Article(id=1320435173385159474, tenantId=1146029695717560320, journalId=1319373162502258839, issueId=1320435149154669015, articleNumber=null, orderNo=null, doi=10.16016/j.2097-0927.202510035, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=null, receivedDate=1760457600000, receivedDateStr=2025-10-15, revisedDate=1767024000000, revisedDateStr=2025-12-30, acceptedDate=null, acceptedDateStr=null, onlineDate=1792745877833, onlineDateStr=2026-10-23, pubDate=1769702400000, pubDateStr=2026-01-30, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1792745877833, onlineIssueDateStr=2026-10-23, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1792745877833, creator=13701087609, updateTime=1792745877833, updator=13701087609, issue=Issue{id=1320435149154669015, tenantId=1146029695717560320, journalId=1319373162502258839, year='2026', volume='48', issue='2', pageStart='107', pageEnd='250', issueExtLink='null', onlineDate='null', pubDate='1769702400000', pubDateStr='2026-01-30', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1792745872057, creator='13701087609', updateTime=1792749692151, updator='13701087609', preIssue=null, nextIssue=null, articleTotal=null, ext={EN=IssueExt(id=1320451171886682715, tenantId=1146029695717560320, journalId=1319373162502258839, issueId=1320435149154669015, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1320451171886682716, tenantId=1146029695717560320, journalId=1319373162502258839, issueId=1320435149154669015, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null, downloadFileDto=null}, startPage=169, endPage=180, ext={EN=ArticleExt(id=1320435175088046901, articleId=1320435173385159474, tenantId=1146029695717560320, journalId=1319373162502258839, language=EN, title=Signature pathogenic bacteria in tongue coating drive the progression of chronic hepatitis B complicated with fatty liver disease via the “Oral-gut-liver axis” by regulating glucose and lipid metabolism, columnId=1320435174995772212, journalTitle=Journal of Army Medical University, columnName=Clinical Medicine, runingTitle=null, highlight=null, articleAbstract=
Objective

To compare the characteristics of tongue coating flora in healthy people, patients with chronic hepatitis B (CHB) and patients with CHB complicated with non-alcoholic fatty liver disease (NAFLD), and to explore the characteristics of tongue coating flora in patients with CHB complicated with NAFLD and its comorbidity mechanism.

Methods

From January 2024 to December 2024, 42 with CHB complicated with NAFLD patients with CHB complicated with NAFLD(CHB complicated with NAFLD group) and 34 patients with simple CHB (simple CHB group) were subjected in our department, and 28 healthy individuals were selected and served as control group. Baseline clinical data were collected from all the participants, and intergroup differences were analyzed. After tongue coating samples were harvested for 16S ribosomal RNA (16S rRNA) sequencing using the PacBio Sequel sequencing platform. After sequence quality control, denoising, and amplicon sequence variant (ASV) clustering, the microbial composition, α diversity, and β diversity were analyzed in the 3 sample groups. Linear discriminant analysis effect size (LEfSe) was used to screen for intergroup differential marker species, by linear discriminant analysis (LDA) score >2 and P<0.05, and Kyoto encyclopedia of genes and genomes (KEGG) and MetaCyc functional prediction analyses were also performed.

Results

Significant differences were observed among the 3 groups in terms of age, gender, body mass index (BMI), alanine aminotransferase (ALT), fasting plasma glucose (FPG) and free fatty acids (FFA) (P<0.05). There were totally 48 236 ASVs obtained, with CHB complicated with NAFLD group having the highest number of unique ASVs. Compared to the healthy control group, the CHB complicated with NAFLD group exhibited an increasing trend in species richness, community heterogeneity, and distribution uniformity of the tongue coating microbiota. At the phylum level, the proportions of Firmicutes (32%) and Proteobacteria (26%) were increased, while the proportion of Bacteroidota (27%) was decreased in the CHB complicated with NAFLD group than the healthy control group. In comparison to the CHB-only group, the proportion of Bacteroidota was increased. At the genus level, the proportions of Veillonella (18%) and Neisseria (18%) were increased, while that of Prevotella_7 (15%) was decreased in the CHB complicated with NAFLD group than the healthy control group, and that of Neisseria and Prevotella_7 was increased in the CHB complicated with NAFLD group when compared with the CHB-only group. LEfSe analysis revealed that Actinobacteriota, Neisseria_elongata, and Streptococcus_oralis were potential marker species in the CHB complicated with NAFLD group. Both KEGG and MetaCyc functional prediction analyses indicated that the differentially expressed functions of the tongue coating microbiota among the 3 groups were primarily enriched in pathways such as cofactor and vitamin metabolism, carbohydrate metabolism, and lipid metabolism.

Conclusion

CHB complicated with NAFLD patients exhibit characteristic changes in their tongue coating microbiota. The flora of tongue coating may affect the glucose and lipid metabolism of CHB patients through the "Oral-gut-liver axis", and promote the process and progress of NAFLD.

