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Article(id=1308816736632267151, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1308816657934541639, articleNumber=null, orderNo=null, doi=10.16438/j.0513-4870.2025-0735, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1750953600000, receivedDateStr=2025-06-27, revisedDate=1758038400000, revisedDateStr=2025-09-17, acceptedDate=null, acceptedDateStr=null, onlineDate=1789975826652, onlineDateStr=2026-09-21, pubDate=1768147200000, pubDateStr=2026-01-12, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1789975826652, onlineIssueDateStr=2026-09-21, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1789975826652, creator=13701087609, updateTime=1789975826652, updator=13701087609, issue=Issue{id=1308816657934541639, tenantId=1146029695717560320, journalId=1189982191388893191, year='2026', volume='61', issue='1', pageStart='1', pageEnd='320', issueExtLink='null', onlineDate='null', pubDate='1768147200000', pubDateStr='2026-01-12', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1789975807888, creator='13701087609', updateTime=1789984282344, updator='13701087609', preIssue=null, nextIssue=null, articleTotal=null, ext={EN=IssueExt(id=1308852202421191556, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1308816657934541639, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1308852202421191557, tenantId=1146029695717560320, journalId=1189982191388893191, issueId=1308816657934541639, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null, downloadFileDto=null}, startPage=194, endPage=203, ext={EN=ArticleExt(id=1308816736858759568, articleId=1308816736632267151, tenantId=1146029695717560320, journalId=1189982191388893191, language=EN, title=Neuroprotective role and underlying mechanisms of benzoylpaeoniflorin in rat cerebral ischemia-reperfusion injury, columnId=1190335348761793317, journalTitle=Acta Pharmaceutica Sinica, columnName=Original Articles, runingTitle=null, highlight=null, articleAbstract=

To explore the protective effect of benzoylpaeoniflorin (BP), an active component of Paeonia lactiflora, on cerebral ischemic reperfusion injury (CIRI) in rats and its molecular mechanism, middle cerebral artery occlusion and reperfusion (MCAO/R) model was established, and oxygen-glucose deprivation/recovery (OGD/R) model was established by using rat adrenal pheochromocytoma nerve cells (PC12) to carry out experimental research. The results show that BP can significantly improve the neuronal apoptosis induced by CIRI, reduce the area of cerebral infarction, the neuronal damage in hippocampus, the loss of Nissl bodies and the mitochondrial damage. Western blot results showed that BP could significantly down-regulate the expression of pro-apoptosis proteins (Caspase3, Bax) and up-regulate the expression of anti-apoptosis protein (Bcl-2). qRT-PCR showed that BP down-regulated the gene expression levels of pro-inflammatory factors (TNF-α, IL-1β, IL-6) and pro-apoptosis proteins (Caspase3, Bax), and up-regulated the expression level of anti-apoptosis protein (Bcl-2). Transcriptome analysis showed that the differential genes regulated by BP were mainly concentrated in TNF signaling pathway, rheumatoid arthritis, IL-17 signaling pathway and so on. To sum up, BP can reduce inflammatory reaction and oxidative stress, inhibit neuronal apoptosis, and alleviate the nerve injury of CIRI in rats, which provides experimental basis for the clinical application of BP in the treatment of ischemic stroke. All experimental procedures were approved by the Experimental Animal Ethics Committee of Zhejiang Chinese Medical University (approval number: 20241209-15).

, authors=Tong LIN, Xu-ting WEI, Huang-qing ZHU, Yu SUN, Zhen-hong ZHU*, authorsList=Tong LIN, Xu-ting WEI, Huang-qing ZHU, Yu SUN, Zhen-hong ZHU, authorCompany=null, correspAuthors=Zhen-hong ZHU, authorNote=null, correspAuthorsNote=null, copyrightStatement=Copyright ©2026 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, fund=null), CN=ArticleExt(id=1308816740516192670, articleId=1308816736632267151, tenantId=1146029695717560320, journalId=1189982191388893191, language=CN, title=苯甲酰芍药苷对大鼠脑缺血再灌注损伤神经保护作用及机制的研究, columnId=1190335348896011050, journalTitle=药学学报, columnName=研究论文, runingTitle=null, highlight=null, articleAbstract=

本研究旨在探讨赤芍活性成分苯甲酰芍药苷(benzoylpaeoniflorin, BP) 对大鼠脑缺血再灌注损伤(cerebral ischemic reperfusion injury, CIRI) 的保护作用及其分子机制。通过建立大脑中动脉栓塞/再灌注(middle cerebral artery occlusion and reperfusion, MCAO/R) 模型, 利用大鼠肾上腺嗜铬细胞瘤神经细胞(PC12) 建立氧糖剥夺/再灌注(oxygen-glucose deprivation/recovery, OGD/R) 模型展开实验研究。结果显示, BP显著改善CIRI引起的神经细胞凋亡, 减少脑梗死面积, 减轻海马区神经元损伤、减少尼氏体丢失和减轻线粒体损伤。Western blot结果表明BP可显著下调促凋亡蛋白(Caspase3、Bax) 表达, 上调抗凋亡蛋白(Bcl-2) 的表达。qRT-PCR显示, BP下调促炎因子(TNF-α、IL-1β、IL-6) 和促凋亡蛋白(Caspase3、Bax) 的基因表达水平, 上调抗凋亡蛋白(Bcl-2) 的基因表达水平。转录组学分析发现, BP显著调控的差异基因主要富集在TNF信号通路、类风湿关节炎、IL-17信号通路等。综上所述, BP可降低炎症反应和氧化应激, 抑制神经元凋亡, 减轻大鼠CIRI的神经损伤, 为BP治疗缺血性脑卒中的临床应用提供了实验依据。本实验得到浙江中医药大学实验动物伦理委员会的批准(批准号: 20241209-15)。

, authors=林彤, 魏旭婷, 祝黄清, 孙羽, 朱振洪*, authorsList=林彤, 魏旭婷, 祝黄清, 孙羽, 朱振洪, authorCompany=null, correspAuthors=朱振洪, authorNote=

作者贡献:

