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Article(id=1322923237469986952, tenantId=1146029695717560320, journalId=1255847919539208197, issueId=1322923011518628427, articleNumber=null, orderNo=null, doi=10.13193/j.issn.1673-7717.2025.03.017, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=null, receivedDateStr=null, revisedDate=null, revisedDateStr=null, acceptedDate=null, acceptedDateStr=null, onlineDate=1793339078538, onlineDateStr=2026-10-30, pubDate=1741536000000, pubDateStr=2025-03-10, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1793339078538, onlineIssueDateStr=2026-10-30, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1793339078538, creator=13701087609, updateTime=1793339078538, updator=13701087609, issue=Issue{id=1322923011518628427, tenantId=1146029695717560320, journalId=1255847919539208197, year='2025', volume='43', issue='3', pageStart='1', pageEnd='258', issueExtLink='null', onlineDate='null', pubDate='1741536000000', pubDateStr='2025-03-10', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=0, createTime=1793339024666, creator='13701087609', updateTime=1793340602374, updator='13701087609', preIssue=null, nextIssue=null, articleTotal=null, ext={EN=IssueExt(id=1322929628976906432, tenantId=1146029695717560320, journalId=1255847919539208197, issueId=1322923011518628427, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1322929628981100737, tenantId=1146029695717560320, journalId=1255847919539208197, issueId=1322923011518628427, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null, downloadFileDto=null}, startPage=102, endPage=106, ext={EN=ArticleExt(id=1322923238128492681, articleId=1322923237469986952, tenantId=1146029695717560320, journalId=1255847919539208197, language=EN, title=Mechanism of Electroacupuncture Preconditioning Improving Myocardial Ischemia-Reperfusion Injury Based on mTOR/NLRP3 Signaling Pathway, columnId=1256183361521967297, journalTitle=Chinese Archives of Traditional Chinese Medicine, columnName=Target on National Project, runingTitle=null, highlight=null, articleAbstract=
Objective

To investigate the effects of electroacupuncture（EA）preconditioning on myocardial ischemia-reperfusion injury（MIRI）and its regulation of the inflammatory response through the mTOR/NLRP3 signaling pathway.

Methods

Seventy SD rats were randomly divided into five groups：sham operation group，model group，EA group，EA+saline group and EA+mTOR agonists（EA+MHY1485）group，with 14 rats in each group. After the intervention，it evaluated cardiac function by measuring the left ventricular ejection fraction（LVEF）and left ventricular fraction shortening（LVFS）. Additionally，it performed hematoxylin-eosin（HE）staining to observe histopathological changes in the myocardium. The levels of tumor necrosis factor-α（TNF-α）in myocardial tissue were measured using an enzyme-linked immunosorbent assay（ELISA）. Furthermore，it assessed the gene expression levels of apoptosis-associated speck-like protein containing a CARD（ASC），cysteinyl aspartate specific proteinase-1（Caspase-1），interleukin-1β（IL-1β）and interleukin-18（IL-18）in cardiac tissue using quantitative polymerase chain reaction（RT-qPCR）. Finally，it analyzed the protein expression levels of mammalian target of rapamycin（mTOR），NOD-like receptor thermal protein domain associated protein 3（NLRP3）and nuclear factor kappa-B（NF-κB）by Western blotting.

Results

Compared to the sham operation group，the rats in the model group showed decreased values of LVFS and LVEF（P<0.05）. Myocardial tissue exhibited muscle fiber rupture，blurred structure and a significant infiltration of inflammatory cells. Additionally，the TNF-αlevel in the myocardial tissue increased（P<0.05）. The relative gene expression levels of Caspase-1，ASC，IL-1βand IL-18 were significantly increased（P<0.05），along with increased protein expression levels of mTOR，NLRP3 and NF-κB. In contrast，the rats in the EA group showed increased FSand EF values（P<0.05）. The myocardial tissue structure improved，inflammatory cell infiltration decreased and the expression of TNF-αin the myocardial tissue decreased（P<0.05）. The relative expression levels of Caspase-1，ASC，IL-1βand IL-18 genes also decreased（P<0.05），as well as the protein expressions of mTOR，NLRP3 and NF-κB（P<0.05）. However，compared to the EA+saline group，the EA+MHY1485 group showed worsened rats myocardial tissue，increased TNF-αlevel in the myocardial tissue（P<0.05），increased relative expression levels of Caspase-1，ASC，IL-1βand IL-18 genes（P<0.05），and increased protein expression levels of mTOR，NLRP3 and NF-κB（P<0.05）.

Conclusion

The mTOR agonist MHY1485 can weaken the protective effect of electricity against myocardial tissue damage in MIRI rats，and EA pretreatment can reduce the inflammatory response of MIRI rat myocardial cells，thereby improving myocardial tissue damage caused by ischemia-reperfusion. Therefore，it is inferred that the mechanism of EA pretreatment to improve MIRI injury in rats may be related to the mTOR/NLRP3 signaling pathway.

