Article(id=1193674742768042263, tenantId=1146029695717560320, journalId=1190317699101192196, issueId=1193674740352119804, articleNumber=1001-2494(2025)06-0604-08, orderNo=null, doi=10.11669/cpj.2025.06.006, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1731513600000, receivedDateStr=2024-11-14, revisedDate=null, revisedDateStr=null, acceptedDate=null, acceptedDateStr=null, onlineDate=1762523836080, onlineDateStr=2025-11-07, pubDate=1742572800000, pubDateStr=2025-03-22, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1762523836080, onlineIssueDateStr=2025-11-07, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1762523836080, creator=13701087609, updateTime=1762523836080, updator=13701087609, issue=Issue{id=1193674740352119804, tenantId=1146029695717560320, journalId=1190317699101192196, year='2025', volume='60', issue='6', pageStart='553', pageEnd='662', issueExtLink='null', onlineDate='null', pubDate='null', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1762523835503, creator=13701087609, updateTime=1762524041683, updator=13701087609, preIssue=null, nextIssue=null, ext={EN=IssueExt(id=1193675605205025683, tenantId=1146029695717560320, journalId=1190317699101192196, issueId=1193674740352119804, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1193675605205025684, tenantId=1146029695717560320, journalId=1190317699101192196, issueId=1193674740352119804, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null}, startPage=604, endPage=611, ext={EN=ArticleExt(id=1193674742998728986, articleId=1193674742768042263, tenantId=1146029695717560320, journalId=1190317699101192196, language=EN, title=Nalmefene Alleviates Postoperative Cognitive Dysfunction in Aged Rats via the Sirt1/TLR4/NF-κB Pathway, columnId=null, journalTitle=Chinese Pharmaceutical Journal, columnName=null, runingTitle=null, highlight=null, articleAbstract=

OBJECTIVE To explore the effect and molecular mechanism of nalmefene on postoperative cognitive dysfunction (POCD) in aged rats. METHODS Seventeen-month-old SD rats were randomly divided into control group (Control), model group (Model), model + nalmefene group (Model+Nal), model+nalmefene+EX527 group (Model+Nal+EX527) with 8 rats in each group after one week of acclimatization. The POCD animal models (excluding the Control group) were established by sevoflurane anesthesia and exploratory laparotomy. The rats in Model+Nal group were subcutaneously injected with 0.1 mg·kg-1 nalmefene hydrochloride 30 min before anesthesia induction. The rats in Model+Nal+EX527 group were injected subcutaneously with 0.1 mg·kg-1 nalmefene hydrochloride and intraperitoneally with 10 mg·kg-1 EX527 (Sirt1 inhibitor) 30 min before anesthesia induction. Morris water maze experiment was conducted to analyze the cognitive abilities of rats. Hippocampal tissues were collected 3 d after surgery. The pathological changes of the hippocampus were observed by HE staining. Neuron apoptosis was analyzed by Tunel staining. ELISA method was used to detect the contents of TNF-α and IL-6. The protein expression levels of Bax, Bcl-2, Sirt1, TLR4 and NF-κB p65 were determined by Western blot. RESULTS Compared with the Control group, the cognitive dysfunction was seen, neuronal damage and the proportion of apoptotic neurons in hippocampal DG region were increased, TNF-α and IL-6 contents as well as the expression of Bax, TLR4 and NF-κB p65 were elevated, while the expression of Bcl-2 and Sirt1 was decreased in the Model group (P<0.001). Compared with the Model group, the cognitive ability was enhanced, neuronal damage and the proportion of apoptotic neurons in hippocampal DG region were decreased, TNF-α and IL-6 contents as well as the expression of Bax, TLR4 and NF-κB p65 were reduced, whereas the expression of Bcl-2 and Sirt1 was elevated in the Model+Nal group (P<0.001). Compared with the Model+Nal group, the cognitive ability was suppressed, neuronal damage and the proportion of apoptotic neurons in hippocampal DG region were increased, TNF-α and IL-6 contents as well as the expression of Bax, TLR4 and NF-κB p65 were elevated, while the expression of Bcl-2 and Sirt1 was decreased in the Model+Nal+EX527 group (P<0.001). CONCLUSION Nalmefene can reduce neuroinflammation and neuronal damage, and improve cognitive dysfunction in aged POCD rats through regulating Sirt1/TLR4/NF-κB pathway.

, correspAuthors=Hongfang GENG, 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=Lin ZHANG, Fudong TANG, Yuedan LIU, Hongfang GENG), CN=ArticleExt(id=1193675147505795911, articleId=1193674742768042263, tenantId=1146029695717560320, journalId=1190317699101192196, language=CN, title=纳美芬通过调控Sirt1/TLR4/NF-κB通路改善老年大鼠术后认知功能障碍, columnId=1190352405612040510, journalTitle=中国药学杂志, columnName=论著, runingTitle=null, highlight=null, articleAbstract=