, authors=Qing ZHAO, Xinju CHEN, Lihui ZHANG, Sutong LIU, Dongfang SHANG, Qiang ZHANG, Wenxia ZHAO, authorsList=Qing ZHAO, Xinju CHEN, Lihui ZHANG, Sutong LIU, Dongfang SHANG, Qiang ZHANG, Wenxia ZHAO, authorCompany=null, correspAuthors=Wenxia ZHAO, authorNote=null, correspAuthorsNote=null, copyrightStatement=© 2026 Journal of Army Medical University, copyrightOwner=Editorial Office of Journal of Army Medical University, 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=1320435180884575053, articleId=1320435173385159474, tenantId=1146029695717560320, journalId=1319373162502258839, language=CN, title=舌苔标志性致病菌通过“口-肠-肝轴”调控糖脂代谢驱动慢性乙型肝炎合并脂肪肝疾病进展, columnId=1320435175822050102, journalTitle=陆军军医大学学报, columnName=论著·临床医学, runingTitle=null, highlight=null, articleAbstract=
目的

通过比较健康人群、慢性乙型肝炎(chronic hepatitis B,CHB)患者、CHB合并非酒精性脂肪性肝病(non-alcoholic fatty liver disease,NAFLD)患者的舌苔菌群变化特点,探讨CHB合并NAFLD患者舌苔菌群特征及共病机制。

方法

选取2024年1-12月就诊于河南中医药大学第一附属医院消化科的CHB合并NAFLD患者42例(CHB合并NAFLD组)、单纯CHB患者34例(单纯CHB组),另选取体检科同期体检的健康人群(健康对照组)28例作为对照。收集所有研究对象的基线临床资料并分析组间差异。留取舌苔标本,采用PacBio Sequel测序平台对其16S核糖体核糖核酸(16S ribosomal RNA, 16S rRNA)基因进行测序,经序列质控、去噪及ASV聚类后,分析3组样本的菌群组成、α多样性与β多样性,利用线性判别分析效应大小(linear discriminant analysis effect size, LEfSe)分析筛选组间差异标志物种,并进行京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes, KEGG)及MetaCyc功能预测分析。

结果

3组研究对象在年龄、性别、BMI、谷丙转氨酶、空腹血糖、游离脂肪酸的差异均具有统计学意义(P<0.05)。共获得48 236个ASV,CHB合并NAFLD组独有ASV最多。与健康对照组相比,CHB合并NAFLD组舌苔菌群的物种丰富度、群落异质性及分布均匀度均呈上升趋势。在门水平上,CHB合并NAFLD组厚壁菌门(Firmicutes,32%)、变形菌门(Proteobacteria,26%)占比较健康对照组增加,拟杆菌门(Bacteroidota,27%)占比减少;与单纯CHB组相比,其Bacteroidota占比增加。在属水平上,CHB合并NAFLD组韦荣球菌属(Veillonella,18%)、奈瑟菌属(Neisseria,18%)占比较健康对照组增加,普雷沃菌属_7(Prevotella_7,15%)占比减少;与单纯CHB组相比,其Neisseria、Prevotella_7占比增加。LEfSe分析显示,放线菌门(Actinobacteriota)、长奈瑟菌(Neisseria_elongata)及口腔链球菌(Streptococcus_oralis)是CHB合并NAFLD组的潜在标志物种。KEGG与MetaCyc功能预测分析均提示,3组舌苔菌群的功能差异主要富集于辅因子与维生素代谢、糖代谢及脂质代谢等通路。

结论

CHB合并NAFLD患者的舌苔菌群结构表现出特征性变化,其特定致病菌丰度增加可能通过“口-肠-肝轴”调控糖脂代谢,从而促进NAFLD的进程。

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赵文霞,E-mail:
ZHAO Wenxia,E-mail:
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Comparison of 3 groups of general data [M (Q₁, Q₃), n(%),x¯±s]

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ItemsCHB complicated with
NAFLD group(n=42)
Simple CHB group(n=34)Healthy control group(n=28)χ²/FP
Age/years38.50(35.25,43.00)41.00(35.00,51.00)24.00 (24.00,25.00)55.25<0.001
Gender16.23<0.001
Male33 (78.57)16 (47.06)9 (32.14)
Female9 (21.43)18 (52.94)19 (67.86)
BMI/(kg/m²)26.00 ± 2.9122.59 ± 3.3321.21 ± 2.5224.97<0.001
Hypertension-0.063
Yes7 (16.67)3 (8.82)0
No35 (83.33)31 (91.18)28 (100.00)
Diabetes mellitus-0.171
Yes2 (4.76)4 (11.76)0
No40 (95.24)30 (88.24)28 (100.00)
ALT/(U/L)29.40 (26.00,42.40)26.55 (16.20,37.35)18.90 (14.97,27.55)13.680.001
AST/(U/L)27.00 (20.50,32.30)23.90 (20.80,33.83)22.35 (13.65,31.65)2.990.224
FPG/(mmol/L)5.70 (5.19,5.89)5.36 (5.04,6.08)5.00 (4.60,5.54)11.690.003
FFA/(mmol/L)0.48 ± 0.190.39 ± 0.160.37 ± 0.154.300.016
), ArticleFig(id=1320435188610483093, tenantId=1146029695717560320, journalId=1319373162502258839, articleId=1320435173385159474, language=CN, label=表1, caption=