, authors=Chunhong JIANG, Lili HE, Shunchang LIU, Hua BAI, Yihuang GU, Kaiping LI, authorsList=Chunhong JIANG, Lili HE, Shunchang LIU, Hua BAI, Yihuang GU, Kaiping LI, authorCompany=null, correspAuthors=Kaiping LI, 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, fund=null), CN=ArticleExt(id=1322923241630736530, articleId=1322923237469986952, tenantId=1146029695717560320, journalId=1255847919539208197, language=CN, title=电针预处理基于mTOR/NLRP3信号通路改善心肌缺血再灌注损伤的作用机制, columnId=1256183362172142382, journalTitle=中华中医药学刊, columnName=国家项目点击, runingTitle=null, highlight=null, articleAbstract=
目的

基于mTOR/NLRP3信号通路探讨电针预处理抗大鼠心肌缺血再灌注损伤的作用机制。

方法

70只SD大鼠随机分为假手术组、模型组、电针组、电针+生理盐水组，电针+MHY1485（mTOR激动剂）组，各14只。采用超声心动图评估大鼠心脏功能指标心室射血分数（LVEF）和左心室短轴缩短率分数（LVFS），苏木精-伊红（HE）染色法观察心肌组织病理学改变，酶联免疫吸附法（ELISA）检测心肌组织中肿瘤坏死因子-α（TNF-α）水平变化，实时荧光定量PCR（RT-qPCR）检测心肌组织中凋亡相关斑点样蛋白（ASC）、含半胱氨酸的天冬氨酸蛋白水解酶-1（Caspase-1）、白细胞介素-1β（IL-1β）、白细胞介素-18（IL-18）的基因表达水平，蛋白质免疫印迹法（Western blot）检测心肌组织中哺乳动物雷帕霉素靶蛋白（mTOR）、NOD样受体家族蛋白3（NLRP3）及核因子-κB（NF-κB）的表达水平。

结果

与假手术组相比，模型组大鼠FS、EF值下降（P<0.05）；心肌组织出现肌纤维断裂、结构模糊、炎性细胞浸润等现象，心肌组织内TNF-α含量升高（P<0.05），Caspase-I、ASC、IL-1β、IL-18的基因相对表达水平（P<0.05）与mTOR、NLRP3、NFκB的蛋白表达水平均上调（P<0.05）；与模型组相比，电针组大鼠FS、EF值上升（P<0.05），心肌组织肌纤维结构改善，炎性细胞浸润减少，心肌组织内的TNF-α含量降低（P<0.05），Caspase-I、ASC、IL-1β、IL-18的基因相对表达水平（P<0.05）与mTOR、NLRP3、NF-κB的蛋白表达水平（P<0.05）均下调；与电针+生理盐水组比，激动剂组大鼠心肌组织损伤加重，心肌组织内TNF-α含量升高（P<0.05），Caspase-I、ASC、IL-1β、IL-18的基因相对表达水平（P<0.05）与mTOR与NLRP3、NF-κB的蛋白表达水平（P<0.05）均上调。

结论

mTOR激动剂MHY1485可减弱电针对心肌缺血再灌注损伤（MIRI）大鼠心肌组织损伤的保护作用，电针预处理能减轻MIRI大鼠心肌细胞炎性反应，进而改善缺血再灌注引起的心肌组织损伤；因此推断电针预处理改善大鼠MIRI损伤的机制可能与mTOR/NLRP3信号通路有关。

, authors=姜春宏, 贺丽丽, 刘顺畅, 白桦, 顾一煌, 李开平, authorsList=姜春宏, 贺丽丽, 刘顺畅, 白桦, 顾一煌, 李开平, authorCompany=null, correspAuthors=李开平, authorNote=

姜春宏（1997-），女，山东烟台人，硕士在读，研究方向：中医养生方法技术研究与应用。

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李开平（1967-），男，安徽马鞍山人，副教授，硕士研究生导师，博士，研究方向：中医养生治未病的基础与研究。E-mail：。
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姜春宏（1997-），女，山东烟台人，硕士在读，研究方向：中医养生方法技术研究与应用。

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姜春宏（1997-），女，山东烟台人，硕士在读，研究方向：中医养生方法技术研究与应用。

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注：***与假手术组比，P<0.001；###与模型组比，P<0.001；**与电针+生理盐水组比，P<0.01。

