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Article(id=1216517522048864499, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1216517514570417012, articleNumber=null, orderNo=null, doi=10.19812/j.cnki.jfsq11-5956/ts.20250218006, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1739808000000, receivedDateStr=2025-02-18, revisedDate=null, revisedDateStr=null, acceptedDate=null, acceptedDateStr=null, onlineDate=1767969979058, onlineDateStr=2026-01-09, pubDate=1755187200000, pubDateStr=2025-08-15, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1767969979058, onlineIssueDateStr=2026-01-09, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1767969979058, creator=13701087609, updateTime=1767969979058, updator=13701087609, issue=Issue{id=1216517514570417012, tenantId=1146029695717560320, journalId=1149652044408987649, year='2025', volume='16', issue='15', pageStart='1', pageEnd='322', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1767969977276, creator=13701087609, updateTime=1768211590858, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1217530915467743720, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1216517514570417012, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1217530915467743721, tenantId=1146029695717560320, journalId=1149652044408987649, issueId=1216517514570417012, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=232, endPage=238, ext={EN=ArticleExt(id=1216517523277795634, articleId=1216517522048864499, tenantId=1146029695717560320, journalId=1149652044408987649, language=EN, title=Protective effects of Chuanxiong tea on oxidative stress induced by 2,2'-azobis(2-methylpropionamidine) dihydrochloride in LLC-PK1 cells, columnId=1151923894560060071, journalTitle=Journal of Food Safety & Quality, columnName=Food Nutrition and Functional Foods, runingTitle=null, highlight=null, articleAbstract=

Objective To investigate the protective effects of ethanol extract of Chuangxiong tea on the 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPH) (1 mmol/L) on oxidative stress injury of proximal renal tubule epithelial cells of pigs. Methods LLC-PK1 cells were co-cultured with ethanol extract of Chuanxiong tea (labeled CXTEE) with different mass concentrations ranging from 10 to 100 μg/mL for 24 h, and then placed in Dulbecco's modified eagle medium (DMEM) containing AAPH for 4 h to establish the cell damage model. The cell survival rate was measured by tetramethyl azazole blue (MTT) method. The content of malondialdehyde (MDA) and the level of total reactive oxygen species (ROS) were determined by thiobarbituric acid colorimetry and 2',7'-dihydrodichlorofluorescein yellow diacetate (DCFH-DA) probe technique, respectively. In addition, the activity of several antioxidant enzymes, including catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), glutathione S-transferase (GST), γ-glutamylcysteine synthetase (γ-GCs), and the concentration of glutathione (GSH), were also evaluated using specific kits. Results The results showed that the cells pretreated with CXTEE showed a higher survival rate, and the total ROS level and MDA production in the cells were significantly reduced. At the same time, the extract also enhanced the activity of antioxidant enzymes (such as CAT, SOD, GSH-Px and GST) in the damaged cells, promoted the activity of γ-GCS and increased the content of GSH. Further experiments showed that CXTEE could also up-regulate the mRNA expression levels of SOD, GSH-Px and CAT in oxidative damage LLC-PK1 cells. Conclusion The ethanol extract of Chuangxiong tea may reduce the levels of MDA and ROS by strengthening the antioxidant defense system of cells, thus alleviating the oxidative stress damage caused by AAPH to LLC-PK1 cells. This study can provide a basis for further application and development of Chuangxiong tea.

, correspAuthors=Hong-Bin ZHAN, 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=Si-Min LIN, Li-Dan SHI, Hong-Bin ZHAN), CN=ArticleExt(id=1216517527660843525, articleId=1216517522048864499, tenantId=1146029695717560320, journalId=1149652044408987649, language=CN, title=川芎茶对2,2'-盐酸脒基丙烷诱发LLC-PK1细胞氧化应激的保护作用, columnId=1151923894698472105, journalTitle=食品安全质量检测学报, columnName=食品营养及功能性食品, runingTitle=null, highlight=null, articleAbstract=

