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Article(id=1153429501351547238, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1153429493357203682, articleNumber=null, orderNo=null, doi=10.19812/j.cnki.jfsq11-5956/ts.20241120002, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1732032000000, receivedDateStr=2024-11-20, revisedDate=null, revisedDateStr=null, acceptedDate=null, acceptedDateStr=null, onlineDate=1752928622806, onlineDateStr=2025-07-19, pubDate=1741968000000, pubDateStr=2025-03-15, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1752928622806, onlineIssueDateStr=2025-07-19, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1752928622806, creator=13701087609, updateTime=1752928622806, updator=13701087609, issue=Issue{id=1153429493357203682, tenantId=1146029695717560320, journalId=1149652044408987649, year='2025', volume='16', issue='5', pageStart='1', pageEnd='326', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=0, createTime=1752928620900, creator=13701087609, updateTime=1758690311058, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1177595773500932351, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1153429493357203682, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1177595773500932352, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1153429493357203682, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=69, endPage=75, ext={EN=ArticleExt(id=1153429501905195383, articleId=1153429501351547238, tenantId=1146029695717560320, journalId=1149652044408987649, language=EN, title=Effects of B-type proanthocyanidins trimer from Litchi chinensis Sonn pulp on regulating lipid metabolism in hepatocytes, columnId=1151895322591638525, journalTitle=Journal of Food Safety & Quality, columnName=Special Topic: Functional Foods and Functional Components, runingTitle=null, highlight=null, articleAbstract=

Objective To explore the dose effects and molecular mechanism of B-type proanthocyanidins trimer, the main active component of Litchi chinensis Sonn pulp phenolics, on hepatocyte triglyceride (TG) deposition. Methods HepG2 cell steatosis model induced by oleic acid (OA) was treated with different mass concentrations (0.5-10.0 μg/mL) of B-type proanthocyanidins trimer. The content of TG and the expressions of genes related to lipid absorption, transport and oxidation, together with apoptosis of hepatocytes, were detected in oleic acid loaded hepatocytes. Results B-type proanthocyanidins trimer (0.5-10.0 μg/mL) all significantly inhibited TG accumulation in oleic acid-loaded hepatocytes, while no dose dependence was observed. Low-dose (1.0 μg/mL) B-type proanthocyanidins trimer inhibited hepatocyte apoptosis by increasing the relative expression ratio of Bcl-2 to Bax, thereby reducing TG accumulation in hepatocytes. In addition to the above-mentioned pathway, medium/high-dose (5.0 μg/mL, 10.0 μg/mL) B-type proanthocyanidins trimer also inhibited lipid absorption in hepatocytes by down-regulating CD36 and FATP2 expression, and promoted hepatocytes lipolysis by up-regulating ACSL1 and CPT1α expression, hence reducing TG accumulation in hepatocytes. Conclusion B-type proanthocyanidins trimer of Litchi chinensis Sonn pulp can inhibit lipid absorption, promote β-oxidation of fatty acids and inhibit excessive apoptosis of liver tissue cells, thereby improving lipid metabolism and preventing fatty liver.

, correspAuthors=Kun HU, authorNote=null, correspAuthorsNote=null, copyrightStatement=null, 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=Guang-Yi LUO, Ye-Jie WEN, Rui-Fen ZHANG, Mei DENG, Ming-Wei ZHANG, Xu-Chao JIA, Kun HU), CN=ArticleExt(id=1153429529201725463, articleId=1153429501351547238, tenantId=1146029695717560320, journalId=1149652044408987649, language=CN, title=荔枝果肉B型原花青素三聚体调节肝细胞脂代谢作用研究, columnId=1151895323909124661, journalTitle=食品安全质量检测学报, columnName=本期专题：功能性食品与功能性成分, runingTitle=null, highlight=null, articleAbstract=

目的 探究荔枝果肉多酚主要活性组分B型原花青素三聚体抑制肝细胞甘油三酯(triglyceride, TG)沉积的剂量效应及其分子机制。方法 采用不同质量浓度(0.5~10.0 μg/mL) B型原花青素三聚体处理油酸(oleic acid, OA)诱导的HepG2细胞脂肪变性模型, 检测OA负荷肝细胞内TG含量及其与脂质吸收、转运、氧化和肝细胞凋亡相关基因表达。结果 0.5~10.0 μg/mL B型原花青素三聚体均能显著抑制OA负荷肝细胞内TG沉积, 但不呈现剂量依赖性; 低剂量(1.0 μg/mL) B型原花青素三聚体主要通过增强肝细胞Bcl-2和Bax表达量的比值, 抑制肝细胞凋亡进而减轻细胞TG沉积; 除上述途径外, 中、高剂量(5.0 μg/mL、10.0 μg/mL) B型原花青素三聚体还可通过下调肝细胞CD36和FATP2的表达抑制细胞脂质吸收, 同时上调肝细胞ACSL1和CPT1α的表达促进细胞脂质分解, 进而减轻肝细胞TG沉积。结论 荔枝果肉B型原花青素三聚体可以通过抑制肝细胞脂质吸收, 促进脂肪酸的β氧化以及抑制肝细胞过度凋亡, 进而改善肝脏脂质代谢紊乱。

