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Article(id=1199786456123277465, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1199786450628735631, articleNumber=null, orderNo=null, doi=10.16438/j.0513-4870.2024-0482, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1716134400000, receivedDateStr=2024-05-20, revisedDate=1720108800000, revisedDateStr=2024-07-05, acceptedDate=null, acceptedDateStr=null, onlineDate=1763980982094, onlineDateStr=2025-11-24, pubDate=1726070400000, pubDateStr=2024-09-12, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1763980982094, onlineIssueDateStr=2025-11-24, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1763980982094, creator=13701087609, updateTime=1763980982094, updator=13701087609, issue=Issue{id=1199786450628735631, tenantId=1146029695717560320, journalId=1189982191388893191, year='2024', volume='59', issue='9', pageStart='2417', pageEnd='2676', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1763980980784, creator=13701087609, updateTime=1764225057364, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1200810182063280632, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1199786450628735631, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1200810182063280633, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1199786450628735631, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=2529, endPage=2538, ext={EN=ArticleExt(id=1199786456559485105, articleId=1199786456123277465, tenantId=1146029695717560320, journalId=1189982191388893191, language=EN, title=Metabolomic study of the improvement of nitazoxanide on Western-diet induced hepatic steatosis in ApoE-/- mice, columnId=null, journalTitle=Acta Pharmaceutica Sinica, columnName=null, runingTitle=null, highlight=null, articleAbstract=

Nitazoxanide is an FDA-approved antiprotozoal drug. Our previous study found that oral administration of nitazoxanide inhibited Western diet (WD)-induced hepatic steatosis in ApoE-/- mice. However, the specific mechanism remains to be elucidated. In the present study, we performed an untargeted metabolomics approach to reveal the effect of nitazoxanide on the liver metabolic profiles in WD-fed ApoE-/- mice, and carried out the cellular experiments to elucidate the underlying mechanisms. UPLC-MS-based untargeted metabolomics analysis was used to investigate the effect of nitazoxanide on global metabolite changes in liver tissues. The differential metabolites were screened for enrichment analysis and pathway analysis. Hepatocytes were treated with tizoxanide, the metabolite of nitazoxanide, to investigate the underlying mechanism based on the findings in metabolomics study. The improvement of liver lipid metabolism disorders by nitazoxanide treatment in WD-fed ApoE-/- mice was mainly through regulating glycerophospholipid metabolism, D-glutamine and glutamate metabolism, glutathione metabolism, and arginine biosynthesis metabolism. Tizoxanide, the active metabolite of nitazoxanide, increased glutathione (GSH) contents and glutamate-cysteine ligase catalytic subunit (Gcl-c) and glutathione reductase (Gsr) mRNA expressions in HepG2 cells. Tizoxanide increased cystathionine β-synthase (CBS) and phosphatidylethanolamine N-methyltransferase (PEMT) protein levels, inhibited lipid accumulation in hepatocytes induced by free fatty acid (FFA). Tizoxanide increased S-adenosyl-L-homocysteine hydrolase (SAHH) protein levels in HepG2 cells and mouse primary liver cells stimulated with free fatty acid (FFA). Tizoxanide increased N-acetyl glutamate synthase (Nags) and carbamoylphosphate synthetase 1 (Cps1) mRNA expressions in HepG2 cells. In conclusion, nitazoxanide improves WD-induced hepatic steatosis in ApoE-/- mice and the underlying mechanisms include increasing CBS expression, GSH content, PEMT protein expression, Nags and Cps1 mRNA expression in hepatocytes.