目的 探讨川芎茶的乙醇提取物对由2,2'-盐酸脒基丙烷[2,2'-azobis (2-methylpropionamidine) dihydrochloride, AAPH, 1 mmol/L]诱导产生的LLC-PK1猪肾近端小管上皮细胞氧化应激损伤的保护效果。方法 将LLC-PK1细胞与不同质量浓度范围(10~100 μg/mL)的川芎茶乙醇提取物(记作CXTEE)共培养24 h后, 再将其置于含有AAPH的杜尔贝科改良伊格尔培养基(Dulbecco's modified eagle medium, DMEM)中继续培养4 h以建立细胞受损模型。通过四甲基偶氮唑蓝(tetramethyl azazole blue, MTT)法检测细胞存活率; 利用硫代巴比妥酸比色分析法和2',7'-二氢二氯荧光黄双乙酸钠(2',7'-dihydrodichlorofluorescein yellow diacetate, DCFH-DA)探针技术分别测定细胞内丙二醛(malondialdehyde, MDA)含量及总活性氧(reactive oxygen species, ROS)水平; 此外, 还采用特定试剂盒评估了包括过氧化氢酶(catalase, CAT)、超氧化物歧化酶(superoxide dismutase, SOD)、谷胱甘肽过氧化物酶(glutathione peroxidase, GSH-Px)、谷胱甘肽S-转移酶(glutathione S-transferase, GST)、γ-谷氨酰半胱氨酸合成酶(γ-glutamylcysteine synthetase, γ-GCS)在内的多种抗氧化酶活性以及谷胱甘肽(glutathione, GSH)的浓度。结果 经过CXTEE预处理后的细胞表现出更高的存活率, 并且细胞内的总ROS水平与MDA生成量显著降低。同时, 该提取物还能增强受损细胞中的抗氧化酶活性(如CAT、SOD、GSH-Px和GST), 并促进γ-GCS活性及提高GSH含量。进一步的实验显示CXTEE还可以上调氧化损伤LLC-PK1细胞中SOD、GSH-Px和CAT的mRNA表达水平。结论 川芎茶乙醇提取物可能通过强化细胞自身的抗氧化防御系统来减少MDA和ROS水平, 从而减轻AAPH对LLC-PK1细胞造成的氧化应激损害。本研究可为川芎茶的进一步应用开发提供依据。

, correspAuthors=占洪斌, authorNote=null, correspAuthorsNote=
*占洪斌(1989—), 男, 硕士, 主治医师, 主要研究方向为肾脏医学。E-mail:
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林思敏(1995—), 女, 医师, 主要研究方向为肾脏医学。E-mail:

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注: 不同小写字母标记代表各组之间存在显著性差异(P<0.05), 下同。

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Effects of CXTEE on the activities of CAT, SOD, GSH-Px and GST in LLC-PK1 cells induced by AAPH

, figureFileSmall=null, figureFileBig=null, tableContent=
组别 质量浓度/(μg/mL) CAT活力/(U/mg prot) SOD活力/(U/mg prot) GSH-Px活力/(U/mg prot) GST活力/(U/mg prot)
正常组 / 2.02±0.21a 5.52±0.37a 3.66±0.26a 1.02±0.12a
CXTEE 0 0.65±0.07e 1.51±0.20e 1.31±0.14e 0.33±0.04e
10 0.89±0.08d 2.66±0.26d 1.93±0.16d 0.49±0.04d
50 1.26±0.16c 3.12±0.23c 2.32±0.18c 0.65±0.07c
100 1.63±0.15b 4.33±0.32b 2.89±0.21b 0.83±0.05b
), ArticleFig(id=1217127897865835031, tenantId=1146029695717560320, journalId=1149652044408987649, articleId=1216517522048864499, language=CN, label=表1, caption=

CXTEE对AAPH诱导的LLC-PK1细胞中CAT、SOD、GSH-Px及GST活性的影响

, figureFileSmall=null, figureFileBig=null, tableContent=
组别 质量浓度/(μg/mL) CAT活力/(U/mg prot) SOD活力/(U/mg prot) GSH-Px活力/(U/mg prot) GST活力/(U/mg prot)
正常组 / 2.02±0.21a 5.52±0.37a 3.66±0.26a 1.02±0.12a
CXTEE 0 0.65±0.07e 1.51±0.20e 1.31±0.14e 0.33±0.04e
10 0.89±0.08d 2.66±0.26d 1.93±0.16d 0.49±0.04d
50 1.26±0.16c 3.12±0.23c 2.32±0.18c 0.65±0.07c
100 1.63±0.15b 4.33±0.32b 2.89±0.21b 0.83±0.05b
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川芎茶对2,2'-盐酸脒基丙烷诱发LLC-PK1细胞氧化应激的保护作用
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林思敏 , 施丽丹 , 占洪斌 *
食品安全质量检测学报 | 食品营养及功能性食品 2025,16(15): 232-238
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食品安全质量检测学报 | 食品营养及功能性食品 2025, 16(15): 232-238
川芎茶对2,2'-盐酸脒基丙烷诱发LLC-PK1细胞氧化应激的保护作用
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林思敏 , 施丽丹, 占洪斌*
作者信息
  • 宁德师范学院附属宁德市医院, 宁德 355001