, figureFileSmall=P8qeVZiR/G9YVQvVSNiBSg==, figureFileBig=0goKTRUWJ+bua+nIxtA6Hw==, tableContent=null), ArticleFig(id=1322923246718427326, tenantId=1146029695717560320, journalId=1255847919539208197, articleId=1322923237469986952, language=EN, label=null, caption=null, figureFileSmall=DE6ZZOVB8bC3J84z6Fj82Q==, figureFileBig=rWYLIQztteP/4/wofwmYRw==, tableContent=null), ArticleFig(id=1322923246814896319, tenantId=1146029695717560320, journalId=1255847919539208197, articleId=1322923237469986952, language=CN, label=图2, caption=各组大鼠心肌病理学形态HE染色检测（200x）, figureFileSmall=DE6ZZOVB8bC3J84z6Fj82Q==, figureFileBig=rWYLIQztteP/4/wofwmYRw==, tableContent=null), ArticleFig(id=1322923248471646400, tenantId=1146029695717560320, journalId=1255847919539208197, articleId=1322923237469986952, language=EN, label=null, caption=null, figureFileSmall=3Witnma5VHxvALPsyPPt6w==, figureFileBig=x/RXkRJRofyqwIjS8sYCNQ==, tableContent=null), ArticleFig(id=1322923248559726785, tenantId=1146029695717560320, journalId=1255847919539208197, articleId=1322923237469986952, language=CN, label=图3, caption=各组大鼠心肌组织中mTOR、NF-κB和NLRP3蛋白水平表达

注：*与假手术组比，P<0.05；#与模型组比，P<0.05，**与模型组比，P<0.01；##与电针+生理盐水组比，P<0.01；***与电针+生理盐水组比，P<0.001。

, figureFileSmall=3Witnma5VHxvALPsyPPt6w==, figureFileBig=x/RXkRJRofyqwIjS8sYCNQ==, tableContent=null), ArticleFig(id=1322923248622641346, tenantId=1146029695717560320, journalId=1255847919539208197, articleId=1322923237469986952, language=EN, label=null, caption=null, figureFileSmall=4UebRzxMnJGqKbQjMtiPxA==, figureFileBig=Ll+TLuTCO8giopa+PxSiww==, tableContent=null), ArticleFig(id=1322923248735887555, tenantId=1146029695717560320, journalId=1255847919539208197, articleId=1322923237469986952, language=CN, label=图4, caption=各组大鼠心肌组织中Caspsae-1、ASC、IL-1β和IL-18mRNA相对表达量

注：**与假手术组比，P<0.01；*与模型组比，P<0.01；#与电针+生理盐水组比较，P<0.05；##与电针+生理盐水组比较，P<0.01。

, figureFileSmall=4UebRzxMnJGqKbQjMtiPxA==, figureFileBig=Ll+TLuTCO8giopa+PxSiww==, tableContent=null), ArticleFig(id=1322923248823967940, tenantId=1146029695717560320, journalId=1255847919539208197, articleId=1322923237469986952, language=EN, label=null, caption=null, figureFileSmall=null, figureFileBig=null, tableContent=
基因名称引物序列（5-3）
ASCF：TTGGCTGCCGACTGAGGAGG
R：AGTGAAGTAAGGCCGGAAT
Caspase-1F：CAGATGCCAACCACTGAA
R：CATGATTCCCAACACAGGT
IL-1βF：TGACAGGCAACCACTTACC
R：CCCATACACACGGACAACT
IL-18F：AACGAATCCCAGACCAGAC
R：AGAGGGTAGACATCCTTCCAT
β-actinF：CGTCATCCATGGCGAACT
R：CCCGCGAGTACAACCTTCT
), ArticleFig(id=1322923248924631237, tenantId=1146029695717560320, journalId=1255847919539208197, articleId=1322923237469986952, language=CN, label=表1, caption=

内参及各目的基因的引物序列

, figureFileSmall=null, figureFileBig=null, tableContent=
基因名称引物序列（5-3）
ASCF：TTGGCTGCCGACTGAGGAGG
R：AGTGAAGTAAGGCCGGAAT
Caspase-1F：CAGATGCCAACCACTGAA
R：CATGATTCCCAACACAGGT
IL-1βF：TGACAGGCAACCACTTACC
R：CCCATACACACGGACAACT
IL-18F：AACGAATCCCAGACCAGAC
R：AGAGGGTAGACATCCTTCCAT
β-actinF：CGTCATCCATGGCGAACT
R：CCCGCGAGTACAACCTTCT
), ArticleFig(id=1322923249012711622, tenantId=1146029695717560320, journalId=1255847919539208197, articleId=1322923237469986952, language=EN, label=null, caption=null, figureFileSmall=null, figureFileBig=null, tableContent=
组别TNF-α/（pg/mg）
假手术组133.79±7.388
模型组251.12±22.50**
电针组177.38±16.53*
电针+生理盐水组173.14±28.98
电针+MHY1485组188.92±14.33
), ArticleFig(id=1322923249084014791, tenantId=1146029695717560320, journalId=1255847919539208197, articleId=1322923237469986952, language=CN, label=表2, caption=

