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

作　　者：徐亚洁[1] 杜岩 方步武[3] XU Yajie;DU Yan;FANG Buwu(Department of Pharmacy,The Second Affiliated Hospital of Tianjin University of Traditional Chinese Medicine,Tianjin 300250,China;Department of Pharmacy,Tianjin Jizhou District People’s Hospital,Tianjin 301900,China;Department of Pharmacology,Tianjin Medical University,Tianjin 300070,China)

机构地区：[1]天津中医药大学第二附属医院药学部,天津300250 [2]天津市蓟州区人民医院药剂科,天津301900 [3]天津医科大学药理学教研室,天津300070

出　　处：《天津中医药》2025年第5期630-637,共8页Tianjin Journal of Traditional Chinese Medicine

基　　金：国家自然科学基金项目(30772856)。

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 250μmol/L组、Art 350μmol/L组、Art 450μmol/L组、丙米嗪(Imi)10μmol/L组、Imi 30μmol/L组、Imi 10μmol/L+Art 450μmol/L组(加入Art 450μmol/L 30 min前加入Imi 10μmol/L)、Imi 30μmol/L+Art 450μmol/L组(加入Art 450μmol/L 30 min前加入Imi 30μmol/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 10μmol/L组、Imi 30μmol/L组,油酰乙醇胺(NOE)50μmol/L组、PD98059 100μmol/L组、Imi 10μmol/L+Art450μmol/L组、Imi 30μmol/L+Art 450μmol/L组、Art 450μmol/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 450μmol/L组可提高ASMase活性(P<0.01)并促进ASMase蛋白的表达(P<0.01)。Imi 30μmol/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�[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 250μmol/L group,Art 350μmol/L group,Art 450μmol/L group,promethazine(Imi)10μmol/L group,Imi 30μmol/L group,Imi 10μmol/L+Art 450μmol/L group(Imi 10μmol/L was added 30 min before the addition of Art 450μmol/L),Imi 30μmol/L+Art 450μmol/L group(Imi 30μmol/L+Art 450μmol/L was added 30 min before the addition of Art 450μmol/L),and Imi 30μmol/L+Art 450μmol/L group(Imi 30μmol/L+Art 450μmol/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 10μmol/L group,Imi 30μmol/L group,oleoylethanolamine(NOE)50μmol/L group,PD98059100μmol/L group,Imi 10μmol/L+Art 450μmol/L group,Imi 30μmol/L+Art 450μmol/L group,Art 450μmol/L group,a

关 键 词：青蒿琥酯 肝纤维化 酸性鞘磷脂酶 神经酰胺 ERK1/2 

分 类 号：R575.2[医药卫生—消化系统]