林彤负责实验设计、实验操作、数据分析和文章撰写; 魏旭婷、祝黄清和孙羽参与部分实验设计; 朱振洪负责课题设计、实验指导、数据分析、文章写作与修改。

, correspAuthorsNote=
朱振洪, Tel: 86-571-86613726, E-mail:
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Front Genet, 2023, 14: 997383., articleTitle=null, refAbstract=null)], funds=null, companyList=[AuthorCompany(id=1308816741283750304, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308816736632267151, xref=null, ext=[AuthorCompanyExt(id=1308816741329887649, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308816736632267151, companyId=1308816741283750304, language=EN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=School of Life Sciences, Zhejiang Chinese Medical University, Hangzhou 310053, China), AuthorCompanyExt(id=1308816741380219298, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308816736632267151, companyId=1308816741283750304, language=CN, country=null, province=null, city=null, postcode=null, companyName=null, departmentName=null, remark=浙江中医药大学生命科学学院, 浙江 杭州 310053)])], figs=[ArticleFig(id=1308816745926844871, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308816736632267151, language=EN, label=null, caption=null, figureFileSmall=OFfLtWgQbLdrM7KjSDJi0Q==, figureFileBig=SjShJfRGj+sVacMxmxavqQ==, tableContent=null), ArticleFig(id=1308816747197718984, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308816736632267151, language=CN, label=Fig.1, caption=Pathological changes of brain tissue stained by BP in CIRI rats. A: Neurological function scores of rats in each group; B: Observation on cerebral infarction area of rats in each group; C: Area ratio of cerebral infarction; D: HE staining (100×); E: Nissl staining (100×); F: Ultrastructure of hippocampal neurons in ischemic side of rats in each group (30 000×); Magnification ×100; scale bar = 100 μm. Magnification ×30 000; scale bar = 1 μm. n = 3, x ± s. **P < 0.01, ****P < 0.000 1 vs Sham; ###P < 0.001 vs CIRI. CIRI: Cerebral ischemia-reperfusion injury; BP: Benzoylpaeoniflorin; ED: Edaravone, figureFileSmall=OFfLtWgQbLdrM7KjSDJi0Q==, figureFileBig=SjShJfRGj+sVacMxmxavqQ==, tableContent=null), ArticleFig(id=1308816747432600009, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308816736632267151, language=EN, label=null, caption=null, figureFileSmall=fRSmyyMTs+D7I6NBVAj12A==, figureFileBig=PErsQ/iwtM/Jz2IK3/+NsA==, tableContent=null), ArticleFig(id=1308816747520680394, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308816736632267151, language=CN, label=Fig.2, caption=Evaluation of serum oxidative stress index and Western blot index in hippocampus of CIRI rats by BP. A: Glutathione peroxidase (GSH-Px), catalase (CAT), malondialdehyde (MDA), superoxide dismutase (SOD); B: The expressions of Bcl-2, Bax, Caspase3 were detected by Western blot; C: Quantitative analysis of Bcl-2, Bax, Caspase3 protein. n = 3, x ± s. *P < 0.05, **P < 0.01, ****P < 0.0001 vs Sham; #P < 0.05, ##P < 0.01, ###P < 0.001, ####P < 0.000 1 vs CIRI. 2.5 BP: 2.5 mg·kg-1 BP; 5 BP: 5 mg·kg-1 BP; 10 BP: 10 mg·kg-1 BP, figureFileSmall=fRSmyyMTs+D7I6NBVAj12A==, figureFileBig=PErsQ/iwtM/Jz2IK3/+NsA==, tableContent=null), ArticleFig(id=1308816747596177867, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308816736632267151, language=EN, label=null, caption=null, figureFileSmall=WWaePcIxIB53BjpamUxNgA==, figureFileBig=iUlGD1qBIGwINlxIMfI0QA==, tableContent=null), ArticleFig(id=1308816747671675340, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308816736632267151, language=CN, label=Fig.3, caption=Effect of BP on PC12 neuron apoptosis after OGD/R. A: Effects of different concentrations of BP drugs on PC12 cells after OGD/R; B: Effect of BP on apoptosis of OGD/R model cells; C: The effect of BP on apoptosis after OGD/R was detected by flow cytometry; D: Crystal violet staining of PC12 cells after OGD/R modeling. Magnification ×100; scale bar = 100 μm. n = 3, x ± s. ***P < 0.001, ****P < 0.000 1 vs Sham; ###P < 0.001, ####P < 0.000 1 vs OGD/R. OGD/R: Oxygen-glucose deprivation/recovery, figureFileSmall=WWaePcIxIB53BjpamUxNgA==, figureFileBig=iUlGD1qBIGwINlxIMfI0QA==, tableContent=null), ArticleFig(id=1308816747730395597, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308816736632267151, language=EN, label=null, caption=null, figureFileSmall=JOcDZd0Ti6uNnaC9iS3CRg==, figureFileBig=ZDTcZVGJyGZ/t0olQCT4aA==, tableContent=null), ArticleFig(id=1308816747805893070, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308816736632267151, language=CN, label=Fig.4, caption=Expression of apoptosis-related proteins in OGD/R model by BP. The expressions of Bcl-2, Bax, Caspase3 were detected by Western blot and quantitative analysis of Bcl-2, Bax, Caspase3 protein. n = 3, x ± s. *P < 0.05, ***P < 0.001 vs Sham; ##P < 0.01, ###P < 0.001 vs OGD/R, figureFileSmall=JOcDZd0Ti6uNnaC9iS3CRg==, figureFileBig=ZDTcZVGJyGZ/t0olQCT4aA==, tableContent=null), ArticleFig(id=1308816747961082319, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308816736632267151, language=EN, label=null, caption=null, figureFileSmall=NYH3raScZoZQM+5Ipr9GHA==, figureFileBig=MVjr1d76CZmGCimkfSkCwg==, tableContent=null), ArticleFig(id=1308816748074328529, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308816736632267151, language=CN, label=Fig.5, caption=Transcriptome study on the protective effect of BP on CIRI rats. A: mRNA volcanogram of differential expression; B: KEGG analysis of differentially expressed genes, figureFileSmall=NYH3raScZoZQM+5Ipr9GHA==, figureFileBig=MVjr1d76CZmGCimkfSkCwg==, tableContent=null), ArticleFig(id=1308816748200157650, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308816736632267151, language=EN, label=null, caption=null, figureFileSmall=LM5f/YhAo60bDZCSeBEI8A==, figureFileBig=JbIotnDilhiXhmp2kg+NvQ==, tableContent=null), ArticleFig(id=1308816748279849427, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308816736632267151, language=CN, label=Fig.6, caption=qRT-PCR detection of Caspase3, IL-1β, TNF-α, Bax, Bcl-2, IL-6. n = 3, x ± s. **P < 0.01, ***P < 0.001, ****P < 0.000 1 vs Sham; ##P < 0.01, ###P < 0.001, ####P < 0.000 1 vs OGD/R, figureFileSmall=LM5f/YhAo60bDZCSeBEI8A==, figureFileBig=JbIotnDilhiXhmp2kg+NvQ==, tableContent=null), ArticleFig(id=1308816748401484244, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308816736632267151, language=EN, label=null, caption=null, figureFileSmall=null, figureFileBig=null, tableContent=
GenePrimer sequence (5′-3′)
Bcl-2-FCGG GAG AAC AGG GTA TGA T
Bcl-2-RTCA GGC TGG AAG GAG AAG AT
Bax-FGCG ATG AAC TGG ACA ACA A
Bax-RTCC CGA AGT AGG AAA GGA G
IL-6-FCCA CTC GGT CAG AGC ACT CC
IL-6-RCCA TCG CCG TCA CCA CTC C
IL-1β-FCCT GCA GCT GGA GAG TGT GGA T
IL-1β-RTGC TCT GCT TGT GAG GTG CTG
TNF-α-FACC GCA GAA AGA AGC CGT GG
TNF-α-RTGC CCC GCT TAC AGT TCC TC
Caspase3-FCTG GAC TGC GGT ATT GAG AC
Caspase3-RCCG GGT GCG GTA GAG TAA GC
β-actin-FCCG TAA AGA CCT CTA TGC CAA CA
β-actin-RTAG GAG CCA GGG CAG TAA TC
), ArticleFig(id=1308816748502147541, tenantId=1146029695717560320, journalId=1189982191388893191, articleId=1308816736632267151, language=CN, label=Tab.1, caption=