各组大鼠心肌组织中TNF-α含量表达

, figureFileSmall=null, figureFileBig=null, tableContent=
组别TNF-α/（pg/mg）
假手术组133.79±7.388
模型组251.12±22.50**
电针组177.38±16.53*
电针+生理盐水组173.14±28.98
电针+MHY1485组188.92±14.33
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电针预处理基于mTOR/NLRP3信号通路改善心肌缺血再灌注损伤的作用机制
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姜春宏 , 贺丽丽 , 刘顺畅 , 白桦 , 顾一煌 , 李开平
中华中医药学刊 | 国家项目点击 2025,43(3): 102-106
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中华中医药学刊 |国家项目点击 2025 , 43 (3) : 102 -106
电针预处理基于mTOR/NLRP3信号通路改善心肌缺血再灌注损伤的作用机制
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姜春宏, 贺丽丽, 刘顺畅, 白桦, 顾一煌, 李开平
作者信息
  • 南京中医药大学，江苏 南京 210000
通讯作者:
李开平（1967-），男，安徽马鞍山人，副教授，硕士研究生导师，博士，研究方向：中医养生治未病的基础与研究。E-mail：。
作者简介:

姜春宏（1997-），女，山东烟台人，硕士在读，研究方向：中医养生方法技术研究与应用。

Mechanism of Electroacupuncture Preconditioning Improving Myocardial Ischemia-Reperfusion Injury Based on mTOR/NLRP3 Signaling Pathway
Chunhong JIANG, Lili HE, Shunchang LIU, Hua BAI, Yihuang GU, Kaiping LI
Affiliations
  • Nanjing University of Chinese Medicine，Nanjing 210000，Jiangsu，China
出版时间: 2025-03-10 doi: 10.13193/j.issn.1673-7717.2025.03.017
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摘要
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目的

基于mTOR/NLRP3信号通路探讨电针预处理抗大鼠心肌缺血再灌注损伤的作用机制。

方法

70只SD大鼠随机分为假手术组、模型组、电针组、电针+生理盐水组，电针+MHY1485（mTOR激动剂）组，各14只。采用超声心动图评估大鼠心脏功能指标心室射血分数（LVEF）和左心室短轴缩短率分数（LVFS），苏木精-伊红（HE）染色法观察心肌组织病理学改变，酶联免疫吸附法（ELISA）检测心肌组织中肿瘤坏死因子-α（TNF-α）水平变化，实时荧光定量PCR（RT-qPCR）检测心肌组织中凋亡相关斑点样蛋白（ASC）、含半胱氨酸的天冬氨酸蛋白水解酶-1（Caspase-1）、白细胞介素-1β（IL-1β）、白细胞介素-18（IL-18）的基因表达水平，蛋白质免疫印迹法（Western blot）检测心肌组织中哺乳动物雷帕霉素靶蛋白（mTOR）、NOD样受体家族蛋白3（NLRP3）及核因子-κB（NF-κB）的表达水平。

结果

与假手术组相比，模型组大鼠FS、EF值下降（P<0.05）；心肌组织出现肌纤维断裂、结构模糊、炎性细胞浸润等现象，心肌组织内TNF-α含量升高（P<0.05），Caspase-I、ASC、IL-1β、IL-18的基因相对表达水平（P<0.05）与mTOR、NLRP3、NFκB的蛋白表达水平均上调（P<0.05）；与模型组相比，电针组大鼠FS、EF值上升（P<0.05），心肌组织肌纤维结构改善，炎性细胞浸润减少，心肌组织内的TNF-α含量降低（P<0.05），Caspase-I、ASC、IL-1β、IL-18的基因相对表达水平（P<0.05）与mTOR、NLRP3、NF-κB的蛋白表达水平（P<0.05）均下调；与电针+生理盐水组比，激动剂组大鼠心肌组织损伤加重，心肌组织内TNF-α含量升高（P<0.05），Caspase-I、ASC、IL-1β、IL-18的基因相对表达水平（P<0.05）与mTOR与NLRP3、NF-κB的蛋白表达水平（P<0.05）均上调。

结论

mTOR激动剂MHY1485可减弱电针对心肌缺血再灌注损伤（MIRI）大鼠心肌组织损伤的保护作用，电针预处理能减轻MIRI大鼠心肌细胞炎性反应，进而改善缺血再灌注引起的心肌组织损伤；因此推断电针预处理改善大鼠MIRI损伤的机制可能与mTOR/NLRP3信号通路有关。

心肌缺血再灌注损伤  /  电针预处理  /  炎症  /  mTOR/NLRP3信号通路
Objective

To investigate the effects of electroacupuncture（EA）preconditioning on myocardial ischemia-reperfusion injury（MIRI）and its regulation of the inflammatory response through the mTOR/NLRP3 signaling pathway.