, correspAuthors=De-li DONG, authorNote=null, correspAuthorsNote=null, copyrightStatement=Copyright ©2024 Acta Pharmaceutica Sinica. All rights reserved., copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=null, magXml=null, pdfUrl=null, pdf=null, pdfFileSize=null, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=null, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=null, mapNumber=null, authorCompany=null, fund=null, authors=null, authorsList=Hu-tai-long ZHU, Xiao-fan CHENG, Xin GUO, Le CHANG, Yin-di ZHAO, Shang-ze WU, De-li DONG), CN=ArticleExt(id=1199786458333675819, articleId=1199786456123277465, tenantId=1146029695717560320, journalId=1189982191388893191, language=CN, title=硝唑尼特改善西方饮食诱导的ApoE-/-小鼠脂肪肝作用的代谢组学研究, columnId=1190335348896011050, journalTitle=药学学报, columnName=研究论文, runingTitle=null, highlight=null, articleAbstract=

硝唑尼特(nitazoxanide) 是一种临床抗原虫药物, 本课题组前期研究发现口服途径给予硝唑尼特可改善西方饮食(Western diet, WD) 诱导的ApoE-/-小鼠肝脏脂肪样变, 尚待深入阐明其机制。本文通过非靶向代谢组学研究硝唑尼特对西方饮食诱导的ApoE-/-小鼠肝脏代谢谱的影响, 进而通过细胞水平实验研究其潜在机制。采用UPLC-MS联用法对小鼠的肝脏组织代谢物进行非靶向代谢组学研究; 筛选差异代谢物, 并对差异代谢物进行富集分析和通路分析; 采用硝唑尼特的体内代谢产物替唑尼特处理肝细胞, 在代谢组学发现的基础上研究硝唑尼特改善肝脏脂代谢异常的潜在机制。硝唑尼特改善西方饮食诱导的ApoE-/-小鼠肝脏代谢紊乱, 主要通过调控甘油磷脂代谢、D-谷氨酰胺和谷氨酸代谢、谷胱甘肽代谢和精氨酸生物合成。硝唑尼特的体内活性代谢产物替唑尼特(tizoxanide) 增加HepG2细胞谷胱甘肽(glutathione, GSH) 含量并促进谷氨酸半胱氨酸连接酶(glutamate-cysteine ligase catalytic subunit, Gcl-c) 和谷胱甘肽还原酶(glutathione reductase, Gsr) 的mRNA表达; 增加肝细胞胱硫醚β合酶(cystathionine β-synthase, CBS) 和磷脂酰乙醇胺N-甲基转移酶(phosphatidylethanolamine N-methyltransferase, PEMT) 蛋白水平, 抑制游离脂肪酸(free fatty acid, FFA) 诱导的肝细胞脂质蓄积; 替唑尼特可增加FFA刺激下HepG2细胞及小鼠原代肝细胞中S-腺苷同型半胱氨酸水解酶(S-adenosyl-L-homocysteine hydrolase, SAHH) 蛋白表达; 替唑尼特提高HepG2细胞N-乙酰谷氨酸合酶(N-acetyl glutamate synthase, Nags) 和氨基甲酰磷酸合成酶1 (carbamoylphosphate synthetase 1, Cps1) mRNA表达水平。综上所述, 硝唑尼特改善西方饮食诱导的ApoE-/-小鼠肝脏脂肪样变的潜在机制包括增加CBS表达、提高肝细胞内谷胱甘肽含量, 增加肝细胞PEMT蛋白水平, 增加肝细胞Nags和Cps1的表达。

, correspAuthors=董德利, authorNote=null, correspAuthorsNote=
*董德利，E-mail:
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J Hepatol, 2016, 64: 823-833., articleTitle=null, refAbstract=null), Reference(id=1200378866918355522, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1199786456123277465, doi=null, pmid=null, pmcid=null, year=null, volume=null, issue=null, pageStart=null, pageEnd=null, url=null, language=null, rfNumber=[34], rfOrder=33, authorNames=null, journalName=null, refType=null, unstructuredReference=Eriksen PL, Vilstrup H, Rigbolt K, et al. Non-alcoholic fatty liver disease alters expression of genes governing hepatic nitrogen conversion [J]. Liver Int, 2019, 39: 2094-2101., articleTitle=null, refAbstract=null)], funds=[Fund(id=1200378862300426693, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1199786456123277465, awardId=null, language=CN, fundingSource=中国药科大学人才引进科研启动基金, fundOrder=null, country=null)], companyList=[AuthorCompany(id=1200378853601439789, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1199786456123277465, xref=null, ext=[AuthorCompanyExt(id=1200378853609828396, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1199786456123277465, companyId=1200378853601439789, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=1. 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Lc-Bio Technologies Co., Ltd., Hangzhou 310018, China), AuthorCompanyExt(id=1200378853790183485, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1199786456123277465, companyId=1200378853777600572, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=2.杭州联川生物技术股份有限公司, 浙江 杭州 310018)])], figs=[ArticleFig(id=1200378860480098673, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1199786456123277465, language=EN, label=null, caption=null, figureFileSmall=vlZrnrHFjq/xVViq2Exz5Q==, figureFileBig=zLz60swFxVJT9B2ebAXJKA==, tableContent=null), ArticleFig(id=1200378860618510711, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1199786456123277465, language=CN, label=Figure 1, caption= Oral nitazoxanide improved Western diet-induced liver metabolic profiles in ApoE<sup>-/-</sup> mice. A: Scores plot of the PCA model of mouse livers; B: Scores plot of the PLS-DA model of mouse livers; C: Heatmap showing tested metabolites in livers. <i>n</i> = 8. Nit: Nitazoxanide; ND: Normal diet; WD: Western diet; PCA: Principal component analysis; PLS-DA: Partial least squares discrimination analysis , figureFileSmall=vlZrnrHFjq/xVViq2Exz5Q==, figureFileBig=zLz60swFxVJT9B2ebAXJKA==, tableContent=null), ArticleFig(id=1200378860819837312, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1199786456123277465, language=EN, label=null, caption=null, figureFileSmall=JZLMoTPhxbITTQlcGmB9sQ==, figureFileBig=Vj2fjJ52gZHY3a0Wrr9pyw==, tableContent=null), ArticleFig(id=1200378860903723399, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1199786456123277465, language=CN, label=Figure 2, caption= Oral nitazoxanide altered the metabolic pathways in WD-fed ApoE<sup>-/-</sup> mice. A: Enrichment analysis based on metabolites that displayed significant variation in livers; B: Pathway analysis of the differential metabolites; C: Overview of the differential metabolites and the related metabolic pathways; D: The analyzed data of different metabolites in livers. <i>n</i> = 8, <i>x</i> ± SEM. <sup>*</sup><i>P</i> < 0.05, <sup>**</sup><i>P</i> < 0.01 <i>vs</i> ND+WT; <sup>#</sup><i>P</i> < 0.05, <sup>##</sup><i>P</i> < 0.01 <i>vs</i> WD-fed ApoE<sup>-/-</sup> mice. WT: Wild type; NAG: <i>N</i>-Acetyl glutamate; SAH: <i>S</i>-Adenosyl-<i>L</i>-homocysteine; PE: Phosphatidylethanolamine; GPC: Glycerophosphocholine; GSSG: Oxidized glutathione , figureFileSmall=JZLMoTPhxbITTQlcGmB9sQ==, figureFileBig=Vj2fjJ52gZHY3a0Wrr9pyw==, tableContent=null), ArticleFig(id=1200378861012775310, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1199786456123277465, language=EN, label=null, caption=null, figureFileSmall=lF9Jwg2sAkClEHt6mifxLg==, figureFileBig=Yqj0Qpw/F9fXb428I2lNtw==, tableContent=null), ArticleFig(id=1200378861117632912, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1199786456123277465, language=CN, label=Figure 3, caption= Tizoxanide treatment increased liver glutathione (GSH) contents. A: The analyzed data of cell viability in HepG2 treated with different concentration of tizoxanide for 24 h; B: The analyzed data of cell viability in HepG2 treated with different concentration of FFA for 24 h; C: The analyzed data of GSH contents in HepG2 cells treated with tizoxanide (2, 5, 10 μmol·L<sup>-1</sup>) for 24 h; D: <i>Gcl-c</i> and <i>Gsr</i> mRNA expressions in HepG2 cells treated with tizoxanide (2, 5, 10 μmol·L<sup>-1</sup>) for 24 h. <i>n</i> = 6, <i>x</i> ± SEM. <sup>*</sup><i>P</i> < 0.05, <sup>**</sup><i>P</i> < 0.01 <i>vs</i> CTL; E: The representative Western blots and analyzed data of CBS protein levels in the livers of mice. <i>n</i> = 8, <i>x</i> ± SEM. <sup>#</sup><i>P</i> < 0.05 <i>vs</i> WD+ApoE<sup>-/-</sup>; F, G: The representative Western blots and analyzed data of CBS protein levels in HepG2 cells and primary hepatocytes. <i>n</i> = 6, <i>x</i> ± SEM. <sup>*</sup><i>P</i> < 0.05 <i>vs</i> CTL; <sup>#</sup><i>P</i> < 0.05, <sup>##</sup><i>P</i> < 0.01 <i>vs</i> FFA (200 μmol·L<sup>-1</sup>). FFA: Free fatty acid; Tiz: Tizoxanide; GSSG: Oxidized glutathione; CBS: Cystathionine <i>β</i>-synthase; GCL-C: Glutamate-cysteine ligase catalytic subunit; GSR: Glutathione reductase; CTL: Control , figureFileSmall=lF9Jwg2sAkClEHt6mifxLg==, figureFileBig=Yqj0Qpw/F9fXb428I2lNtw==, tableContent=null), ArticleFig(id=1200378861218296216, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1199786456123277465, language=EN, label=null, caption=null, figureFileSmall=QbrrfJPK/PUsW4OsiQ8kLw==, figureFileBig=+AUkJg+10JzpTZHh2hk+VQ==, tableContent=null), ArticleFig(id=1200378861381874077, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1199786456123277465, language=CN, label=Figure 4, caption= Tizoxanide treatment increased PEMT protein expression and reduced FFA-induced lipid accumulation in hepatocytes. A: The representative Western blots and analyzed data of PEMT protein levels in the livers of mice. <i>n</i> = 8, <i>x</i> ± SEM. <sup>#</sup><i>P</i> < 0.05 <i>vs</i> WD+ApoE<sup>-/-</sup>; B: The representative Western blots and analyzed data of PEMT protein levels in HepG2 cells treated by tizoxanide for 24 h; C, D: The TG content in HepG2 cells and primary hepatocytes treated with tizoxanide for 24 h. <i>n</i> = 6, <i>x</i> ± SEM. <sup>*</sup><i>P</i> < 0.05, <sup>**</sup><i>P</i> < 