Article(id=1316714709223592235, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1316714454184739470, articleNumber=null, orderNo=null, doi=10.13343/j.cnki.wsxb.20260440, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1779638400000, receivedDateStr=2026-05-25, revisedDate=null, revisedDateStr=null, acceptedDate=1783008000000, acceptedDateStr=2026-07-03, onlineDate=1791858850055, onlineDateStr=2026-10-13, pubDate=1791043200000, pubDateStr=2026-10-04, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1791858850055, onlineIssueDateStr=2026-10-13, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1791858850055, creator=13701087609, updateTime=1791858850055, updator=13701087609, issue=Issue{id=1316714454184739470, tenantId=1146029695717560320, journalId=1192105938417971205, year='2026', volume='66', issue='10', pageStart='4661', pageEnd='5310', issueExtLink='null', onlineDate='null', pubDate='1791043200000', pubDateStr='2026-10-04', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=1, specialIssue=null, createTime=1791858789249, creator='13701087609', updateTime=1791885400527, updator='13701087609', preIssue=null, nextIssue=null, articleTotal=null, ext={EN=IssueExt(id=1316826070066774161, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1316714454184739470, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1316826070066774162, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1316714454184739470, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null, downloadFileDto=null}, startPage=5223, endPage=5236, ext={EN=ArticleExt(id=1316714710431551790, articleId=1316714709223592235, tenantId=1146029695717560320, journalId=1192105938417971205, language=EN, title=Shouwu Pill ameliorates doxorubicin-induced premature aging by modulating gut microbiota and bile acid metabolism in mice, columnId=1192149543992045670, journalTitle=Acta Microbiologica Sinica, columnName=Research Article, runingTitle=null, highlight=null, articleAbstract=

[Objective] To investigate the effects of Shouwu Pill (SWW) on physical performance, gut microbiota composition, and bile acid metabolism in the mouse model of doxorubicin (DOXO)-induced premature aging, and to preliminarily explore its potential mechanisms. [Methods] A mouse model of premature aging was established by intraperitoneal injection of DOXO. Mice were randomized into Control, DOXO, DOXO+Shouwu Pill low-dose (SWW-L), and DOXO+Shouwu Pill high-dose (SWW-H) groups. Grip strength test, hanging endurance test, and treadmill exercise test were performed to evaluate physical performance. Gut microbiota composition and diversity were analyzed by 16S rRNA gene sequencing, followed by PICRUSt functional prediction. Fecal bile acid profiling was performed via UHPLC-QQQ-MS. Spearman correlation analysis was further conducted to explore the association between differentially abundant gut microbial taxa and bile acid metabolism. [Results] Compared with the Control group, DOXO-treated mice exhibited increased systemic inflammatory cytokine levels and significantly decreased forelimb grip strength, hanging endurance, and maximal running speed, indicating successful establishment of the premature aging model. SWW treatment significantly reduced systemic inflammation and improved physical performance, with the DOXO+SWW-H group demonstrating greater efficacy than the DOXO+SWW-L group. The results of 16S rRNA gene sequencing revealed that DOXO induced gut microbiota dysbiosis characterized by altered microbial diversity and disrupted microbiota structure, including decreased abundance of Muribaculaceae and increased abundance of opportunistic pathogenic genera such as Desulfovibrio and Helicobacter. SWW treatment partially restored the microbiota structure by increasing the abundance of potentially beneficial bacteria, including Akkermansia, Lactobacillus, Bifidobacterium, and Roseburia. PICRUSt functional prediction indicated significant alterations in bile acid metabolism-related pathways following SWW intervention. Fecal bile acid profiling further demonstrated that DOXO treatment led to the accumulation of several conjugated bile acids, including T-α-MCA and T-β-MCA, accompanied by reduced levels of unconjugated bile acids. SWW intervention significantly lowered the levels of conjugated bile acids and partially restored the levels of unconjugated bile acids. Correlation analysis showed that several beneficial bacteria were positively correlated with unconjugated bile acids but negatively correlated with conjugated bile acids. [Conclusion] SWW ameliorates physical decline and gut microbiota dysbiosis in the mouse model of DOXO-induced premature aging. Its anti-aging effects may be associated with modulation of gut microbiota-related bile acid metabolism and restoration of bile acid homeostasis.