3组一般资料比较 [M (Q₁, Q₃), 例(%),x¯±s]

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ItemsCHB complicated with
NAFLD group(n=42)
Simple CHB group(n=34)Healthy control group(n=28)χ²/FP
Age/years38.50(35.25,43.00)41.00(35.00,51.00)24.00 (24.00,25.00)55.25<0.001
Gender16.23<0.001
Male33 (78.57)16 (47.06)9 (32.14)
Female9 (21.43)18 (52.94)19 (67.86)
BMI/(kg/m²)26.00 ± 2.9122.59 ± 3.3321.21 ± 2.5224.97<0.001
Hypertension-0.063
Yes7 (16.67)3 (8.82)0
No35 (83.33)31 (91.18)28 (100.00)
Diabetes mellitus-0.171
Yes2 (4.76)4 (11.76)0
No40 (95.24)30 (88.24)28 (100.00)
ALT/(U/L)29.40 (26.00,42.40)26.55 (16.20,37.35)18.90 (14.97,27.55)13.680.001
AST/(U/L)27.00 (20.50,32.30)23.90 (20.80,33.83)22.35 (13.65,31.65)2.990.224
FPG/(mmol/L)5.70 (5.19,5.89)5.36 (5.04,6.08)5.00 (4.60,5.54)11.690.003
FFA/(mmol/L)0.48 ± 0.190.39 ± 0.160.37 ± 0.154.300.016
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舌苔标志性致病菌通过“口-肠-肝轴”调控糖脂代谢驱动慢性乙型肝炎合并脂肪肝疾病进展
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赵晴 , 陈欣菊 , 张丽慧 , 刘素彤 , 尚东方 , 张强 , 赵文霞
陆军军医大学学报 | 论著·临床医学 2026,48(2): 169-180
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陆军军医大学学报 |论著·临床医学 2026 , 48 (2) : 169 -180
舌苔标志性致病菌通过“口-肠-肝轴”调控糖脂代谢驱动慢性乙型肝炎合并脂肪肝疾病进展
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赵晴, 陈欣菊, 张丽慧, 刘素彤, 尚东方, 张强, 赵文霞
作者信息
  • 1河南中医药大学第一附属医院消化科,河南 郑州
通讯作者:
赵文霞,E-mail:
ZHAO Wenxia,E-mail:
Signature pathogenic bacteria in tongue coating drive the progression of chronic hepatitis B complicated with fatty liver disease via the “Oral-gut-liver axis” by regulating glucose and lipid metabolism
Qing ZHAO, Xinju CHEN, Lihui ZHANG, Sutong LIU, Dongfang SHANG, Qiang ZHANG, Wenxia ZHAO
Affiliations
  • 1Department of Gastroenterology, the First Affiliated Hospital of Henan University of Chinese Medicine, Zhengzhou, Henan, China
出版时间: 2026-01-30 doi: 10.16016/j.2097-0927.202510035
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目的

通过比较健康人群、慢性乙型肝炎(chronic hepatitis B,CHB)患者、CHB合并非酒精性脂肪性肝病(non-alcoholic fatty liver disease,NAFLD)患者的舌苔菌群变化特点,探讨CHB合并NAFLD患者舌苔菌群特征及共病机制。

方法

选取2024年1-12月就诊于河南中医药大学第一附属医院消化科的CHB合并NAFLD患者42例(CHB合并NAFLD组)、单纯CHB患者34例(单纯CHB组),另选取体检科同期体检的健康人群(健康对照组)28例作为对照。收集所有研究对象的基线临床资料并分析组间差异。留取舌苔标本,采用PacBio Sequel测序平台对其16S核糖体核糖核酸(16S ribosomal RNA, 16S rRNA)基因进行测序,经序列质控、去噪及ASV聚类后,分析3组样本的菌群组成、α多样性与β多样性,利用线性判别分析效应大小(linear discriminant analysis effect size, LEfSe)分析筛选组间差异标志物种,并进行京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes, KEGG)及MetaCyc功能预测分析。