Methods

Seventy SD rats were randomly divided into five groups：sham operation group，model group，EA group，EA+saline group and EA+mTOR agonists（EA+MHY1485）group，with 14 rats in each group. After the intervention，it evaluated cardiac function by measuring the left ventricular ejection fraction（LVEF）and left ventricular fraction shortening（LVFS）. Additionally，it performed hematoxylin-eosin（HE）staining to observe histopathological changes in the myocardium. The levels of tumor necrosis factor-α（TNF-α）in myocardial tissue were measured using an enzyme-linked immunosorbent assay（ELISA）. Furthermore，it assessed the gene expression levels of apoptosis-associated speck-like protein containing a CARD（ASC），cysteinyl aspartate specific proteinase-1（Caspase-1），interleukin-1β（IL-1β）and interleukin-18（IL-18）in cardiac tissue using quantitative polymerase chain reaction（RT-qPCR）. Finally，it analyzed the protein expression levels of mammalian target of rapamycin（mTOR），NOD-like receptor thermal protein domain associated protein 3（NLRP3）and nuclear factor kappa-B（NF-κB）by Western blotting.

Results

Compared to the sham operation group，the rats in the model group showed decreased values of LVFS and LVEF（P<0.05）. Myocardial tissue exhibited muscle fiber rupture，blurred structure and a significant infiltration of inflammatory cells. Additionally，the TNF-αlevel in the myocardial tissue increased（P<0.05）. The relative gene expression levels of Caspase-1，ASC，IL-1βand IL-18 were significantly increased（P<0.05），along with increased protein expression levels of mTOR，NLRP3 and NF-κB. In contrast，the rats in the EA group showed increased FSand EF values（P<0.05）. The myocardial tissue structure improved，inflammatory cell infiltration decreased and the expression of TNF-αin the myocardial tissue decreased（P<0.05）. The relative expression levels of Caspase-1，ASC，IL-1βand IL-18 genes also decreased（P<0.05），as well as the protein expressions of mTOR，NLRP3 and NF-κB（P<0.05）. However，compared to the EA+saline group，the EA+MHY1485 group showed worsened rats myocardial tissue，increased TNF-αlevel in the myocardial tissue（P<0.05），increased relative expression levels of Caspase-1，ASC，IL-1βand IL-18 genes（P<0.05），and increased protein expression levels of mTOR，NLRP3 and NF-κB（P<0.05）.

Conclusion

The mTOR agonist MHY1485 can weaken the protective effect of electricity against myocardial tissue damage in MIRI rats，and EA pretreatment can reduce the inflammatory response of MIRI rat myocardial cells，thereby improving myocardial tissue damage caused by ischemia-reperfusion. Therefore，it is inferred that the mechanism of EA pretreatment to improve MIRI injury in rats may be related to the mTOR/NLRP3 signaling pathway.