, authors=Bingbing FENG1, 2, Zheng CAO1, 2, Yao YI1, 2, Jing LI1, *, authorsList=Bingbing FENG, Zheng CAO, Yao YI, Jing LI, authorCompany=null, correspAuthors=Jing LI, authorNote=null, correspAuthorsNote=
*E-mail:
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【目的】 探讨首乌丸对阿霉素(doxorubicin, DOXO)诱导早衰小鼠运动能力、肠道菌群及胆汁酸代谢的影响,并初步阐明其潜在作用机制。 【方法】 小鼠随机分为Control组、DOXO组、首乌丸低剂量组(Shouwu Pill low-dose, SWW-L)及首乌丸高剂量组(Shouwu Pill high-dose, SWW-H);除Control组外,其余各组采用DOXO腹腔注射建立早衰小鼠模型。通过前肢握力实验、悬挂网实验及跑台实验评价小鼠运动能力变化;采用16S rRNA基因测序分析肠道菌群结构及多样性变化,并结合PICRUSt进行菌群功能预测;采用UHPLC-QQQ-MS检测粪便胆汁酸谱变化;进一步通过Spearman相关性分析探讨差异菌群与胆汁酸代谢之间的关系。 【结果】 与Control组相比,DOXO组小鼠血清炎症因子水平升高,握力、悬挂耐力及最大跑步速度显著下降,提示早衰模型构建成功;首乌丸干预后,血清炎症因子水平降低,上述运动能力指标明显改善,其中高剂量组效果更为显著。16S rRNA基因测序结果显示,DOXO诱导小鼠肠道菌群失衡,表现为菌群多样性改变及群落结构紊乱,鼠杆状菌属(Muribaculaceae_unclassified)等优势菌丰度下降,而脱硫弧菌属(Desulfovibrio)、Helicobacter等条件致病菌相对丰度升高;首乌丸干预后,阿克曼氏菌属(Akkermansia)、乳杆菌属(Lactobacillus)、双歧杆菌属(Bifidobacterium)及罗斯拜瑞氏菌属(Roseburia)等潜在有益菌丰度增加,菌群结构逐渐恢复。PICRUSt功能预测显示,首乌丸显著调节胆汁酸代谢相关通路。胆汁酸谱分析结果表明,DOXO组多种结合型胆汁酸(如T-α-MCA、T-β-MCA)显著积累,而部分非结合型胆汁酸水平下降;首乌丸干预后,结合型胆汁酸明显下降,非结合型胆汁酸水平部分恢复。相关性分析显示,多种有益菌与非结合型胆汁酸呈正相关,而与结合型胆汁酸呈负相关。 【结论】 首乌丸能够改善DOXO诱导的早衰小鼠运动功能下降及肠道菌群失衡,并可能通过调节肠道菌群相关胆汁酸代谢恢复胆汁酸稳态,从而发挥延缓早衰的作用。

, authors=冯兵兵1, 2, 曹政1, 2, 易瑶1, 2, 李婧1, *, authorsList=冯兵兵, 曹政, 易瑶, 李婧, authorCompany=null, correspAuthors=李婧, authorNote=

作者贡献声明

冯兵兵:参与实验和原始数据收集,整理并分析了所有原始数据,解释实验结果、绘制论文图片并撰写手稿;曹政:参与实验,收集原始数据及肠道菌群数据分析;易瑶:参与实验和收集原始数据;李婧:设计实验、修改手稿。

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A: Experimental design [Premature aging was induced by intraperitoneal injection of doxorubicin (DOXO, 10 mg/kg) on day 0 and 10. Shouwu Pill was administered daily by gavage at low dose (200 mg/kg) or high dose (400 mg/kg) throughout the experimental period]; B: Serum tumor necrosis factor-α (TNF-α) levels in each group (n=6 per group); C: Serum interleukin-1β (IL-1β) levels in each group (n=6 per group); D: Hanging endurance time in each group (n=6 per group); E: Maximal running speed in each group (n=6 per group); F: Forelimb grip strength in each group (n=6 per group). Data are presented as mean±SD. ns: P≥0.05; *: P<0.05; **: P<0.01; ***: P<0.001., figureFileSmall=w7/+XGq5luOoSYDr/f16+Q==, figureFileBig=3fPJrNvBKOeWKf3Au9K3aQ==, tableContent=null), ArticleFig(id=1316829969800614511, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1316714709223592235, language=CN, label=图1, caption=首乌丸改善DOXO诱导早衰小鼠的衰老表型, figureFileSmall=w7/+XGq5luOoSYDr/f16+Q==, figureFileBig=3fPJrNvBKOeWKf3Au9K3aQ==, tableContent=null), ArticleFig(id=1316829969918055024, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1316714709223592235, language=EN, label=Figure 2, caption=Shouwu Pill ameliorates DOXO-induced gut microbiota dysbiosis (n=6 per group). A, B: Alpha diversity analysis of gut microbiota in each group, including Chao1 and Simpson indices; C: Principal coordinates analysis (PCoA) based on Bray-Curtis distances showing differences in microbial beta diversity among groups; D: Relative abundance of gut microbiota at the phylum level; E: Relative abundance of gut microbiota at the genus level. Data are presented as mean±SD. ns: P≥0.05; *: P<0.05; **: P<0.01; ***: P<0.001., figureFileSmall=umZHbT7qrbRFAgnl0sw7tg==, figureFileBig=4D+b9NBOcoy/AAsZqJB6Lg==, tableContent=null), ArticleFig(id=1316829969997746801, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1316714709223592235, language=CN, label=图2, caption=首乌丸改善DOXO诱导的肠道菌群稳态, figureFileSmall=umZHbT7qrbRFAgnl0sw7tg==, figureFileBig=4D+b9NBOcoy/AAsZqJB6Lg==, tableContent=null), ArticleFig(id=1316829970073244274, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1316714709223592235, language=EN, label=Figure 3, caption=Shouwu Pill modulates gut microbiota-associated metabolic functions and bile acid homeostasis in premature aging mice. A: PICRUSt functional prediction analysis based on 16S rRNA gene sequencing showing significantly altered microbial functional pathways in the DOXO+SWW-H group compared with the DOXO group (Colors indicate different functional categories, and the x-axis represents -log10P); B: Fecal concentrations of conjugated bile acids, including primary and secondary bile acids, in each group (n=6 per group); C: Fecal concentrations of unconjugated bile acids, including primary and secondary bile acids, in each group (n=6 per group). Data are presented as mean±SD. *: P<0.05; **: P<0.01, ***: P<0.001., figureFileSmall=xegqvTrDUyqgYPHW8+zejg==, figureFileBig=GLulxMKcP1vVNdzF3sxq+Q==, tableContent=null), ArticleFig(id=1316829970140353139, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1316714709223592235, language=CN, label=图3, caption=首乌丸调控早衰小鼠肠道菌群相关代谢功能及胆汁酸稳态, figureFileSmall=xegqvTrDUyqgYPHW8+zejg==, figureFileBig=GLulxMKcP1vVNdzF3sxq+Q==, tableContent=null), ArticleFig(id=1316829970220044916, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1316714709223592235, language=EN, label=Figure 4, caption=Spearman correlation heatmap showing the associations between differential bacterial genera and differential bile acids. Red indicates positive correlations, whereas blue indicates negative correlations. 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首乌丸对阿霉素诱导的早衰小鼠生理功能和肠道微生物的影响