, correspAuthors=胡坤, authorNote=null, correspAuthorsNote=
* 胡坤(1975—), 男, 博士, 教授, 主要研究方向为功能性食品。E-mail:
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罗广怡(1999—), 女, 硕士研究生, 主要研究方向为功能性食品。E-mail:

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罗广怡(1999—), 女, 硕士研究生, 主要研究方向为功能性食品。E-mail:

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罗广怡(1999—), 女, 硕士研究生, 主要研究方向为功能性食品。E-mail:

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注: 不同字母表示组间具有显著性差异(P<0.05), 下同。

, figureFileSmall=WoQz+NXCTUYSFCaqw8QPNA==, figureFileBig=0kkUhdG5e8weH/vB7WkQQA==, tableContent=null), ArticleFig(id=1177619646636241318, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153429501351547238, language=EN, label=Fig.3, caption=Effects of different mass concentrations of B-type proanthocyanidins trimer on the expression of genes involved in lipid uptake and transport in HepG2 cells stimulated by OA, figureFileSmall=uTbYifb9XS9zvSvBZrL45g==, figureFileBig=NtqASh9RHraidnh3P4mOtQ==, tableContent=null), ArticleFig(id=1177619646694961575, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153429501351547238, language=CN, label=图3, caption=不同质量浓度的B型原花青素三聚体对OA刺激的HepG2细胞内脂质吸收和转运相关基因表达的影响, figureFileSmall=uTbYifb9XS9zvSvBZrL45g==, figureFileBig=NtqASh9RHraidnh3P4mOtQ==, tableContent=null), ArticleFig(id=1177619646762070440, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153429501351547238, language=EN, label=Fig.4, caption=Effects of different mass concentrations of B-type proanthocyanidins trimer on the expression of genes related to lipid β-oxidation in HepG2 cells stimulated by OA, figureFileSmall=junCxAxhyvrunbabf0sLMQ==, figureFileBig=RAvJfxxTQZ62e+H4zqJ8Cg==, tableContent=null), ArticleFig(id=1177619646829179305, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153429501351547238, language=CN, label=图4, caption=不同质量浓度的B型原花青素三聚体对OA刺激的HepG2细胞内脂质β氧化相关基因表达的影响, figureFileSmall=junCxAxhyvrunbabf0sLMQ==, figureFileBig=RAvJfxxTQZ62e+H4zqJ8Cg==, tableContent=null), ArticleFig(id=1177619646883705258, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153429501351547238, language=EN, label=Fig.5, caption=Effects of different mass concentrations of B-type proanthocyanidins trimer on the expression of apoptosis-related genes in HepG2 cells stimulated by OA, figureFileSmall=/fb+EjjVQ8DsMIlyqYYtkA==, figureFileBig=1Bytdn9KsZ9+UZV+ZwO2Zg==, tableContent=null), ArticleFig(id=1177619646942425515, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153429501351547238, language=CN, label=图5, caption=不同质量浓度的B型原花青素三聚体对OA刺激的HepG2细胞凋亡相关基因表达的影响, figureFileSmall=/fb+EjjVQ8DsMIlyqYYtkA==, figureFileBig=1Bytdn9KsZ9+UZV+ZwO2Zg==, tableContent=null), ArticleFig(id=1177619647009534380, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153429501351547238, language=EN, label=Table 1, caption=

Primer sequence

, figureFileSmall=null, figureFileBig=null, tableContent=
基因名称 引物(5'-3') 引物长度/bp
18S rRNA
(内源参照
基因)		 正向 CGGCGGCTTTGGTGACTCTAGA 201
反向 CCTGCTGCCTTCCTTGGATGTG
FATP2 正向 GTGAGACTGGCACTGGGAAATGG 105
反向 GCCTTCAGTGGCAGCATAGAACT
FATP5 正向 GGGTAGGGATCAAAGCCAGCCA 107
反向 CAGCCCACTGAGGTTGAGGGTA
CPT1α 正向 GCGATGGCTGAACTGTTGGAGT 216
反向 GGCATGGTGTTGGGCGTGTAAT
BAX 正向 TCCACCAAGAAGCTGAGCGAGT 95
反向 TCTGTGTCCACGGCGGCAAT
ACSL1 正向 CGTGCGTACTCTTCCGACCAAC 138
反向 CTACCCGCCACTTCCACTGACT
CD36 正向 TAACCCAGGACGCTGAGGACAAC 122
反向 TGCCACAGCCAGATTGAGAACTG
BCL-2 正向 TGGACAACATCGCCCTGTGGAT 110
反向 TCAGCCCAGACTCACATCACCA
), ArticleFig(id=1177619647101809069, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1153429501351547238, language=CN, label=表1, caption=