    • 林思敏(1995—), 女, 医师, 主要研究方向为肾脏医学。E-mail:

通讯作者:

*占洪斌(1989—), 男, 硕士, 主治医师, 主要研究方向为肾脏医学。E-mail:
Protective effects of Chuanxiong tea on oxidative stress induced by 2,2'-azobis(2-methylpropionamidine) dihydrochloride in LLC-PK1 cells
Si-Min LIN , Li-Dan SHI, Hong-Bin ZHAN*
Affiliations
  • Ningde Hospital Affiliated to Ningde Normal University, Ningde 355001, China
出版时间: 2025-08-15 doi: 10.19812/j.cnki.jfsq11-5956/ts.20250218006
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目的 探讨川芎茶的乙醇提取物对由2,2'-盐酸脒基丙烷[2,2'-azobis (2-methylpropionamidine) dihydrochloride, AAPH, 1 mmol/L]诱导产生的LLC-PK1猪肾近端小管上皮细胞氧化应激损伤的保护效果。方法 将LLC-PK1细胞与不同质量浓度范围(10~100 μg/mL)的川芎茶乙醇提取物(记作CXTEE)共培养24 h后, 再将其置于含有AAPH的杜尔贝科改良伊格尔培养基(Dulbecco's modified eagle medium, DMEM)中继续培养4 h以建立细胞受损模型。通过四甲基偶氮唑蓝(tetramethyl azazole blue, MTT)法检测细胞存活率; 利用硫代巴比妥酸比色分析法和2',7'-二氢二氯荧光黄双乙酸钠(2',7'-dihydrodichlorofluorescein yellow diacetate, DCFH-DA)探针技术分别测定细胞内丙二醛(malondialdehyde, MDA)含量及总活性氧(reactive oxygen species, ROS)水平; 此外, 还采用特定试剂盒评估了包括过氧化氢酶(catalase, CAT)、超氧化物歧化酶(superoxide dismutase, SOD)、谷胱甘肽过氧化物酶(glutathione peroxidase, GSH-Px)、谷胱甘肽S-转移酶(glutathione S-transferase, GST)、γ-谷氨酰半胱氨酸合成酶(γ-glutamylcysteine synthetase, γ-GCS)在内的多种抗氧化酶活性以及谷胱甘肽(glutathione, GSH)的浓度。结果 经过CXTEE预处理后的细胞表现出更高的存活率, 并且细胞内的总ROS水平与MDA生成量显著降低。同时, 该提取物还能增强受损细胞中的抗氧化酶活性(如CAT、SOD、GSH-Px和GST), 并促进γ-GCS活性及提高GSH含量。进一步的实验显示CXTEE还可以上调氧化损伤LLC-PK1细胞中SOD、GSH-Px和CAT的mRNA表达水平。结论 川芎茶乙醇提取物可能通过强化细胞自身的抗氧化防御系统来减少MDA和ROS水平, 从而减轻AAPH对LLC-PK1细胞造成的氧化应激损害。本研究可为川芎茶的进一步应用开发提供依据。