目的 探究纳美芬对老年大鼠术后认知功能障碍(postoperative cognitive dysfunction,POCD)的影响及其分子机制。方法 将17月龄的SD大鼠适应饲养一周后,随机分成对照(Control)组、模型(Model)组、模型+纳美芬(Model+Nal)组、模型+纳美芬+EX527(Model+Nal+EX527)组,每组8只。除Control组外,使用七氟烷麻醉和剖腹探查术建立POCD模型。Model+Nal组大鼠在麻醉诱导前30 min皮下注射0.1 mg·kg-1盐酸纳美芬;Model+Nal+EX527组大鼠在麻醉诱导前30 min皮下注射0.1 mg·kg-1盐酸纳美芬,并腹腔注射10 mg·kg-1 EX527 [沉默信息调节因子1(Sirt1)抑制剂]。Morris水迷宫实验评估大鼠认知能力。术后第3天处死大鼠,苏木素-伊红(HE)染色观察海马组织病理学改变,Tunel染色分析神经元凋亡,酶联免疫吸附测定(ELISA)方法分析肿瘤坏死因子α(TNF-α)和白细胞介素6(IL-6)含量,蛋白质免疫印迹(Western blot)法检测B-细胞淋巴瘤因子2(Bcl-2)、Bcl-2 相关X蛋白(Bax)、Sirt1、Toll样受体4(TLR4)和核因子κB亚基p65(NF-κB p65)蛋白表达水平。结果 与Control组相比,Model组大鼠出现认知障碍,海马DG区神经元出现损伤,DG区神经元凋亡比例增多,TNF-α和IL-6含量增加,Bax、TLR4和NF-κB p65蛋白表达增加,Bcl-2和Sirt1表达降低(P<0.001);与Model组相比,Model+Nal组大鼠认知能力增强,海DG区神经元损伤减轻,DG区神经元凋亡比例减少,TNF-α和IL-6含量降低,Bax、TLR4和NF-κB p65蛋白表达减少,Bcl-2和Sirt1表达增加(P<0.001);与Model+Nal组相比,Model+Nal +EX527组大鼠认知能力减弱,海马DG区神经元损伤增加,DG区神经元凋亡比例增多,TNF-α和IL-6含量增加,Bax、TLR4和NF-κB p65蛋白表达增加,Bcl-2和Sirt1表达降低(P<0.001)。结论 纳美芬可通过Sirt1/TLR4/NF-κB通路减轻POCD老年大鼠神经炎症和神经元损伤,改善认知功能障碍。

, correspAuthors=耿红芳, authorNote=null, correspAuthorsNote=
*耿红芳,女,硕士,副主任医师 研究方向:麻醉基础及临床 Tel:(0371)65580445
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张琳,女,硕士,副主任医师 研究方向:小儿麻醉基础与临床

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articleId=1193674742768042263, language=CN, orderNo=5, keyword=Toll 样受体 4/核因子κB)], refs=[Reference(id=1193713127960309785, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1193674742768042263, doi=null, pmid=null, pmcid=null, year=2024, volume=18, issue=null, pageStart=1328790, pageEnd=null, url=null, language=null, rfNumber=[1], rfOrder=0, authorNames=ZHAO Q, WAN H, PAN H, journalName=Front Behav Neurosci, refType=null, unstructuredReference=ZHAO Q, WAN H, PAN H, et al. 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Nal-nalmefene; EX527-Sirt1 inhibitor.

, figureFileSmall=hHAmXf2iOXVOfieRqF4Nqg==, figureFileBig=79AWiFUFUGe62hcn0O/e3A==, tableContent=null), ArticleFig(id=1193713126777516045, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1193674742768042263, language=CN, label=图1, caption=各组大鼠定位航行实验和空间探索实验中游泳轨迹的变化。n=8

Nal-纳美芬;EX527-沉默信息调节因子1(Sirt1)抑制剂。

, figureFileSmall=hHAmXf2iOXVOfieRqF4Nqg==, figureFileBig=79AWiFUFUGe62hcn0O/e3A==, tableContent=null), ArticleFig(id=1193713126899150862, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1193674742768042263, language=EN, label=Fig.2, caption=Pathological changes of neurons in the hippocampal DG regions of rats were detected by HE staining(×40 and ×200), figureFileSmall=0GVyPyaL7//ZLioJybVHKA==, figureFileBig=+7zy1179Rs8iuRX54dYZ6A==, tableContent=null), ArticleFig(id=1193713126974648335, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1193674742768042263, language=CN, label=图2, caption=苏木素-伊红(HE)染色检测大鼠海马齿状回(DG)区神经元病理变化(×40和×200), figureFileSmall=0GVyPyaL7//ZLioJybVHKA==, figureFileBig=+7zy1179Rs8iuRX54dYZ6A==, tableContent=null), ArticleFig(id=1193713127062728720, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1193674742768042263, language=EN, label=Fig.3, caption=Comparison of neuronal apoptosis in the hippocampal DG regions of rats in different groups.n=4,$\bar{x} \pm s$

A-Tunel staining for hippocampal neurons (×200 and ×400); B-apoptotic rate of neurons in hippocampal DG region; C-Western blot assay for the protein levels of Bax and Bcl-2 in hippocampal tissues; D-statistical analysis for Bax protein relative expression; E-statistical analysis for Bcl-2 protein relative expression; 1)P<0.001, compared with control group; 2)P<0.001, compared with model group; 3)P<0.001, compared with model+Nal group.