引物序列

, figureFileSmall=null, figureFileBig=null, tableContent=
基因名称 引物(5'-3') 引物长度/bp
18S rRNA
(内源参照
基因)		 正向 CGGCGGCTTTGGTGACTCTAGA 201
反向 CCTGCTGCCTTCCTTGGATGTG
FATP2 正向 GTGAGACTGGCACTGGGAAATGG 105
反向 GCCTTCAGTGGCAGCATAGAACT
FATP5 正向 GGGTAGGGATCAAAGCCAGCCA 107
反向 CAGCCCACTGAGGTTGAGGGTA
CPT1α 正向 GCGATGGCTGAACTGTTGGAGT 216
反向 GGCATGGTGTTGGGCGTGTAAT
BAX 正向 TCCACCAAGAAGCTGAGCGAGT 95
反向 TCTGTGTCCACGGCGGCAAT
ACSL1 正向 CGTGCGTACTCTTCCGACCAAC 138
反向 CTACCCGCCACTTCCACTGACT
CD36 正向 TAACCCAGGACGCTGAGGACAAC 122
反向 TGCCACAGCCAGATTGAGAACTG
BCL-2 正向 TGGACAACATCGCCCTGTGGAT 110
反向 TCAGCCCAGACTCACATCACCA
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荔枝果肉B型原花青素三聚体调节肝细胞脂代谢作用研究
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罗广怡 1 , 温叶杰 2 , 张瑞芬 2 , 邓梅 2 , 张名位 2 , 贾栩超 2 , 胡坤 1, *
食品安全质量检测学报 | 本期专题：功能性食品与功能性成分 2025,16(5): 69-75
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食品安全质量检测学报 | 本期专题：功能性食品与功能性成分 2025, 16(5): 69-75
荔枝果肉B型原花青素三聚体调节肝细胞脂代谢作用研究
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罗广怡1 , 温叶杰2, 张瑞芬2, 邓梅2, 张名位2, 贾栩超2, 胡坤1, *
作者信息
  • 1.广东药科大学食品科学学院, 中山 528400
  • 2.广东省农业科学院蚕业与农产品加工研究所, 农业农村部功能食品重点实验室, 广东省农产品加工重点实验室, 广州 510000

    • 罗广怡(1999—), 女, 硕士研究生, 主要研究方向为功能性食品。E-mail:

通讯作者:

* 胡坤(1975—), 男, 博士, 教授, 主要研究方向为功能性食品。E-mail:
Effects of B-type proanthocyanidins trimer from Litchi chinensis Sonn pulp on regulating lipid metabolism in hepatocytes
Guang-Yi LUO1 , Ye-Jie WEN2, Rui-Fen ZHANG2, Mei DENG2, Ming-Wei ZHANG2, Xu-Chao JIA2, Kun HU1, *
Affiliations
  • 1. College of Food Science, Guangdong Pharmaceutical University, Zhongshan 528400, China
  • 2. Sericultural & Agri-food Research Institute Guangdong Academy of Agricultural Sciences, Key Laboratory of Functional Foods, Ministry of Agriculture, Guangdong Key Laboratory of Agricultural Products Processing, Guangzhou 510000, China
出版时间: 2025-03-15 doi: 10.19812/j.cnki.jfsq11-5956/ts.20241120002
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摘要
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目的 探究荔枝果肉多酚主要活性组分B型原花青素三聚体抑制肝细胞甘油三酯(triglyceride, TG)沉积的剂量效应及其分子机制。方法 采用不同质量浓度(0.5~10.0 μg/mL) B型原花青素三聚体处理油酸(oleic acid, OA)诱导的HepG2细胞脂肪变性模型, 检测OA负荷肝细胞内TG含量及其与脂质吸收、转运、氧化和肝细胞凋亡相关基因表达。结果 0.5~10.0 μg/mL B型原花青素三聚体均能显著抑制OA负荷肝细胞内TG沉积, 但不呈现剂量依赖性; 低剂量(1.0 μg/mL) B型原花青素三聚体主要通过增强肝细胞Bcl-2和Bax表达量的比值, 抑制肝细胞凋亡进而减轻细胞TG沉积; 除上述途径外, 中、高剂量(5.0 μg/mL、10.0 μg/mL) B型原花青素三聚体还可通过下调肝细胞CD36和FATP2的表达抑制细胞脂质吸收, 同时上调肝细胞ACSL1和CPT1α的表达促进细胞脂质分解, 进而减轻肝细胞TG沉积。结论 荔枝果肉B型原花青素三聚体可以通过抑制肝细胞脂质吸收, 促进脂肪酸的β氧化以及抑制肝细胞过度凋亡, 进而改善肝脏脂质代谢紊乱。

