酸性鞘磷脂酶-神经酰胺-ERK1/2信号通路在青蒿琥酯抗肝纤维化中的作用

Artesunate inhibits liver fiber generation through the acid sphingomyelinase-ceramide-ERK1/2 signal pathway

关键词:  青蒿琥酯  肝纤维化  酸性鞘磷脂酶  神经酰胺  ERK1/2

Key words:  artesunate  hepatic fibrosis  sphingomyelinase  ceramide  ERK1/2

摘要:

[目的] 探讨神经酰胺（Cer）在青蒿琥酯（Art）抑制肝纤维发生中的作用，并初步揭示酸性鞘磷脂酶（ASMase）-Cer-细胞外信号调节激酶（ERK）1/2信号通路在此过程中的作用。[方法] 将人源肝星状细胞LX-2分为对照组与实验组，对照组包括细胞对照组、1‰ DMSO组、溶酶组（1‰ DMSO预处理30 min后，加入5% NaHCO₃），实验组包括Art 250 μmol/L组、Art 350 μmol/L组、Art 450 μmol/L组、丙米嗪（Imi）10 μmol/L组、Imi 30 μmol/L组、Imi 10 μmol/L+Art 450 μmol/L组（加入Art 450 μmol/L 30 min前加入Imi 10 μmol/L）、Imi 30 μmol/L+Art 450 μmol/L组（加入Art 450 μmol/L 30 min前加入Imi 30 μmol/L），应用MTT法检测各实验组对LX-2细胞增殖的影响；比色法检测Art组对LX-2细胞培养上清液中乳酸脱氢酶活性的影响；细胞消化法测定各实验组LX-2细胞培养上清液中羟脯氨酸含量；高效液相-荧光法（HPLC-FLD）测定各实验组LX-2细胞培养上清液中Cer含量，荧光法测定ASMase的活性，蛋白免疫印记（Western blot）法测定ASMase蛋白表达的变化情况。采用LX-2细胞为实验对象，分为细胞对照组、Imi 10 μmol/L组、Imi 30 μmol/L组，油酰乙醇胺（NOE）50 μmol/L组、PD98059 100 μmol/L组、Imi 10 μmol/L+Art 450 μmol/L组、Imi 30 μmol/L+Art 450 μmol/L组、Art 450 μmol/L组，Western blot法观察ERK1/2磷酸化蛋白表达的变化情况。[结果] Art各实验组可显著抑制LX-2细胞的增殖（P＜0.05），且其对LX-2细胞增殖的抑制作用呈剂量-效应依赖性及时间-效应依赖性；Art作用24 h后，各实验组LX-2细胞培养上清液中乳酸脱氢酶活性与对照组比较，差异无统计学意义（P>0.05）；Art各浓度组可有效降低LX-2细胞培养上清液中羟脯氨酸含量（P＜0.05）且均可显著增加LX-2细胞培养上清液中Cer含量（P＜0.05）。Art 450 μmol/L组可提高ASMase活性（P＜0.01）并促进ASMase蛋白的表达（P＜0.01）。Imi 30 μmol/L作用24 h后，与DMSO组比较，可促进LX-2细胞的增殖，降低LX-2细胞培养上清液中Cer的含量，并升高LX-2细胞培养上清液中羟脯氨酸的含量（P＜0.05）。Imi 30 μmol/L可降低ASMase活性（P＜0.01）并降低ASMase蛋白的表达（P＜0.01）。与Art 450 μmol/L组比较，Imi 30 μmol/L预处理组可明显降低Art对LX-2细胞增殖的抑制作用、明显减弱其对羟脯氨酸含量的降低作用、并明显降低其对Cer含量的升高作用（P＜0.05）。Imi 30 μmol/L预处理组可明显减弱Art对ASMase活性的促进作用（P＜0.01）及对ASMase蛋白表达的促进作用（P＜0.01）。与对照组比较，Art 450 μmol/L、NOE 50 μmol/L、PD98059 100 μmol/L均能够抑制ERK1/2磷酸化蛋白的表达（P＜0.05）；Imi 30 μmol/L能够促进ERK1/2磷酸化蛋白的表达（P＜0.05）。与Art 450 μmol/L比较，Imi 30 μmol/L预处理组可减弱Art对ERK1/2磷酸化蛋白表达的抑制作用（P＜0.05）。[结论] Art可能通过上调LX-2细胞中的Cer含量来发挥抗肝纤维作用，且其可能通过ASMase-Cer-ERK1/2信号通路来发挥此作用。

Abstract:

[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.