结果

3组研究对象在年龄、性别、BMI、谷丙转氨酶、空腹血糖、游离脂肪酸的差异均具有统计学意义(P<0.05)。共获得48 236个ASV,CHB合并NAFLD组独有ASV最多。与健康对照组相比,CHB合并NAFLD组舌苔菌群的物种丰富度、群落异质性及分布均匀度均呈上升趋势。在门水平上,CHB合并NAFLD组厚壁菌门(Firmicutes,32%)、变形菌门(Proteobacteria,26%)占比较健康对照组增加,拟杆菌门(Bacteroidota,27%)占比减少;与单纯CHB组相比,其Bacteroidota占比增加。在属水平上,CHB合并NAFLD组韦荣球菌属(Veillonella,18%)、奈瑟菌属(Neisseria,18%)占比较健康对照组增加,普雷沃菌属_7(Prevotella_7,15%)占比减少;与单纯CHB组相比,其Neisseria、Prevotella_7占比增加。LEfSe分析显示,放线菌门(Actinobacteriota)、长奈瑟菌(Neisseria_elongata)及口腔链球菌(Streptococcus_oralis)是CHB合并NAFLD组的潜在标志物种。KEGG与MetaCyc功能预测分析均提示,3组舌苔菌群的功能差异主要富集于辅因子与维生素代谢、糖代谢及脂质代谢等通路。

结论

CHB合并NAFLD患者的舌苔菌群结构表现出特征性变化,其特定致病菌丰度增加可能通过“口-肠-肝轴”调控糖脂代谢,从而促进NAFLD的进程。

慢性乙型肝炎  /  非酒精性脂肪性肝病  /  共病  /  舌  /  微生物群
Objective

To compare the characteristics of tongue coating flora in healthy people, patients with chronic hepatitis B (CHB) and patients with CHB complicated with non-alcoholic fatty liver disease (NAFLD), and to explore the characteristics of tongue coating flora in patients with CHB complicated with NAFLD and its comorbidity mechanism.

Methods

From January 2024 to December 2024, 42 with CHB complicated with NAFLD patients with CHB complicated with NAFLD(CHB complicated with NAFLD group) and 34 patients with simple CHB (simple CHB group) were subjected in our department, and 28 healthy individuals were selected and served as control group. Baseline clinical data were collected from all the participants, and intergroup differences were analyzed. After tongue coating samples were harvested for 16S ribosomal RNA (16S rRNA) sequencing using the PacBio Sequel sequencing platform. After sequence quality control, denoising, and amplicon sequence variant (ASV) clustering, the microbial composition, α diversity, and β diversity were analyzed in the 3 sample groups. Linear discriminant analysis effect size (LEfSe) was used to screen for intergroup differential marker species, by linear discriminant analysis (LDA) score >2 and P<0.05, and Kyoto encyclopedia of genes and genomes (KEGG) and MetaCyc functional prediction analyses were also performed.

Results

Significant differences were observed among the 3 groups in terms of age, gender, body mass index (BMI), alanine aminotransferase (ALT), fasting plasma glucose (FPG) and free fatty acids (FFA) (P<0.05). There were totally 48 236 ASVs obtained, with CHB complicated with NAFLD group having the highest number of unique ASVs. Compared to the healthy control group, the CHB complicated with NAFLD group exhibited an increasing trend in species richness, community heterogeneity, and distribution uniformity of the tongue coating microbiota. At the phylum level, the proportions of Firmicutes (32%) and Proteobacteria (26%) were increased, while the proportion of Bacteroidota (27%) was decreased in the CHB complicated with NAFLD group than the healthy control group. In comparison to the CHB-only group, the proportion of Bacteroidota was increased. At the genus level, the proportions of Veillonella (18%) and Neisseria (18%) were increased, while that of Prevotella_7 (15%) was decreased in the CHB complicated with NAFLD group than the healthy control group, and that of Neisseria and Prevotella_7 was increased in the CHB complicated with NAFLD group when compared with the CHB-only group. LEfSe analysis revealed that Actinobacteriota, Neisseria_elongata, and Streptococcus_oralis were potential marker species in the CHB complicated with NAFLD group. Both KEGG and MetaCyc functional prediction analyses indicated that the differentially expressed functions of the tongue coating microbiota among the 3 groups were primarily enriched in pathways such as cofactor and vitamin metabolism, carbohydrate metabolism, and lipid metabolism.

Conclusion

CHB complicated with NAFLD patients exhibit characteristic changes in their tongue coating microbiota. The flora of tongue coating may affect the glucose and lipid metabolism of CHB patients through the "Oral-gut-liver axis", and promote the process and progress of NAFLD.