荔枝  /  B型原花青素三聚体  /  肝细胞脂肪变性  /  分子机制

Objective To explore the dose effects and molecular mechanism of B-type proanthocyanidins trimer, the main active component of Litchi chinensis Sonn pulp phenolics, on hepatocyte triglyceride (TG) deposition. Methods HepG2 cell steatosis model induced by oleic acid (OA) was treated with different mass concentrations (0.5-10.0 μg/mL) of B-type proanthocyanidins trimer. The content of TG and the expressions of genes related to lipid absorption, transport and oxidation, together with apoptosis of hepatocytes, were detected in oleic acid loaded hepatocytes. Results B-type proanthocyanidins trimer (0.5-10.0 μg/mL) all significantly inhibited TG accumulation in oleic acid-loaded hepatocytes, while no dose dependence was observed. Low-dose (1.0 μg/mL) B-type proanthocyanidins trimer inhibited hepatocyte apoptosis by increasing the relative expression ratio of Bcl-2 to Bax, thereby reducing TG accumulation in hepatocytes. In addition to the above-mentioned pathway, medium/high-dose (5.0 μg/mL, 10.0 μg/mL) B-type proanthocyanidins trimer also inhibited lipid absorption in hepatocytes by down-regulating CD36 and FATP2 expression, and promoted hepatocytes lipolysis by up-regulating ACSL1 and CPT1α expression, hence reducing TG accumulation in hepatocytes. Conclusion B-type proanthocyanidins trimer of Litchi chinensis Sonn pulp can inhibit lipid absorption, promote β-oxidation of fatty acids and inhibit excessive apoptosis of liver tissue cells, thereby improving lipid metabolism and preventing fatty liver.

