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| Artesunate inhibits liver fiber generation through the acid sphingomyelinase-ceramide-ERK1/2 signal pathway |
| Hits 506 Download times 251 Received:January 13, 2025 |
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| DOI
10.11656/j.issn.1672-1519.2025.05.14 |
| Key Words
artesunate;hepatic fibrosis;sphingomyelinase;ceramide;ERK1/2 |
| Author Name | Affiliation | E-mail | | XU Yajie | Department of Pharmacy, The Second Affiliated Hospital of Tianjin University of Traditional Chinese Medicine, Tianjin 300250, China | | | DU Yan | Department of Pharmacy, Tianjin Jizhou District People's Hospital, Tianjin 301900, China | | | FANG Buwu | Department of Pharmacology, Tianjin Medical University, Tianjin 300070, China | fangdch@yahoo.com.cn |
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| Abstract
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| [Objective] To investigate the role of ceramide（Cer） in the inhibition of hepatic fibrogenesis by artesunate（Art） and to preliminarily reveal the role of the acid sphingomyelinase（ASMase）-Cer-extracellular signal-regulated kinase（ERK） 1/2 signaling pathway in this process. [Methods] Human-derived hepatic stellate cell LX-2 cells were divided into control and experimental groups，the control group included cell control group，1‰ DMSO group，lysostaphin group（1% NaHCO3 was added after 1‰ DMSO pretreatment for 30 min），and the experimental group included Art 250 μmol/L group，Art 350 μmol/L group，Art 450 μmol/L group，promethazine（Imi） 10 μmol/L group，Imi 30 μmol/L group，Imi 10 μmol/L+Art 450 μmol/L group（Imi 10 μmol/L was added 30 min before the addition of Art 450 μmol/L），Imi 30 μmol/L+Art 450 μmol/L group（Imi 30 μmol/L+Art 450 μmol/L was added 30 min before the addition of Art 450 μmol/L），and Imi 30 μmol/L+Art 450 μmol/L group（Imi 30 μmol/L+Art 450 μmol/L）. The effects of each experimental group on the proliferation of LX-2 cells were detected by MTT assay；the effects of Art group on the activity of lactate dehydrogenase in the supernatant of LX-2 cell culture were detected by colorimetric assay；the content of hydroxyproline in the supernatant of LX-2 cell culture in each experimental group was determined by cell digestion；and the content of C-terminaldehyde（C-terminaldehyde） in the supernatant of LX-2 cell culture in each experimental group was determined by high-performance liquid chromatography-fluorescence（HPLC-FLD） assay. The 2 cell culture supernatants，fluorescence method to determine Cer content，fluorescence method to determine ASMase activity，and protein immunoblotting（Western blot） method to determine the changes of ASMase protein expression. LX-2 cells were used as experimental subjects，which were divided into cell control group，Imi 10 μmol/L group，Imi 30 μmol/L group，oleoylethanolamine（NOE） 50 μmol/L group，PD98059 100 μmol/L group，Imi 10 μmol/L+Art 450 μmol/L group，Imi 30 μmol/L+Art 450 μmol/L group，Art 450 μmol/L group，and Western blot method to observe the changes of ERK1/2 phosphorylated protein expression. [Results] The proliferation of LX-2 cells in each experimental group was significantly inhibited by Art（P＜0.05），and the inhibitory effect on the proliferation of LX-2 cells was dose-effect-dependent and time-effect-dependent；after 24 h of Art action，there was no significant difference in the lactate dehydrogenase activity in the supernatant of LX-2 cell culture of each experimental group compared with that of the control group（P>0.05）；the concentration of Art in each experimental group effectively reduced the content of hydroxyproline in the supernatant of LX-2 cell culture（P＜0.05） and significantly increased the content of Cer in the supernatant of LX-2 cell culture（P＜0.05）. Art 450 μmol/L group could effectively reduce the hydroxyproline content in LX-2 cell culture supernatant（P＜0.05） and significantly increase the Cer content in LX-2 cell culture supernatant（P＜0.05）. Art 450 μmol/L group could increase the activity of ASMase（P＜0.01） and promote the expression of ASMase protein（P＜0.01）.Imi 30 μmol/L was used to increase the activity of lactate dehydrogenase（LDH） and DMSO for 24 h，which was significantly different from that of control group（P>0.05）. Imi 30 μmol/L increased ASMase activity（P＜0.01） and decreased ASMase protein expression（P＜0.01） in the LX-2 cell culture supernatant compared with that in the DMSO group after 24 h. Imi 30 μmol/L increased ASMase activity（P＜0.01） and decreased ASMase protein expression（P＜0.01）. Compared with the Art 450 μmol/L group，the Imi 30 μmol/L pretreatment group significantly reduced the inhibitory effect of Art on the proliferation of LX-2 cells，significantly attenuated its effect on the reduction of hydroxyproline content，and significantly reduced its effect on the elevation of Cer content（P＜0.05）. The Imi 30 μmol/L pretreatment group significantly attenuated the promotional effect of Art on the ASMase activity（P＜0.05）. activity（P＜0.01） and ASMase protein expression（P＜0.01）. Compared with the control group，Art 450 μmol/L，NOE 50 μmol/L，and PD98059 100 μmol/L inhibited the expression of ERK1/2 phosphorylated proteins（P＜0.05）；Imi 30 μmol/L promoted the expression of ERK1/2 phosphorylated proteins（P＜0.05）. Compared with Art 450 μmol/L，Imi 30 μmol/L pretreatment group attenuated the inhibitory effect of Art on ERK1/2 phosphorylated protein expression（P＜0.05）. [Conclusion] Art may exert its anti-hepatic fibrotic effect by up-regulating Cer content in LX-2 cells，and it may exert this effect through the ASMase-Cer-ERK1/2 signaling pathway. |
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