Primer sequence. Bcl-2: B-cell lymphoma-2; Bax: Bcl-2-associated X; IL-6: Interleukin-6; IL-1β: Interleukin-1β; TNF-α: Tumor necrosis factor alpha; Caspase3: Cysteinyl aspartate specific proteinase 3

, figureFileSmall=null, figureFileBig=null, tableContent=
GenePrimer sequence (5′-3′)
Bcl-2-FCGG GAG AAC AGG GTA TGA T
Bcl-2-RTCA GGC TGG AAG GAG AAG AT
Bax-FGCG ATG AAC TGG ACA ACA A
Bax-RTCC CGA AGT AGG AAA GGA G
IL-6-FCCA CTC GGT CAG AGC ACT CC
IL-6-RCCA TCG CCG TCA CCA CTC C
IL-1β-FCCT GCA GCT GGA GAG TGT GGA T
IL-1β-RTGC TCT GCT TGT GAG GTG CTG
TNF-α-FACC GCA GAA AGA AGC CGT GG
TNF-α-RTGC CCC GCT TAC AGT TCC TC
Caspase3-FCTG GAC TGC GGT ATT GAG AC
Caspase3-RCCG GGT GCG GTA GAG TAA GC
β-actin-FCCG TAA AGA CCT CTA TGC CAA CA
β-actin-RTAG GAG CCA GGG CAG TAA TC
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苯甲酰芍药苷对大鼠脑缺血再灌注损伤神经保护作用及机制的研究
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林彤 , 魏旭婷 , 祝黄清 , 孙羽 , 朱振洪 *
药学学报 | 研究论文 2026,61(1): 194-203
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药学学报 |研究论文 2026 , 61 (1) : 194 -203
苯甲酰芍药苷对大鼠脑缺血再灌注损伤神经保护作用及机制的研究
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林彤, 魏旭婷, 祝黄清, 孙羽, 朱振洪*
作者信息
  • 浙江中医药大学生命科学学院, 浙江 杭州 310053
通讯作者:
朱振洪, Tel: 86-571-86613726, E-mail:
作者简介:

作者贡献:

林彤负责实验设计、实验操作、数据分析和文章撰写; 魏旭婷、祝黄清和孙羽参与部分实验设计; 朱振洪负责课题设计、实验指导、数据分析、文章写作与修改。

Neuroprotective role and underlying mechanisms of benzoylpaeoniflorin in rat cerebral ischemia-reperfusion injury
Tong LIN, Xu-ting WEI, Huang-qing ZHU, Yu SUN, Zhen-hong ZHU*
Affiliations
  • School of Life Sciences, Zhejiang Chinese Medical University, Hangzhou 310053, China
出版时间: 2026-01-12 doi: 10.16438/j.0513-4870.2025-0735
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摘要
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本研究旨在探讨赤芍活性成分苯甲酰芍药苷(benzoylpaeoniflorin, BP) 对大鼠脑缺血再灌注损伤(cerebral ischemic reperfusion injury, CIRI) 的保护作用及其分子机制。通过建立大脑中动脉栓塞/再灌注(middle cerebral artery occlusion and reperfusion, MCAO/R) 模型, 利用大鼠肾上腺嗜铬细胞瘤神经细胞(PC12) 建立氧糖剥夺/再灌注(oxygen-glucose deprivation/recovery, OGD/R) 模型展开实验研究。结果显示, BP显著改善CIRI引起的神经细胞凋亡, 减少脑梗死面积, 减轻海马区神经元损伤、减少尼氏体丢失和减轻线粒体损伤。Western blot结果表明BP可显著下调促凋亡蛋白(Caspase3、Bax) 表达, 上调抗凋亡蛋白(Bcl-2) 的表达。qRT-PCR显示, BP下调促炎因子(TNF-α、IL-1β、IL-6) 和促凋亡蛋白(Caspase3、Bax) 的基因表达水平, 上调抗凋亡蛋白(Bcl-2) 的基因表达水平。转录组学分析发现, BP显著调控的差异基因主要富集在TNF信号通路、类风湿关节炎、IL-17信号通路等。综上所述, BP可降低炎症反应和氧化应激, 抑制神经元凋亡, 减轻大鼠CIRI的神经损伤, 为BP治疗缺血性脑卒中的临床应用提供了实验依据。本实验得到浙江中医药大学实验动物伦理委员会的批准(批准号: 20241209-15)。