Litchi chinensis Sonn  /  B-type proanthocyanidins trimer  /  steatosis of hepatocytes  /  molecular mechanism
罗广怡, 温叶杰, 张瑞芬, 邓梅, 张名位, 贾栩超, 胡坤. 荔枝果肉B型原花青素三聚体调节肝细胞脂代谢作用研究. 食品安全质量检测学报, 2025 , 16 (5) : 69 -75 . DOI: 10.19812/j.cnki.jfsq11-5956/ts.20241120002
Guang-Yi LUO, Ye-Jie WEN, Rui-Fen ZHANG, Mei DENG, Ming-Wei ZHANG, Xu-Chao JIA, Kun HU. Effects of B-type proanthocyanidins trimer from Litchi chinensis Sonn pulp on regulating lipid metabolism in hepatocytes[J]. Journal of Food Safety & Quality, 2025 , 16 (5) : 69 -75 . DOI: 10.19812/j.cnki.jfsq11-5956/ts.20241120002
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0 引言
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随着社会节奏的加快和人民生活水平的提高, 我国非酒精性脂肪性肝病(nonalcoholic fatty liver disease, NAFLD)的患病率逐年攀升, 且从2016年到2030年, 我国NAFLD相关死亡率将增加22%, NAFLD已成为严重威胁人类健康的公共卫生事件[1-2]。NAFLD是一种进行性疾病, 其发病机制尚未完全阐明, 缺乏特异性治疗药物, 因此寻找安全高效的防治方法已成为公共卫生目标。越来越多的证据表明, 植物膳食中的多酚[3-4]、多糖[5]、皂苷[6]、生物碱[7]等活性成分在调节NAFLD中呈现积极作用。CUI等[8]采用葡萄果皮酚处理棕榈酸(palmitic acid, PA)诱导的HepG2细胞脂质变性模型, 结果表明葡萄果皮多酚能显著降低PA刺激HepG2细胞内甘油三酯(triglyceride, TG)水平并上调脂质分解因子CPT1α和PPARA的mRNA表达, 进而减轻HepG2细胞内脂质蓄积。SU等[9]采用原花青素B2处理游离脂肪酸(free fatty acid, FFAs)诱导的HepG2细胞脂质积累模型, 发现原花青素B2可减轻FFAs引发的细胞内活性氧自由基(reactive oxide species, ROS)及超氧阴离子(superoxide anion, O2-)的水平, 提高超氧化物歧化酶(superoxide dismutase, SOD)与过氧化氢酶(catalase, CAT)的活性。这表明, 原花青素B2通过调控抗氧化酶体系, 有效抑制了FFAs引起的细胞氧化应激反应, 进而减轻HepG2细胞内脂质蓄积。可见, 不同类型的多酚改善NAFLD作用机制并不相同。
荔枝(Litchi chinensis Sonn)属无患子科植物, 是亚热带地区特色水果, 也是广东省最主要的经济作物水果。荔枝果肉富含酚类物质, 主要为原花青素类物质[10]。荔枝果肉原花青素主要包括B型原花青素二聚体和三聚体, 其中三聚体在果肉中含量较高。课题组前期研究发现, 荔枝果肉原花青素具有抗氧化[11]、调节脂质代谢[3,12]等多种生物活性; 进一步通过半制备液相分离制备荔枝果肉多酚提取物中主要酚类成分群, 比较各酚类成分群对油酸(oleic acid, OA)负荷肝细胞内TG含量的影响, 进而结合高效液相色谱-串联质谱法(high performance liquid chromatography- tandem mass spectrometry, HPLC-MS/MS)对活性酚类成分群进行结构解析, 发现荔枝果肉B型原花青素三聚体为其抑制肝细胞TG沉积的主效成分, 但B型原花青素三聚体调节肝细胞脂质代谢的具体机制尚未可知。因此, 本研究拟通过OA诱导的肝细胞脂肪变性模型, 探究荔枝果肉主要活性成分(即B型原花青素三聚体)改善肝细胞脂肪变性的分子机制。研究结果有助于阐明荔枝的健康效应机制, 亦为荔枝的精准营养加工提供理论依据。
1 材料与方法
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1.1 材料与试剂
荔枝果肉B型原花青素三聚体由课题组前期分离制备[13]; HepG2细胞(中国科学院细胞库); 磷酸缓冲盐溶液(phosphate buffered saline, PBS)、胰酶、必需基础培养基(minimum essential medium, MEM)、非必需氨基酸溶液及胎牛血清(fetal bovine serum, FBS)(美国Thermo Fisher Scientific公司); 噻唑蓝[3-(4,5-dimethyl-2-thiazolyl)- 2,5-diphenyl-2-H-tetrazolium bromide‌‌, MTT]细胞增殖及细胞毒性检测试剂盒、甘油三酯检测试剂盒、二辛可酸(bicinchoninic acid, BCA)法蛋白质浓度测定试剂盒(南京建成生物工程研究所); M-MLV逆转录酶(reverse transcriptase M-MLV, RNase H-)试剂盒(北京TAKARA公司); OA及牛血清白蛋白(bovine serum albumin, BSA)(美国Sigma公司) 二甲基亚砜(dimethyl sulfoxide, DMSO)(天津大茂化学试剂厂)。