0.01 <i>vs</i> CTL; <sup>#</sup><i>P</i> < 0.05, <sup>##</sup><i>P</i> < 0.01 <i>vs</i> FFA (200 μmol·L<sup>-1</sup>). PEMT: Phosphatidylethanolamine <i>N</i>-methyltransferase; TG: Triglyceride , figureFileSmall=QbrrfJPK/PUsW4OsiQ8kLw==, figureFileBig=+AUkJg+10JzpTZHh2hk+VQ==, tableContent=null), ArticleFig(id=1200378861545451939, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1199786456123277465, language=EN, label=null, caption=null, figureFileSmall=V31qBAph+duP5BKF7N18bw==, figureFileBig=VxTObtirayZyRH0NGXx4vA==, tableContent=null), ArticleFig(id=1200378861742584239, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1199786456123277465, language=CN, label=Figure 5, caption= Tizoxanide treatment increased SAHH protein expression in hepatocytes. HepG2 cells and primary hepatocytes were treated by tizoxanide and FFA for 24 h. The representative Western blots and analyzed data of SAHH protein levels in HepG2 cells were shown in (A) and primary hepatocytes were shown in (B). <i>n</i> = 6, <i>x</i> ± SEM. <sup>#</sup><i>P</i> < 0.05, <sup>##</sup><i>P</i> < 0.01 <i>vs</i> FFA (200 μmol·L<sup>-1</sup>). SAHH: <i>S</i>-Adenosyl-<i>L</i>-homocysteine hydrolase , figureFileSmall=V31qBAph+duP5BKF7N18bw==, figureFileBig=VxTObtirayZyRH0NGXx4vA==, tableContent=null), ArticleFig(id=1200378861889384881, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1199786456123277465, language=EN, label=null, caption=null, figureFileSmall=dmxl99VFqyk2ViEoP0leQQ==, figureFileBig=OOkcWwodRm70t8LOqfq7fg==, tableContent=null), ArticleFig(id=1200378862019408314, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1199786456123277465, language=CN, label=Figure 6, caption= Tizoxanide treatment increased <i>Nags</i> and <i>Cps1</i> mRNA expressions in HepG2 cells. HepG2 cells were treated by tizoxanide for 24 h. A: <i>Nags</i> mRNA expressions in HepG2 cells; B: <i>Cps1</i> mRNA expressions in HepG2 cells. <i>n</i> = 6, <i>x</i> ± SEM. <sup>**</sup><i>P</i> < 0.01 <i>vs</i> CTL; <sup>#</sup><i>P</i> < 0.05, <sup>##</sup><i>P</i> < 0.01 <i>vs</i> FFA (200 μmol·L<sup>-1</sup>). NAGS: <i>N</i>-Acetyl glutamate synthase; CPS1: Carbamoylphosphate synthetase , figureFileSmall=dmxl99VFqyk2ViEoP0leQQ==, figureFileBig=OOkcWwodRm70t8LOqfq7fg==, tableContent=null)], attaches=null, journal=Journal(id=1189982048455397383, delFlag=0, nameCn=药学学报, nameEn=Acta Pharmaceutica Sinica, nameHistory1=null, nameHistory2=null, issn=0513-4870, eissn=null, cn=11-2163/R, coden=null, periodic=0, language=CN, oaType=null, ccby=null, superviseOffice=null, ownerOffice=null, pubOffice=null, editorOffice=null, officeType=null, aims=null, clcCode=null, officeProv=null, officeCity=null, officeAddr=null, officeZip=null, officeEmail=null, officePhone=null, editDirector=null, officeDirector=null, officeDirectorPhone=null, officeStaffNum=null, officeEmpNum=null, coverPicUrl=BTxjudbJDVO4PqdBR6On6Q==, journalPrice=null, startedYear=null, abbrevIsoEn=null, journalRemark=null, publicationField=null, createdTime=1761643429151, updatedTime=1761735768113, createdBy=18614031015, updatedBy=13701087609, 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硝唑尼特改善西方饮食诱导的ApoE-/-小鼠脂肪肝作用的代谢组学研究
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朱胡太龙 1 , 程晓范 1 , 郭欣 2 , 常乐 1 , 赵银娣 1 , 吴尚泽 1 , 董德利 1, *
药学学报 | 研究论文 2024,59(9): 2529-2538
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药学学报 | 研究论文 2024, 59(9): 2529-2538
硝唑尼特改善西方饮食诱导的ApoE-/-小鼠脂肪肝作用的代谢组学研究
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朱胡太龙1, 程晓范1, 郭欣2, 常乐1, 赵银娣1, 吴尚泽1, 董德利1, *
作者信息
  • 1.中国药科大学药学院, 江苏 南京 211198
  • 2.杭州联川生物技术股份有限公司, 浙江 杭州 310018

通讯作者:

*董德利，E-mail:
Metabolomic study of the improvement of nitazoxanide on Western-diet induced hepatic steatosis in ApoE-/- mice
Hu-tai-long ZHU1, Xiao-fan CHENG1, Xin GUO2, Le CHANG1, Yin-di ZHAO1, Shang-ze WU1, De-li DONG1, *
Affiliations
  • 1. School of Pharmacy, China Pharmaceutical University, Nanjing 211198, China
  • 2. Lc-Bio Technologies Co., Ltd., Hangzhou 310018, China
出版时间: 2024-09-12 doi: 10.16438/j.0513-4870.2024-0482
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摘要