苯甲酰芍药苷  /  脑缺血再灌注损伤  /  炎症反应  /  细胞凋亡  /  转录组测序

To explore the protective effect of benzoylpaeoniflorin (BP), an active component of Paeonia lactiflora, on cerebral ischemic reperfusion injury (CIRI) in rats and its molecular mechanism, middle cerebral artery occlusion and reperfusion (MCAO/R) model was established, and oxygen-glucose deprivation/recovery (OGD/R) model was established by using rat adrenal pheochromocytoma nerve cells (PC12) to carry out experimental research. The results show that BP can significantly improve the neuronal apoptosis induced by CIRI, reduce the area of cerebral infarction, the neuronal damage in hippocampus, the loss of Nissl bodies and the mitochondrial damage. Western blot results showed that BP could significantly down-regulate the expression of pro-apoptosis proteins (Caspase3, Bax) and up-regulate the expression of anti-apoptosis protein (Bcl-2). qRT-PCR showed that BP down-regulated the gene expression levels of pro-inflammatory factors (TNF-α, IL-1β, IL-6) and pro-apoptosis proteins (Caspase3, Bax), and up-regulated the expression level of anti-apoptosis protein (Bcl-2). Transcriptome analysis showed that the differential genes regulated by BP were mainly concentrated in TNF signaling pathway, rheumatoid arthritis, IL-17 signaling pathway and so on. To sum up, BP can reduce inflammatory reaction and oxidative stress, inhibit neuronal apoptosis, and alleviate the nerve injury of CIRI in rats, which provides experimental basis for the clinical application of BP in the treatment of ischemic stroke. All experimental procedures were approved by the Experimental Animal Ethics Committee of Zhejiang Chinese Medical University (approval number: 20241209-15).

