基于LC- MS的四氯化碳致大鼠急性肝损伤的脂质组学研究
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摘要
目的:基于液相色谱-质谱联用(LC-MS)技术从脂质组学的角度探讨四氯化碳(CCl_4)致大鼠急性肝损伤的作用机制。方法:取Wistar大鼠12只,随机分为正常组和模型组,每组6只。模型组大鼠单次腹腔注射50%CCl_4诱导急性肝损伤,给药剂量为1 ml/kg;正常组大鼠腹腔注射等体积橄榄油。给药24 h后,采集血清样本,分离肝大叶。生化分析仪检测血清ALT、AST、TG、HDL和LDL水平;HE染色后光镜下观察肝组织病理形态学变化;LC-MS检测血清样本,采用多元统计分析(主成分分析和正交偏最小二乘法判别分析)比较两组间脂质代谢谱差异,并筛选差异性脂质代谢物。结果:与正常组比较,模型组大鼠ALT和AST水平显著升高(P<0.01),TG含量明显降低(P<0.05)。HE染色可见模型组大鼠的肝细胞肿大、坏死,伴有明显的炎症浸润。脂质组学分析显示,两组间脂质代谢谱差异明显,共找出80个差异性脂类成分;进一步经S-plot图比对,筛选出34个差异最为显著的脂质代谢物,其中15个属于磷脂酰胆碱类,6个属于三酰甘油类,4个属于磷脂酰乙醇胺类,2个属于溶血磷脂酰胆碱类,2个属于鞘氨醇类,5个属于血小板激活因子。结论:脂质组学研究揭示CCl_4诱导急性肝损伤的机制与磷脂及三酰甘油代谢紊乱相关。
Objective: To investigate the mechanism of carbon tetrachloride(CCl_4)-induced acute liver injury in rats using lipidomics based on liquid chromatography-mass spectrometry(LC-MS). Methods: A total of 12 Wistar rats were randomly divided into the normal group and model group, 6 rats in each group. The model group was treated with 50%CCl_4 at dose of 1 ml/kg by single intraperitoneal injection to induce the acute liver injury, and the normal group was treated with olive oil at equivalent volume. Twenty-four hours after modeling,the blood and liver tissue were collected. The serum levels of alanine aminotransferase(ALT), aspartate aminotransferase(AST), triglyceride(TG), high-density lipoprotein(HDL) and low-density lipoprotein(LDL) were detected by automatic biochemical analyzer. The pathological changes of liver tissue were observed by light microscopy after HE staining. The serum samples were detected by LC-MS. Multivariate statistical analysis(principal component analysis and orthogonal partial least square discriminate analysis) was used to compare the differences of lipid metabolism profiles between the two groups, and the differential lipid metabolites were screened. Results: Compared with the normal group, the levels of ALT and AST were significantly increased in the model group(P<0.01), and the level of TG was obviously decreased(P<0.05). HE staining showed that the hepatocytes swelling and necrosis and the obvious inflammatory infiltration were found in the model group. Lipidomics analysis showed that there were significant differences in lipid metabolism profiles between the two groups. A total of 80 differential lipid metabolites were identified, and 34 lipid metabolites with the most significant differences were further screened by S-plot comparison. Among, 15 metabolites were phosphatidylcholine(PC), 6 metabolites were triglyceride(TG), 4 metabolites were phosphatidyl ethanolamine(PE), 2 metabolites were lysophosphatidylcholine(LPC), 2 metabolites were sphingomyelin(SM), and 5 metabolites were platelet activating factor(PAF). Conclusion: Lipidomics study reveals that the mechanism of CCl_4-induced acute liver injury may be related to disorders of phospholipid and triglyceride metabolism.