myocardial ischemia reperfusion injury  /  electroacupuncture preconditioning  /  inflammation  /  mTOR/NLRP3 signaling pathway
姜春宏, 贺丽丽, 刘顺畅, 白桦, 顾一煌, 李开平. 电针预处理基于mTOR/NLRP3信号通路改善心肌缺血再灌注损伤的作用机制. 中华中医药学刊, 2025 , 43 (3) : 102 -106 . DOI: 10.13193/j.issn.1673-7717.2025.03.017
Chunhong JIANG, Lili HE, Shunchang LIU, Hua BAI, Yihuang GU, Kaiping LI. Mechanism of Electroacupuncture Preconditioning Improving Myocardial Ischemia-Reperfusion Injury Based on mTOR/NLRP3 Signaling Pathway[J]. Chinese Archives of Traditional Chinese Medicine, 2025 , 43 (3) : 102 -106 . DOI: 10.13193/j.issn.1673-7717.2025.03.017
正文
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心血管疾病是全球病死率最高的疾病之一，据世界卫生组织调查，每年约有1860万人因心血管疾病失去生命[1]。心肌梗死是其高发疾病之一，急性心肌梗死在经过治疗重新恢复灌注后，缺血区心肌组织会出现比再灌注前更严重的结构损伤和功能障碍，这被称为心肌缺血再灌注损伤[2]（MIRI）。MIRI是心功能损害、预后变差及死亡率上升的主要因素之一。研究发现，MIRI的严重程度与炎症因子的表达水平之间存在直接的关联[3]。心肌缺血过程中，机体产生大量炎症因子损伤心肌细胞，再灌注期间，随着血液和氧气的回流，促使TNF-α等产生增加[35]，中性粒细胞在血管内皮表面聚集黏附，加重组织和血管腔水肿，引起微循环障碍，加重心肌损伤程[29]。哺乳动物雷帕霉素靶蛋白（mTOR）能够促进炎症因子的表达，持续活化的mTOR会导致细胞炎症水平不断上升。最近的研究发现mTOR与NLRP3之间存在一种交互调控关系，mTOR能上调NF-κB[34]的活性，从而激活NLRP3炎性小体，增加促炎细胞因子的分泌。
针法作为中医疗法之一，在防治疾病方面具有独特优势[4]。内关穴是心包经之络穴，通于三焦经，又是八脉交会穴，是治疗心胸疾患的首选穴。有研究表明，电针内关穴预处理能有效调控炎性因子水平，减轻心肌缺血再灌注损伤[5-9]。基于课题组前期研究已证实，电针内关穴预处理对MIRI大鼠心肌具有保护作用[35]，但mTOR/NLRP3信号通路对MIRI的调控机制尚不清楚。因此，本实验拟对此进行研究，补充和完善电针预处理对心肌缺血再灌注损伤的保护机制。
1　材料与方法
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1.1　实验动物与分组
70只雄性SD大鼠，体质量（220±20）g，购于北京维通利华实验动物技术有限公司［SCXK（京）2021-0011］。于南京中医药大学SPF级实验动物中心适应性饲养一周，昼夜节律为12 h光照/12 h黑暗，自由饮食，屏障温度20～26℃，湿度40%～70%。一周后采取随机数字表法随机分为假手术组、模型组、电针组、电针+生理盐水组、电针+mTOR激动剂MHY1485（以下简称电针+MHY1485）组，每组14只。实验过程中对动物的处置符合中华人民共和国科学技术部于2006年颁布的《关于善待实验动物的指导性意见》。
1.2　主要仪器及试剂
主要仪器：小动物呼吸机（深圳市瑞沃德生命科技有限公司），异氟烷麻醉机（美国VMR matrx），MyLab多普勒超声仪（意大利ESAOTE），台式高速冰冻离心机（上海安亭科学仪器厂），切片机（德国Microm），韩氏电针仪（南京济生医疗科技有限公司），酶标仪（美国Biotek），电泳仪、电泳槽（美国Bio-Rad）。主要试剂：MHY1485（HY-B0795，美国MCE），4%多聚甲醛固定液（BL539A，中国 Biosharp），异氟烷麻醉剂（1973015，深圳市瑞沃德生命科技有限公司），PBS缓冲液（AR0030，美国Boster），ECL发光液（BMU102-S，亚科因武汉生物技术有限公司），marker（WJ102，上海雅酶生物医药科技有限公司），一抗NF-κB（AF5006，杭州AffinityBiosciences），mTOR、NLRP3（28273-1-AP，27458-1-AP，武汉三鹰生物技术有限公司），兔二抗（ab150077，英国Abcam），TNFαELISA试剂盒（目录号：AF20230420）等。
1.3　干预方法
所有组均常规饲养，予相同时长和方式的抓取、固定。假手术组只穿线不结扎；模型组穿线结扎冠状动脉左前降支近分支30 min，再灌注4 h；电针组采用0.18 mm×13 mm一次性不锈钢毫针制成双极电针，在双侧内关穴直刺2～3 mm，穴位定位法参照《实验针灸学》[22]，接韩氏电针仪，频率2 Hz/100 Hz，电流强度2 mA，每次20 min，每日1次，连续4 d，第5天进行模型制备；电针+生理盐水组，电针内关穴同时注射0.9%的生理盐水（5 mg/kg），每日1次，连续4 d，第5天进行模型制备；电针+MHY1485组，电针内关穴同时注射MHY1485（5 mg/kg）[21]，每日1次，连续4 d，第5天进行模型制备。