, figureFileSmall=UXQMx82kBb/fnqbcwA7lzg==, figureFileBig=IyfqnvFP1hLv3gBYo0whTw==, tableContent=null), ArticleFig(id=1193713127188557841, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1193674742768042263, language=CN, label=图3, caption=各组大鼠海马DG区神经元凋亡情况比较。n=4,$\bar{x} \pm s$

A-海马DG区神经元Tunel染色(×200和×400);B-海马DG区神经元凋亡率统计;C-Western blot检测海马组织中Bax和Bcl-2蛋白表达情况;D-Bax蛋白表达量的统计分析;E-Bcl-2蛋白表达量的统计分析;与对照组比较,1)P<0.001;与模型组比较,2)P<0.001;与模型+纳美芬组比较,3)P<0.001。

, figureFileSmall=UXQMx82kBb/fnqbcwA7lzg==, figureFileBig=IyfqnvFP1hLv3gBYo0whTw==, tableContent=null), ArticleFig(id=1193713127259861010, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1193674742768042263, language=EN, label=Fig.4, caption=ELISA assay for TNF-α and IL-6 levels in hippocampal tissues of rats in different groups.n=4,$\bar{x} \pm s$

A-ELISA analysis for TNF-α levels; B-ELISA analysis for IL-6 levels; 1)P<0.001, compared with control group; 2)P<0.001, compared with model group; 3)P<0.001, compared with model+Nal group.

, figureFileSmall=b3t0ZsITHFJlEg6ItUj09Q==, figureFileBig=emoP7RAHj0auwN3v8+MNUg==, tableContent=null), ArticleFig(id=1193713127335358483, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1193674742768042263, language=CN, label=图4, caption=ELISA检测各组大鼠海马组织中肿瘤坏死因子α(TNF-α)和白细胞介素6(IL-6)表达。n=4,$\bar{x} \pm s$

A-ELISA检测TNF-α表达;B-ELISA检测IL-6表达;与对照组比较,1)P<0.001;与模型组比较,2)P<0.001;与模型+纳美芬组比较,3)P<0.001。

, figureFileSmall=b3t0ZsITHFJlEg6ItUj09Q==, figureFileBig=emoP7RAHj0auwN3v8+MNUg==, tableContent=null), ArticleFig(id=1193713127436021780, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1193674742768042263, language=EN, label=Fig.5, caption=Comparison of Sirt1, TLR4, and NF-κB p65 protein levels in hippocampal tissues of rats in each group. n=4,$\bar{x} \pm s$

A-Western blot assay for the protein levels of Sirt1, TLR4, and NF-κB p65; B-statistical analysis for Sirt1 protein relative expression; C-statistical analysis for TLR4 protein relative expression; D-statistical analysis for NF-κB p65 protein relative expression; 1)P<0.001, compared with control group; 2)P<0.001, compared with model group; 3)P<0.001, compared with model+Nal group.

, figureFileSmall=E0A5OG34Bf1FZ3bAMzKjhg==, figureFileBig=N8RUYMNfqExJm6GhGfLtIg==, tableContent=null), ArticleFig(id=1193713127511519253, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1193674742768042263, language=CN, label=图5, caption=各组大鼠海马组织中沉默信息调节因子1 (Sirt1)、Toll 样受体 4(TLR4)和核因子κB亚基p65(NF-κB p65)蛋白表达比较。n=4,$\bar{x} \pm s$

A-Western blot检测Sirt1、TLR4和NF-κB p65蛋白表达情况;B-Sirt1蛋白表达量的统计分析;C-TLR4蛋白表达量的统计分析;D-NF-κB p65蛋白表达量的统计分析;与对照组比较,1)P<0.001;与模型组比较,2)P<0.001;与模型+纳美芬组比较,3)P<0.001。

, figureFileSmall=E0A5OG34Bf1FZ3bAMzKjhg==, figureFileBig=N8RUYMNfqExJm6GhGfLtIg==, tableContent=null), ArticleFig(id=1193713127578628118, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1193674742768042263, language=EN, label=Tab.1, caption=

Morris water maze experiment assay for the learning and memory abilities of rats.n=8,$\bar{x} \pm s$

, figureFileSmall=null, figureFileBig=null, tableContent=
Group Escape latency/s Platform crossing
times
Time spent in target
quadrant/s
1 d before surgery 1st day after surgery 3rd day after surgery
Control 13.88±2.85 12.12±2.90 10.87±3.48 7.12±1.13 46.75±4.06
Model 13.62±2.45 52.87±4.581) 42.50±4.751) 1.75±1.031) 24.75±5.341)
Model+Nal 13.25±2.82 40.12±5.222) 31.25±3.542) 5.37±0.922) 42.25±4.172)
Model+Nal+EX527 14.12±2.17 50.75±5.573) 40.12±3.683) 2.37±1.063) 31.50±4.043)
), ArticleFig(id=1193713127649931287, tenantId=1146029695717560320, journalId=1190317699101192196, articleId=1193674742768042263, language=CN, label=表1, caption=