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冯兵兵 1, 2 , 曹政 1, 2 , 易瑶 1, 2 , 李婧 1, *
微生物学报 | 研究报告 2026,66(10): 5223-5236
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微生物学报 |研究报告 2026 , 66 (10) : 5223 -5236
首乌丸对阿霉素诱导的早衰小鼠生理功能和肠道微生物的影响
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冯兵兵1, 2, 曹政1, 2, 易瑶1, 2, 李婧1, *
作者信息
  • 1.湖北省中医院,湖北中医药大学附属医院,湖北 武汉
  • 2.湖北中医药大学基础医学院,湖北 武汉
通讯作者:
李婧
作者简介:

作者贡献声明

冯兵兵:参与实验和原始数据收集,整理并分析了所有原始数据,解释实验结果、绘制论文图片并撰写手稿;曹政:参与实验,收集原始数据及肠道菌群数据分析;易瑶:参与实验和收集原始数据;李婧:设计实验、修改手稿。

Shouwu Pill ameliorates doxorubicin-induced premature aging by modulating gut microbiota and bile acid metabolism in mice
Bingbing FENG1, 2, Zheng CAO1, 2, Yao YI1, 2, Jing LI1, *
Affiliations
  • 1.Hubei Provincial Hospital of Traditional Chinese Medicine, Affiliated Hospital of Hubei University of Chinese Medicine, Wuhan, Hubei, China
  • 2.School of Basic Medical Sciences, Hubei University of Chinese Medicine, Wuhan, Hubei, China
  • Corresponding Author:
出版时间: 2026-10-04 doi: 10.13343/j.cnki.wsxb.20260440
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【目的】 探讨首乌丸对阿霉素(doxorubicin, DOXO)诱导早衰小鼠运动能力、肠道菌群及胆汁酸代谢的影响,并初步阐明其潜在作用机制。 【方法】 小鼠随机分为Control组、DOXO组、首乌丸低剂量组(Shouwu Pill low-dose, SWW-L)及首乌丸高剂量组(Shouwu Pill high-dose, SWW-H);除Control组外,其余各组采用DOXO腹腔注射建立早衰小鼠模型。通过前肢握力实验、悬挂网实验及跑台实验评价小鼠运动能力变化;采用16S rRNA基因测序分析肠道菌群结构及多样性变化,并结合PICRUSt进行菌群功能预测;采用UHPLC-QQQ-MS检测粪便胆汁酸谱变化;进一步通过Spearman相关性分析探讨差异菌群与胆汁酸代谢之间的关系。 【结果】 与Control组相比,DOXO组小鼠血清炎症因子水平升高,握力、悬挂耐力及最大跑步速度显著下降,提示早衰模型构建成功;首乌丸干预后,血清炎症因子水平降低,上述运动能力指标明显改善,其中高剂量组效果更为显著。16S rRNA基因测序结果显示,DOXO诱导小鼠肠道菌群失衡,表现为菌群多样性改变及群落结构紊乱,鼠杆状菌属(Muribaculaceae_unclassified)等优势菌丰度下降,而脱硫弧菌属(Desulfovibrio)、Helicobacter等条件致病菌相对丰度升高;首乌丸干预后,阿克曼氏菌属(Akkermansia)、乳杆菌属(Lactobacillus)、双歧杆菌属(Bifidobacterium)及罗斯拜瑞氏菌属(Roseburia)等潜在有益菌丰度增加,菌群结构逐渐恢复。PICRUSt功能预测显示,首乌丸显著调节胆汁酸代谢相关通路。胆汁酸谱分析结果表明,DOXO组多种结合型胆汁酸(如T-α-MCA、T-β-MCA)显著积累,而部分非结合型胆汁酸水平下降;首乌丸干预后,结合型胆汁酸明显下降,非结合型胆汁酸水平部分恢复。相关性分析显示,多种有益菌与非结合型胆汁酸呈正相关,而与结合型胆汁酸呈负相关。 【结论】 首乌丸能够改善DOXO诱导的早衰小鼠运动功能下降及肠道菌群失衡,并可能通过调节肠道菌群相关胆汁酸代谢恢复胆汁酸稳态,从而发挥延缓早衰的作用。

首乌丸  /  肠道菌群  /  早衰  /  胆汁酸代谢

[Objective] To investigate the effects of Shouwu Pill (SWW) on physical performance, gut microbiota composition, and bile acid metabolism in the mouse model of doxorubicin (DOXO)-induced premature aging, and to preliminarily explore its potential mechanisms. [Methods] A mouse model of premature aging was established by intraperitoneal injection of DOXO. Mice were randomized into Control, DOXO, DOXO+Shouwu Pill low-dose (SWW-L), and DOXO+Shouwu Pill high-dose (SWW-H) groups. Grip strength test, hanging endurance test, and treadmill exercise test were performed to evaluate physical performance. Gut microbiota composition and diversity were analyzed by 16S rRNA gene sequencing, followed by PICRUSt functional prediction. Fecal bile acid profiling was performed via UHPLC-QQQ-MS. Spearman correlation analysis was further conducted to explore the association between differentially abundant gut microbial taxa and bile acid metabolism. [Results] Compared with the Control group, DOXO-treated mice exhibited increased systemic inflammatory cytokine levels and significantly decreased forelimb grip strength, hanging endurance, and maximal running speed, indicating successful establishment of the premature aging model. SWW treatment significantly reduced systemic inflammation and improved physical performance, with the DOXO+SWW-H group demonstrating greater efficacy than the DOXO+SWW-L group. The results of 16S rRNA gene sequencing revealed that DOXO induced gut microbiota dysbiosis characterized by altered microbial diversity and disrupted microbiota structure, including decreased abundance of Muribaculaceae and increased abundance of opportunistic pathogenic genera such as Desulfovibrio and Helicobacter. SWW treatment partially restored the microbiota structure by increasing the abundance of potentially beneficial bacteria, including Akkermansia, Lactobacillus, Bifidobacterium, and Roseburia. PICRUSt functional prediction indicated significant alterations in bile acid metabolism-related pathways following SWW intervention. Fecal bile acid profiling further demonstrated that DOXO treatment led to the accumulation of several conjugated bile acids, including T-α-MCA and T-β-MCA, accompanied by reduced levels of unconjugated bile acids. SWW intervention significantly lowered the levels of conjugated bile acids and partially restored the levels of unconjugated bile acids. Correlation analysis showed that several beneficial bacteria were positively correlated with unconjugated bile acids but negatively correlated with conjugated bile acids. [Conclusion] SWW ameliorates physical decline and gut microbiota dysbiosis in the mouse model of DOXO-induced premature aging. Its anti-aging effects may be associated with modulation of gut microbiota-related bile acid metabolism and restoration of bile acid homeostasis.