benzoylpaeoniflorin  /  cerebral ischemia-reperfusion injury  /  inflammatory reaction  /  apoptosis  /  transcriptome sequencing
林彤, 魏旭婷, 祝黄清, 孙羽, 朱振洪. 苯甲酰芍药苷对大鼠脑缺血再灌注损伤神经保护作用及机制的研究. 药学学报, 2026 , 61 (1) : 194 -203 . DOI: 10.16438/j.0513-4870.2025-0735
Tong LIN, Xu-ting WEI, Huang-qing ZHU, Yu SUN, Zhen-hong ZHU. Neuroprotective role and underlying mechanisms of benzoylpaeoniflorin in rat cerebral ischemia-reperfusion injury[J]. Acta Pharmaceutica Sinica, 2026 , 61 (1) : 194 -203 . DOI: 10.16438/j.0513-4870.2025-0735
正文
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脑卒中是全世界第二大死亡原因, 具有高发病率、高致残率、高复发率、高死亡率等特点, 每年的死亡率约为550万, 全世界有1/4的人在其一生中经历过脑卒中[1]。随着人口老龄化进程的加快, 脑卒中患者年龄标准化率大幅下降, 这一趋势在70岁以上的人群中尤为明显, 其中急性缺血性脑卒中(acute ischemic stroke, AIS) 约占全部卒中的80%[2]。与此同时, 我国的脑卒中年患病率为202.20万, 年发病率为39.4万, 年死亡率为21.9万, 因此如何有效地防治脑卒中疾病的发生与发展在社会发展与进步中显得尤为重要[3]。在现代临床实践中, 脑卒中首选的治疗方法是血管再通和再灌注, 然而再灌注可进一步引起脑神经元的死亡, 加重脑损伤, 即脑缺血再灌注损伤(cerebral ischemia reperfusion injury, CIRI)。
缺血性脑卒中属于中医脑梗死的范畴, 中医称之为“卒中”或“中风”。外风学说认为: 脑卒中是由于体虚外邪入里、致使营卫不和、肢体失养所致; 内风学说认为: 脑卒中是由于脏腑阴阳失调、内风妄动、体内阴、阳、气、血逆乱所致[4]。中药可以通过改善氧化应激、凋亡、炎症反应、钙超载等因素来缓解脑缺血后造成的损伤, 在治疗AIS上具有独特疗效和开发价值[3]。赤芍作为地道中药的一个品种具有活血化瘀、清热止痛的作用[5]。现代药理学相关研究证实, 赤芍具有抗炎、抗病毒、免疫调节等作用[6]。苯甲酰芍药苷(benzoylpaeoniflorin, BP) 是赤芍的有效活性成分, 可以缓解脑缺血再灌注损伤引起的大鼠脑组织细胞凋亡[7], 同时它还可以通过Nrf2/HO-1信号通路来抑制大鼠急性心肌梗死[8]、缓解脂多糖引起的小鼠急性肺损伤[9]、调控JAK-STAT信号通路从而抑制滑膜细胞分泌异常来治疗风湿类关节炎[10]。
缺血性脑卒中发生时细胞中产生了大量的活性氧导致与体内的抗氧化水平失衡, 破坏了机体的抗氧化防御模式, 进而诱发一系列负面的细胞反应, 产生过量的活性氧, 引发氧化应激反应[11]。BP是赤芍中重要的活性物质, 具有抑制氧化应激反应和炎性反应的作用[12], 而氧化应激是缺血性脑卒中发生时不可忽视的问题。然而, BP对于脑缺血再灌注大鼠的脑保护作用尚不明确, BP抗CIRI的分子机制尚未系统阐明。近年来, 转录组学技术的迅速发展, 其在基因挖掘、代谢调控、药物研发、揭示疾病分子机制等方面发挥日益重要的作用。本研究通过转录组学揭示BP的作用靶点及通路, 筛选脑组织中的差异表达基因, 并对测序结果进行生物信息学分析及实时荧光定量逆转录聚合酶链反应(quantitative reverse transcription polymerase chain reaction, qRT-PCR) 验证, 以期阐明BP改善CIRI预后的可能靶基因及相关通路, 进而阐明BP治疗CIRI的潜在分子作用机制。
材料与方法
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实验动物    SPF级雄性SD大鼠, 体质量250 ± 10 g, 8~11周龄, 共30只, 由上海BK公司提供, 许可证号SCXK (沪) 2023-0009。适应性喂养1周, 自由进食进水, 室温22~26 ℃, 湿度55%, 定时通风换气, 模拟标准昼夜系统即12 h光照/12 h黑夜。实验中所有操作均获得浙江中医药大学实验动物伦理委员会的批准(批准号: 20241209-15)。
细胞系    PC12细胞来自本课题组冻存, 用含10%胎牛血清的DMEM培养基, 置于37 ℃、5% CO2培养箱中培养, 取处于对数生长期的细胞用于实验。
实验试剂    苯甲酰芍药苷购于成都曼斯特生物科技有限公司(CAS: MUST-22112805, 纯度≥ 98%), 以DMSO (终浓度 < 5%) 为溶剂溶解, 用生理盐水稀释到工作浓度; 依达拉奉(edaravone, ED) 购于中国食品药品检定研究院(CAS: 89-25-8, 纯度≥ 99%), 以DMSO (终浓度 < 5%) 为溶剂溶解, 用生理盐水稀释到工作浓度; 脑中动脉栓塞/再灌注损伤(middle cerebral artery embolization/reperfusion injury, MCAO/R) 线栓(批号: 2838-50/A4, 北京西浓科技有限公司); RNA逆转录试剂盒(赛默飞世尔科技公司, 货号3079237); 戊巴比妥(浙江中医药大学动物实验研究中心); AnnexinV-FITC细胞凋亡检测试剂盒(C1062M)、结晶紫染色液(C0121) 购于上海碧云天生物技术有限公司; 丙二醛(malondialdehyde, MDA) 测试盒(A003-1-1)、超氧化物歧化酶(superoxide dismutase, SOD) 测定试剂盒(A001-3-1)、谷胱甘肽过氧化物酶(glutathione peroxidase, GSH-Px) 测定试剂盒(A005-1-2)、过氧化氢酶(catalase, CAT) 测定试剂盒(A007-1-1) 均购于南京建成生物科技有限公司; 乙醇(杭州双林化工试剂厂); TRIzol (ThermoFisher公司, 15596018); 镁离子打断试剂盒(美国NEB公司, NEBNextR magnesium RNA fragmentation module, cat. E6150S); 逆转录酶(Invitrogen公司, SuperScriptTM Ⅱ Reverse Transcriptase, cat.1896649)。
仪器    台式高速离心机(北京六一生物); RM2255型切片机(Leica Microsystems公司); UC7型超薄切片机(德国徕卡公司); TE2000-S型倒置相差荧光显微镜(日本Nikon公司); illumina NovaseqTM 6000 (LC Bio Technology CO., Ltd.); BS323S型电子天平(北京赛多利斯仪器系统); Bioanalyzer 2100 (美国Agilent公司); oligo (dT) 磁珠[Dynabeads Oligo (dT), cat.25-61005, 美国Thermo Fisher公司]。