1.2 仪器与设备
UV-1800型紫外可见分光光度计(日本岛津公司); Infinite M200pro酶标仪(瑞士Tecan公司); HeraCell 240i CO2培养箱(美国Thermo Fisher Scientific公司); DMI 3000B型倒置荧光显微镜(德国Leica公司); ABI ViiA7荧光定量聚合酶链反应(polymerase chain reaction, PCR)仪(美国ABI公司); 5702R台式低速冷冻离心机(德国Eppendorf公司)。
1.3 实验方法
1.3.1 细胞培养
在37 ℃、5% (V:V) CO2的细胞培养条件下, 将HepG2细胞培养于含有10% FBS的MEM培养基中。待细胞贴壁生长融合接近80%后, 弃去培养液, 用PBS清洗后加入一定量的胰酶消化, 当显微镜下细胞慢慢皱缩出现间隙时, 立即向培养瓶中加入培养液终止消化, 1000 r/min离心5 min, 取适量细胞进行传代或实验, 每次传代或实验均使用对数生长期的细胞。
1.3.2 MTT法测定细胞活力
取100 μL对数生长期的细胞, 将浓度为5×104个/mL的细胞接种至96孔培养板, 随后在37 ℃、5% (V:V) CO2的细胞培养环境中孵育24 h, 吸去培养液, PBS清洗后, 分别加入含不同质量浓度B型原花青素三聚体(0、10、50、80、100、200、400、600、1000 μg/mL)的1% BSA无血清培养液, 在37 ℃、5% (V:V) CO2的细胞培养环境中孵育24 h后, 向各孔添加50 μL MTT试剂, 继续在37 ℃条件下培养4 h。随后, 移除培养液, 每孔加入150 μL DMSO, 振荡使其形成的结晶完全溶解后在570 nm的波长下测定每孔的吸光值。B型原花青素三聚体对细胞的毒性用细胞存活率表示, 计算公式如公式(1)所示。
细胞存活率/%=[(OD加样细胞-OD空白)/
(OD对照细胞-OD空白)]×100%
1.3.3 B型原花青素三聚体对OA刺激的HepG2细胞内TG含量的影响
参照董丽红等[14-15]的方法, 采用OA诱导肝细胞脂肪变性模型, 进而探究B型原花青素三聚体对脂肪变性肝细胞内TG含量的影响。以5×105个/mL的浓度, 将HepG2细胞接种至12孔培养板内, 并置于培养箱中孵育24 h。移除原有培养液, 经PBS洗涤后, 加入含有1% BSA的无血清培养基, 继续培养14 h后, 弃去培养液。空白组(NC)加入1 mL 1% BSA无血清培养液、模型组(MC)加入1 mL含有40 mmol/L OA的1% BSA无血清培养液、样品组加入1 mL含有40 mmol/L OA和不同质量浓度B型原花青素三聚体的1% BSA无血清培养, 置于培养箱中培养24 h, 弃去培养液, PBS清洗后, 按照测定TG试剂盒的操作方法, 每孔加入130 μL细胞裂解液, 裂解10 min, 取裂解液用于TG和蛋白含量的测定, 其中蛋白含量的测定遵循BCA蛋白浓度测定试剂盒的操作指南进行。以每mg蛋白浓度为基础, 对细胞内的TG含量进行校正, 并选取OA模型组作为参照, 进一步计算各组中TG的相对含量。
1.3.4 实时荧光定量聚合酶链式反应测定脂代谢相关基因的表达
参照赵广河[16]的方法, 收集细胞, 用TrizoL试剂提取总RNA。通过分光光度计检测RNA的浓度及其纯度, 当A260/A280的比值落在1.8至2.0区间时, RNA适合进行荧光定量检测。随后, 依据RNase H-试剂盒的说明书执行逆转录步骤并开展实时荧光定量PCR分析。
逆转录体系的配制方法如下: 准备1.0 μg RNA、0.5 μL Oligo (dT) 18引物、0.5 μL随机引物, 以及10 μL无RNase的ddH2O。混合以上成分, 在70 ℃下加热10 min, 然后迅速冷却1 min。向体系中依次加入0.5 μL 10 mmol/L的dNTP混合物、0.25 μL 40 U/μL的RNase抑制剂、4.0 μL 5倍浓度的M-MLV缓冲液、以及0.5 μL RNase H-。用ddH2O将总体积调整至20 μL, 充分混匀。在42 ℃下恒温孵育60 min, 然后在72 ℃下处理15 min以灭活酶。将反应产物置于-20 ℃保存, 以备后续使用。
设计特异性的正反向引物, 以18S rRNA为内源参照基因(引物见表1)。采用荧光染料SYBR Green I方法, 其反应组成为: 正向引物、反向引物各0.5 μL, 10 μL AceQTM PCR SYBR® Green Master Mix, 2 μL稀释10倍的cDNA, 7 μL ddH2O。使用ViiA7系统软件设定反应程序: 95 ℃ 1 min, 95 ℃ 10 s, 59 ℃ 20 s, 45个循环; 60~95 ℃进行溶解曲线分析。利用Applied Biosystems ViiA7实时荧光定量PCR仪检测各个基因的循环阈(cycle threshold, Ct)值, 所有反应均设3个复孔。根据Comparative Delta-Delta Ct法△△Ct=(Ct处理组基因-Ct内参基因)-(Ct对照组基因-Ct内参基因), 使用2-△△Ct计算各个处理组基因相对于MC组的表达量。