Shouwu Pill  /  gut microbiota  /  premature aging  /  bile acid metabolism
冯兵兵, 曹政, 易瑶, 李婧. 首乌丸对阿霉素诱导的早衰小鼠生理功能和肠道微生物的影响. 微生物学报, 2026 , 66 (10) : 5223 -5236 . DOI: 10.13343/j.cnki.wsxb.20260440
Bingbing FENG, Zheng CAO, Yao YI, Jing LI. Shouwu Pill ameliorates doxorubicin-induced premature aging by modulating gut microbiota and bile acid metabolism in mice[J]. Acta Microbiologica Sinica, 2026 , 66 (10) : 5223 -5236 . DOI: 10.13343/j.cnki.wsxb.20260440
衰老作为机体生理功能随时间推移而发生的渐进性衰退过程[1],是神经退行性疾病、代谢综合征及心血管疾病等多种老年性疾病发生的重要原因[2]。随着全球人口老龄化进程的加速,探索安全有效的抗衰老干预策略已成为生命科学领域的重大课题。尽管现代医学在衰老机制研究方面取得了显著进展,如发现端粒缩短[3]、线粒体功能障碍[4]等是促进衰老的因素,但针对单一靶点开发的化学合成抗衰老药物,常因作用局限性及潜在的毒副作用而难以满足临床需求[5]。相比之下,中医药凭借“整体调节”与“多靶点协同”的治疗优势,在延缓衰老及改善衰老相关表型方面展现出独特潜力。
首乌丸源自清代《景岳全书》中的何首乌散加减,是以制何首乌为主药,辅以茯苓、牛膝等成分的传统补益方剂[6],临床常用于治疗肝肾亏虚所致的须发早白、头晕耳鸣等早衰症状[7]。现代药理学研究初步证实,首乌丸中的二苯乙烯苷可通过激活SIRT1长寿蛋白通路减轻氧化应激损伤[8],而蒽醌类成分则能调节脂质代谢紊乱[9]。然而,现有研究多聚焦于单一成分或特定组织器官的效应,缺乏对药物干预下机体整体生理功能改善的系统性评价,尤其忽视了肠道微生态在中药药效发挥中的关键作用。
近年来,肠道菌群作为“第二基因组”在宿主健康与疾病中起到关键调控作用[10]。衰老进程中普遍伴随肠道菌群结构的显著紊乱,表现为共生有益菌丰度降低、机会致病菌增加以及菌群多样性下降[11]。这种生态失衡不仅破坏肠道屏障完整性,导致内毒素入血及慢性低度炎症,更通过调节宿主代谢稳态影响全身生理功能[12]。特定功能菌群及其代谢产物在延缓衰老进程中扮演着重要角色。例如,嗜黏蛋白阿克曼氏菌(Akkermansia muciniphila)作为一种关键的共生菌,能够通过降解黏蛋白促进肠道屏障修复,其丰度与机体代谢健康及寿命呈正相关[13];研究发现,向早衰模型小鼠进行粪菌移植可延长其健康寿命和总寿命[14]。此外,肠道菌群发酵膳食纤维产生的短链脂肪酸(如丁酸、乙酸)等代谢物也被证实具有调节免疫、抑制炎症及维持肠道稳态的功能,是连接菌群结构与宿主健康的关键分子[15]。
中药活性成分的生物利用度通常依赖于肠道菌群的代谢转化,而中药干预又能反向重塑菌群结构,形成“中药-菌群”的双向调控关系。然而,关于首乌丸是否通过调节特定功能菌群及其代谢功能来延缓衰老目前尚未见系统报道。基于此,本研究采用阿霉素(doxorubicin, DOXO)构建早衰小鼠模型,聚焦于首乌丸对模型小鼠行为学表型的改善作用,并深入探究其对肠道菌群结构及菌群代谢功能的调控效应。通过解析药物干预下关键菌群及代谢物的变化规律,旨在揭示首乌丸延缓衰老的菌群相关机制,为传统中药的现代药理研究及健康老龄化干预策略提供新的实验依据。
本实验选用24只3-4月龄C57BL/6J小鼠(鼠来宝生物科技有限公司),所有小鼠均在12 h光照/12 h黑暗、温度为(23±1) ℃、相对湿度为50%-60%的条件下适应1周。适应期间所有小鼠均自由摄食常规鼠粮和饮水,本研究中所有动物实验均获得武汉华联科生物技术有限公司动物伦理委员会批准(HLK-20250818-001)。适应性饲养结束后,小鼠随机分为4组,每组6只,包括:Control组、DOXO组、首乌丸低剂量组(Shouwu Pill low-dose, SWW-L)及首乌丸高剂量组(Shouwu Pill high-dose, SWW-H)。除Control组外,其余各组采用阿霉素(DOXO)建立早衰模型。DOXO以无菌生理盐水配制,分别于实验第0天和第10天按照10 mg/kg剂量进行腹腔注射,Control组同期腹腔注射等体积生理盐水。在实验期间,DOXO+SWW-L组及DOXO+SWW-H组分别给予低剂量(200 mg/kg)和高剂量(400 mg/kg)首乌丸干预。首乌丸以无菌蒸馏水配制成混悬液,每日灌胃1次,连续干预40 d;Control组及DOXO组同期灌胃等体积蒸馏水。实验期间每日观察小鼠精神状态、活动能力、饮食饮水及体质量变化。首乌丸(Shouwu Pill, SWW)购自雷允上药业集团有限公司,主要由制何首乌、茯苓、牛膝等中药组成,以蒸馏水配制成混悬液,于实验期间每日新鲜配制后灌胃给药。
小鼠禁食12 h后,经异氟烷麻醉,眼眶取血收集全血样本,于室温静置30 min后,4 ℃、3 000 r/min离心15 min,收集血清并保存于-80 ℃备用。采用酶联免疫吸附测定法(enzyme-linked immunosorbent assay, ELISA)检测血清中肿瘤坏死因子-α (tumor necrosis factor-α, TNF-α)和白细胞介素-1β (interleukin-1β, IL-1β)水平。ELISA试剂盒购自武汉伊莱瑞特生物科技股份有限公司,实验操作严格按照试剂盒说明书进行。采用酶标仪(BioTek公司)于450 nm波长下测定各孔吸光度,根据标准曲线计算样品中TNF-α和IL-1β的浓度,结果以pg/mL表示。