脑缺血再灌注损伤大鼠模型的制备    参考Longa对SD大鼠进行MCAO/R模型制作[13]。大鼠称重后用2%戊巴比妥通过腹腔注射麻醉。仔细分离出右侧颈总动脉、颈外动脉和颈内动脉。手术线结扎颈总动脉的近心端和颈外动脉, 在颈总动脉近分叉处打一活结, 动脉夹夹住颈内动脉。于颈总动脉结扎部位上方剪口, 能容纳栓线插入, 将栓线插入至颈内动脉前段, 松开动脉夹, 当栓线遇轻微阻力时即停止继续插入, 即模型成功。缺血1 h后将栓线缓慢取出, 结扎颈总动脉远心端, 缝合伤口并给予碘伏消毒。
大鼠分组与给药    根据前期预实验的结果及查阅的文献, 将SD大鼠随机分为假手术组(Sham组)、模型组(CIRI组)、BP组(2.5、5、10 mg·kg-1)、ED组(3 mg·kg-1), 每组10只, 于再灌注后2 h首次腹腔注射给予BP和ED药物, 每天1次, 连续7天, 假手术组与模型组按照文献[9]方法进行腹腔注射给予同体积生理盐水。
大鼠神经功能缺损症状评分    在实验大鼠再灌注后第1天和第7天, 按照Zea-Longa 5级评分制[14], 对各组大鼠进行神经行为学评分, 评分在1~3分者定为造模成功大鼠, 0分和4分者弃去。标准如下: 无神经损伤症状, 0分; 大鼠提尾悬空时, 不能完全伸展对侧前爪, 1分; 动物自由行走时, 表现为向对侧转圈, 2分; 动物自由行走时, 表现为向对侧倾倒, 3分; 不能自由行走, 意识丧失, 4分。实验中评分为1~3分的大鼠共35只, 造模成功率为87.5%, 可以进行实验测定。
TTC染色    各组大鼠经0.9% NaCl心脏灌注后, 取出的完整脑组织置于预冷的大鼠脑槽中, 将脑槽放入-80 ℃冰箱内速冻至脑组织微硬, 将其切成冠状切片后, 用2% TTC染液浸泡脑片, 37 ℃避光孵育。每隔5 min摇晃组织, 重复3次。
HE染色    各组大鼠经0.9% NaCl和多聚甲醛心脏灌注后, 取出完整的脑组织放入4%多聚甲醛中进行固定。然后包埋切片后, 分别用二甲苯、无水乙醇、95%、85%、70%乙醇浸泡, 并在蒸馏水中浸泡脱蜡。分别用苏木素、伊红染色, 并用不同浓度梯度的乙醇溶液脱水, 二甲苯透明处理, 滴加中性树脂封片固定。利用光学显微镜观察脑组织海马区病理变化并记录图像。
Nissl染色    各组大鼠经0.9% NaCl和多聚甲醛心脏灌注后, 取出完整的脑组织放入4%多聚甲醛中进行固定。然后包埋切片, 分别用二甲苯、无水乙醇、95%、85%、70%乙醇浸泡, 并在蒸馏水中浸泡脱蜡。蒸馏水冲洗3次, 加入1%甲苯胺蓝60 ℃温箱孵育染色40 min, 蒸馏水洗净, 不同浓度梯度的乙醇溶液中脱水, 再用二甲苯透明处理, 最后用中性树胶封片。
大鼠海马体超微结构透射电镜    各组大鼠经心脏灌注后, 冰上取出脑组织中的海马体, 切割成1 mm3大小, 放入2.5%戊二醛溶液中, 4 ℃冰箱固定。ddH2O漂洗3次, 1%锇酸溶液固定样品。ddH2O漂洗3次, 用不同梯度浓度乙醇溶液对样品程序脱水, 再用无水乙醇和纯丙酮处理; 用包埋剂混合液处理样品, 最后加入纯包埋剂常温避光过夜, 第二天取出样品70 ℃加热聚合过夜, 最后进行超薄切片并使用双铅染色, 最后透射电镜观察并记录图像。
大鼠造模部位脑蛋白提取与Western blot    各组大鼠经0.9% NaCl心脏灌注后, 冰上分离出造模部位脑组织海马体, 取部分海马组织, 按20倍体积加入RIPA裂解液, 取2颗研磨球加入离心管, 将仪器设置为: 60 Hz频率研磨60 s, 重复两次。研磨结束后, 冰上裂解30 min, 每10 min用涡旋振荡器震荡一次。离心(4 ℃、12 000 r·min-1离心15 min) 后取上清液至新的离心管中, 将每个样品稀释10倍, BCA法测定蛋白浓度; 根据蛋白浓度, 进行备样, 将样品100 ℃煮沸5 min进行变性, -80 ℃保存。以80 V电压跑完SDS-PAGE电泳, 随后以400 mA、40 min条件进行转膜, 完成后用5%脱脂奶粉封闭1 h, 按照要求孵育一抗、二抗, 显影。
大鼠血清中GSH-Px、CAT、MDA、SOD含量测定    抽取各组大鼠动脉血, 室温静置10 min后, 3 000 r·min-1离心10 min, 取上清后冻存于-80 ℃冰箱中, 按照GSH-PX、CAT、MDA、SOD试剂盒要求, 进行血清指标检测。
氧糖剥夺/再灌注(oxygen-glucose deprivation/recovery, OGD/R)模型的构建    取对数生长期细胞, 进行细胞消化, 用血球计数板计数, 将细胞悬液稀释为每毫升1×105个, 置于37 ℃、5% CO2培养箱中培养28 h。将孔板取出, PBS润洗3次, 加入无糖无血清DMEM培养基, 将96孔板放置于充满95% N2、5% CO2的缺氧小室中培养6 h。重新换成完全培养基, 在37 ℃、5% CO2培养箱中继续培养18 h。
CCK8法探究不同BP浓度对细胞的活力影响    将细胞接种至96孔板内进行铺板造模, 将细胞分为Sham组、OGD/R组、BP组, 置于培养箱培养至对数生长期; 取出96孔板, 用PBS轻轻润洗3次, 然后向孔中分别加入含0、25、50、100、125、150 μmol·L-1 BP的DMEM完全培养液, 至培养箱中培养18 h; 使用酶标仪测定吸光度(A450) 值, 计算细胞活力值。细胞活力值= (A给药组 - A空白组) / (ASham组 - A空白组) × 100%。
结晶紫染色    将细胞接种至96孔板内进行铺板造模, 将细胞分为Sham组、OGD/R组、BP组, 置于培养箱培养至对数生长期; 取出6孔板, 用PBS轻轻润洗3次, 然后向BP组的孔中加入含50、100、150 μmol·L-1 BP的DMEM完全培养液, 至培养箱中培养18 h, 取出培养板, 按照说明书要求加入等量结晶紫染, 在倒置显微镜下观察拍照。
流式细胞术检测不同浓度BP对OGD/R后PC12细胞凋亡的影响    将细胞接种至96孔板内进行铺板造模, 将细胞分为Sham组、OGD/R组、BP组, 置于培养箱培养至对数生长期; 取出6孔板, 用PBS轻轻润洗3次, 然后向BP组的孔中加入含50、100、150 μmol·L-1 BP的DMEM完全培养液, 至培养箱中培养18 h, 按照Annexin V-FITC细胞凋亡检测试剂盒说明书处理样本。