Objective: To investigate the mechanism of carbon tetrachloride(CCl_4)-induced acute liver injury in rats using lipidomics based on liquid chromatography-mass spectrometry(LC-MS). Methods: A total of 12 Wistar rats were randomly divided into the normal group and model group, 6 rats in each group. The model group was treated with 50%CCl_4 at dose of 1 ml/kg by single intraperitoneal injection to induce the acute liver injury, and the normal group was treated with olive oil at equivalent volume. Twenty-four hours after modeling,the blood and liver tissue were collected. The serum levels of alanine aminotransferase(ALT), aspartate aminotransferase(AST), triglyceride(TG), high-density lipoprotein(HDL) and low-density lipoprotein(LDL) were detected by automatic biochemical analyzer. The pathological changes of liver tissue were observed by light microscopy after HE staining. The serum samples were detected by LC-MS. Multivariate statistical analysis(principal component analysis and orthogonal partial least square discriminate analysis) was used to compare the differences of lipid metabolism profiles between the two groups, and the differential lipid metabolites were screened. Results: Compared with the normal group, the levels of ALT and AST were significantly increased in the model group(P<0.01), and the level of TG was obviously decreased(P<0.05). HE staining showed that the hepatocytes swelling and necrosis and the obvious inflammatory infiltration were found in the model group. Lipidomics analysis showed that there were significant differences in lipid metabolism profiles between the two groups. A total of 80 differential lipid metabolites were identified, and 34 lipid metabolites with the most significant differences were further screened by S-plot comparison. Among, 15 metabolites were phosphatidylcholine(PC), 6 metabolites were triglyceride(TG), 4 metabolites were phosphatidyl ethanolamine(PE), 2 metabolites were lysophosphatidylcholine(LPC), 2 metabolites were sphingomyelin(SM), and 5 metabolites were platelet activating factor(PAF). Conclusion: Lipidomics study reveals that the mechanism of CCl_4-induced acute liver injury may be related to disorders of phospholipid and triglyceride metabolism.
引文
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[19] GORDEN D L,MYERS D S,IVANOVA P T,et al.Biomarkers of NAFLD progression:a lipidomics approach to an epidemic[J].J Lipid Res,2015,56(3):722- 736.
[20] ISHIKAWA M,SAITO K,YAMADA H,et al.Plasma lipid profiling of different types of hepatic fibrosis induced by carbon tetrachloride and lomustine in rats[J].Lipids Health Dis,2016,15(1):74.
[21] 吕晓梅,卢任玲,马月宏,等.红花对四氯化碳致大鼠急性肝损伤的保护作用及其机制[J].北京中医药大学学报,2018,41(11):943- 949.
[22] 王跃峰,张可锋,周雨晴,等.拳卷地钱总黄酮对四氯化碳致急性肝损伤大鼠的保护作用及其作用机制[J].时珍国医国药,2017,28(2):27- 29.
[23] MARSLIN G,PRAKASH J,QI S,et al.Oral Delivery of Curcumin Polymeric Nanoparticles Ameliorates CCl4- Induced Subacute Hepatotoxicity in Wistar Rats[J].Polymers(Basel),2018,10(5):541.
[2] 林景超.化学性肝损伤及肝癌的代谢组学研究[D].上海:上海交通大学,2008.
[3] 周迎春.四氯化碳诱导的小鼠急性肝损伤模型的实验研究[J].黑龙江中医药,2018,47(1):90- 91.
[4] ZHANG W,DONG Z,CHANG X J,et al.Protective effect of the total flavonoids from Apocynum venetum L.on carbon tetrachloride- induced hepatotoxicity in vitro and in vivo[J].J Physiol Biochem,2018,74(2):301- 312.
[5] AHN M,KIM J,HONG S,et al.Black Radish (Raphanus sativus L.var.niger) Extract Mediates ItsHepatoprotective Effect on Carbon Tetrachloride- Induced Hepatic Injury by Attenuating Oxidative Stress[J].J Med Food,2018,21(9):866- 875.