川芎茶  /  氧化损伤  /  抗氧化  /  LLC-PK1细胞  /  mRNA表达

Objective To investigate the protective effects of ethanol extract of Chuangxiong tea on the 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPH) (1 mmol/L) on oxidative stress injury of proximal renal tubule epithelial cells of pigs. Methods LLC-PK1 cells were co-cultured with ethanol extract of Chuanxiong tea (labeled CXTEE) with different mass concentrations ranging from 10 to 100 μg/mL for 24 h, and then placed in Dulbecco's modified eagle medium (DMEM) containing AAPH for 4 h to establish the cell damage model. The cell survival rate was measured by tetramethyl azazole blue (MTT) method. The content of malondialdehyde (MDA) and the level of total reactive oxygen species (ROS) were determined by thiobarbituric acid colorimetry and 2',7'-dihydrodichlorofluorescein yellow diacetate (DCFH-DA) probe technique, respectively. In addition, the activity of several antioxidant enzymes, including catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), glutathione S-transferase (GST), γ-glutamylcysteine synthetase (γ-GCs), and the concentration of glutathione (GSH), were also evaluated using specific kits. Results The results showed that the cells pretreated with CXTEE showed a higher survival rate, and the total ROS level and MDA production in the cells were significantly reduced. At the same time, the extract also enhanced the activity of antioxidant enzymes (such as CAT, SOD, GSH-Px and GST) in the damaged cells, promoted the activity of γ-GCS and increased the content of GSH. Further experiments showed that CXTEE could also up-regulate the mRNA expression levels of SOD, GSH-Px and CAT in oxidative damage LLC-PK1 cells. Conclusion The ethanol extract of Chuangxiong tea may reduce the levels of MDA and ROS by strengthening the antioxidant defense system of cells, thus alleviating the oxidative stress damage caused by AAPH to LLC-PK1 cells. This study can provide a basis for further application and development of Chuangxiong tea.