RNA-seq转录组测序分析    用TRIzol对总样品的RNA进行分离和纯化。NanoDrop ND-1000对总RNA的量与纯度进行质控, 并通过Bioanalyzer 2100对RNA的完整性进行检测; 浓度 > 50 ng·μL-1, RIN值> 7.0, total RNA > 1 μg满足下游实验。使用oligo (dT) 磁珠通过两轮纯化对其中带有PolyA的mRNA进行特异性捕获。捕获到的mRNA在高温条件下利用镁离子打断试剂盒进行片段化, 94 ℃、5~7 min。将片段化的RNA通过逆转录酶的作用合成cDNA, 然后使用E. coli DNA polymerase I与RNase H进行双链合成。将这些DNA与RNA的复合双链转化成DNA双链, 同时在双链中掺入dUTP solution, 将双链DNA的末端补齐为平末端, 再在其两端各加上一个A碱基, 使其能够与末端带有T碱基的接头进行连接, 并利用磁珠对其片段大小进行筛选和纯化。以UDG酶消化双链, 通过PCR预变性95 ℃保持3 min, 98 ℃变性8个循环, 每次15 s, 退火到60 ℃保持15 s, 72 ℃下延伸30 s, 最后延伸72 ℃保留5 min, 使其形成片段大小为300 ± 50 bp的文库。最后, 使用illumina NovaseqTM 6000对其进行双端测序, 测序模式为PE150。
KEGG富集分析    根据差异倍数(fold change, FC) ≥ 1.2、P adjust < 0.05为标准进行差异基因筛选, 筛选出Sham vs CIRI和BP vs CIRI的差异基因。以大鼠全基因组为背景, P < 0.05为标准进行GO富集分析和KEGG代谢通路富集分析, 从而获取关键通路信息, 并通过R语言将相关信息进行可视化。
qRT-PCR验证    取各组大鼠缺血半球脑组织标本, 液氮速冻, -80 ℃保存。用qRT-PCR验证预测的关键差异基因: 肿瘤坏死因子-α (tumor necrosis factor alpha, TNF-α)、白细胞介素-1β (interleukin-1β, IL-β)、白细胞介素-6 (interleukin-6, IL-6)、半胱氨酸的天冬氨酸蛋白水解酶3 (cysteinyl aspartate specific proteinase 3, Caspase3)、Bcl-2 (B-cell lymphoma-2) 和Bax (Bcl-2 associated X protein)。根据试剂盒说明书操作, 使用TRIzol提取脑组织中的总RNA, 随后进行逆转录合成cDNA。以β肌动蛋白(β-actin) 为内源性参照基因, 定量检测目的基因的表达水平。各引物序列见表1。反应条件为95 ℃预变性30 s, 95 ℃变性5 s, 60 ℃退火延伸1 min, 反应完毕, 计算各基因的相对表达量。
统计学分析    实验数据采用SPSS 22.0统计软件分析, 计数资料均以平均值±标准差(x ± s) 表示, 组间多个样本均数比较采用单因素方差分析(one way ANOVA) 和LSD检验, 显著性检验采用双侧检验, P < 0.05表示有显著性差异。
结果
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1　BP对CIRI大鼠脑组织染色的病理性变化
为探究BP对CIRI大鼠是否具有治疗作用, 在造模手术后的第1天和第7天, 本研究对不同组别的大鼠进行了神经功能评分, 实验结果显示(图1A), 评分为1~3分的大鼠为35只, 造模成功率为87.5%, 7天后, 与CIRI组相比, BP组神经功能损伤评分显著下降, 具显著性差异, 说明BP可以减轻脑缺血再灌注神经功能损伤。同时, TTC染色结果显示(图1B), 红色部分为脑组织正常区域, 白色部分为脑组织梗死区域, 与CIRI组相比, BP组的脑梗死体积减小, 且具有药物剂量依赖性, 依达拉奉阳性对照组的脑梗死体积减少(图1C)。HE染色结果显示(图1D), BP组较CIRI组, 海马区细胞边界明显清晰, 排列整齐, 没有明显的细胞坏死等病理变化。Nissl染色结果显示(图1E), CIRI组尼氏体减少、皱缩, 神经元数量减少, 呈空泡性; BP组尼氏体数量明显增多, 神经元数量明显增加, 尼氏体无明显皱缩。此外, 本研究观察了CIRI大鼠脑组织海马体中线粒体状态, 通过透射电镜对大鼠缺血侧海马区超微结构进行观察。结果显示(图1F), CIRI组线粒体出现异常, 线粒体发生肿胀、变形, 嵴断裂并且数量有一定的减少, BP组形态规则, 大小适中, 线粒体嵴较清晰。上述结果表明, BP对CIRI大鼠具有明显的治疗作用, 能够抑制CIRI大鼠脑部神经细胞的损伤, 起到保护神经细胞的作用。
2　BP对CIRI大鼠血清氧化应激指标和海马组织Western blot指标评价
GSH-Px是一种催化过氧化氢分解的酶, 与模型组相比, BP组GSH-Px表达量升高(图2A)。CAT是一种过氧化氢酶, 通过计算其活力可以反映细胞氧化损伤, 与模型组相比, BP组CAT表达量升高。MDA可以表征机体内脂质过氧化的程度, 从而间接反映出细胞损伤的程度, 与模型组相比, BP给药组MDA的含量明显下降。SOD活力可以反映机体清除氧自由基的能力, 与正常组相比, 模型组SOD含量明显下降, 而BP组SOD含量升高。此外, 通过Western blot检测CIRI后大鼠细胞凋亡相关的蛋白表达情况, 结果显示(图2B、C), SD大鼠通过MCAO/R造模后, Bcl-2蛋白表达量降低, 而Bax和Caspase3蛋白表达量上调, 与Sham组相比, 均存在显著性差异; 经BP给药治疗后, Bax和Caspase3蛋白表达量下调, Bcl-2蛋白表达量上调, 与CIRI组存在显著性差异。以上结果表明, BP一定程度能够抑制CIRI大鼠的氧化应激反应, 并且可能对CIRI后大鼠神经细胞凋亡有明显的改善作用。
3　BP对OGD/R后PC12神经细胞凋亡的影响
不同浓度BP对OGD/R后PC12细胞存活率的结果显示(图3A), 与Sham组细胞相比, OGD/R组(0 μmol·L-1) 细胞存活率约为61%, 具有显著性差异; 因此, 后续实验选择100 μmol·L-1为最佳给药浓度。利用流式细胞术验证细胞凋亡, 给予0、50、100、150 μmol·L-1 BP药物培养, 由图可知(图3B、C), 与Sham组相比, OGD/R组凋亡细胞显著增多; 与OGD/R组相比, BP组凋亡细胞减少。通过结晶紫染色观察细胞凋亡情况, 如图所示(图3D), 深紫色为正常细胞, OGD/R组有较少的深紫色细胞, 说明大量贴壁细胞凋亡脱落; 而BP组基本与Sham组无明显差异, 表明细胞凋亡较少。以上结果表明, BP可以抑制OGD/R后PC12细胞的凋亡。随后, 通过Western blot检测细胞凋亡相关的蛋白表达情况, 结果如图所示(图4), PC12细胞通过OGD/R造模后, Bcl-2蛋白表达量降低, 而Bax和Caspase3蛋白表达量上调, 与Sham组相比, 均存在显著性差异; 经BP给药治疗后, Bax和Caspase3蛋白表达量下调, Bcl-2蛋白表达量上调, 与模型组存在显著性差异。以上结果表明, BP对OGD/R后神经细胞凋亡有明显的改善作用, BP可能抑制细胞凋亡, 从而保护细胞。
4　BP对CIRI大鼠保护作用的转录组学数据
