摘要
目的:探讨1-磷酸-鞘氨醇受体2(sphingosine-1-phosphate receptor 2,S1PR2)、前蛋白转化酶枯草溶菌素9(proprotein convertase subtilisin/kexin type 9,PCSK9)对非酒精性脂肪性肝炎(nonalcoholic steatohepatitis,NASH)肝脏损伤可能的调控机制。方法:将12只C57BL/6J小鼠随机分为对照组和实验组,对照组给予普通饮食,实验组给予蛋氨酸-胆碱缺乏饮食(methionine choline deficiency diet,MCD),MCD 6周建立NASH模型。6周末ELISA检测血清和肝脏生化指标丙氨酸氨基转移酶(alanine aminotransferase,ALT)、天门冬氨酸氨基转移酶(aspartate aminotransferase,AST)、谷氨酰转移酶(glutamyl transferase,GGT)及炎症因子肿瘤坏死因子α(tumor necrosis factor alpha,TNF-α)、白细胞介素6(interleukin-6,IL-6)、γ-干扰素(interferon-γ,IFN-γ)的水平;HE染色、油红O染色和天狼星红染色分别观察肝脏炎症、脂质沉积和纤维化情况。小鼠原代肝细胞和HepG2细胞分别予棕榈酸和/或JTE-013(S1PR2特异性抑制剂)处理,油红O染色观察肝细胞内脂质沉积情况,蛋白免疫印迹实验检测S1PR2、PCSK9和低密度脂蛋白受体(low density lipoprotein cholesterol receptor,LDLR)的蛋白表达。构建S1PR2干扰、过表达质粒,分别转染HepG2细胞,进行转录组测序寻找S1PR2潜在关联基因,并在NASH造模小鼠中检测筛选出的基因的表达情况。结果:喂养6周后实验组小鼠较对照组肝组织S1PR2表达减少、PCSK9表达增加;使用JTE-013可加重小鼠肝原代细胞和HepG2细胞内脂质沉积,使ERK磷酸化增加,PCSK9表达升高、LDLR降低;S1PR2干扰、过表达质粒分别转染的HepG2细胞中,共有343种基因发生改变,其中早期生长反应蛋白1(early growth response protein 1,Egr1)在S1PR2过表达时增加、干扰时降低,并在NASH模型小鼠中表达减少。结论:S1PR2参与NASH的脂质沉积和炎症损伤过程,这可能与炎症状态下S1PR2被抑制后促进PCSK9表达,降低LDLR表达有关。
Objective:This study aims to investigate the role of sphingosine-1-phosphate receptor 2(S1 PR2),proprotein convertase subtilisin/kexin type 9(PCSK9)in nonalcoholic steatohepatitis(NASH).Methods:Twelve C57 BL/6 J mice were randomly assigned to control group and experimental group.After NASH models were successfully established by methionine choline deficiency diet(MCD)diet for six weeks,biochemical indices and inflammatory factors in serum and liver,including alanine aminotransferase(ALT),aspartate aminotransferase(AST),glutamyl transferase(GGT),tumor necrosis factor alpha(TNF-α),interleukin-6(IL-6)and interferon-γ(IFN-γ)were detected by ELISA.HE staining,oil red O staining and sirius red staining were used to observe liver inflammatory damage,lipid deposition and fibrosis.In vitro,the primary hepatocytes of C57 BL/6 J mice and HepG2 cells were challenged with palmiticacid and/or JTE-013(S1 PR2 specific inhibitor),respectively;the lipid deposition was observed by oil red O staining,and the protein expression of S1 PR2,PCSK9 and low-density lipoprotein cholesterol receptor(LDLR)was detected by Western blot.HepG2 cells were transfected with S1 PR2 interference or overexpression plasmids to find potential related genes by gene chip technology,and genes with significant expression differences were screened out in NASH mice.Results:Compared with the control group,expression level of liver S1 PR2 was decreased and PCSK9 increased in experimetal group.In mouse primary hepatocytes and HepG2 cells,JTE-013 could increase lipid deposition,and increase the expression of phosphorylated ERK and PCSK9,while the expression of LDLR was decreased.A total of 343 genes were changed in HepG2 cells transfected with S1 PR2 knockdown and overexpression plasmids.The levels of early growth response protein 1(Egr1)increased when S1 PR2 was overexpressed and decreased when S1 PR2 was interfered,and decreased in NASH model mice.Conclusion:S1 PR2 is involved in the lipid metabolism and inflammatory injury of NASH,which may be related to the suppression of S1 PR2 in inflammatory state,increaseing the expression of PCSK9 and decreaseing the expression of LDLR.
作者
张培
张园园
严君君
李梦羽
周希乔
ZHANG Pei;ZHANG Yuanyuan;YAN Junjun;LI Mengyu;ZHOU Xiqiao(Department of Gastroenterology,the First Affiliated Hospital of Nanjing Medical University,Nanjing 210029,China)
出处
《南京医科大学学报（自然科学版）》
CAS
CSCD
北大核心
2020年第6期810-815,845,共7页
Journal of Nanjing Medical University(Natural Sciences)
基金
国家自然科学基金(81570522)
江苏省卫建委强卫工程青年人才(QNRC2016568)。