Morris水迷宫实验分析各组大鼠学习和记忆能力。n=8,$\bar{x} \pm s$

, figureFileSmall=null, figureFileBig=null, tableContent=
Group Escape latency/s Platform crossing
times
Time spent in target
quadrant/s
1 d before surgery 1st day after surgery 3rd day after surgery
Control 13.88±2.85 12.12±2.90 10.87±3.48 7.12±1.13 46.75±4.06
Model 13.62±2.45 52.87±4.581) 42.50±4.751) 1.75±1.031) 24.75±5.341)
Model+Nal 13.25±2.82 40.12±5.222) 31.25±3.542) 5.37±0.922) 42.25±4.172)
Model+Nal+EX527 14.12±2.17 50.75±5.573) 40.12±3.683) 2.37±1.063) 31.50±4.043)
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纳美芬通过调控Sirt1/TLR4/NF-κB通路改善老年大鼠术后认知功能障碍
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张琳 , 唐富东 , 刘跃丹 , 耿红芳 *
中国药学杂志 | 论著 2025,60(6): 604-611
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中国药学杂志 | 论著 2025, 60(6): 604-611
纳美芬通过调控Sirt1/TLR4/NF-κB通路改善老年大鼠术后认知功能障碍
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张琳, 唐富东, 刘跃丹, 耿红芳*
作者信息
  • 河南省人民医院 麻醉与围术期医学科, 郑州大学人民医院, 河南大学人民医院, 郑州 450003
  • 张琳,女,硕士,副主任医师 研究方向:小儿麻醉基础与临床

通讯作者:

*耿红芳,女,硕士,副主任医师 研究方向:麻醉基础及临床 Tel:(0371)65580445
Nalmefene Alleviates Postoperative Cognitive Dysfunction in Aged Rats via the Sirt1/TLR4/NF-κB Pathway
Lin ZHANG, Fudong TANG, Yuedan LIU, Hongfang GENG*
Affiliations
  • Department of Anesthesiology and Perioperative Medicine, Henan Provincial People's Hospital, Zhengzhou University People's Hospital, Henan University People's Hospital, Zhengzhou 450003, China
出版时间: 2025-03-22 doi: 10.11669/cpj.2025.06.006
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目的 探究纳美芬对老年大鼠术后认知功能障碍(postoperative cognitive dysfunction,POCD)的影响及其分子机制。方法 将17月龄的SD大鼠适应饲养一周后,随机分成对照(Control)组、模型(Model)组、模型+纳美芬(Model+Nal)组、模型+纳美芬+EX527(Model+Nal+EX527)组,每组8只。除Control组外,使用七氟烷麻醉和剖腹探查术建立POCD模型。Model+Nal组大鼠在麻醉诱导前30 min皮下注射0.1 mg·kg-1盐酸纳美芬;Model+Nal+EX527组大鼠在麻醉诱导前30 min皮下注射0.1 mg·kg-1盐酸纳美芬,并腹腔注射10 mg·kg-1 EX527 [沉默信息调节因子1(Sirt1)抑制剂]。Morris水迷宫实验评估大鼠认知能力。术后第3天处死大鼠,苏木素-伊红(HE)染色观察海马组织病理学改变,Tunel染色分析神经元凋亡,酶联免疫吸附测定(ELISA)方法分析肿瘤坏死因子α(TNF-α)和白细胞介素6(IL-6)含量,蛋白质免疫印迹(Western blot)法检测B-细胞淋巴瘤因子2(Bcl-2)、Bcl-2 相关X蛋白(Bax)、Sirt1、Toll样受体4(TLR4)和核因子κB亚基p65(NF-κB p65)蛋白表达水平。结果 与Control组相比,Model组大鼠出现认知障碍,海马DG区神经元出现损伤,DG区神经元凋亡比例增多,TNF-α和IL-6含量增加,Bax、TLR4和NF-κB p65蛋白表达增加,Bcl-2和Sirt1表达降低(P<0.001);与Model组相比,Model+Nal组大鼠认知能力增强,海DG区神经元损伤减轻,DG区神经元凋亡比例减少,TNF-α和IL-6含量降低,Bax、TLR4和NF-κB p65蛋白表达减少,Bcl-2和Sirt1表达增加(P<0.001);与Model+Nal组相比,Model+Nal +EX527组大鼠认知能力减弱,海马DG区神经元损伤增加,DG区神经元凋亡比例增多,TNF-α和IL-6含量增加,Bax、TLR4和NF-κB p65蛋白表达增加,Bcl-2和Sirt1表达降低(P<0.001)。结论 纳美芬可通过Sirt1/TLR4/NF-κB通路减轻POCD老年大鼠神经炎症和神经元损伤,改善认知功能障碍。