hepatitis B, chronic  /  non-alcoholic fatty liver disease  /  comorbidity  /  tongue  /  microbiota
赵晴, 陈欣菊, 张丽慧, 刘素彤, 尚东方, 张强, 赵文霞. 舌苔标志性致病菌通过“口-肠-肝轴”调控糖脂代谢驱动慢性乙型肝炎合并脂肪肝疾病进展. 陆军军医大学学报, 2026 , 48 (2) : 169 -180 . DOI: 10.16016/j.2097-0927.202510035
Qing ZHAO, Xinju CHEN, Lihui ZHANG, Sutong LIU, Dongfang SHANG, Qiang ZHANG, Wenxia ZHAO. Signature pathogenic bacteria in tongue coating drive the progression of chronic hepatitis B complicated with fatty liver disease via the “Oral-gut-liver axis” by regulating glucose and lipid metabolism[J]. Journal of Army Medical University, 2026 , 48 (2) : 169 -180 . DOI: 10.16016/j.2097-0927.202510035
慢性乙型肝炎(chronic hepatitis B,CHB)和非酒精性脂肪性肝病(non-alcoholic fatty liver disease,NAFLD)是全球常见的两种肝脏疾病,也是向肝硬化、肝癌进展的常见肝病,随着NAFLD的全球大流行[1],一项荟萃分析显示亚洲成人CHB合并NAFLD的患病率为36.5%[2]。这种共病状态不仅加速肝病进展[3],而且增加了肝细胞癌(hepatocellular carcinoma, HCC)发生风险[4-5],更是目前导致肝脏疾病相关并发症发生和死亡的重要原因[6-7],给临床诊断和治疗决策带来了巨大挑战。因此,二者的共病研究已成为肝病领域热点。随着微生物学研究的不断深入,研究显示肝病患者容易出现口腔菌群失调[8-10],从而引发肠道菌群结构失衡,对疾病治疗和预后产生负面影响[11-12]。而舌苔作为大量微生物聚集的场所,舌苔菌群是口腔微生物的重要组成部分,其结构变化能够反映机体代谢状态,可作为某些全身疾病的微生物学指征[13]。现阶段,关于该共病机制的研究存在3个明显局限性:①既往的共病机制研究多简单归因于乙肝病毒(hepatitis B virus,HBV)与代谢异常的叠加效应,忽略了微生物群落在其中的重要作用[14-16];②即使关注到菌群的影响,在探讨微生物群作用时,研究多集中于肠道菌群[17-19],而忽略了口腔菌群在“口-肠-肝轴”中的关键“始动”作用;③现有的口腔菌群研究也往往仅聚焦于CHB或者NAFLD单一疾病状态下的口腔菌群变化[20-22],缺乏对二者共病状态下口腔菌群特征的系统研究。这种研究空白使得我们难以回答一个关键临床问题:CHB合并NAFLD患者是否具有独特的口腔菌群特征?这些特征能否为阐明“口-肠-肝轴”参与共病机制提供依据?基于此,本研究针对CHB合并NAFLD 患者的舌苔菌群变化,探索此类患者群体的新型生物标志物和可能的共病机制,以期为CHB合并NAFLD的综合治疗提供一定的理论依据。
选取河南中医药大学第一附属医院2024年1-12月消化科就诊的CHB合并NAFLD患者(CHB合并NAFLD组)、单纯CHB患者(单纯CHB组),另选取同期体检科体检的健康人群(健康对照组)作为对照。单纯CHB组纳入标准:①年龄18~65岁;②诊断符合《慢性乙型肝炎防治指南(2022年版)》[23],且不符合《非酒精性脂肪性肝病防治指南(2018更新版)》[24]。CHB合并NAFLD组纳入标准:①年龄18~65岁;②诊断符合《慢性乙型肝炎防治指南(2022年版)》,且同时符合《非酒精性脂肪性肝病防治指南(2018更新版)》。健康对照组纳入标准:①既往及当前无明确的躯体疾病;②18~65岁。排除标准:①近1个月使用过可能对舌苔菌群产生干扰的药物,如抗生素、微生态调节剂、免疫抑制剂等;②患有可能对舌苔菌群产生干扰的口腔局部疾病,如牙周炎等,或近1个月内接受过口腔洁治、刮治等口腔治疗操作;③戒烟史≤1年;④严重的感染未控制者;⑤合并其他类型肝病;⑥有严重心、肝、肺疾病或其他恶性疾病;⑦精神障碍者;⑧妊娠期或哺乳期妇女。同时,收集所有纳入研究对象的一般人口学资料(年龄、性别)、人体测量学指标[体质指数(BMI)]及临床生化指标[谷丙转氨酶(alanine aminotransferase,ALT)、天冬氨酸氨基转移酶(aspartate aminotransferase,AST)、空腹血糖(fasting plasma glucose,FPG)、游离脂肪酸(free fatty acids,FFA)]。本研究经河南中医药大学第一附属医院伦理委员会批准(2025HL-247),委员会对研究自起始(2024年1月)以来的全过程予以追认。所有参与者在入选前签署了知情同意书。
所有纳入研究对象在晨起早餐前采集舌苔样本,采样前8 h内禁食并保持口腔清洁。采样前用纯净水漱口,5 min后使用3个无菌拭子在舌苔中部表面各连续刮取5次,随后将拭子放入保存管,置于干冰泡沫采样盒中,并于5 h内送至实验室,立即冻存于-80 ℃冰箱。