本实验所建立的测序文库中, 9个样品的原始数据在4.1×107~5.4×107, 总计4.09×108。每个cDNA文库平均获得4.5×107个原始数据, 对应于61.37 Gb的原始数据。经过原始数据过滤后, 约有3.87×108 (94.6%) 的原始数据作为高质量的读段(clean reads) 被保留下来。9个样品的reads差异较小, 质量值≥ 20的碱基所占百分比(Q20) ≥ 99.59%, 质量值≥ 30的碱基所占百分比(Q30) ≥ 98.18%, 表明测序结果较好, 可用于后续的分析。同时, 采用DEseq软件, 筛选出具有统计学意义的差异表达基因(| log2Fold change |≥ 2, P < 0.05)。数据分析结果表明, 与假手术组比较, 模型组显著上调的差异基因为151个, 显著下调的差异基因为308个; 与模型组比较, BP组显著上调的差异基因为176个, 显著下调的差异基因为57个(图5A)。
本研究对筛选出的差异基因进行KEGG富集分析, 并设置P < 0.05为筛选标准结果(图5B)。假手术组vs模型组的KEGG富集分析结果显示基因主要富集在细胞因子-细胞因子相互作用、钙离子通路、AGE-RAGE信号通路; 而BP与CIRI相比, 基因主要富集在抗体加工与呈递、TNF信号通路、类风湿关节炎、IL-17信号通路、NOD样受体信号通路等。为了验证BP对脑缺血再灌注损伤大鼠凋亡相关通路基因表达是否具有调控作用, 采用qRT-PCR法对转录组测序分析结果的TNF-α通路中3个炎症关键因子TNF-α、IL-1β、IL-6及凋亡相关的Caspase3、Bax、Bcl-2关键因子进行表达水平的验证。结果表明(图6), 与Sham组比较, 模型组TNF-α、IL-1β、IL-6、Caspase3、Bax表达上调, Bcl-2表达下调, 差异有统计学意义; 与模型组比较, BP可降低TNF-α、IL-6、IL-1β、Caspase3、Bax的表达, 提高Bcl-2的表达, 差异有统计学意义。
讨论
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本研究通过建立大鼠MCAO/R模型[15], 探讨了BP对CIRI的保护作用及其潜在的分子机制。结合转录组学分析, 发现BP通过调控多个信号通路, 显著减轻了CIRI引起的神经功能损伤、脑梗死面积扩大以及细胞凋亡, 并通过神经功能评分、TTC染色、HE染色、Nissl染色和线粒体超微结构分析等方法, 证实了BP对CIRI大鼠的神经功能具有显著的保护作用。与模型组相比, BP治疗组大鼠的神经功能评分显著降低, 脑梗死面积明显缩小, 且海马区神经元形态和尼氏体数量得到显著改善, 在超微显微镜下观察相较于CIRI组, BP组形态规则, 大小适中, 线粒体嵴较清晰。在体外实验中, 在OGD/R模型中, BP能够显著提高PC12细胞的存活率, 减少细胞凋亡。结晶紫染色和流式细胞术的结果进一步证实了BP的抗凋亡作用。脑缺血再灌注损伤会导致细胞产生各种破坏性的细胞毒性介质。GSH-Px和SOD具有清除自由基和衍生物的作用, 减少脂质过氧化物的形成, 增强机体抗氧化损伤的能力; MDA则是膜脂质过氧化最重要的产物之一, 其大量产生会使细胞膜的脂质发生过氧化作用而引起膜裂变, 导致细胞损伤甚至死亡[16]。CAT是一种过氧化氢酶, 其活力可以反映细胞氧化损伤, CAT累积越多, 表明氧化损伤越轻。本实验结果显示, BP能够有效减轻CIRI引起的脑组织损伤, 保护神经元免受缺血再灌注的损害。
细胞凋亡是在基因严格控制下主动的细胞“自杀”行为, 又称为细胞程序性死亡[17], 是CIRI后神经元死亡的主要机制之一。本研究通过转录组学分析发现, BP显著调控了多个与细胞凋亡相关的基因和信号通路。线粒体可以通过促凋亡蛋白Bax与Caspases的相互作用来调节凋亡, Caspases也可以通过Bcl-2家族蛋白来调控线粒体释放促凋亡蛋白的作用, 从而放大凋亡级联反应[18]。其中, 通过Western blot和qRT-PCR的结果可知, Bcl-2家族蛋白(如Bcl-2和Bax) 和Caspase3的表达在BP治疗后发生了显著变化。Bcl-2是一种抗凋亡蛋白, 当细胞受到凋亡刺激后, Bcl-2蛋白被激活[19], 而Bax是Bcl-2家族蛋白的关键成员, 促进细胞凋亡。BP通过上调Bcl-2的表达并下调Bax的表达, 抑制了Caspase3的激活, 从而减少神经元的凋亡。这一结果与先前的研究一致, 表明BP通过调控Bcl-2、Bax、Caspase3信号通路, 发挥其抗凋亡作用。
缺血性脑卒中发生后, 大脑内氧糖供应不足会引起神经元的死亡并释放大量炎症因子, 诱发神经炎症, 进而加重神经损伤程度和炎症反应[20], 尤其是TNF-α、IL-1β和IL-6等蛋白炎症因子的过度表达会加剧脑损伤。TNF-α是一种具有广泛生物学功能的多肽类细胞因子, 若持续释放, 则会引起机体发热、休克、恶病质和组织损伤[21]。本研究选取TNF-α通路中3个炎症关键因子TNF-α、IL-1β、IL-6及凋亡相关的Caspase3、Bax、Bcl-2关键因子进行表达水平的验证。发现BP显著降低了TNF-α、IL-1β和IL-6的表达水平, 表明BP具有抗炎作用。转录组是指特定的组织或细胞在某一生长阶段或功能状态下所转录出来RNA总和, 包括编码蛋白质的mRNA和各种非编码RNA[22]。转录组学是功能基因组学研究的重要组成部分, 是一门在整体水平研究基因功能和基因结构, 揭示基因转录、转录调控规律以及疾病发生过程中的分子机制的学科[23]。通过RNA-seq技术, 本研究筛选出了BP治疗CIRI大鼠后的差异表达基因, 结果显示, BP主要通过调控细胞凋亡、炎症反应和TNF信号通路等关键通路, 发挥其脑保护作用。这些发现为进一步研究BP的药理机制提供了重要的分子基础, 结合其抗炎、抗凋亡的多重作用机制, BP有望成为治疗脑卒中的潜在药物。然而, BP的具体作用机制仍需进一步研究, 尤其是在临床前和临床试验中验证其安全性和有效性。
本研究发现BP可通过调控Bcl-2/Bax通路改善CIRI, 表明BP可能是CIRI脑损伤的潜在治疗药物。然而, BP的具体作用靶点和信号通路仍需进一步研究。未来的工作可以聚焦BP的临床应用转化, 如优化给药方案、评估长期疗效和安全性, 以及探索其与其他治疗药物的协同作用。此外, 基于转录组学的结果, 可以进一步挖掘BP调控的关键基因和通路, 为开发新型神经保护剂提供理论支持。

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2026年第61卷第1期
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doi: 10.16438/j.0513-4870.2025-0735
  • 接收时间：2025-06-27
  • 首发时间：2026-09-21
  • 出版时间：2026-01-12
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  • 收稿日期：2025-06-27
  • 修回日期：2025-09-17
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    浙江中医药大学生命科学学院, 浙江 杭州 310053

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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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