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硝唑尼特(nitazoxanide) 是一种临床抗原虫药物, 本课题组前期研究发现口服途径给予硝唑尼特可改善西方饮食(Western diet, WD) 诱导的ApoE-/-小鼠肝脏脂肪样变, 尚待深入阐明其机制。本文通过非靶向代谢组学研究硝唑尼特对西方饮食诱导的ApoE-/-小鼠肝脏代谢谱的影响, 进而通过细胞水平实验研究其潜在机制。采用UPLC-MS联用法对小鼠的肝脏组织代谢物进行非靶向代谢组学研究; 筛选差异代谢物, 并对差异代谢物进行富集分析和通路分析; 采用硝唑尼特的体内代谢产物替唑尼特处理肝细胞, 在代谢组学发现的基础上研究硝唑尼特改善肝脏脂代谢异常的潜在机制。硝唑尼特改善西方饮食诱导的ApoE-/-小鼠肝脏代谢紊乱, 主要通过调控甘油磷脂代谢、D-谷氨酰胺和谷氨酸代谢、谷胱甘肽代谢和精氨酸生物合成。硝唑尼特的体内活性代谢产物替唑尼特(tizoxanide) 增加HepG2细胞谷胱甘肽(glutathione, GSH) 含量并促进谷氨酸半胱氨酸连接酶(glutamate-cysteine ligase catalytic subunit, Gcl-c) 和谷胱甘肽还原酶(glutathione reductase, Gsr) 的mRNA表达; 增加肝细胞胱硫醚β合酶(cystathionine β-synthase, CBS) 和磷脂酰乙醇胺N-甲基转移酶(phosphatidylethanolamine N-methyltransferase, PEMT) 蛋白水平, 抑制游离脂肪酸(free fatty acid, FFA) 诱导的肝细胞脂质蓄积; 替唑尼特可增加FFA刺激下HepG2细胞及小鼠原代肝细胞中S-腺苷同型半胱氨酸水解酶(S-adenosyl-L-homocysteine hydrolase, SAHH) 蛋白表达; 替唑尼特提高HepG2细胞N-乙酰谷氨酸合酶(N-acetyl glutamate synthase, Nags) 和氨基甲酰磷酸合成酶1 (carbamoylphosphate synthetase 1, Cps1) mRNA表达水平。综上所述, 硝唑尼特改善西方饮食诱导的ApoE-/-小鼠肝脏脂肪样变的潜在机制包括增加CBS表达、提高肝细胞内谷胱甘肽含量, 增加肝细胞PEMT蛋白水平, 增加肝细胞Nags和Cps1的表达。

硝唑尼特  /  替唑尼特  /  非酒精性脂肪肝  /  代谢组学  /  谷胱甘肽

Nitazoxanide is an FDA-approved antiprotozoal drug. Our previous study found that oral administration of nitazoxanide inhibited Western diet (WD)-induced hepatic steatosis in ApoE-/- mice. However, the specific mechanism remains to be elucidated. In the present study, we performed an untargeted metabolomics approach to reveal the effect of nitazoxanide on the liver metabolic profiles in WD-fed ApoE-/- mice, and carried out the cellular experiments to elucidate the underlying mechanisms. UPLC-MS-based untargeted metabolomics analysis was used to investigate the effect of nitazoxanide on global metabolite changes in liver tissues. The differential metabolites were screened for enrichment analysis and pathway analysis. Hepatocytes were treated with tizoxanide, the metabolite of nitazoxanide, to investigate the underlying mechanism based on the findings in metabolomics study. The improvement of liver lipid metabolism disorders by nitazoxanide treatment in WD-fed ApoE-/- mice was mainly through regulating glycerophospholipid metabolism, D-glutamine and glutamate metabolism, glutathione metabolism, and arginine biosynthesis metabolism. Tizoxanide, the active metabolite of nitazoxanide, increased glutathione (GSH) contents and glutamate-cysteine ligase catalytic subunit (Gcl-c) and glutathione reductase (Gsr) mRNA expressions in HepG2 cells. Tizoxanide increased cystathionine β-synthase (CBS) and phosphatidylethanolamine N-methyltransferase (PEMT) protein levels, inhibited lipid accumulation in hepatocytes induced by free fatty acid (FFA). Tizoxanide increased S-adenosyl-L-homocysteine hydrolase (SAHH) protein levels in HepG2 cells and mouse primary liver cells stimulated with free fatty acid (FFA). Tizoxanide increased N-acetyl glutamate synthase (Nags) and carbamoylphosphate synthetase 1 (Cps1) mRNA expressions in HepG2 cells. In conclusion, nitazoxanide improves WD-induced hepatic steatosis in ApoE-/- mice and the underlying mechanisms include increasing CBS expression, GSH content, PEMT protein expression, Nags and Cps1 mRNA expression in hepatocytes.

nitazoxanide  /  tizoxanide  /  non-alcoholic fatty liver disease  /  metabolomics  /  glutathione
朱胡太龙, 程晓范, 郭欣, 常乐, 赵银娣, 吴尚泽, 董德利. 硝唑尼特改善西方饮食诱导的ApoE-/-小鼠脂肪肝作用的代谢组学研究. 药学学报, 2024 , 59 (9) : 2529 -2538 . DOI: 10.16438/j.0513-4870.2024-0482
Hu-tai-long ZHU, Xiao-fan CHENG, Xin GUO, Le CHANG, Yin-di ZHAO, Shang-ze WU, De-li DONG. Metabolomic study of the improvement of nitazoxanide on Western-diet induced hepatic steatosis in ApoE-/- mice[J]. Acta Pharmaceutica Sinica, 2024 , 59 (9) : 2529 -2538 . DOI: 10.16438/j.0513-4870.2024-0482
正文
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代谢相关脂肪性肝病(metabolic dysfunction-associated steatotic liver disease, MAFLD) 是一种由非过量饮酒、非病毒感染引起的以肝脏脂肪变性为特征的疾病[1]。近年来, MAFLD患病率不断上升, 并同时伴肥胖和2型糖尿病[2]。MAFLD包括从非酒精性脂肪肝(non-alcoholic fatty liver, NAFL) 到非酒精性脂肪性肝炎(non-alcoholic steatohepatitis, NASH) 的一系列肝脏疾病, 是肝硬化、肝衰竭甚至肝细胞癌等慢性肝病的重要危险因素。同时, MAFLD增加患者的高血压、高脂血症、动脉粥样硬化和心力衰竭等心血管事件风险[3]。尽管MAFLD的发病机制和治疗靶点已受到广泛关注, 但目前尚无FDA批准的治疗药物[4, 5]。