1.4 数据处理
实验数据以平均值±标准偏差表示, 采用SPSS 18.0软件和Origin 8.0软件进行统计分析和绘图。利用单因素方差分析进行组间比较, P<0.05将认为有统计学差异。
2 结果与分析
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2.1 B型原花青素三聚体的细胞毒性
采用MTT法测定B型原花青素三聚体的细胞毒性, 结果如图1所示。采用0~1000 μg/mL质量浓度范围的B型原花青素三聚体处理HepG2细胞24 h, 细胞存活率均在90%以上, 说明该质量浓度范围下B型原花青素三聚体对HepG2细胞增殖无明显影响和毒性作用。
2.2 B型原花青素三聚体对OA刺激的HepG2细胞内TG含量的影响
B型原花青素三聚体对OA刺激的HepG2细胞内TG含量的影响如图2所示, 与NC组相比, OA刺激的HepG2细胞内TG含量显著增加(P<0.05), 是前者的9.23倍, 而不同质量浓度的B型原花青素三聚体处理均能显著降低OA刺激的HepG2细胞内TG含量(P<0.05), 但不呈现剂量效应。PARK等[17]采用荔枝果肉原花青素低聚体处理PA诱导的脂肪变性的HepG2细胞, 发现荔枝果肉原花青素低聚体显著减轻了PA刺激的HepG2细胞内TG的沉积。LI等[18]采用苹果多酚提取物处理OA+PA诱导的HepG2细胞脂质蓄积, 结果显示苹果多酚提取物可以显著降低细胞内TG含量, 改善细胞脂滴沉积。在剂量上, LI等[18]使用的浓度为2.0 mg/mL, 本研究使用最大浓度为10.0 μg/mL即可明显改善脂质沉积。
2.3 B型原花青素三聚体调节肝细胞脂质代谢的分子机制
2.3.1 B型原花青素三聚体对HepG2细胞脂质吸收和转运的影响
如前所述, 0.5~10.0 μg/mL荔枝果肉B型原花青素三聚体均能显著抑制OA刺激的肝细胞内TG沉积, 本研究进一步选取了1.0、5.0、10.0 μg/mL B型原花青素三聚体处理组, 分别为低(L-PC)、中(M-PC)、高剂量(H-PC)处理组, 探究B型原花青素三聚体改善肝细胞脂质代谢的分子机制。CD36、FATP是脂肪酸吸收的相关基因[19]。CD36是一种重要的脂肪酸转运体, 其在肿瘤细胞中高表达, 通过参与脂肪酸摄取、脂质从头合成等促进脂质沉积[20]。NAFLD患者肝脏中CD36表达及胞膜定位相较于健康人群显著增加[21]。脂肪酸转运蛋白(fatty acid transport protein, FATP)家族是重要的脂肪摄取蛋白质家族, 主要是将外周循环中的脂肪酸转入细胞内, FATP2和FATP5基因主要在肝细胞中表达, 其中FATP2在HepG2细胞中具有较高的表达[22]。不同剂量B型原花青素三聚体处理OA负荷的HepG2细胞后, 与脂质吸收转运相关基因表达结果如图3所示。与MC组相比, 低剂量B型原花青素三聚体显著上调了CD36和FATP2的表达, 中、高剂量B型原花青素三聚体则显著下调了CD36和FATP2的表达(P<0.05), 但上述剂量B型原花青素三聚体对FATP5的表达无显著影响, 说明中、高剂量B型原花青素三聚体可以有效下调OA刺激的脂肪变性肝细胞中CD36和FATP2的表达, 进而抑制肝细胞脂质吸收。TANAKA等[23]采用没食子酸处理PA诱导的HepG2细胞脂肪变性模型, 发现低(8.5 μg/mL)、中剂量(17.0 μg/mL)没食子酸处理不能显著下调脂肪变性肝细胞中FATP2基因表达, 而高剂量(34.0 μg/mL)没食子酸能显著下调FATP2基因表达。钟艳花等[24]采用厚朴酚处理OA+PA诱导的HepG2细胞脂肪变性模型, 发现中剂量(2.7 μg/mL)、高剂量(10.7 μg/mL)厚朴酚可显著抑制CD36、FATP2等脂质合成和转运相关基因表达, 低剂量(0.7 μg/mL)无此作用, 由此可知, 酚类物质需达到一定剂量才能发挥调节肝细胞脂质代谢相关基因表达的作用, 这与本研究结果相一致。
2.3.2 B型原花青素三聚体对HepG2细胞脂质β氧化的影响
组织中脂肪酸β氧化是脂质分解转化的重要途径。在脂质β氧化途径中, 首要步骤涉及脂肪酸向脂酰辅酶A (coenzyme A, CoA)的活化过程。随后, 该活化产物进入线粒体基质内, 并在脂肪酸β氧化酶系的逐步催化作用下, 经历脱氢、加水、再次脱氢及硫解这一系列反应。此过程最终导致一分子乙酰CoA与一分子碳链缩短两个碳单位的脂酰CoA的生成与释放。‌当这一过程出现异常时, ‌可能会导致脂肪肝的形成。‌ACSL1和CPT1α是脂质β氧化的相关基因[25]。ACSL1主要把细胞内的长链脂肪酸活化进而生成脂酰CoA[22]。CPT1α则是脂肪酸β-氧化的限速酶, 可以和脂酰辅酶A合成酶(acyl-CoA synthetase, ACS)共同作用, 将脂酰CoA转移到线粒体中完成脂肪酸的β氧化[26]。不同剂量B型原花青素三聚体处理OA负荷的HepG2细胞后, 细胞ACSL1和CPT1α基因表达结果如图4所示。与MC组相比, 低剂量B型原花青素三聚体显著下调ACSL1的表达, 而中、高剂量B型原花青素三聚体显著上调ACSL1的表达(P<0.05)。低、中剂量B型原花青素三聚体对CPT1α表达无显著影响, 但高剂量B型原花青素三聚体能显著上调CPT1α表达(P<0.05)。由此可知, 中、高剂量B型原花青素三聚体可通过上调ACSL1和CPT1α等参与肝细胞脂质β氧化基因的表达, 促进肝细胞脂质分解转化, 进而减轻细胞内TG沉积。前人研究指出, 芒果苷[27]、槲皮素[28]等酚类物质也可以通过上调肝脏组织和细胞中PPARα及CPT1α等基因的表达来促进脂质分解转化, 进而调节脂质代谢平衡, 上述研究结果与本研究相一致。