[6] YANG C F,LI L,MA Z H,et al.Hepatoprotective effect of methyl ferulic acid against carbon tetrachloride- induced acute liver injury in rats[J].Exp Ther Med,2018,15(3):2228- 2238.
[7] 段冷昕,高杨,胡举,等.四氯化碳诱导小鼠内质网应激所致急性肝损伤的作用研究[J].中国临床药理学杂志,2018,34(12):1440- 1443.
[8] YANG K,HAN X L.Lipidomics:Techniques,Applications,and Outcomes Related to Biomedical Sciences[J].Trends Biochem Sci,2016,41(11):954- 969.
[9] 潘喆敏,李丽,杨婵,等.脂质组学分析方法进展及其在癌症研究中的应用[J].现代生物医学进展,2017,17(9):1793- 1797.
[10] 赵鑫,苟小军,陈龙,等.花旗松素预防CCl4致大鼠急性肝损伤的代谢组学研究[J].上海中医药杂志,2017,51(8):82- 86.
[11] WU J Y,WU Q,WANG D,et al.Common lipid features of lethal ventricular tarchyarrhythmias (LVTAs) induced by myocardial infarction and myocardial ion channel diseases[J].Sci Rep,2017,7(1):4220- 4230.
[12] ZHAO N N,SUN Y F,ZONG L,et al.Serum lipidomics study of Ding- Zhi- Xiao- Wan effect on Alzheimer’s disease using online liquid extraction surface analysis coupled to direct infusion mass spectrometry[J].Int J Mass Spectrom,2018,434:29- 36.
[13] 洪赟,厉有名,虞朝晖.脂质组学技术在非酒精性脂肪性肝病中的研究进展[J].国际消化病杂志,2013,33(6):384- 385.
[14] 蒋莹莹,郑素军.肝细胞癌的鞘脂组学研究进展[J].世界华人消化杂志,2018,26(36):2109- 2114.
[15] TU L N ,SHOWALTER M R ,CAJKA T ,et al.Metabolomic characteristics of cholesterol- induced non- obese nonalcoholic fatty liver disease in mice[J].Sci Rep,2017,7(1):6120- 6134.
[16] 叶梓,王世杰,李辉,等.血管内皮细胞中的凝血物质:理论研究新进展[J].中国组织工程研究,2017,21(4):627- 632.
[17] CHAO Y F,GAO S Y,WANG X L,et al.Untargeted lipidomics based on UPLC- QTOF- MS/MS and structural characterization reveals dramatic compositional changes in serum and renal lipids in mice with glyoxylate- induced nephrolithiasis[J].J Chromatogr B,2018,1095:258- 266.
[18] 孟胜喜,冯琴,胡义扬.脂质组学——非酒精性脂肪性肝病中医药防治的新平台[J].新中医,2013,45(9):3- 6.
[19] GORDEN D L,MYERS D S,IVANOVA P T,et al.Biomarkers of NAFLD progression:a lipidomics approach to an epidemic[J].J Lipid Res,2015,56(3):722- 736.
[20] ISHIKAWA M,SAITO K,YAMADA H,et al.Plasma lipid profiling of different types of hepatic fibrosis induced by carbon tetrachloride and lomustine in rats[J].Lipids Health Dis,2016,15(1):74.
[21] 吕晓梅,卢任玲,马月宏,等.红花对四氯化碳致大鼠急性肝损伤的保护作用及其机制[J].北京中医药大学学报,2018,41(11):943- 949.
[22] 王跃峰,张可锋,周雨晴,等.拳卷地钱总黄酮对四氯化碳致急性肝损伤大鼠的保护作用及其作用机制[J].时珍国医国药,2017,28(2):27- 29.
[23] MARSLIN G,PRAKASH J,QI S,et al.Oral Delivery of Curcumin Polymeric Nanoparticles Ameliorates CCl4- Induced Subacute Hepatotoxicity in Wistar Rats[J].Polymers(Basel),2018,10(5):541.