纳美芬  /  老年大鼠  /  术后认知功能障碍  /  沉默信息调节因子1  /  Toll 样受体 4/核因子κB

OBJECTIVE To explore the effect and molecular mechanism of nalmefene on postoperative cognitive dysfunction (POCD) in aged rats. METHODS Seventeen-month-old SD rats were randomly divided into control group (Control), model group (Model), model + nalmefene group (Model+Nal), model+nalmefene+EX527 group (Model+Nal+EX527) with 8 rats in each group after one week of acclimatization. The POCD animal models (excluding the Control group) were established by sevoflurane anesthesia and exploratory laparotomy. The rats in Model+Nal group were subcutaneously injected with 0.1 mg·kg-1 nalmefene hydrochloride 30 min before anesthesia induction. The rats in Model+Nal+EX527 group were injected subcutaneously with 0.1 mg·kg-1 nalmefene hydrochloride and intraperitoneally with 10 mg·kg-1 EX527 (Sirt1 inhibitor) 30 min before anesthesia induction. Morris water maze experiment was conducted to analyze the cognitive abilities of rats. Hippocampal tissues were collected 3 d after surgery. The pathological changes of the hippocampus were observed by HE staining. Neuron apoptosis was analyzed by Tunel staining. ELISA method was used to detect the contents of TNF-α and IL-6. The protein expression levels of Bax, Bcl-2, Sirt1, TLR4 and NF-κB p65 were determined by Western blot. RESULTS Compared with the Control group, the cognitive dysfunction was seen, neuronal damage and the proportion of apoptotic neurons in hippocampal DG region were increased, TNF-α and IL-6 contents as well as the expression of Bax, TLR4 and NF-κB p65 were elevated, while the expression of Bcl-2 and Sirt1 was decreased in the Model group (P<0.001). Compared with the Model group, the cognitive ability was enhanced, neuronal damage and the proportion of apoptotic neurons in hippocampal DG region were decreased, TNF-α and IL-6 contents as well as the expression of Bax, TLR4 and NF-κB p65 were reduced, whereas the expression of Bcl-2 and Sirt1 was elevated in the Model+Nal group (P<0.001). Compared with the Model+Nal group, the cognitive ability was suppressed, neuronal damage and the proportion of apoptotic neurons in hippocampal DG region were increased, TNF-α and IL-6 contents as well as the expression of Bax, TLR4 and NF-κB p65 were elevated, while the expression of Bcl-2 and Sirt1 was decreased in the Model+Nal+EX527 group (P<0.001). CONCLUSION Nalmefene can reduce neuroinflammation and neuronal damage, and improve cognitive dysfunction in aged POCD rats through regulating Sirt1/TLR4/NF-κB pathway.