1.4　模型制备
操作：5%异氟烷伴70%氮气+30%氧气诱导麻醉大鼠后，1%～2%的浓度维持，将动物仰卧固定在（37±3）℃的实验板上，剔除动物颈部及左侧胸毛，擦拭消毒，横行切开皮肤约1 cm，逐层分离暴露气管，于第3、4气管软骨环间作倒“T”型切口，置入气管导管，接呼吸机，安装心电监护仪，标准Ⅱ导联监测心电图。剪开胸骨左侧第3、4肋间，钝性扩张暴露胸腔，挤出心脏后用6.0的丝线结扎冠状动脉左前降支，宽度深度约2 mm，缺血30 min后打开丝线结，关胸缝合再灌注4 h[19-20]。
1.5　观察指标与检测方法
1.5.1　超声心动图观察大鼠心功能变化情况
术后4 h使用多普勒超声仪采集各组大鼠超声心动图数据。通过二维及M型模式测量心室舒张末期内径及左心室收缩末期内径，通过公式计算获取左心室射血分数及左心室缩短分数。
1.5.2　苏木素-伊红（HE）染色
术后4 h，各组随机挑选4只大鼠异氟烷深度麻醉后摘取心脏，取结扎线以下左心室前壁组织，4℃PBS缓冲液冲洗干净，4%多聚甲醛中固定，石蜡包埋，5μm厚度切片，二甲苯Ⅰ、Ⅱ脱蜡后，梯度乙醇（100%、95%、80%、75%）及蒸馏水水化。苏木素染色5 min，蒸馏水冲洗，伊红染液中浸泡2 min，梯度乙醇脱水、透明中性树胶封片，风干过夜。显微镜观察图像并拍片。
1.5.3　ELISA检测心肌组织中TNF-a含量水平
术后4 h，各组随机挑选4只大鼠异氟烷深度麻醉后摘取心脏，取结扎线以下左心室前壁组织，4℃PBS缓冲液中清洗干净，加入定量PBS（pH 7.4），手工将标本匀浆充分，离心20 min（3000 r/min），收上清液，标号分装备用。将样品加入各反应孔，使用大鼠特异性ELISA试剂盒检测心肌组织中TNF-α的水平，操作步骤严格按照说明书进行，用酶标仪检测450 nm处吸光度值，计算表达含量。
1.5.4　Western
blot法检测大鼠缺血心肌组织中mTOR、NFκB、NLRP3蛋白表达术后4 h，各组随机挑选3只大鼠异氟烷深度麻醉后，取下结扎线下左心室前壁心肌组织，PBS缓冲溶液冲洗干净，液氮速冻并研磨，使用RIPA裂解液提取心肌组织中蛋白，BCA法定量蛋白浓度及上样量。后续依次制胶，上样，跑胶，转膜，封闭，孵育过夜，TBST洗膜3次×10 min，加入二抗，摇床常温孵育1.5 h，TBST洗膜3次×10 min，最后在凝胶成像仪对条带进行成像与分析。
1.5.5　实时荧光定量PCR检测
术后4 h，各组随机挑选3只大鼠在同样的时间点取结扎线以下位置心肌组织并于-80℃冰箱保存。使用Trizol提取心肌总RNA，逆转录合成cDNA，采用实时荧光定量PCR系统扩增cDNA。引物序列列表于表1。实验均以β-actin作为内参基因。
1.6　统计学处理
采用SPSS 27.0软件进行实验数据统计分析，符合正态分布的计量资料以均数±标准差表示，若各组间所测数据均符合正态分布且满足方差齐性，则多组间比较用单因素方差分析，多重比较用LSD检验。结果以P<0.05为差异有统计学意义。
2　实验结果
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2.1　电针预处理对MIRI大鼠心功能的影响
假手术组左心室壁运动协调正常，模型组左心室壁无明显运动。与假手术组比较，模型组大鼠FS、EF值下降（P<0.001）；与模型组比较，电针组大鼠FS、EF值升高（P<0.001）；与电针+生理盐水组相比，电针+MHY1485组大鼠FS、EF值降低（P<0.01）。见插页ⅩⅩⅦ图1。
2.2　电针预处理对MIRI大鼠心肌组织病理学形态的影响
研究发现假手术组大鼠心肌纤维排列规则、细胞结构清晰，可见清晰横纹。与假手术组相比，模型组大鼠心肌纤维排列不规则，且纤维断裂结构模糊，细胞结构较疏松，观察到炎性细胞浸润；与模型组相比，电针组大鼠心肌组织的病理改变均有所减轻，纤维断裂减少，炎性细胞浸润减少；电针组与电针+生理盐水组两组大鼠心肌组织病理改变几乎一致，无明显差异。见插页ⅩⅩⅧ图2。
2.3　电针预处理对MIRI大鼠心肌组织中TNF-α表达水平的影响
与假手术组相比较，模型组大鼠心肌组织中的TNF-α的含量显著升高（P<0.05）；与模型组相比较，电针组大鼠心肌组织中的TNF-α的含量明显降低（P<0.05）。见表2。
2.4　电针预处理对MIRI大鼠心肌细胞中mTOR、NLRP3以及NF-κB蛋白表达的影响
与假手术组相比较，模型组中mTOR、NLRP3以及NF-κB蛋白表达均明显升高（P<0.05）；与模型组相比较，电针组mTOR、NLRP3及NF-κB蛋白表达明显降低（P<0.01，P<0.05）；与电针组+生理盐水组相比，电针+MHY1485组的mTOR、NLRP3及NF-κB蛋白表达明显升高（P<0.01，P<0.001）；电针组与电针+生理盐水组之间无统计学意义。见插页ⅩⅩⅧ图3。
2.5　电针预处理对MIRI大鼠心肌细胞中Caspase-1、ASC、IL-1β、IL-18mRNA水平的影响
与假手术组比，模型组心肌细胞中Caspase-1、ASC、IL-1β、IL-18mRNA表达水平上调（P<0.01）；与模型组比，电针组心肌细胞中Caspase-1、ASC、IL-1β、IL-18mRNA表达水平下调（P<0.01）；与电针+生理盐水组相比，电针+MHY1485组心肌细胞中Caspase-1、ASC、IL-1β、IL-18mRNA表达水平上调（P<0.05）。见插页ⅩⅩⅨ图4。
3　讨论