腺苷酸活化蛋白激酶(AMP-activated protein kinase, AMPK) 是机体能量代谢的关键调节因子, 参与糖类、脂质及蛋白质代谢的调节, 是肥胖、2型糖尿病等多种代谢性疾病的治疗靶点。研究表明, AMPK可通过磷酸化乙酰辅酶A羧化酶(acetyl CoA carboxylase, ACC) 抑制脂肪的从头合成, 进而促进脂肪酸氧化分解[6]。此外, AMPK激活也可提高自噬水平减轻肝脏脂毒性[7]。
硝唑尼特(nitazoxanide) 是FDA批准用于治疗隐孢子虫、贾第鞭毛虫、阿米巴原虫引起腹泻的药物[8, 9]。本课题组前期研究发现, 硝唑尼特及其活性代谢产物替唑尼特(tizoxanide) 可通过线粒体解偶联机制激活肝细胞AMPK, 口服途径给予硝唑尼特可显著改善西方饮食诱导的ApoE-/-小鼠肝脏脂肪变性[10]。代谢组学是对生物系统中小分子代谢物鉴定和定量的方法[11], 可将药物作用与细胞信号联系起来, 深入认识疾病发生机制和药物作用机制[12]。代谢组学已越来越多地应用于代谢调控、疾病发生机制、药物靶点发现等研究中[13]。本研究首先使用基于超高效液相色谱-串联质谱(UPLC/MS) 的非靶向代谢组学方法研究硝唑尼特对西方饮食诱导的ApoE-/-小鼠肝脏代谢谱的影响, 进而在代谢组学发现的基础上, 通过硝唑尼特的体内代谢产物替唑尼特在细胞水平上研究硝唑尼特改善肝脏脂代谢异常的潜在机制。
材料与方法
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基于UPLC/MS的代谢组学分析    称取20 mg小鼠肝脏组织至1.5 mL离心管中, 加入100 μL预冷超纯水后匀浆(-20 ℃), 再加入-80 ℃预冷的800 μL甲醇溶液(含0.2 μg·mL-1 4-氯苯丙氨酸) 涡旋振荡10 min, 18 000 r·min-1, 4 ℃离心10 min, 取上清, 在真空下干燥直至使用。使用100 μL甲醇∶水溶液(50∶50, v/v) 复溶, 18 000 r·min-1, 4 ℃离心10 min, 取上清进样检测。色谱柱: XBridge BEH Amide HPLC柱(100 mm × 4.6 mm, id 3.5 μm), 柱温: 45 ℃, 进样量: 2 μL, 流速: 0.4 mL·min-1。流动相: A相由95% 5 mmol·L-1乙酸铵缓冲液和5%乙腈(pH = 9) 组成, B相为乙腈。洗脱梯度: 0~2 min, 85% B; 2~8 min, 85%~30% B; 8~11 min, 30% B; 11~14 min, 30%~85% B; 14~20 min, 85% B。
离子源以正、负电喷雾电离(electrospray ionization, ESI) 模式运行。参数设置如下: 离子源气体1 (gas ion source, GSⅠ) 0 psi, 离子源气体2 (GSⅡ) 30 psi, 幕帘气体(curtain gas, CUR) 30 psi; 正、负离子模式下喷雾电压分别设置为+5 500 V和-4 500 V; 碰撞能量(collision energy, CE) 分别设置为+40和-35 V; 干燥气体温度分别设置为550和500 ℃; TOF-MS扫描范围m/z 50~1 000 Da, 乘积离子扫描范围m/z 50~900 Da。
使用质谱仪进行数据采集, 由Progenesis QI (version 2.0, Waters) 对采集的数据进行识别, 包括新实验创建、数据导入、审核对齐、实验设计设置、峰选取、审核反褶积和化合物序列识别[14]。对于识别出的峰采用MetaScope进行初步检索确定可能的代谢物, 进一步将MS/MS数据与METLIN数据库进行片段匹配, 选取母离子得分 > 40同时子离子得分 > 10的化合物作为该峰的鉴定结果, 筛选变量投射重要性(variable importance in the projection, VIP) > 1和差异倍数(fold change, FC) > 1.5的物质为差异代谢物。MetaScope参数设置: 前驱体公差(10 ppm); 保留时间(0.1 min内); 片段耐受性(15 ppm)。METLIN参数设置: 前驱体公差(10 ppm); 片段耐受性(15 ppm)。
细胞培养    HepG2细胞(人肝母细胞瘤细胞) 购自中国上海中乔新舟生物技术有限公司。HepG2细胞在添加10%胎牛血清(FBS, 美国Gibco公司)、1%青霉素/链霉素(上海Beyotime公司) 和1%丙酮酸钠(100 mmol·L-1) 溶液的最低必需培养基(MEM, 上海中乔新舟生物技术有限公司) 中培养。所有细胞均在37 ℃含5% CO2的湿化培养箱中培养。
小鼠原代肝细胞提取于C57BL/6J小鼠的肝脏, 采用改良的两步灌注法制备[15]。肝脏灌流冲洗血液, 胶原酶消化促进细胞分散, 然后将肝细胞释放到含10% FBS和1%青霉素/链霉素的DMEM中, 通过70 μm细胞过滤器过滤到15 mL管中。将悬浮液在50 ×g下4 ℃离心5 min, 沉淀物为肝细胞。将肝细胞(8×105个/孔) 重新悬浮在胶原包被的6孔细胞培养板上, 用于后续实验。
MTT法检测细胞活力    取对数生长期HepG2细胞以每孔2×104个细胞的密度接种于96孔板中, 每孔体积100 μL。待培养至汇合度为60%~70%后, 用无血清培养基饥饿8~12 h, 加入不同浓度替唑尼特和游离脂肪酸(free fatty acid, FFA) 处理24 h, 弃去培养基, 每孔加入100 μL MTT溶液(0.5 mg·mL-1) 在培养箱中孵育3~4 h后终止培养, 将孔内液体全部吸出, 每孔加入100 μL DMSO, 摇床低速震荡5~10 min使结晶物完全溶解, 最终在490 nm波长处测定吸光度, 计算细胞活力。
细胞内甘油三酯含量测定    将收集的细胞用PBS洗涤1次, 然后每管加入1 mL PBS重悬细胞, 取200 μL细胞悬液, 1 000 r·min-1离心5 min收集细胞用于蛋白浓度测定; 取700 μL细胞悬液, 1 000 r·min-1离心5 min收集细胞, 加入100 μL 1% TritonX100充分裂解细胞, 根据商用甘油三酯含量检测试剂盒的说明测定细胞内甘油三酯含量, 以蛋白浓度校准甘油三酯含量。
细胞内谷胱甘肽(glutathione, GSH) 含量测定    取对数生长期HepG2细胞以每孔6×105个细胞的密度接种于6孔板中, 每孔体积2 mL, 培养至汇合度为60%~70%后, 用无血清培养基饥饿8~12 h, 加入不同浓度替唑尼特处理细胞24 h。吸去培养基后用PBS洗涤2次, 每孔加入500 μL胰酶消化细胞3 min后加入500 μL MEM完全培养基终止消化, 收集细胞至1.5 mL离心管中, 用PBS洗涤1次后每管加入1 mL PBS重悬细胞, 取200 μL细胞悬液, 1 000 r·min-1离心5 min收集细胞用于蛋白浓度测定; 取700 μL细胞悬液, 1 000 r·min-1离心5 min, 收集细胞沉淀, 根据说明使用商用GSH含量检测试剂盒测定细胞内GSH含量。
蛋白印迹(Western blot)    细胞培养板弃培养基后用PBS洗涤2次, 将细胞培养板置于冰上, 加入RIPA裂解液(含1 mmol·L-1 PMSF和磷酸酶抑制剂) 裂解细胞30 min, 期间每10 min涡旋振荡1次。使用细胞刮刀收集细胞至1.5 mL离心管中, 13 500 r·min-1、4 ℃离心15 min, 收集上清。组织样本: 称取小鼠肝组织30 mg, 至2 mL研磨管中, 加入RIPA裂解液(含1 mmol·L-1 PMSF和磷酸酶抑制剂) 匀浆, 13 500 r·min-1、4 ℃离心15 min, 收集上清。
采用BCA蛋白浓度测定法进行蛋白定量, 将各蛋白样本稀释至相同浓度后与蛋白上样缓冲液混合均匀, 使用金属浴100 ℃变性10 min。蛋白样品(40 μg) 在10%或15%分离胶上进行SDS-PAGE电泳分离, 采用湿转法将蛋白转移至NC膜上, 将NC膜置于5%脱脂奶粉中室温封闭2 h, TBST洗涤去封闭液后加入一抗, 在摇床上4 ℃孵育过夜。用TBST洗涤3次, 每次10 min, 然后加入荧光标记的二抗(IRDye800), 在摇床上室温避光孵育1 h, 用TBST洗涤3次, 每次10 min。使用Odyssey红外成像系统进行图像采集, Image Studio在800 nm通道对条带进行定量分析。
实时定量PCR    采用Trizol (南京Vazyme公司) 试剂从HepG2细胞中分离总RNA并进行定量。按照说明, 使用HiScriptⅢRT SuperMix进行qPCR (南京Vazyme公司), 将1 μg总RNA逆转录为cDNA。采用QuantStudio 1 real-time PCR系统(美国Thermo Fisher Scientific公司) 进行实时定量PCR反应, 并将其标准化为肌动蛋白β (ACTB)。采用2-ΔΔCT定量公式计算基因相对表达量。引物序列如下: ATCB-F, 5'-TGAGCGCGGCTACAGCTT-3'; ATCB-R, 5'-CCTTAATGTCA CACACGATT-3'; GCL-C-F, 5'-AATGAGATTTAAGC-CCCCTCCTC-3'; GCL-C-R, 5'-AAAGTCTGTTAATTGCACCTCC-3'; GSR-F, 5'-TTCACGAGTGATCCCAA-GCC-3'; GSR-R, 5'-ATTCCTAAGCTGGCACCGGG-3'; NAGS-F, 5'-ACGTCTCCGAGGGGTACAA-3'; NAGS-R, 5'-AATTTGTCCAGGTACGGGGTG-3'; CPS1-F, 5'-GTGGCTTGCTTTGGTGAAGGTA-3'; CPS1-R, 5'-TTGGCCGGAATGATTGCTGGATG-3'。