2.3.3 B型原花青素三聚体对HepG2细胞凋亡的影响
肝细胞凋亡与肝损伤、炎症和纤维化有关, 是造成脂肪肝发生发展的重要因素[29]。Bax和Bcl-2是控制细胞凋亡的基因, 在哺乳动物细胞凋亡过程中, Bax和Bcl-2是一对正、负调控剂。在正常情况下, 位于胞质的Bax在接收到死亡信息后, 可以通过改变自身的构象穿插到线粒体膜中进而形成Bax-Bax二聚体, 开启线粒体通透性的转换孔, 从而影响线粒体跨膜电位和细胞色素C, 细胞色素C与凋亡蛋白活化因子结合形成凋亡小体完成凋亡, 而凋亡的肝细胞将会促进肝纤维化的发生。位于线粒体外膜上的Bcl-2可以与Bax蛋白竞争性结合进而形成Bax-Bcl-2二聚体, 终止凋亡。Bcl-2和Bax基因表达量的比值(Bcl-2/Bax)可以用于评价细胞凋亡[30]。若Bcl-2/Bax比值上升, 表明抑制细胞凋亡, 反之则促进细胞凋亡。不同剂量B型原花青素三聚体处理OA负荷的HepG2细胞后, Bcl-2和Bax表达量的比值结果如图5所示。与MC组相比, 不同剂量B型原花青素三聚体均能显著增加Bcl-2/Bax的比值(P<0.05), 但低剂量和中剂量组效果相当, 均显著强于高剂量组(P<0.05), 说明不同剂量B型原花青素三聚体可以通过抑制HepG2细胞凋亡减轻肝细胞脂质蓄积。禹萍[31]采用蓝莓提取原浆灌胃NAFLD大鼠, 发现蓝莓提取原浆能够有效增加NAFLD大鼠肝组织Bcl-2表达, 降低Bax表达, 增加Bcl-2/Bax的比值, 提高机体的抗氧化应激能力从而抑制肝细胞凋亡, 研究结果与本研究相一致。
3 结论
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本研究以40 mmol/L OA诱导人肝癌HepG2细胞发生脂质沉积, 建立单纯性肝细胞脂肪变性模型, 探究了荔枝果肉多酚主要活性成分B型原花青素三聚体改善肝细胞脂肪变性的分子机制。研究结果表明, 0.5~10.0 μg/mL B型原花青素三聚体均能显著抑制OA负荷肝细胞内TG沉积, 但不呈现剂量依赖性; 低剂量B型原花青素三聚体主要通过增强肝细胞Bcl-2和Bax表达量的比值, 抑制肝细胞凋亡进而减轻细胞TG沉积; 除上述途径外, 中、高剂量B型原花青素三聚体还可通过下调肝细胞CD36和FATP2的表达抑制细胞脂质吸收, 同时上调肝细胞ACSL1和CPT1α的表达促进细胞脂质分解, 进而减轻肝细胞TG沉积。本研究结果将丰富酚类成分改善脂质代谢紊乱的理论基础, 并为荔枝的精准营养加工提供理论依据。
基金
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  • 2024年广东省级乡村振兴战略专项(2024KJ15)
  • 广东省特支计划本土创新团队项目(2019BT02N112)
文献
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2025年第16卷第5期
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doi: 10.19812/j.cnki.jfsq11-5956/ts.20241120002
  • 接收时间：2024-11-20
  • 首发时间：2025-07-19
  • 出版时间：2025-03-15
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  • 收稿日期：2024-11-20
基金
2024年广东省级乡村振兴战略专项(2024KJ15)
广东省特支计划本土创新团队项目(2019BT02N112)
作者信息
    1.广东药科大学食品科学学院, 中山 528400
    2.广东省农业科学院蚕业与农产品加工研究所, 农业农村部功能食品重点实验室, 广东省农产品加工重点实验室, 广州 510000

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* 胡坤(1975—), 男, 博士, 教授, 主要研究方向为功能性食品。E-mail:
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2种不同金属材料的力学参数
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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
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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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