Chuanxiong tea  /  oxidative damage  /  antioxidation  /  LLC-PK1 cells  /  mRNA expression
林思敏, 施丽丹, 占洪斌. 川芎茶对2,2'-盐酸脒基丙烷诱发LLC-PK1细胞氧化应激的保护作用. 食品安全质量检测学报, 2025 , 16 (15) : 232 -238 . DOI: 10.19812/j.cnki.jfsq11-5956/ts.20250218006
Si-Min LIN, Li-Dan SHI, Hong-Bin ZHAN. Protective effects of Chuanxiong tea on oxidative stress induced by 2,2'-azobis(2-methylpropionamidine) dihydrochloride in LLC-PK1 cells[J]. Journal of Food Safety & Quality, 2025 , 16 (15) : 232 -238 . DOI: 10.19812/j.cnki.jfsq11-5956/ts.20250218006
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0 引言
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川芎茶是一种以川芎和茶叶为主要成分的药膳饮品, 常被用作日常代茶饮品饮用, 因其配方中还包括白芷、薄荷、防风、甘草、荆芥、羌活以及细辛等多种草本植物, 适宜中老年人饮用[1]。这种茶饮源于四川地区, 其中所含有的川芎性质温和且味道辛辣, 能够进入人体的肝经与胆经之中发挥作用。它具有调和血液、促进气血流通及缓解因风热引起的头痛症状等功效, 在调节相关病症时也被作为日常保健饮品配合药物治疗进行广泛应用[2-3]。研究显示川芎茶中含有橙皮苷和蒙花苷等黄酮类物质, 黄酮类物质具有很好的抗氧化效果, 川芎茶也因此可能具有较好的抗氧化效果[4]。
在生物体执行有氧呼吸过程中, 会自然生成一系列生理代谢副产物, 如超氧阴离子(O2−)、过氧根离子(O22−)、羟自由基(·OH)、过氧化氢(H2O2)以及有机过氧自由基(ROO·)和脂过氧自由基(LOO·), 这些物质统称为活性氧(reactive oxygen species, ROS)[5-6]。普遍认为, 当体内或特定组织中ROS积累过多时, 可能会引发细胞或组织的氧化应激反应, 并进一步导致疾病的发生[7]。对于某些慢性肾病来说, 由于肾脏内抗氧化酶水平相对较低, 过量的ROS不仅能够促进多不饱和脂肪酸的脂质过氧化, 还会造成蛋白质与DNA等生物大分子结构的变化, 从而对肾小管上皮细胞产生氧化损伤, 最终可能导致整个肾脏组织的坏死[8-9]。因此, ROS被认为是引起慢性肾病的重要因素之一。在中国悠久的饮食文化里, 茶叶饮品因其独特的风味及营养价值而被认为具有一定的保健效果, 这类食物通常富含抗氧化成分, 具备良好的抗氧化性能。适量食用这类食品有助于增强机体的抗氧化防御系统, 减轻氧化应激对身体各部位特别是关键器官带来的负面影响, 进而预防相关疾病[10-12]。为了探讨川芎茶干预抗氧化应激反应造成生理损伤的作用及分子机制, 本研究利用2,2'-盐酸脒基丙烷[2,2'-azobis(2-methylpropionamidine) dihydrochloride, AAPH]建立LLC-PK1细胞氧化应激损伤, 通过细胞模型进行体外验证, 旨在为川芎茶应用于预防氧化应激所致肾脏疾病的理论基础提供参考依据。
1 材料与方法
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1.1 材料与试剂
川芎茶(泽群中药饮片有限公司); LLC-PK1猪肾近曲小管上皮细胞系(厦门逸漠生物科技有限公司)。
AAPH、四甲基偶氮唑蓝(tetramethyl azazole blue, MTT)、2',7'-二氢二氯荧光黄双乙酸钠(2',7'-dihydrodichlorofluorescein yellow diacetate, DCFH-DA)、青链霉素双抗(美国默克生命科学公司); 杜尔贝科改良伊格尔培养基(Dulbecco's modified eagle medium, DMEM)、胎牛血清、Trizol、OligodT18、核糖核酸酶(ribonuclease, RNase)、脱氧核糖核苷三磷酸(deoxyribonucleoside triphosphate, dNTP)、鼠白血病病毒(murine leukemia virus, MLV)逆转录酶(美国赛默飞世尔科技有限公司); 羧基-X-罗丹明(carboxy-X-rhodamine, ROX)参考染料、SYBR Premix Ex Taq II(日本TAKARA公司); 二甲基亚砜(dimethyl sulfoxide, DMSO)、磷酸盐缓冲液(phosphate buffered saline, PBS)、超氧化物歧化酶(superoxide dismutase, SOD)、过氧化氢酶(catalase, CAT)、谷胱甘肽过氧化物酶(glutathione peroxidase, GSH-Px)、谷胱甘肽S-转移酶(glutathione S-transferase, GST)、γ-谷氨酰半胱氨酸合成酶(γ-glutamylcysteine synthetase, γ-GCS)、谷胱甘肽(glutathione, GSH)测定试剂盒(北京索莱宝公司)。