首先采用数字式握力计(BIOSEB公司)测定前肢握力。测试时,轻提小鼠尾部使其前爪自然抓握网格拉杆,随后缓慢向后水平轻拉,记录小鼠完全松开前的最大拉力值(g)。每次测定间隔20 min,每只小鼠共测定3次,取平均值作为最终结果。其次,通过悬挂网实验评估肌肉耐力:将小鼠置于倒置的金属网格(孔径1 cm×1 cm)上,记录其从悬挂开始至掉落的时间(s),若超过180 s则强制结束并记为180 s。所有测试均在安静环境中进行,网下铺设软垫防止摔伤。最后,利用小动物跑步机评估最大速度及力竭时间。实验前进行为期3 d的适应性训练(速度5-15 m/min)。正式测试采用递增负荷模式:起始速度15 m/min运行30 min,随后逐步提升至18 m/min及21 m/min直至力竭。力竭标准定义为小鼠连续10 s停留在电刺激区且经轻戳刺激后无法恢复奔跑。记录指标包括最大维持速度(m/min)、力竭时间及总运动距离。实验过程中环境温度控制在22-25 ℃,数据由配套软件自动采集并分析。行为学实验结束后小鼠禁食12 h,经异氟烷麻醉后处死,迅速收集粪便及盲肠内容物样本,液氮速冻后保存于-80 ℃,用于后续16S rRNA基因测序及胆汁酸代谢检测。
取盲肠内容物(约100 mg),使用土壤DNA提取试剂盒(FastDNA Spin Kit for Soil,MP Biomedicals公司)进行DNA提取。采用细菌通用引物338F (5′-ACTCCTACGGGAGGCAGCAG-3′)和806R (5′-GGACTACHVGGGTWTCTAAT-3′)扩增细菌16S rRNA基因的V3-V4可变区。PCR反应(20 μL):5×TransStart FastPfu Buffer 4.0 μL,dNTPs (2.5 mmol/L) 2.0 μL,正、反向引物(5 μmol/L)各0.8 μL,TransStart FastPfu DNA Polymerase (2.5 U/μL) 0.4 μL,BSA 0.2 μL,模板DNA 10 ng,无菌去离子水补足至20 μL。PCR扩增条件:95 ℃预变性3 min;95 ℃变性30 s,55 ℃退火30 s,72 ℃延伸45 s,共27个循环,72 ℃终延伸10 min。每个样本设置3个平行扩增反应。PCR产物经2%琼脂糖凝胶电泳检测后,采用AxyPrep DNA Gel Extraction Kit进行纯化,并使用Quantus Fluorometer进行定量。纯化后的扩增产物按等摩尔比例混合后,由上海美吉生物医药科技有限公司进行Illumina平台双端测序。使用QIIME 2 (v2022.11)进行样本拆分(demultiplex)、正反向序列拼接以及去除嵌合体。基于SILVA_134数据库进行序列比对并构建操作分类单元(operational taxonomic unit, OTU)表。最后,使用R软件基于OTU表对微生物多样性数据进行分析。
粪便样本中的胆汁酸提取参考先前发表的实验步骤[16]。实验选用了氘代胆酸(CA-D4)、氘代石胆酸(LCA-D4)、氘代鹅去氧胆酸(CDCA-D4)、氘代甘氨鹅去氧胆酸(GCDCA-D4)、氘代去氧胆酸(DCA-D4)以及氘代甘氨胆酸(GCA-D4)作为内标。所有样本均进行了随机化处理,并使用配备C18色谱柱(100 mm×2.1 mm, 2.6 μm) (Phenomenex Inc.公司)的超高效液相色谱-三重四极杆质谱(UHPLC-QQQ-MS,安捷伦科技有限公司,Santa Clara, CA)进行检测。UHPLC-QQQ-MS的色谱条件设定为:流动相A (含0.005%甲酸的超纯水)和流动相B (含0.05%甲酸的HPLC级乙腈)。质谱检测采用负离子多反应监测(multiple reaction monitoring, MRM)模式,并通过积分峰面积和校准曲线来完成胆汁酸的定量分析。实验共使用了35种胆汁酸标准品(Steraloids有限公司),相关数据已上传至ScienceDB (www.scidb.cn),CSTR编号为31253.11.sciencedb.j00231.00081。
所有图形的绘制均在GraphPad Prism 10.4.1和Adobe Illustrator 2025中进行。实验结果均以mean±SD表示。两组间比较采用非配对双尾Student’s t检验;多组间比较采用单因素方差分析(one-way ANOVA),随后采用Tukey’s multiple comparisons test进行组间差异分析。当数据不符合正态分布时采用非参数检验进行分析。P<0.05认为差异具有统计学意义。16S rRNA基因测序数据采用QIIME 2 (v2022.11)进行处理与分析。菌群α多样性采用Chao1指数及Simpson指数进行评价;β多样性基于Bray-Curtis距离矩阵进行主坐标分析(principal coordinates analysis, PCoA)。PICRUSt用于预测肠道菌群潜在功能通路。胆汁酸代谢组学数据经标准化处理后进行统计分析。