实验流程包括微生物组总脱氧核糖核酸(deoxyribonucleic acid, DNA)提取、目标片段PCR扩增、扩增产物回收纯化、荧光定量、测序文库制备及上机测序。采用Nanodrop微量核酸蛋白测定仪定量和0.8%琼脂糖凝胶电泳检测DNA质量;通过两步PCR法为样本添加Barcode,使用Q5高保真DNA聚合酶扩增,并设置阴性对照;扩增产物经2%琼脂糖凝胶电泳切胶回收后,利用Quant-iT PicoGreen dsDNA定量检测试剂盒进行荧光定量,按比例混合样本;使用PacBio测序模板制备试剂盒(版本1.0)制备测序文库,经损伤修复、末端修复、磁珠纯化、平端连接及外切酶处理后,通过PacBio Sequel测序仪完成测序。其中Q5高保真DNA聚合酶购自美国NEB公司,荧光试剂Quant-iT PicoGreen dsDNA Assay Kit购自美国Thermo Fisher公司,Template Prep Kit 1.0试剂购自美国PacBio公司。
使用CCS软件处理PacBio测序原始数据,获得高质量FASTQ序列,对问题样本进行重测或补测。根据Barcode信息划分样本并去除引物序列,将反向互补序列转为正向。采用改进的QIIME2流程中的DADA2算法进行序列去噪,生成高精度扩增子序列变体(amplicon sequence variant, ASV),并基于SILVA数据库对ASV进行物种注释。
基于上述ASV表,进行以下分析。①物种丰度与分布评估:绘制序列长度分布图、丰度等级曲线及Shannon指数稀疏曲线,以评估测序数据质量与覆盖度;绘制Venn图展示3组间共有和独有的ASV数目。②α多样性分析:计算Chao1指数(物种丰富度)、Simpson指数(群落异质性)和Pielou’s evenness指数(分布均匀度),评估组内微生物多样性。③β多样性分析:基于Jaccard距离矩阵进行主坐标分析(principal co-ordinates analysis, PCoA),并使用Adonis检验进行组间差异统计学检验。④物种组成分析:分别在门和属水平计算物种的相对丰度,并绘制柱形图展示优势菌群分布。⑤差异物种与标志物分析:采用线性判别分析效应大小(linear discriminant analysis effect size, LEfSe)方法(基于Python LEfSe包,版本1.0)筛选组间差异显著的标志物种,设定线性判别分析(linear discriminant analysis, LDA)得分>2且P<0.05作为显著性阈值。⑥功能预测分析:基于16S核糖体核糖核酸(16S ribosomal RNA, 16S rRNA)基因测序结果,使用PICRUSt2软件,参照京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes, KEGG)通路数据库(https://www.kegg.jp/)和MetaCyc数据库(https://metacyc.org/)预测菌群代谢功能并分析差异通路。
统计数据分析基于SPSS 22.0完成,计量资料符合正态分布以x¯±s表示,方差齐性采用t检验,方差不齐采用t'检验;不符合正态分布的计量资料以M(Q1,Q3)表示,采用Mann-Whitney U检验。分类资料以n(%)表示,组间比较采用χ²检验或Fisher确切概率法。P<0.05表示差异具有统计学意义。测序数据通过DADA2和R语言处理。
本研究初步评估并筛选了138例包括就诊患者及健康志愿者的潜在研究对象,经过严格的纳入与排除标准审核,其中34例被排除(如近期使用抗生素、存在其他肝病、口腔疾病等原因),最终104例研究对象成功入组,其中CHB合并NAFLD组42例,单纯CHB组34例,健康对照组28例。对3组的年龄、性别、BMI、ALT、FPG、FFA进行比较,差异均具有统计学意义(P<0.05,表1)。
图1为测序后16S rRNA 高质量序列,长度分布在547~2 542 bp。图2为3组样本的丰度等级曲线,其长短及高度不一,说明各样本的物种分布情况具有差异。图3为Shannon指数稀疏曲线,随着样本量的增加,稀疏曲线已经接近平缓,表明所包含的物种多样性已经接近饱和,即本次研究送检样本量充足。
本研究共得到48 236个ASV,绘制Venn图,结果显示,CHB合并NAFLD组独有ASV数目最多,单纯CHB组次之,健康对照组最少;147个ASV为3组所共有(图4)。
通过统一流程计算样本的物种丰富度(Chao1)、群落异质性(Simpson)、分布均匀度(Pielou’s evenness)等核心参数,结果均显示组间差异无统计学意义(图5),但与健康对照组相比,CHB合并NAFLD组的Chao1指数、Simpson指数、Pielou’s evenness指数均呈上升趋势。
对于不同样本β多样性的分析,使用基于Jaccard距离的PCoA将3组人群进行区分,但Adonis检验结果显示,3组人群的舌苔菌群分布差异无统计学意义(图6)。
根据物种丰度表和物种注释表,选取具有明确分类信息且相对丰度最高的分类单元,将每个分组的相对丰度进行展示。其中,门水平柱状图展示了所有具有明确分类信息的菌门(图7);属水平柱状图则选取了相对丰度前20位的菌属进行展示(图8)。