nalmefene  /  aged rat  /  postoperative cognitive dysfunction  /  Sirt1  /  TLR4/NF-κB
张琳, 唐富东, 刘跃丹, 耿红芳. 纳美芬通过调控Sirt1/TLR4/NF-κB通路改善老年大鼠术后认知功能障碍. 中国药学杂志, 2025 , 60 (6) : 604 -611 . DOI: 10.11669/cpj.2025.06.006
Lin ZHANG, Fudong TANG, Yuedan LIU, Hongfang GENG. Nalmefene Alleviates Postoperative Cognitive Dysfunction in Aged Rats via the Sirt1/TLR4/NF-κB Pathway[J]. Chinese Pharmaceutical Journal, 2025 , 60 (6) : 604 -611 . DOI: 10.11669/cpj.2025.06.006
术后认知功能障碍(postoperative cognitive dysfunction,POCD)是手术之后常见的神经系统并发症,主要表现为认知能力显著下降,包括记忆、注意力、协调性、空间定向、语言流畅性和执行能力等方面的障碍[1]。研究[2]发现POCD在患者术后的发病率为17%~43%,且其在老年患者中的发病率要高于年轻患者[3]。研究表明,POCD可持续数周至数年,影响患者康复,延长住院时间,并可能导致额外的身体和精神疾病,增加死亡率,给患者及其家属带来沉重的负担[1]。随着人口老龄化,老年患者的手术需求逐渐增多,POCD的发生率也逐渐增加,这给当前的医疗卫生体系提出了巨大的挑战[4]。POCD是一个涉及多种致病因素的复杂病理过程,其发病机制尚不清楚。目前对于POCD的治疗和预防还处于探索阶段[5]
纳美芬是一种特异性阿片类受体拮抗剂,常用于逆转阿片类药物的毒性。越来越多的研究[6-8]发现纳美芬具有一定的神经保护作用。例如,纳美芬可保护重型颅脑损伤患者的神经系统[6],还可抑制酒精诱导的神经炎症和大脑损伤[8]。体外研究[7]发现纳美芬可逆转高浓度阿片类药物羟考酮对大鼠神经干细胞增殖的抑制作用,以及对凋亡和分化的促进作用,提示纳美芬可改善神经发育障碍。临床研究[9]发现纳美芬可改善行单肺通气的电视辅助胸腔镜手术的老年患者术后认知功能,减少神经认知恢复延迟的发生率。但是目前关于纳美芬与POCD关系的研究甚少,且纳美芬改善POCD的作用机制尚不明确。沉默信息调节因子1 (Sirt1)在POCD模型动物中表达降低,它的激活剂SRT1720通过抑制Toll 样受体 4(TLR4)/核因子κB(NF-κB)通路改善小鼠POCD症状[10]。有研究[11]发现纳美芬可通过调节Sirt1减轻大鼠肺缺血再灌注损伤。因此,本研究使用七氟烷麻醉和剖腹探查术建立老年大鼠POCD模型[12-13],探究纳美芬改善POCD的作用,并探究其作用是否与Sirt1/TLR4/NF-κB通路有关。
32只SPF级SD雄性大鼠[许可证号SYXK(京)2022-0052];所有大鼠于郑州大学实验动物中心SPF级环境下适应饲养一周[温度20~26 ℃,湿度 40%~70%,12 h光/暗循环]。本实验经河南省人民医院动物实验伦理委员会批准(批件号:HNSRMYY2023-019)。
七氟烷(江苏恒瑞医药股份有限公司,批号:22090631);盐酸纳美芬(西安利君制药有限责任公司,批号:230401,规格:1 mL:0.1 mg);Sirt1抑制剂EX527(南京沃博生物科技有限公司,货号:IEP1062);原位末端转移酶标记技术(Tunel)细胞凋亡检测试剂盒(显色法)(上海碧云天生物科技有限公司,货号:C1098);肿瘤坏死因子α (TNF-α)和白细胞介素6(IL-6)酶联免疫吸附测定(ELISA)试剂盒(上海酶联生物公司,货号:ml002953、ml102828);放射免疫沉淀法(RIPA)裂解液、二喹啉甲酸法(BCA)蛋白浓度测定试剂盒、增强型化学发光(ECL)Plus超敏发光液、甘油醛-3-磷酸脱氢酶(GAPDH)小鼠单克隆抗体(北京索莱宝科技有限公司,货号:R0010、PC0020、PE0010、K200057M);B-细胞淋巴瘤因子2(Bcl-2)兔多克隆抗体(美国Abcam公司,批号:ab196495);Bcl-2 相关X蛋白(Bax)兔单克隆抗体、Sirt1兔单克隆抗体和NF-κB p65兔单克隆抗体(美国Cell signaling technology公司,批号:#14796、#9475、#8242); TLR4小鼠单克隆抗体(美国Santa Cruz公司,批号:sc-293072);辣根过氧化物酶(HRP)标记的山羊抗小鼠IgG二抗和山羊抗兔IgG二抗(武汉博士德生物工程有限公司,货号:BA1050、BA1050)。
参考文献[12-13]建立POCD大鼠模型:使用体积分数5%七氟烷麻醉诱导,5 min后改成体积分数2%七氟烷维持,并进行剖腹探查术(腹部消毒后,沿腹部中线做一个约3 cm的切口,然后依次检查肠、肝、脾、肾等脏器),手术时间大约15 min,确定没有出血后,用无菌手术线缝合伤口。缝合伤口后,立即通过腹腔注射60 000 U青霉素,连续注射3 d,每天1次,防止术后感染。将大鼠放置在电热毯上防止其体温过低,待苏醒后放回笼子中。
大鼠随机分成以下4组,每组8只:对照(Control)组、模型(Model)组、模型+纳美芬(Model+Nal)组、模型+纳美芬+EX527(Model+Nal+EX527)组。其中Control组不进行麻醉和手术,另外三组均按照“2.1”项下方法建立POCD模型。纳美芬的注射方式和剂量参考文献[14]和前期的预实验结果,发现皮下注射纳美芬高于0.1 mg·kg-1对大鼠认知功能的改变与0.1 mg·kg-1纳美芬效果差别不显著,因此,纳美芬的浓度选择0.1 mg·kg-1。Model+Nal组大鼠在麻醉诱导前30 min通过皮下注射0.1 mg·kg-1盐酸纳美芬。EX527的注射方式和剂量参考文献[15-16]。EX527的配制过程如下:50 mg EX527冻干粉加入1 mL DMSO,配置成50 mg·mL-1的储备液,然后用生理盐水将其稀释成2.5 mg·mL-1的工作液。Model+Nal+EX527组大鼠在麻醉诱导前30 min通过皮下注射0.1 mg·kg-1盐酸纳美芬,并通过腹腔注射10 mg·kg-1 EX527。其他组给予等量体积分数5%二甲基亚砜(DMSO)溶剂。