为评价首乌丸对DOXO诱导早衰小鼠生理功能的改善作用,本研究检测了小鼠血清炎症因子水平、运动能力及肌肉功能(图1A)。结果显示,与Control组相比,DOXO组小鼠血清肿瘤坏死因子-α (tumor necrosis factor-α, TNF-α)及白细胞介素-1β (interleukin-1β, IL-1β)水平均显著升高(P<0.01或P<0.001),提示DOXO诱导的早衰状态伴随明显炎症反应。给予首乌丸干预后,TNF-α及IL-1β水平明显下降,其中高剂量组改善更为显著(图1B、1C),表明首乌丸能够在一定程度上缓解DOXO诱导的炎症状态。与Control组相比,DOXO组小鼠悬挂时间显著缩短(P<0.001),提示阿霉素诱导的早衰模型小鼠出现明显的肌肉耐力下降。给予首乌丸干预后,低剂量组(DOXO+SWW-L)及高剂量组(DOXO+SWW-H)小鼠悬挂时间均较DOXO组明显延长,其中高剂量组改善效果更为显著(P<0.05),表明首乌丸能够有效缓解早衰导致的肌肉耐力下降(图1D)。在跑步能力测试中,DOXO组小鼠最大跑步速度较Control组显著降低(P<0.01),提示运动能力受损。经首乌丸干预后,低剂量组和高剂量组小鼠最大跑步速度均出现回升趋势,其中高剂量组提升更为明显(P<0.01),表明首乌丸可改善早衰小鼠的运动能力(图1E)。此外,前肢握力检测结果显示,DOXO处理后小鼠握力较Control组显著下降(P<0.001);而首乌丸干预后,低剂量组及高剂量组小鼠握力均显著提高,其中高剂量组恢复效果更加明显(P<0.01) (图1F)。结果提示首乌丸能够缓解DOXO诱导的肌肉力量下降。综上所述,DOXO诱导的早衰小鼠表现出明显的运动功能及肌肉力量减退,而首乌丸干预能够在一定程度上逆转上述变化,且高剂量组整体改善效果优于低剂量组,提示首乌丸具有延缓机体功能衰退、改善早衰相关表型的作用。
为探讨首乌丸对早衰小鼠肠道菌群结构的调控作用,对各组小鼠盲肠内容物进行16S rRNA基因测序分析。α多样性分析结果显示,与Control组相比,DOXO组小鼠菌群α多样性发生明显改变,其中Chao1指数和Simpson指数下降,提示DOXO诱导的早衰状态伴随肠道菌群α多样性下降(图2A、2B)。经首乌丸干预后,菌群多样性逐渐恢复,其中高剂量组改善趋势更加明显,表明首乌丸能够在一定程度上缓解DOXO诱导的肠道微生态失衡。进一步采用β多样性分析评估各组菌群整体结构差异。主坐标分析(principal coordinates analysis, PCoA)结果显示,各组样本间菌群结构存在明显分离,DOXO组与Control组距离较远,而首乌丸干预后菌群结构逐渐向Control组靠近(图2C),提示首乌丸能够部分恢复DOXO诱导的菌群结构异常。在门水平(phylum)分析中,芽孢杆菌门(Bacillota)与拟杆菌门(Bacteroidota)为各组小鼠肠道菌群的主要优势菌门(图2D)。与Control组相比,DOXO组Bacillota相对丰度升高,而Bacteroidota丰度下降,同时变形菌门(Pseudomonadota)及弯曲菌门(Campylobacterota)等与炎症相关菌门呈升高趋势,提示DOXO诱导的早衰状态伴随菌群稳态破坏及潜在炎症反应增强。经首乌丸干预后,Bacillota/Bacteroidota比例逐渐恢复,同时炎症相关菌门丰度下降,提示首乌丸能够改善DOXO诱导的菌群结构紊乱。属水平(genus)分析结果进一步显示,Muribaculaceae_unclassified、Odoribacter及Bacteroides为各组主要优势分类单元(图2E)。DOXO处理后,Muribaculaceae_unclassified丰度明显下降,而脱硫弧菌属(Desulfovibrio)、Helicobacter及Clostridia_UCG-014等菌属相对丰度升高,提示DOXO诱导的肠道环境更倾向于炎症及代谢紊乱状态。首乌丸干预后,Muribaculaceae_unclassified丰度明显恢复,同时阿克曼氏菌属(Akkermansia)、乳杆菌属(Lactobacillus)、双歧杆菌属(Bifidobacterium)及罗斯拜瑞氏菌属(Roseburia)等潜在有益菌丰度增加,而Desulfovibrio及Helicobacter等条件致病菌丰度下降,表明首乌丸能够促进肠道菌群向健康稳态方向恢复。综上结果表明,DOXO诱导的早衰状态可导致明显的肠道菌群失衡,而首乌丸干预能够改善菌群多样性及群落结构异常,促进有益菌恢复并抑制潜在致病菌增殖,从而可能参与其延缓早衰的作用。
为进一步探讨首乌丸改善早衰表型可能涉及的菌群功能变化,本研究基于16S rRNA基因测序结果采用PICRUSt进行功能预测分析。鉴于高剂量首乌丸组在运动能力改善及菌群结构恢复方面表现最为明显,因此选取DOXO组与DOXO+SWW-H组进行功能比较。结果显示,与DOXO组相比,DOXO+SWW-H组多条与物质代谢相关的功能通路发生明显改变(图3A)。其中,胆汁酸代谢相关功能通路显著富集,同时脂质代谢、氨基酸代谢及能量代谢等相关功能也出现不同程度恢复,提示首乌丸不仅能够调节肠道菌群组成,还可能进一步影响菌群潜在代谢功能,从而改善DOXO诱导的代谢功能紊乱。胆汁酸谱分析结果进一步支持了上述变化。DOXO处理后,小鼠粪便中多种结合型胆汁酸显著积累,尤其是牛磺酸结合α-鼠胆酸(tauro-α-muricholic acid, T-α-MCA)及牛磺酸结合β-鼠胆酸(tauro-β-muricholic acid, T-β-MCA)水平明显升高(P<0.01或P<0.001) (图3B),提示DOXO诱导的早衰状态可能伴随肠道菌群胆汁酸脱结合功能受损。研究表明,肠道菌群可通过胆汁酸水解酶(bile salt hydrolase, BSH)介导结合型胆汁酸脱结合,而结合型胆汁酸异常积累通常反映菌群胆汁酸代谢能力下降[17]。经高剂量首乌丸干预后,T-α-MCA及T-β-MCA水平显著下降,并接近Control组水平,提示首乌丸可能恢复了菌群相关胆汁酸脱结合功能,从而改善胆汁酸代谢稳态。