如图7所示,在门水平上,3组样本最大丰度均处于前5位的物种分布为厚壁菌门(Firmicutes)、拟杆菌门(Bacteroidota)、变形菌门(Proteobacteria)、梭杆菌门(Fusobacteria)、弯曲菌门(Campylobacterota),其中以Firmicutes(31%)、Bacteroidota(30%)和Proteobacteria(23%)为主。在CHB合并NAFLD组、单纯CHB组、健康对照组中,这3种菌群相对丰度的总和分别占各组所有菌群的85%、84%、83%。
如图8所示,在属水平上,3组样本占前 5位的优势菌群分别为韦荣球菌属(Veillonella)、奈瑟菌属(Neisseria)、普雷沃菌属_7(Prevotella_7)、嗜血杆菌属(Haemophilus)、链球菌属(Streptococcus),其中以Veillonella(18%)、Neisseria(15%)、Prevotella_7(15%)为主。在CHB合并NAFLD组、单纯CHB组、健康对照组中,这3种菌群相对丰度的总和分别占各组所有菌群的51%、48%、46%。
对3组人群的舌苔菌群丰度进行LEfSe差异分析。在门水平上,差异菌为放线菌门(Actinobacteriota),在种水平上找到的差异菌为长奈瑟菌(Neisseria_elongata)和口腔链球菌(Streptococcus_oralis),且均只富集在CHB合并NAFLD组。
对3组人群舌苔菌群进行两两比较,发现与健康对照组相比,CHB合并NAFLD组舌苔菌群中Bacteroidota、螺旋体门(Spirochaetota)、密螺旋体属(Treponema)的相对丰度较低,而Actinobacteriota、颗粒链球菌属(Granulicatella)、放线菌属(Actinomyces)、罗斯氏菌属(Rothia)、P5D1_392菌属(P5D1_392)、贫养菌属(Abiotrophia)、小单胞菌属(Parvimonas)的相对丰度较高;与健康对照组相比,单纯CHB组舌苔菌群中Bacteroidota、气单胞菌属(Aeromonas)、糖单胞菌目(Saccharimonadales)、邻单胞菌属(Plesiomonas)的相对丰度较低,而Actinobacteriota、Granulicatella、Neisseria、Rothia、金氏菌属(Kingella)、劳特罗普氏菌属(Lautropia)、聚集杆菌属(Aggregatibacter)、丛毛单胞菌属(Comamonas)、阿里西氏菌属(Alysiella)的相对丰度较高;与单纯CHB组相比,CHB合并NAFLD组舌苔菌群中Fusobacteria、Spirochaetota、梭杆菌属(Fusobacterium)、Treponema、Comamonas、Alysiella的相对丰度较低,而Actinomyces、Aeromonas、Plesiomonas的相对丰度较高(图9)。
KEGG和MetaCyc数据库均能通过分析生物代谢通路,预测显著不同舌苔菌群的通路和代谢过程。结果显示:在KEGG通路数据库,3组样本主要富集途径为“代谢二级功能通路”,其中在辅因子与维生素代谢、糖代谢、氨基酸代谢、其他氨基酸代谢、萜类与聚酮代谢、能量代谢、聚糖生物合成与代谢和脂质代谢二级功能通路的相对丰度均较高(图10);MetaCyc通路数据库,3组样本主要富集途径为“生物合成二级功能通路”,其中在辅因子、辅基、电子载体以及维生素的合成、脂肪酸和脂质生物合成、核苷和核苷酸生物合成和氨基酸生物合成二级功能通路的相对丰度均较高(图11)。
CHB合并NAFLD的共病现象在亚洲呈现显著流行趋势,区域流行病学数据显示其患病率达14%~70%[25],我国的发病率为32.83%[26],已成为临床常见的共病模式。随着这类共病患者数量的不断增加,其复杂的共病机制日益受到关注。虽然已有研究揭示口腔菌群通过“口-肠-肝轴”影响CHB、NAFLD疾病进展[27-28]的作用,但对二者共存时的微生物组特征研究尚少。本研究通过对比CHB合并NAFLD、单纯CHB患者及健康人群的舌苔菌群,初步探索了共病状态下舌苔菌群的组成与功能变化,为理解共病机制提供了新的微生物组学视角。
本研究纳入的3组人群在年龄、性别和BMI上的差异,符合CHB与NAFLD各自的流行病学特点。在此基础上,本研究聚焦于CHB合并NAFLD患者的舌苔菌群特征,旨在探寻除年龄、性别、BMI等传统因素外,舌苔菌群是否能够提供具有鉴别意义的微生物标志物。在门水平上,CHB合并NAFLD组表现出Firmicutes相对丰度升高、Bacteroidota相对丰度降低的趋势,这一变化方向与既往研究中肥胖及NAFLD人群常见的“Firmicutes/Bacteroidota比例升高”的特征相符[29]。在属水平上,CHB合并NAFLD组与单纯CHB组均以Veillonella为优势菌,而健康对照组以Prevotella_7为主,提示HBV感染可能通过改变特定优势菌群的相对丰度来影响口腔微生态平衡。本研究结果显示,3组间的FPG、FFA及ALT水平存在显著差异(P<0.05),其中,CHB合并NAFLD组的上述指标均高于健康对照组,这一趋势与舌苔菌群中富集的糖脂代谢功能预测结果相呼应,提示舌苔菌群可能通过调节宿主代谢,参与NAFLD的发生发展。