统计学分析    使用GraphPad Prism 8.0软件对数据进行分析, 所有数据均表示为均值±标准误。两组间的比较采用Student's t-test, 多组间的差异比较采用单因素方差分析(one way ANOVA)。P < 0.05表示有统计学差异。
结果
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1 硝唑尼特改善西方饮食诱导的ApoE-/-小鼠肝脏代谢的代谢谱特征
本课题组前期研究工作已证明口服途径给予硝唑尼特对西方饮食诱导的ApoE-/-小鼠肝脏脂肪样变具有改善作用[10], 因此本研究继续研究硝唑尼特对西方饮食诱导的ApoE-/-小鼠肝脏代谢谱的影响。主成分分析(principal component analysis, PCA) 与偏最小二乘判别分析法(partial least squares discrimination analysis, PLS-DA) 结果显示, 对照组与模型组样本存在显著分离, 提示西方饮食诱导的ApoE-/-小鼠肝脏脂肪样变模型建立成功。口服途径给予硝唑尼特(100和200 mg·kg-1) 的小鼠肝脏样本靠近对照组, 并与模型组存在显著分离(图 1A、B); 同时, 热图结果也证明硝唑尼特(100和200 mg·kg-1) 组ApoE-/-小鼠肝脏代谢特征与对照组相似(图 1C)。以上结果表明, 口服途径给予硝唑尼特可有效改善西方饮食诱导的ApoE-/-小鼠肝脏代谢物谱紊乱。
2 硝唑尼特对西方饮食诱导的ApoE-/-小鼠肝脏代谢通路的调节
VIP > 1及FC > 1.25或 < 0.8的化合物被确定为肝脏代谢差异化合物, 并对差异化合物进行富集分析和通路分析。结果显示, 硝唑尼特参与肝脏代谢的主要通路包括谷胱甘肽代谢、D-谷氨酰胺和D-谷氨酸代谢、甘油磷脂代谢和精氨酸生物合成通路(图 2A、B)。代谢差异化合物相关通路如图 2C所示, 红色代表相较于模型组上升的代谢物, 蓝色则代表含量下降的代谢物。各通路肝脏组织代谢差异化合物含量统计结果如图 2D所示, 口服途径给予硝唑尼特增加GSH水平, 减少GSH的消耗, 促进肝脏组织中GSH水平升高; 促进磷脂酰乙醇胺(phosphatidylethanolamine, PE) 向磷脂酰胆碱(phosphotidylcholine, PC) 的转化和相关代谢产物的生成; 提高肝组织谷氨酸水平从而增加N-乙酰谷氨酸(N-acetyl glutamate, NAG) 的合成, 参与精氨酸生物合成途径和尿素循环。
3 硝唑尼特体内代谢物替唑尼特增加HepG2细胞GSH水平
上述代谢组学结果显示, 口服途径给予硝唑尼特显著提高小鼠肝脏GSH水平。文献报道非酒精性脂肪肝患者肝脏中GSH耗竭, 而口服GSH对NAFLD具有潜在的治疗作用[16, 17]。硝唑尼特体内吸收后迅速完全转化为替唑尼特[18], 因此本研究进一步检测硝唑尼特体内代谢物替唑尼特对HepG2细胞GSH含量的影响。首先确定替唑尼特在细胞实验中的药物浓度, 如图 3A所示, 替唑尼特(2、5、10 μmol·L-1) 对HepG2细胞活力无明显影响。由于细胞实验将给予细胞FFA处理, 本实验也确定了FFA的使用浓度, 如图 3B所示, FFA在200 μmol·L-1浓度内对HepG2细胞活力无明显影响。
替唑尼特(10 μmol·L-1) 显著增加HepG2细胞总谷胱甘肽和还原型谷胱甘肽的含量, 而对氧化型谷胱甘肽(oxidized glutathione, GSSG) 无显著影响; 替唑尼特显著降低GSSG/GSH比值, 提示细胞抗氧化能力增强(图 3C)。谷氨酸半胱氨酸连接酶(glutamate-cysteine ligase catalytic subunit, GCL-C) 是GSH合成的限速酶, 谷胱甘肽还原酶(glutathione reductase, GSR) 在GSSG还原成GSH的过程中起关键作用。替唑尼特显著上调HepG2细胞中Gcl-c和Gsr的mRNA水平(图 3D)。以上结果证明, 替唑尼特可促进谷胱甘肽的合成及氧化型谷胱甘肽的还原, 从而提高细胞内还原型谷胱甘肽的含量。
转硫途径是谷胱甘肽生成的重要途径, 同型半胱氨酸在蛋氨酸循环中通过胱硫醚β合酶(cystathionine β-synthase, CBS) 生成半胱氨酸, 从而促进GSH的合成[19]。在整体动物水平实验中, 口服途径给予硝唑尼特(200 mg·kg-1) 增加ApoE-/-小鼠肝脏组织CBS蛋白水平(图 3E)。在细胞水平实验中, 替唑尼特处理可抑制FFA诱导的HepG2细胞CBS蛋白水平下降(图 3F); 替唑尼特也显著升高小鼠原代肝细胞中CBS蛋白水平(图 3G)。
4 硝唑尼特体内代谢物替唑尼特增加HepG2细胞PEMT蛋白表达并抑制FFA诱导的HepG2细胞脂质蓄积
在肝脏中, PE可通过磷脂酰乙醇胺N-甲基转移酶(phosphatidylethanolamine N-methyltransferase, PEMT) 甲基化生成PC, 而PEMT的表达降低可诱导小鼠肝脏脂肪变性、炎症和纤维化[20]。上述代谢组学结果显示, 口服给予硝唑尼特可增加ApoE-/-小鼠肝脏组织中PC含量并降低PE水平(图 2D), 因此继续测定PEMT在肝脏组织中蛋白表达水平。在整体动物水平实验中, 口服给予硝唑尼特(200 mg·kg-1) 可提高西方饮食诱导的ApoE-/-小鼠肝脏PEMT蛋白水平(图 4A)。在细胞水平实验中, 替唑尼特处理可增加FFA刺激下HepG2细胞中PEMT蛋白表达(图 4B)。本研究继续检测替唑尼特对FFA诱导的肝细胞脂质蓄积的影响, 结果如图 4C、D所示, 与对照组相比较, FFA处理的HepG2细胞及原代小鼠肝细胞TG含量显著增加, 替唑尼特处理24 h显著降低肝细胞TG含量。
5 硝唑尼特体内代谢物替唑尼特增加肝细胞S-腺苷同型半胱氨酸水解酶(S-adenosyl-L-homocysteine hydrolase, SAHH) 表达
S-腺苷高半胱氨酸(S-adenosylhomocysteine, SAH) 水平升高及其下游转硫和甲基化途径的代谢紊乱参与非酒精性脂肪肝的发生和发展。SAHH催化SAH分解为腺苷和同型半胱氨酸(homocysteine, Hcy), 而Hcy可促进GSH的合成[21]。前述代谢组学数据显示, 口服给予硝唑尼特可降低西方饮食诱导的ApoE-/-小鼠肝脏组织中SAH含量升高(图 2D)。在细胞水平实验中, 替唑尼特(10 μmol·L-1) 可增加FFA刺激下HepG2细胞及小鼠原代细胞中SAHH蛋白表达(图 5A、B)。研究表明, 激活AMPK可增加SAHH活性[22, 23]、降低SAH水平。本课题组前期研究已证明口服途径给予硝唑尼特可激活小鼠肝脏组织AMPK, 替唑尼特处理可激活HepG2细胞AMPK[10], 因此推测替唑尼特可激活肝细胞AMPK/SAHH信号。
6 硝唑尼特体内代谢物替唑尼特增加HepG2细胞Nags和Cps1 mRNA表达
谷氨酸可在N-乙酰谷氨酸合酶(N-acetyl glutamate synthase, NAGS) 的作用下与NH3合成NAG, NAG对尿素循环的关键酶氨基甲酰磷酸合成酶1 (carbamoylphosphate synthetase 1, CPS1) 激活具有重要作用[24]。前述代谢组学结果显示, 西方饮食诱导ApoE-/-小鼠肝脏中NAG含量降低, 口服硝唑尼特可恢复肝脏组织NAG水平(图 2D), 提示硝唑尼特可能提高NAG的合成促进CPS1的激活。为进一步证明此假设, 本研究通过qRT-PCR检测替唑尼特处理HepG2细胞后Nags和Cps1 mRNA水平的变化。结果如图 6A、B所示, 在FFA刺激条件下, HepG2细胞Nags和Cps1 mRNA水平显著降低, 而替唑尼特处理可恢复HepG2细胞Nags和Cps1 mRNA表达水平。
讨论
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MAFLD是一类由多种代谢功能紊乱引起的慢性肝病, 发病机制复杂, 脂毒性、氧化应激和胰岛素抵抗等多种因素可共同促进MAFLD/NASH的进展[25]。目前尚未有FDA批准用于治疗MAFLD的药物。硝唑尼特是一种临床抗原虫药物, 前期研究发现硝唑尼特及其活性代谢物替唑尼特具有线粒体解偶联作用, 可激活AMPK; 口服硝唑尼特可减轻高脂饮食诱导的小鼠肝脏脂肪变性, 改善肝脏功能, 其机制涉及AMPK/ACC通路[10]。基于代谢组学研究发现, 西方饮食喂养导致小鼠肝脏代谢谱紊乱, 口服途径给予硝唑尼特可改善小鼠肝脏代谢异常。通过对代谢组学检测出的代谢物进行筛选和通路分析, 进一步发现这些差异代谢物参与的代谢过程主要包括谷胱甘肽代谢、D-谷氨酰胺和D-谷氨酸代谢、甘油磷脂代谢和精氨酸生物合成等。