与此同时,部分非结合型胆汁酸也发生明显变化(图3C)。与Control组相比,DOXO组小鼠胆酸(cholic acid, CA)水平显著下降,而首乌丸干预后CA水平明显回升;ω-MCA等部分菌群相关胆汁酸也呈恢复趋势。由于CA属于典型非结合型初级胆汁酸,其生成与结合型胆汁酸脱结合过程密切相关,因此上述结果进一步提示DOXO可能导致肠道菌群BSH活性下降,而首乌丸可能促进结合型胆汁酸向非结合型胆汁酸转化。此外,部分次级胆汁酸如猪去氧胆酸(hyodeoxycholic acid, HDCA)、猪胆酸(hyocholic acid, HCA)及石胆酸(lithocholic acid, LCA)虽未达到显著差异,但整体呈一定恢复趋势,提示首乌丸可能在一定程度上改善DOXO诱导的菌群胆汁酸转化功能紊乱。综合PICRUSt功能预测及胆汁酸代谢结果可见,DOXO诱导的早衰状态不仅伴随肠道菌群结构失衡,还表现出明显的胆汁酸代谢异常,尤其是结合型胆汁酸积累及菌群相关胆汁酸转化功能受损。首乌丸干预后可能通过调节肠道菌群代谢功能改善胆汁酸组成及代谢稳态,从而可能参与机体早衰表型的改善。
为进一步探讨肠道菌群变化与胆汁酸代谢之间的潜在联系,本研究对差异菌属与差异胆汁酸进行Spearman相关性分析,并采用热图进行可视化展示(图4)。结果显示,多种肠道菌群与胆汁酸代谢之间存在显著相关性,提示DOXO诱导的肠道菌群失衡可能参与胆汁酸稳态异常的发生。相关性分析结果表明,Muribaculaceae_unclassified、Akkermansia、Lactobacillus、Bifidobacterium及Roseburia等潜在有益菌与部分非结合型胆汁酸呈正相关,而与T-α-MCA、T-β-MCA等结合型胆汁酸呈负相关。其中,Lactobacillus及Bifidobacterium与CA等非结合型胆汁酸具有较强正相关性,提示上述菌群可能参与结合型胆汁酸脱结合过程,并与BSH活性相关[18]。相反,Desulfovibrio、Helicobacter等条件致病菌与多种结合型胆汁酸呈正相关,而与部分非结合型及次级胆汁酸呈负相关。由于结合型胆汁酸积累通常提示肠道菌群脱结合功能下降,因此上述结果进一步说明DOXO诱导的菌群紊乱可能导致胆汁酸代谢异常及肠道稳态失衡。此外,部分菌群与次级胆汁酸之间也存在明显相关性。例如,Akkermansia、Roseburia及Ruminococcus等菌属与HDCA、HCA及LCA等菌群相关次级胆汁酸呈一定正相关趋势,提示首乌丸干预后肠道菌群结构恢复可能进一步促进胆汁酸转化功能改善。综合上述结果表明,肠道菌群组成变化与胆汁酸代谢紊乱之间存在密切联系。首乌丸可能通过调节肠道菌群结构,恢复菌群相关胆汁酸代谢功能,从而参与改善DOXO诱导的早衰表型。
衰老是机体多系统功能逐渐退化的复杂生物学过程,常伴随慢性炎症、代谢紊乱及肠道稳态失衡。近年来,越来越多研究表明肠道菌群在衰老发生发展过程中发挥重要调控作用,肠道菌群失衡不仅可加剧机体慢性炎症状态,还可通过影响宿主代谢、免疫及肠屏障功能参与衰老相关疾病进程。本研究采用DOXO建立早衰小鼠模型,结果显示DOXO处理后小鼠运动能力明显下降,表现为握力减弱、悬挂耐力下降及最大跑步速度降低,同时伴随明显肠道菌群紊乱及胆汁酸代谢异常,提示DOXO能够成功诱导早衰相关表型。首乌丸干预后,小鼠运动能力显著改善,并部分恢复肠道菌群结构及胆汁酸稳态,提示其可能通过调节“肠道菌群-胆汁酸轴”发挥延缓早衰作用[19]。
肠道菌群失衡被认为是衰老的重要特征之一。既往研究发现,衰老状态下有益菌减少而条件致病菌增加可进一步诱导慢性低度炎症及肠屏障损伤。本研究16S rRNA基因测序结果显示,DOXO处理后Muribaculaceae等优势菌丰度下降,而Desulfovibrio、Helicobacter等条件致病菌丰度升高。Muribaculaceae是小鼠肠道中重要的核心菌群,与多糖代谢及短链脂肪酸生成密切相关,其下降通常提示肠道稳态受损[20]。Desulfovibrio属于典型硫酸盐还原菌,可产生硫化氢并损伤肠上皮屏障,其升高与炎症及代谢紊乱密切相关[21];Helicobacter升高则常提示肠道炎症状态增强。首乌丸干预后,Muribaculaceae丰度明显恢复,而Desulfovibrio及Helicobacter丰度下降,同时Akkermansia、Lactobacillus、Bifidobacterium及Roseburia等潜在有益菌丰度增加。上述潜在有益菌多与肠屏障维持、短链脂肪酸生成及免疫稳态密切相关,提示首乌丸能够改善DOXO诱导的肠道微生态失衡。
本研究进一步发现DOXO诱导的菌群紊乱伴随明显胆汁酸代谢异常。胆汁酸不仅参与脂质消化吸收,还作为重要信号分子参与代谢及炎症调控。肠道菌群可通过BSH介导结合型胆汁酸脱结合,并进一步参与次级胆汁酸生成,从而影响胆汁酸池组成及宿主代谢稳态。本研究胆汁酸谱分析显示,DOXO处理后T-α-MCA及T-β-MCA等结合型胆汁酸明显积累,而部分非结合型胆汁酸水平下降,提示DOXO可能导致肠道菌群胆汁酸脱结合功能受损。已有研究表明,Lactobacillus、Bifidobacterium及Bacteroides等菌群具有较强BSH活性,可促进结合型胆汁酸向非结合型胆汁酸转化[16]。本研究中首乌丸干预后上述有益菌丰度增加,同时结合型胆汁酸水平明显下降,而部分非结合型胆汁酸水平恢复,进一步提示首乌丸可能通过恢复菌群相关胆汁酸代谢功能改善胆汁酸稳态。