通过LEfSe分析,发现CHB合并NAFLD组的舌苔菌群在物种组成上与其余2组呈现明显分化。在门水平上,Actinobacteriota在CHB合并NAFLD组中显著富集;在种水平上,Neisseria_elongata和Streptococcus_oralis为该组特有的标志物种。此外,与健康对照组相比,CHB合并NAFLD组的Actinomyces和Granulicatella等潜在致病相关菌属丰度升高,而Bacteroidota等有益菌群减少。这种以Actinobacteriota富集,并伴随Firmicutes相对丰度升高、Bacteroidota相对丰度降低为特征的菌群结构,可能构成了CHB合并NAFLD患者特有的口腔微生态失调模式,为其临床识别提供潜在的微生物标志。
KEGG与MetaCyc功能预测分析显示,3组舌苔菌群差异表达的功能主要富集于辅因子与维生素代谢、糖代谢、脂质代谢及氨基酸代谢等通路。CHB合并NAFLD组舌苔菌群中富集的Firmicutes(如Granulicatella)具有参与碳水化合物与脂质代谢的遗传潜能,可能促进葡萄糖醛酸内酯转化为三磷酸甘油酸,进而导致脂质沉积[30]。这提示舌苔菌群不仅结构改变,其代谢功能的偏移也可能通过产生特定的代谢产物,间接影响宿主代谢状态。
本研究发现的舌苔菌群组成与功能改变,为“口-肠-肝轴”理论提供了口腔端的证据。结合已有研究[20,31],本研究推测CHB合并NAFLD患者口腔中富集的特定菌群(如Actinobacteriota、Neisseria_elongata等)可能经由“口-肠-肝轴”加剧了CHB患者的肝脏代谢紊乱,从而参与共病的发生;然而,本研究未直接检测肠道菌群,该关联的完整性(即口腔菌群如何具体影响肠道生态并进而影响肝脏)尚待未来研究通过同步采集粪便样本进行纵向分析来进一步验证。
本研究尚存在若干局限性:①尽管本研究系统描绘了CHB合并NAFLD患者的舌苔菌群特征图谱,但样本来源于单中心且样本量有限,其结果的外推性需在更大规模、多中心的人群中进一步验证。②横断面研究设计无法推断舌苔菌群变化与共病之间的因果关系,其动态交互作用有待后续纵向研究或干预实验揭示。③本研究采用16S rRNA测序进行功能预测,其结论主要基于生物信息学推演,未来可通过宏基因组学等技术进行直接验证。④本研究聚焦于“口-肠-肝轴”的口腔端,虽通过功能预测提示了潜在的代谢影响,但未能同步分析肠道菌群等下游环节以直接证实该轴中的菌群传递与机制联动。正是这些局限,清晰地界定了本研究的贡献边界,并为未来研究指明了方向:扩大样本进行多中心验证,开展纵向队列研究明确因果时序,并整合多组学数据(尤其是肠道微生物组与宿主代谢组)以完整解析“口-肠-肝轴”在CHB合并NAFLD共病中的具体作用机制。
综上所述,本研究初步揭示了CHB合并NAFLD患者舌苔菌群的特征性改变,包括以Actinobacteriota富集、并伴有Firmicutes与Bacteroidota丰度反向变化为标志的生态失衡,以及相关菌群在糖脂代谢通路的功能富集。这些发现为理解CHB与NAFLD的共病机制提供了新的“口-肠-肝轴”微生物组学视角。在转化应用层面,本研究提示舌苔菌群特征有望发展成为识别CHB患者中NAFLD高风险人群的无创生物标志物。未来,探索通过调节口腔微生态以干预疾病进程,或可为CHB合并NAFLD的防治提供新思路。本研究结论仍需在更大规模、多中心的人群中得到验证。后续研究应通过纵向设计明确菌群变化与疾病进展的因果关系,并整合宏基因组、代谢组及肠道菌群等多维度数据,以期系统阐明“口-肠-肝轴”在共病发生发展中的完整作用机制,为临床诊疗提供更坚实的科学依据。

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doi: 10.16016/j.2097-0927.202510035
  • 接收时间:2025-10-15
  • 首发时间:2026-10-23
  • 出版时间:2026-01-30
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  • 收稿日期:2025-10-15
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    1河南中医药大学第一附属医院消化科,河南 郑州

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2种不同金属材料的力学参数
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属数
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genus
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species
占总种数比例
Percentage of
total species (%)
属
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种数
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species
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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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