caspase-3 and downregulated expression of bcl-2 to induce cells apoptosis by increasing the content of ceramide in LX-2 with dose- and time-dependent manner.3. Artesunate inhibited hepatic fibrosis through inhibiting proliferation and inducing apoptosis of LX-2 to reduce the production of collagen.