Spearman相关性分析结果显示,Akkermansia、Lactobacillus、Bifidobacterium等潜在有益菌与部分非结合型胆汁酸呈正相关,而与T-α-MCA、T-β-MCA等结合型胆汁酸呈负相关;相反,Desulfovibrio及Helicobacter等条件致病菌与多种结合型胆汁酸呈正相关。需要指出的是,相关性分析仅反映变量之间的统计关联,无法判断菌群变化与胆汁酸代谢改变之间的因果关系。因此,本研究结果仅提示肠道菌群组成变化与胆汁酸稳态异常之间存在潜在联系,尚不能证明“菌群失衡导致胆汁酸代谢异常并进一步影响早衰表型”的因果链条。未来可结合粪菌移植、抗生素清除菌群或特定菌株定殖等实验策略进一步验证关键菌群在胆汁酸代谢调控及首乌丸抗早衰作用中的功能贡献。
本研究采用DOXO诱导的早衰模型,虽然该模型能够在较短时间内诱导运动能力下降、组织损伤及细胞衰老相关表型,已被广泛用于衰老干预研究,但其本质上属于药物诱导的加速衰老模型,与自然生理性衰老仍存在差异。DOXO可直接引起DNA损伤、氧化应激及细胞毒性反应,因此部分观察到的表型变化可能同时受到药物毒性效应的影响,而不完全代表自然衰老过程中缓慢累积的病理生理改变。因此,本研究结果在外推至生理性衰老时仍需谨慎,并有必要在自然衰老模型或其他衰老模型中进一步验证首乌丸的作用。此外,本研究采用PICRUSt对16S rRNA基因测序数据进行了功能预测分析。PICRUSt基于已知参考基因组推测微生物群的潜在功能,其结果反映的是功能预测而非实际功能活性,容易受到参考数据库覆盖度及菌群组成差异的影响。因此,本研究中的PICRUSt结果仅作为胆汁酸代谢相关变化的辅助支持证据,而不能作为机制性结论的直接依据。未来仍需结合宏基因组测序、宏转录组学以及关键代谢酶活性检测等方法进行验证。
本研究还存在若干局限性。首先,虽然观察到结合型胆汁酸积累及部分有益菌丰度下降,但尚未直接检测胆汁酸水解酶(bile salt hydrolase, BSH)活性,因此菌群介导胆汁酸脱结合功能变化仍缺乏直接证据。其次,本研究未进一步评价肠道屏障功能,包括肠道通透性检测及紧密连接蛋白(ZO-1、Occludin、Claudin-1等)表达变化,因此尚不能明确首乌丸是否通过改善肠屏障发挥作用。此外,本研究主要关注运动能力表型,尚未检测NF-κB、mTOR、AMPK等与衰老及骨骼肌功能密切相关的信号通路活性,因而无法进一步阐明其下游分子机制。最后,本研究仅检测了粪便胆汁酸谱,尚未系统评价肝脏及回肠组织中胆汁酸合成、转运及受体信号相关分子(如CYP7A1、CYP27A1、FXR、FGF15等)的变化。因此,目前研究结果主要支持首乌丸改善DOXO诱导的早衰表型与肠道菌群及胆汁酸代谢变化之间存在关联,而相关机制环节仍有待进一步实验验证。尽管存在上述局限性,本研究仍表明首乌丸能够显著改善DOXO诱导的运动功能下降、肠道菌群失衡及胆汁酸代谢异常,为后续深入解析“肠道菌群-胆汁酸代谢-衰老表型”之间的相互作用提供了实验依据。
本研究表明,首乌丸能够显著改善DOXO诱导的早衰小鼠运动能力下降及肠道菌群失衡,恢复肠道微生态稳态。其作用可能与调节肠道菌群结构、促进有益菌增殖、抑制条件致病菌富集以及改善菌群相关胆汁酸代谢异常密切相关。首乌丸干预后多种结合型胆汁酸水平下降,部分非结合型胆汁酸恢复,同时肠道菌群与胆汁酸代谢之间呈显著相关性,提示“肠道菌群-胆汁酸轴”可能参与其延缓早衰的作用。上述结果为首乌丸抗衰老作用机制研究提供了新的实验依据,也为基于肠道菌群及胆汁酸代谢干预衰老相关疾病提供了新的思路。
  • 湖北省自然科学基金(JCZRQNB202600476)
  • 湖北省中医药创新团队项目(ZY2025J001)
  • 湖北省中医药创新发展联合基金(JCZRLH202601771)
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2026年第66卷第10期
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doi: 10.13343/j.cnki.wsxb.20260440
  • 接收时间:2026-05-25
  • 首发时间:2026-10-13
  • 出版时间:2026-10-04
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  • 收稿日期:2026-05-25
  • 录用日期:2026-07-03
基金
Hubei Provincial Natural Science Foundation(JCZRQNB202600476)
湖北省自然科学基金(JCZRQNB202600476)
Hubei Provincial Traditional Chinese Medicine Innovation Team Project(ZY2025J001)
湖北省中医药创新团队项目(ZY2025J001)
Hubei Provincial Traditional Chinese Medicine Innovation and Development Joint Fund(JCZRLH202601771)
湖北省中医药创新发展联合基金(JCZRLH202601771)
作者信息
    1.湖北省中医院,湖北中医药大学附属医院,湖北 武汉
    2.湖北中医药大学基础医学院,湖北 武汉

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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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