在术前5 d和术后第1、3天进行Morris水迷宫实验,包括定位航行实验和空间探索实验,来检测大鼠的空间学习和记忆能力。本实验在一个圆形的黑色壁水池中(直径120 cm,深度50 cm)进行,平均分成4个象限(Ⅰ、Ⅱ、Ⅲ和Ⅳ)。在第Ⅳ象限的中央位置放置一个圆形平台,淹没在水下约1.5 cm处。大鼠在术前第5天开始训练,连续训练4 d,每天4次。在每个训练日,将大鼠分别从4个象限中的一个放入水中,记录它们找到平台的时间(即为大鼠的逃避潜伏期),如果在60 s内没有找到平台,则引导其至平台,并在平台上停留10 s。于术前1 d和术后第1、3天按照上述方法测试每组大鼠的逃避潜伏期。在术后第3天测试过逃避潜伏期6 h后,移除平台,将大鼠从第Ⅱ象限放入水中,记录60 s内大鼠游过原平台区域的次数(穿越平台次数)以及在原平台所在象限停留时间。
术后第3天进行行为学测试之后,将大鼠用体积分数7%七氟烷麻醉,颈椎脱臼法处死。迅速从大脑中剥离海马组织,每只大鼠有两个海马组织,磷酸盐缓冲液(PBS)清洗干净后,将每只大鼠的单侧海马组织(共32个)用液氮速冻,置于-80 ℃冰箱冻存,用于细胞因子和蛋白质检测,将每只大鼠的另一侧海马组织(共32个)放入质量分数4%多聚甲醛中固定,用于病理学检测。
将冰冻的组织样本解冻后加入适量生理盐水,在冰上用匀浆器充分匀浆之后,3 000 r·min-1离心10 min,收集上清。然后根据ELISA试剂盒说明书操作,检测海马组织中的TNF-α和IL-6含量。
海马组织在质量分数4%多聚甲醛中固定48 h,进行石蜡包埋,制备成5 μm厚度的石蜡切片。二甲苯脱蜡,逐级乙醇复水后,进行HE染色,脱水透明,中性树脂封片后,用显微镜观察。
根据Tunel试剂盒说明书操作检测海马组织中神经元凋亡情况。步骤如下:将5 μm的石蜡切片充分脱蜡和水化后,滴加20 μg·mL-1不含脱氧核糖核酸酶(DNase)的蛋白酶K处理20 min;PBS彻底清洗蛋白酶K,使用质量分数3% H2O2将切片中的内源性过氧化物酶灭活,加入新鲜配制的50 μL Tunel反应混合物避光孵育1 h。滴加50 μL辣根过氧化物酶标记链霉亲和素(Streptavidin-HRP)工作液,室温孵育30 min后加3,3'-二氨基联苯胺(DAB)显色10 min。苏木素对细胞核进行复染之后,显微镜下观察。正常海马神经元细胞核呈蓝色,凋亡细胞核呈棕褐色(Tunel阳性)。计算凋亡细胞占总细胞数的百分比。
冻存的海马组织解冻后,在RIPA裂解液中匀浆提取组织中的蛋白质。用BCA试剂盒测定浓度之后,每组取等量蛋白质用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)分离。然后将胶上分离的蛋白转移到聚偏二氟乙烯(PVDF)膜上,质量分数5%脱脂奶粉室温封闭膜2 h,加特异性一抗Bax(1∶1 000)、Bcl-2(1∶800)、Sirt1(1∶1 000)、TLR4(1∶500)、NF-κB p65(1∶1 000)、GAPDH(1∶3 000)4℃孵育过夜。清洗掉一抗后,加相应的HRP标记山羊抗兔或者小鼠二抗(稀释度均为1∶5 000)室温孵育45 min,使用ECL发光液观察蛋白条带。Image J软件分析条带灰度值来对蛋白表达进行定量。
数据以均数±标准差($\bar{x} \pm s$)表示,用GraphPad Prism8.0软件进行分析。多组间差异比较采用单因素方差分析,两两比较使用Bonferroni检验。P<0.05表明差异有统计学意义。
术前1 d,四组大鼠的逃避潜伏期无显著差异(P>0.05)。但是术后第1天和第3天,与Control组相比,Model组逃避潜伏期延长(P<0.001);与Model组相比,Model+Nal组逃避潜伏期显著缩短(P<0.001);与Model+Nal组相比,Model+Nal+EX527组逃避潜伏期显著延长(P<0.001)。此外,与Control组相比,Model组大鼠穿越平台次数和在原平台所在象限停留时间均显著减少(P<0.001);与Model组相比,Model+Nal组大鼠穿越平台次数和在原平台所在象限停留时间均显著延长(P<0.001);与Model+Nal组相比,Model+Nal+EX527组大鼠穿越平台次数和在原平台所在象限停留时间均显著减少(P<0.001)。见图1表1
HE染色结果见图2。Control组中大鼠的DG区神经元结构清晰,排列整齐;Model组DG区神经元排列紊乱,神经元细胞核固缩;与Model组相比,Model+Nal组大鼠DG区神经元排列整齐,核固缩减少;与Model+Nal组相比,Model+Nal+EX527组大鼠DG区神经元排列紊乱,核固缩增多。
Tunel染色观察各组大鼠海马神经元凋亡情况,200倍和400倍视野下结果见图3A。与Control组相比,Model组大鼠海马DG区凋亡神经元比例显著增加(P<0.001);与Model组相比,Model+Nal组大鼠海马DG区凋亡神经元比例显著减少(P<0.001);与Model+Nal组相比,Model+Nal+ EX527组大鼠海马DG区凋亡神经元比例显著增加(P<0.001)(图3B)。
Western blot检测海马中凋亡相关蛋白表达,结果见图3C~E:与Control组相比,Model组Bax表达上调,Bcl-2表达下调(P<0.001);与Model组相比,Model+Nal组Bax表达下调,Bcl-2表达上调(P<0.001);与Model+Nal组相比,Model+Nal+EX527组Bax表达上调,Bcl-2表达下调(P<0.001)。
ELISA检测海马组织TNF-α和IL-6表达情况,结果见图4。与Control组相比,Model组这些炎性因子在海马中表达均上调(P<0.001);与Model组相比,Model+Nal组这些炎性因子在海马中表达均下调(P<0.001);与Model+Nal组相比,Model+Nal+EX527组这些炎性因子在海马中表达均增加(P<0.001)。