1.2 仪器与设备
ELx808酶标仪(美国Bio-Tek公司); 3111二氧化碳细胞培养箱、Quant StudioTM 6 Flex PCR仪(美国赛默飞世尔科技有限公司); AUW120D天平(感量为0.1 mg, 日本岛津公司)。
1.3 实验方法
1.3.1 川芎茶的提取
将大约100 mg的冷冻干燥川芎茶样本与80% (V:V)乙醇溶液按照质量比1:9混合, 在室温条件下搅拌过夜以完成提取过程, 随后通过抽滤分离固体和液体部分, 此步骤重复3次, 并将所有收集到的滤液合并。对合并后的滤液进行3600 r/min、持续15 min的离心处理, 去除沉淀物后保留上清液。之后, 在50 ℃下利用减压旋转蒸发技术浓缩上清液, 最终制得川芎茶的乙醇提取物(记作CXTEE)。CXTEE被溶解于DMSO中, 配制成质量浓度为20 mg/mL的储备溶液, 并在-80 ℃条件下保存备用。
1.3.2 细胞培养及分组
在含有10%胎牛血清和1%青链霉素双抗溶液的DMEM培养基中, LLC-PK1细胞被置于37 ℃、5% CO2湿度条件下进行培养, 并且每隔2 d更换一次培养液。实验过程中, 将这些细胞以每孔1×104个和2×105个的数量分别接种到96孔板与6孔板上。通过向培养基中添加1 mmol/L AAPH并继续培养4 h的方式, 构建氧化应激损伤模型。之后, 从该模型中取出的细胞再次以每孔1×104个细胞、每孔100 μL的量接种至新的96孔板, 并使用不同质量浓度(分别为10、50和100 μg/mL)的CXTEE处理24 h后进行后续研究。此外, 未经过任何干预措施处理的正常LLC-PK1细胞则作为对照组[13]。
1.3.3 MTT法测定细胞存活率
首先, 将LLC-PK1细胞按照先前确定的分组条件进行处理, 并在接下来的24 h内持续培养。之后, 去除原有的培养基, 向每个孔中加入终质量浓度为0.5 mg/mL的MTT溶液(体积为100 μL), 再继续培养4 h。完成此步骤后, 吸除上清液, 每孔内添加DMSO(同样为100 μL)并在避光条件下摇晃30 min。最后, 通过测量490 nm处的吸光度值, 并依据公式(1)来计算细胞存活率[13]。
细胞存活率/%=OD样品处理组/OD对照组×100%
1.3.4 细胞内丙二醛生成量的测定
完成上述处理步骤之后, 采用硫代巴比妥酸比色法来评估细胞内丙二醛(malondialdehyde, MDA)的生成量[8]。首先, 所有细胞均需经过冷PBS的洗涤处理。随后, 利用细胞刮刀收集这些细胞, 并将其置入已预冷的细胞裂解液中完成裂解过程。在5 mL容量的玻璃试管内, 依次加入500 μL的细胞裂解上清液和400 μL由15%三氯乙酸与0.67%硫代巴比妥酸组成的混合溶液, 充分混匀后置于95 ℃水浴中加热20 min。待样品冷却至室温后, 向其中添加3 mL异丙醇用于色素的提取, 最后测量其在532 nm波长处的吸光度值。此外, 通过Bio-Rad蛋白质试剂盒确定细胞总蛋白含量。最后, 依据公式(2)计算出MDA的具体生成量。
MDA生成量/(ng/mg prot)=MDA含量/(ng/mL)×1.5 mL/ 总蛋白质含量/mg
1.3.5 细胞内ROS生成量的测定
依据1.3.2节中介绍的方法处理6孔细胞培养板内的细胞后, 加入含有20 μmol/L DCFH-DA的DMEM培养基, 并在37 ℃下培养20 mim。之后, 用冷PBS缓冲液清洗细胞两次。然后, 在485 nm和530 nm的波长下测定荧光强度, 并根据公式(3)计算相对ROS水平。
相对ROS含量/%=荧光强度样品处理组/荧光强度对照组×100%
1.3.6 细胞内部抗氧化酶活性、GSH水平及γ-GCS活性的检测
首先, 将细胞以2×105个/孔的密度接种于6孔板中, 并按照1.3.2节所述的方法进行处理。完成处理后, 依据SOD、GSH、CAT、GSH-Px和γ-GCS测定试剂盒相应说明书中的操作步骤完成检测。所有测量结果均需根据细胞总蛋白量进行校正。
1.3.7 实时荧光定量聚合酶链式反应技术探究LLC-PK1细胞中相关基因表达水平