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冠状动脉疾病引起的急性心肌梗死严重影响人们的生活和生命质量[25]。针刺作为一种安全有效地预防性干预措施，可以发挥内源性保护效应[23]。逆针灸即今日所言针灸预处理，逆针灸一词首见于明代高武的《针灸聚英》，即“无病而先针灸曰逆。逆，未至而迎之也”，指在机体无病或疾病发生之前，预先应用针灸方法，激发经气，扶正祛邪，提高机体抵御各种致病因子的能力，防治疾病。腧穴，是指人体脏腑经络之气输注于体表的特殊部位。《灵枢·九针十二原》载：“五脏有疾也，应出十二原”，腧穴能够特异性反映和调节脏腑功能，具有诊断和治疗疾病的作用。内关穴首见《灵枢·经脉》：“手心主之别，名曰内关，去腕二寸，出于两筋之间……实则心痛。”《针灸甲乙经》：“实则心暴痛，虚则烦心，内关主之。”明代朱权所载的《四总穴歌》增补了“心胸内关穴”之说，更凸显了内关与心脏之间特异性联系[24]。
近年来大量临床和基础研究[10-11]都证明电针内关穴可以改善再灌注造成的心肌组织损伤。现代医学研究表明，心肌缺血再灌注损伤涉及许多生物学过程以及信号传导途径，如钙超载与能量代谢障碍、氧自由基增多、凋亡以及炎性反应等[30]。MIRI不仅促进炎症细胞因子的产生，还促进炎症细胞的组织浸润。炎症细胞因子的过度表达是导致心肌组织损伤的主要原因之一[33，36]。初始阶段血液和氧的回流使心脏产生剧烈应激反应，中性粒细胞被激活并发生聚集和浸润，释放大量促炎因子和炎症介质，如IL-6和TNF-α等，进一步扩大炎性反应，导致血管功能障碍和心肌组织损伤[26-28]。通过本实验证明电针预处理能够下调心肌组织内TNF-α的水平，这与过往研究[16]具有一致性。同时观察本实验超声心动图与心肌组织HE染色后的结果得出抑制过度炎症可缩小梗死范围，进而改善MIRI引起的功能障碍和组织损伤。
近年，NLRP3炎性小体因确切的促炎作用而被广泛关注[12-13]。NLRP3炎性小体是由天然免疫感受器NLRP3、接头蛋白ASC和Caspase-1前体组成的多聚蛋白复合物。研究发现NLRP3炎症小体作为机体防御反应的重要调控因子，贯穿于MIRI整个过程中，通过Caspase-1的活化，能促进心肌细胞焦亡、级联放大炎症反应和破坏心肌血管内皮细胞，导致心肌组织的进一步损伤[14]。相关研究表明抑制NLRP3炎性小体能够减少再灌注小鼠的心肌梗死面积[31]。哺乳动物雷帕霉素蛋白（The mammalian target of rapamycin，mTOR）是一种丝氨酸/苏氨酸蛋白激酶，是心脏结构和心肌代谢正常调节的必需品。研究发现褪黑素可以通过AMPK-mTOR信号通路抑制自噬来减弱心肌I/R损伤[32]，可见抑制mTOR能够增强自噬水平，进而增加抗炎反应，产生心肌保护。目前已明确mTOR与NLRP3之间有明显的交互调控[15]，mTOR[21-22]的活化已经被证实可以通过NF-κB将转录信息从细胞质传至细胞核，促进多种炎症因子表达，激活的NF-κB进一步上调NLRP3，Pro-IL-18，Pro-IL-1βmRNA转录水平，高水平的NLRP3，Pro-IL-18，ProIL-1βmRNA是形成有效炎症小体的关键；NLRP3、ASC和Caspase-1前体组成的多聚蛋白复合物，其组装可导致Caspase-1的激活，活化的Caspase-1随后促进IL-1β前体和IL-18前体裂解，从而产生成熟的、有功能的IL-1β和IL-18[17-18]。根据本研究结果显示，与模型组相比，电针组大鼠心肌组织中的mTOR、NF-κB、NLRP3蛋白表达与ASC、Caspase-1、IL-18、IL-1βmRNA相对表达水平均下调。与电针+生理盐水组相比较，电针+MHY1485组大鼠心肌组织中的mTOR蛋白表达明显提高，表明mTOR激动剂产生作用，且心肌组织中mTOR、NF-κB、NLRP3蛋白与ASC、Caspase-1、IL-18、IL-1βmRNA相对表达水平明显上调。证实电针预处理能够通过mTOR/NLRP3信号通路调控炎性反应，揭示电针预处理对减轻缺血再灌注大鼠心肌组织损伤的具体机制；同时正确理解和应用中医治未病的方法技术，为针刺在心血管疾病的防治提供科学的内涵。
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2025年第43卷第3期
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doi: 10.13193/j.issn.1673-7717.2025.03.017
  • 首发时间：2026-10-30
  • 出版时间：2025-03-10
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    南京中医药大学，江苏 南京 210000

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李开平（1967-），男，安徽马鞍山人，副教授，硕士研究生导师，博士，研究方向：中医养生治未病的基础与研究。E-mail：。
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Number of
species		 占总种数比例
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total species (%)		 属
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Number of
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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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