Western blot检测海马中Sirt1、TLR4和NF-κB p65表达,结果见图5:与Control组相比,Model组Sirt1表达降低,TLR4和NF-κB p65表达升高(P<0.05);与Model组相比,Model+Nal组Sirt1表达增加,TLR4和NF-κB p65表达降低(P<0.05);与Model+Nal组相比,Model+Nal+EX527组Sirt1表达降低,TLR4和NF-κB p65表达升高(P<0.05)。
POCD是老年患者术后常见的神经系统并发症。近些年的研究[1,4,17]指出海马神经元凋亡和神经炎症与POCD的发生密切相关。七氟烷是临床常用的吸入性麻醉剂,具有起效迅速、苏醒快、麻醉深度易于调节等特点,在老年人中应用广泛。有研究[12,18]发现,七氟烷可诱导老年大鼠海马神经元凋亡和神经炎症,造成认知功能障碍。因此,本研究使用了不进行麻醉和手术的大鼠做对照,而没有使用假手术组做对照。本研究用七氟烷和剖腹探查术诱导的POCD模型大鼠出现认知障碍,海马DG区神经元发生病理学损伤、凋亡增加,海马组织中炎性因子表达显著增加。这表明本研究中POCD模型成功诱导。
纳美芬是μ-阿片受体和δ-阿片受体的强效拮抗剂,但也是κ-阿片受体的部分激动剂[19]。Li等[20]的研究发现,κ-阿片受体激动剂可通过抑制Janus 激酶2(JAK2)/信号传导和转录激活因子3 (STAT3)通路改善大鼠POCD。Fan等[21]的研究也显示κ-阿片受体激动剂U50488H 可通过PI3K/AKT/Nrf2/HO-1通路缓解心肺流转术大鼠的POCD症状,减少大脑损伤。临床上纳美芬不仅能够缩短全麻患者术后苏醒时间[22],也能够改善老年胸腔镜手术患者的认知能力[9]。本研究结果发现纳美芬改善了老年大鼠术后认知能力,减轻了神经元损伤。凋亡在POCD的发病机制中发挥着重要作用。研究发现促凋亡蛋白Bax在手术和麻醉诱导的POCD模型动物中表达显著上调,而抗凋亡蛋白Bcl-2表达显著下调[23-24]。本研究通过Tunel染色发现纳美芬可减少POCD模型大鼠海马DG区神经元凋亡,通过Western blot实验检测凋亡相关蛋白表达发现在POCD模型大鼠中,纳美芬可降低Bax表达,增加Bcl-2的表达。神经炎症在POCD的发生中发挥着关键作用[5],炎性因子TNF-α和IL-6在POCD模型动物的海马组织中表达明显增加[25]。本研究结果发现纳美芬可减少海马组织中TNF-α和IL-6表达。TLR4/NF-κB通路是介导炎症反应的重要信号通路,POCD的发生与海马组织中此信号通路的活化密切相关[26]。同样,本研究中手术和七氟烷麻醉诱导的POCD模型大鼠中TLR4和NF-κB p65表达均增加,证实了TLR4/NF-κB通路发生活化。在大鼠肺缺血再灌注损伤模型中纳美芬可抑制TLR4/NF-κB通路的活化[27]。本研究中发现纳美芬在POCD大鼠中也可抑制TLR4/NF-κB通路活化。
Sirt1是一种烟酰胺腺嘌呤二核苷酸(NAD+)依赖的组蛋白去乙酰化酶,广泛存在于大脑中。它通过调节包括NF-κB在内的多个通路来调控多种细胞过程。Sirt1被认为具有神经保护作用[13],可抑制TLR4/NF-κB通路活化改善大鼠认知障碍和海马神经炎症[28]。有研究发现纳美芬通过调节Sirt1减轻大鼠肺缺血再灌注损伤[11],本研究发现纳美芬处理POCD模型大鼠后Sirt1表达增加。使用Sirt1抑制剂EX527进一步探究了纳美芬是否通过调节Sirt1表达在POCD中发挥神经保护作用。结果发现,与Model+Nal组相比,Model+Nal+EX527组大鼠认知能力减弱,海马DG区神经元损伤增多,凋亡和炎性因子表达增多,TLR4/NF-κB通路活化增强。这表明纳美芬是通过上调Sirt1表达发挥神经保护作用的。
综上所述,本研究结果发现纳美芬通过Sirt1/TLR4/NF-κB通路改善大鼠POCD的分子机制,为临床上纳美芬用于POCD的治疗和预防提供了理论基础。当然,纳美芬是否通过其他信号通路发挥改善POCD的作用尚需进一步探究。
  • 河南省医学科技攻关联合共建项目资助(LHGJ20220065)
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2025年第60卷第6期
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doi: 10.11669/cpj.2025.06.006
  • 接收时间:2024-11-14
  • 首发时间:2025-11-07
  • 出版时间:2025-03-22
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  • 收稿日期:2024-11-14
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河南省医学科技攻关联合共建项目资助(LHGJ20220065)
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    河南省人民医院 麻醉与围术期医学科, 郑州大学人民医院, 河南大学人民医院, 郑州 450003

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*耿红芳,女,硕士,副主任医师 研究方向:麻醉基础及临床 Tel:(0371)65580445
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2种不同金属材料的力学参数

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种数
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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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