按照Trizol试剂的操作指南, 从待测细胞中提取总RNA, 并通过紫外光谱法对其纯度进行了测定。随后, 将各组样品中的总RNA浓度统一调整至一致水平。对于每组样本, 均选取等量的2 μg RNA加入到含有1 μL OligodT18、1 μL RNase、1 μL dNTP以及1 μL MLV逆转录酶和10 μL 5×Buffer的无菌聚合酶链式反应(polymerase chain reaction, PCR)管内, 在37 ℃下反应120 min, 接着在99 ℃处理4 min后, 最后于4 ℃维持3 min以完成cDNA的合成过程。采用实时荧光定量PCR (quantitative real-time PCR, qRT-PCR)技术来检测抗氧化酶CAT、GSH-Px与SOD的表达水平。每个qRT-PCR反应体系(总体积20 μL)包含2 μL cDNA模板、各1 μL浓度为10 μmol/L的上下游引物、10 μL SYBR Premix Ex Taq II (2×)、0.4 μL ROX reference Dye (50×), 并补充5.6 μL灭菌双蒸水至最终体积。充分混合后, 将上述溶液置于QuantStudioTM 6 Flex PCR仪中执行扩增程序。该程序设定如下: 首先95 ℃预变性30 s, 然后进入循环阶段, 其中包括95 ℃ 5 s、55~59 ℃ 30 s、95 ℃ 15 s、60 ℃ 60 s, 此循环共重复40次。对每个基因的cDNA样本进行3次平行扩增, 根据平均Ct值评估目标基因的相对表达量[14]。
1.4 数据处理
每项实验均独立进行3次, 所得数据以平均值±标准偏差的形式呈现。本研究采用SPSS 19.0软件进行数据的统计处理, 并通过Duncan's检验执行了多重比较分析, 将P<0.05作为判断显著性差异的标准。
2 结果与分析
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2.1 CXTEE对LLC-PK1细胞增殖的影响
如图1所示, 质量浓度分别为10、50和100 μg/mL的CXTEE作用在LLC-PK1细胞后, 细胞的存活率均超过90%。实验结果表明CXTEE对体外培养的LLC-PK1细胞没有表现出显著的毒性作用。基于此, 在后续实验中选取了10、50、100 μg/mL作为安全浓度进行研究, 以确保不会影响细胞活性。此外, 与未经处理的正常培养LLC-PK1细胞组(对照组)相比, 当细胞被AAPH直接作用4 h后, LLC-PK1细胞的生存率显著下降(P<0.05)。然而, 如果先用不同浓度的CXTEE预处理24 h, 则可以观察到受损细胞的存活率有所提高, 并且这种保护效应随着CXTEE浓度的增加而增强, 显示出明显的剂量依赖性差异(P<0.05)。
2.2 CXTEE对受损LLC-PK1细胞中MDA生成量及ROS水平的影响
相比未受处理的对照组, 在被AAPH作用诱导氧化应激后, LLC-PK1细胞中的MDA水平显著提升(P<0.05)。但是在10、50和100 μg/mL质量浓度的CXTEE干预下, 诱导氧化应激损伤LLC-PK1细胞内的MDA水平则显著下降(P<0.05); 尤其当使用最高质量浓度即100 μg/mL时, MDA生成量达到最低点(图2A)。此外, AAPH还导致了LLC-PK1细胞内的ROS水平大幅上升。但经过24 h的不同质量浓度(10、50、100 μg/mL) CXTEE预处理后, 发现受损细胞中ROS水平呈现逐渐减少的趋势, 并且与仅接受AAPH处理的模型组(0 μg/mL CXTEE组)相比存在显著差异(P<0.05)。值得注意的是, 在100 μg/mL这一最高测试浓度下, CXTEE表现出最强的抑制ROS产生的效果(图2B)。由此可以看出, CXTEE在体外细胞层面, 展示出对氧化应激损伤的抑制作用, 对氧化应激损伤的标志性物质MDA和ROS有显著影响。
2.3 CXTEE对对受损LLC-PK1细胞中CAT、SOD、GSH-Px及GST活性的影响
如表1所示, AAPH诱导氧化应激后, LLC-PK1细胞的CAT、GSH-Px、SOD和GST抗氧化酶的活性出现下降的趋势。然而, 当这些细胞预先接受了不同质量浓度(10、50、100 μg/mL)的川芎茶乙醇提取物处理24 h后, 观察到抗氧化酶活性有所恢复, 与未接受该提取物处理的损伤模型组(0 μg/mL CXTEE组)相比, 差异具有统计学意义(P<0.05)。实验结果显示CXTEE对LLC-PK1细胞的抗氧化酶有显著的提升作用, 由此提示CXTEE具有通过调控细胞中抗氧化酶活力起到抗氧化效果的作用。
2.4 CXTEE对LLC-PK1细胞中γ-GCS活性及GSH水平的影响
AAPH处理不仅显著抑制了细胞内γ-GCS的活性, 还导致GSH合成量下降(图3)。然而, 在使用CXTEE(质量浓度范围为10~100 μg/mL)作用在LLC-PK1细胞中后, 氧化应激损伤细胞的γ-GCS酶活性出现提升的现象。同时, 氧化应激损伤细胞的GSH含量也出现相同趋势并呈现出上升的现象。与未经CXTEE处理组相比, CXTEE对受损LLC-PK1细胞中γ-GCS酶活性及GSH水平的影响具有统计学意义(P<0.05)。实验结果显示CXTEE可能特异性地对细胞中GSH水平及其合成酶具有调控作用, 从而起到抗氧化效果。
2.5 CXTEE对LLC-PK1细胞内氧化相关基因表达水平的影响