谷胱甘肽是由谷氨酸(glutamate, Glu)、半胱氨酸(cysteine, Cys) 和甘氨酸组成的三肽, Glu和Cys由谷氨酰半胱氨酸连接酶(glutamate-cysteine ligase, GCL) 催化形成γ-谷氨酰半胱氨酸(γ-GC), GSH合酶再将甘氨酸连接到γ-GC上生成GSH。γ-谷氨酰基转移酶催化GSH的γ-谷氨酰胺键断裂, Cys-Gly被细胞外的二肽酶水解, 谷氨酰基则转移到一个氨基酸上生成γ-谷氨酰氨基酸[19]。氧化应激在MAFLD和NASH的发病中具有重要作用, GSH不仅是体内重要的自由基清除剂, 也是细胞存活与凋亡的重要调控分子[17]。本研究结果显示, 与西方饮食喂养ApoE-/-小鼠相比较, 口服途径给予硝唑尼特的小鼠肝脏中谷氨酸和谷胱甘肽代谢相关通路变化最为显著。模型组小鼠肝脏L-Glu水平显著下降, 硝唑尼特可恢复肝脏L-Glu含量, 并显著提高GSH水平, 提示硝唑尼特可能通过影响谷胱甘肽代谢减轻西方饮食诱导的ApoE-/-小鼠肝脏氧化应激。细胞学实验结果也显示, 替唑尼特处理可降低HepG2细胞GSSG/GSH比值并增加总谷胱甘肽和还原型谷胱甘肽的含量, 但对氧化型谷胱甘肽含量无显著影响。替唑尼特显著提高HepG2细胞中谷胱甘肽合成限速酶和谷胱甘肽还原酶的mRNA水平, 提示替唑尼特可通过增加谷胱甘肽的合成和氧化型谷胱甘肽的还原, 提高还原型谷胱甘肽水平。另一方面, SAH通过SAHH水解为腺苷和Hcy, Hcy可由CBS通过转硫途径生成Cys, 从而促进谷胱甘肽合成[26]。健康状态下, 蛋氨酸中硫的摄入与Hcy通过转硫途径转移到Cys的硫动态平衡, 转硫途径的关键酶CBS和胱硫醚-γ-裂解酶(cystathionine-γ-lyase, CSE) 在肝脏中高度表达, CBS和CSE缺乏会导致高脂饮食诱导的MAFLD动物模型中肝脏脂肪变性、氧化应激、炎症和纤维化[27]。本研究结果显示, 硝唑尼特口服途径给药可提高小鼠肝组织中CBS表达, 替唑尼特处理肝细胞可抑制游离脂肪酸诱导的肝细胞CBS蛋白水平降低, 提示硝唑尼特可通过转硫途径增加谷胱甘肽的合成。
肝细胞中脂肪酸可通过线粒体β-氧化或再酯化反应生成TG代谢, TG可以脂滴形式储存在肝细胞中或通过VLDL运输到血液中。当肝细胞中脂肪酸的摄取和代谢失调, 过量的脂肪酸在肝内蓄积可产生磷脂酸、甘油二酯、溶血磷脂酰胆碱和神经酰胺等脂毒性物质, 导致胰岛素信号传导受损和肝细胞损伤, 甘油磷脂代谢是产生这些脂毒性物质的主要过程[28]。有研究表明, FFA酯化生成TG可减轻FFA积聚导致的脂毒性, 抑制肝脏TG合成虽然减轻肝脏脂肪样变, 但也可促进肝内FFAs蓄积, 加重肝损伤、炎症和肝纤维化[29]。西方饮食喂养的ApoE-/-小鼠肝脏中白三烯B4的β氧化代谢产物18-carboxy dinor leukotriene B4水平显著升高, 硝唑尼特给药可显著降低其水平。同时, 本课题组发现中长链脂肪酸12-methyl myristic acid、methoprene acid、2-cis-hexadecenoic acid、3-hydroxy-hexadecanoic acid、methoprene acid与甘油磷脂代谢中间产物1-palmitoyl-2-linoleoyl PE、lysoPI(20:4(5Z, 8Z, 11Z, 14Z)/0:0)、O-arachidonoyl glycidol的水平在西方饮食诱导的ApoE-/-小鼠肝脏中的含量显著升高, 硝唑尼特可抑制脂质相关内源性代谢物水平的增加, 提示硝唑尼特可改善FFA在肝脏积聚引起的甘油磷脂代谢紊乱。
肝细胞内脂质过度蓄积导致肝细胞脂肪变性, VLDL将肝脏合成的甘油三酯转运至外周, PC和PE在脂滴的形成以及VLDL的分泌和代谢中具有重要作用。肝脏中PC主要通过胞苷-5'-二磷酸胆碱途径生成, 也可通过PEMT以SAM为甲基供体经PE甲基化生成[30]。有研究发现, 高脂饮食诱导的PEMT-/-小鼠肝脏PC/PE比值降低, 肝脏TG含量是高脂饮食喂养的野生型小鼠的4倍, 表现出肝脏脂肪变性、炎症和纤维化的症状, 提示PEMT可能在维持肝脏PC/PE比值和脂质代谢中发挥重要作用[31]。本实验证明, 硝唑尼特可促进西方饮食诱导的ApoE-/-小鼠肝脏中PEMT蛋白表达。与体内实验一致的是, 硝唑尼特的活性代谢物替唑尼特可抑制FFA诱导的HepG2细胞和小鼠肝原代细胞中甘油三酯水平, 此外, 替唑尼特处理可提高FFA诱导的HepG2细胞PEMT蛋白水平, 表明硝唑尼特活性代谢物替唑尼特可通过提高PEMT表达减少肝细胞内脂质蓄积。
SAM/SAH比值是调控SAM依赖的甲基化反应的关键因素[32], 体内和体外实验结果表明硝唑尼特提高PEMT催化的甲基化反应, 并增加蛋氨酸循环向Cys的转化, 提示硝唑尼特可能通过增强SAHH对SAH的水解, 提高SAM/SAH比值以及Hcy向Cys转化的通量。此外, 研究表明AMPK可通过激活SAHH提高SAM依赖的甲基化反应, 提示硝唑尼特增强SAHH的作用可能是通过激活AMPK实现的。课题组之前的研究证明硝唑尼特及替唑尼特均能激活AMPK, 提示硝唑尼特及替唑尼特可能通过AMPK/SAHH通路提高PEMT对PE/PC的转化, 并增加蛋氨酸循环通过转硫途径生成的Cys水平, 增加Cys的可用性。
尿素循环是将机体内的氨以尿素形式代谢出体外的过程, 肝脏是尿素循环发生及氨清除的主要器官。Gallego-Durán等[24]研究表明MAFLD患者肝脏脂肪酸负荷增加, 扰乱线粒体功能, 尿素循环关键酶鸟氨酸氨甲酰转移酶(ornithine transcarbamylase, OTC) 和CPS1水平降低导致的肝脏氨的积聚, 引起肝星状细胞激活, 最终促进肝纤维化发展[33]。谷氨酰胺通过谷氨酰胺酶(glutaminase, GLS) 分解生成谷氨酸和NH3, NH3在CPS1催化下进一步合成氨基甲酰磷, 最终生成尿素排出体外, 乙酰辅酶A的产物NAG是CPS1的变构激活剂。当MAFLD患尿素循环受损时, 谷氨酰胺合成酶表达升高, 催化谷氨酸和NH3重新生成谷氨酰胺, 代偿性增加氨的清除[34]。西方饮食喂养的ApoE-/-小鼠肝脏谷氨酰胺水平升高, 且NAG水平显著降低, 提示西方饮食喂养的ApoE-/-小鼠肝脏尿素循环可能失调, 硝唑尼特口服途径给药可恢复小鼠肝脏谷氨酰胺和NAG水平。细胞学实验结果显示, FFA诱导的HepG2细胞Nags和Cps1 mRNA水平显著降低, 替唑尼特处理可恢复Nags和Cps1 mRNA水平。因此, 调节NAG水平和CPS1表达也可能是硝唑尼特改善肝脏功能障碍的机制之一。
除ACC外, 脂肪酸合酶、肉毒碱棕榈酰基转移酶1A以及白细胞分化抗原36均参与脂质代谢。本课题组前期研究证明硝唑尼特及替唑尼特可抑制ACC活性[10], 但本文未进一步研究替唑尼特对FAS、CPT1A及CD36的表达及活性的影响, 这是本文的局限性。
作者贡献: 朱胡太龙进行代谢组实验、数据分析和撰写草稿; 程晓范协助参与代谢组实验与数据分析并进行细胞实验; 郭欣负责查阅文献并参与数据分析; 常乐、赵银娣、吴尚泽负责图表制作; 董德利负责实验设计思路指导并修改论文。所有作者阅读并认可终稿。
利益冲突: 所有作者均声明不存在利益冲突。
基金
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  • 中国药科大学人才引进科研启动基金
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参考文献 引证文献
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2024年第59卷第9期
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doi: 10.16438/j.0513-4870.2024-0482
  • 接收时间：2024-05-20
  • 首发时间：2025-11-24
  • 出版时间：2024-09-12
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  • 收稿日期：2024-05-20
  • 修回日期：2024-07-05
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中国药科大学人才引进科研启动基金
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    1.中国药科大学药学院, 江苏 南京 211198
    2.杭州联川生物技术股份有限公司, 浙江 杭州 310018

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*董德利，E-mail:
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2种不同金属材料的力学参数
科
Family		 属数
Number of
genus		 种数
Number of
species		 占总种数比例
Percentage of
total species (%)		 属
Genus		 种数
Number of
species		 占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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