采用qRT-PCR技术进行分析的结果表明, 1 mmol/L AAPH能够导致LLC-PK1细胞中SOD、GSH-Px及CAT这3种关键内源性抗氧化酶的mRNA表达量下降(图4)。然而, 经过CXTEE预处理之后, 这些受损细胞内相关基因的表达水平逐渐恢复。与未经CXTEE处理组(0 μg/mL CXTEE组)相比, 10~100 μg/mL CXTEE显著提升了SOD、GSH-Px和CAT在受损LLC-PK1细胞中的表达量(P<0.05), 显示出其对改善细胞抗氧化状态的有效干预作用。进一步的mRNA表达也验证出CXTEE对抗氧化酶具有调控作用, 结合试剂盒检测结果可以认为CXTEE通过提升抗氧化酶活力抑制LLC-PK1细胞氧化损伤是CXTEE作用的重要机制。
3 讨论与结论
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在生物体内, ROS的过量积累被认为是多种慢性疾病发病机制中的关键因素之一。作为一类重要的自由基来源, 偶氮化合物AAPH能够自发产生过氧化自由基[15]。这些过氧化自由基能够损伤细胞内的关键生物大分子, 如DNA、蛋白质及脂质, 从而引发细胞凋亡[16]。研究显示, 在1 mmol/L AAPH条件下暴露后, LLC-PK1细胞的存活率显著下降; 但若先用不同浓度的川芎茶乙醇提取物处理24 h, 则与未经该提取物预处理的受损细胞相比, 其生存能力有明显增强(P<0.05)。此外, 体内过度生成或累积的ROS还能促使细胞膜发生生理变化, 细胞膜内的不饱和脂肪酸发生变性, 产生脂质过氧化反应的有害产物, 从而影响细胞的正常生理活动[17-18]。所以MDA作为脂质过氧化反应的常见产物, 在生理检测中被用于评价氧化应激损伤的重要指标[19]。对于受到损伤的LLC-PK1细胞而言, 预先使用川芎茶乙醇提取物进行干预可以减少细胞内总ROS水平及MDA含量。这可能归因于川芎茶中富含具有强大抗氧化性能的苯酞类化合物, 比如藁本内酯[20]。现有研究表明, 给予适量的抗氧化剂可以帮助减轻由ROS引起的肾细胞脂质过氧化情况, 并有助于预防肾细胞遭受氧化应激伤害[21]。
在机体和细胞正常生理状态下, 生物体内的包括CAT、SOD、GSH-Px和GST等抗氧化酶和GSH等非酶型抗氧化剂能够清除氧化应激产生的自由基能有害产物, 保持机体和细胞的氧化平衡[22]。这些不同的抗氧化剂作用机制有区别, SOD将过量超氧阴离子转化为过氧化氢, CAT和GSH-Px则能够将过氧化氢分解无害的水[23]。GSH-Px则可以利用GSH作为还原剂转化过氧化氢和烷基过氧化物, 还可以将有机氢过氧化物还原成较安全的羟基化合物。GST可以参与辅助GSH-Px清除过多有机氢过氧化物[24]。本研究的结果显示在不同浓度的川芎茶乙醇提取物预处理后, 受损细胞中主要包括SOD在内的抗氧化酶活性显著增强, 同时非酶抗氧化物质的相应增多。可见川芎茶乙醇提取物可以提升抗氧化酶的活性从而保护LLC-PK1细胞免受氧化应激损害, 同时还可以抑制脂质过氧化反应对细胞膜造成的生理破坏, 并减轻由此引起的附加细胞损伤[25-27]。GSH作为一种重要的非酶抗氧化剂可以通过GSH-Px干预脂质过氧化物和过氧化氢等有毒产物, 同时GSH还能够间接阻止自由基的链式反应, 从而保护细胞不受自由基侵害[28-29]。γ-GCS作为调控GSH合成的关键酶, 对于提高GSH水平至关重要。实验还表明, 川芎茶能够上调受损LLC-PK1细胞中关键抗氧化酶表达水平。加强CAT和GSH-Px基因转录有助于激活细胞内超氧化物歧化酶, 从而缓解氧化应激造成的细胞损伤[30-31]。本研究为探讨川芎茶乙醇提取物作为潜在抗氧化保健饮品提供了理论依据。但是由于本研究还仅限于体外细胞实验层面, 关于川芎茶如何在分子层面上发挥机制作用保护细胞免受氧化应激损伤还有待深入研究。
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2025年第16卷第15期
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doi: 10.19812/j.cnki.jfsq11-5956/ts.20250218006
  • 接收时间：2025-02-18
  • 首发时间：2026-01-09
  • 出版时间：2025-08-15
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  • 收稿日期：2025-02-18
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    宁德师范学院附属宁德市医院, 宁德 355001

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*占洪斌(1989—), 男, 硕士, 主治医师, 主要研究方向为肾脏医学。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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