中国组织工程研究

中国组织工程研究 ›› 2026, Vol. 30 ›› Issue (4): 1058-1072.doi: 10.12307/2025.933

• 组织构建循证医学 evidence-based medicine in tissue construction • 上一篇    

龟鹿二仙胶对膝骨关节炎大鼠肠道菌群的影响：机器学习与16S rDNA分析

顾富城1，2，杨美鑫1，2，吴伟欣1，2，蔡玮俊1，2，钦洋溢1，2，孙铭一1，2，孙  健1，2，耿秋东1，2，李  楠1，2   

  1. 福建中医药大学，1中医学院，2中医骨伤及运动康复教育部重点实验室，福建省福州市  350122
  • 收稿日期:2024-10-21 接受日期:2024-12-06 出版日期:2026-02-08 发布日期:2025-05-23
  • 通讯作者: 李楠，福建中医药大学中医学院副院长，博士生导师，福建中医药大学，中医学院，中医骨伤及运动康复教育部重点实验室，福建省福州市 350122
  • 作者简介:顾富城，男，1999年生，新疆维吾尔自治区石河子市人，汉族，福建中医药大学在读硕士，主要从事中医药防治骨关节病的基础与临床研究。
  • 基金资助:
    福建省省级财政专项(X2022001-财政专项；X2022002-财政专项)，项目负责人：李楠；福建省自然科学基金
    (2023J01865)，项目负责人：李楠；福建省自然科学基金(2022J01364)，项目负责人：耿秋东

Effects of Guilu Erxian Glue on gut microbiota in rats with knee osteoarthritis: machine learning and 16S rDNA analysis

Gu Fucheng1, 2, Yang Meixin1, 2, Wu Weixin1, 2, Cai Weijun1, 2, Qin Yangyi1, 2, Sun Mingyi1, 2, Sun Jian1, 2, Geng Qiudong1, 2, Li Nan1, 2   

  1. 1College of Traditional Chinese Medicine, Fujian University of Traditional Chinese Medicine, Fuzhou 350122, Fujian Province, China; 2Key Laboratory of Traditional Chinese Medicine Orthopedics and Sports Rehabilitation, Ministry of Education, Fujian University of Traditional Chinese Medicine, Fuzhou 350122, Fujian Province, China
  • Received:2024-10-21 Accepted:2024-12-06 Online:2026-02-08 Published:2025-05-23
  • Contact: Li Nan, Doctoral supervisor, College of Traditional Chinese Medicine, Fujian University of Traditional Chinese Medicine, Fuzhou 350122, Fujian Province, China; Key Laboratory of Traditional Chinese Medicine Orthopedics and Sports Rehabilitation, Ministry of Education, Fujian University of Traditional Chinese Medicine, Fuzhou 350122, Fujian Province, China
  • About author:Gu Fucheng, Master candidate, College of Traditional Chinese Medicine, Fujian University of Traditional Chinese Medicine, Fuzhou 350122, Fujian Province, China; Key Laboratory of Traditional Chinese Medicine Orthopedics and Sports Rehabilitation, Ministry of Education, Fujian University of Traditional Chinese Medicine, Fuzhou 350122, Fujian Province, China
  • Supported by:
     Fujian Provincial Financial Special Project, No. X2022001, X2022002 (to LN); Natural Science Foundation of Fujian Province of China, No. 2023J01865 (to LN); Natural Science Foundation of Fujian Province of China, No. 2022J01364 (to GQD)

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摘要：


文题释义：
高效液相色谱法：是以液体为流动相，采用高压输液系统，将具有不同极性的单一溶剂或不同比例的混合溶剂、缓冲液等流动相泵入装有固定相的色谱柱，在柱内各成分被分离后，进入检测器进行检测，从而实现对试样的分析。
16SrDNA检测：利用细菌16SrDNA序列测序的方法对细菌进行种属鉴定，包括细菌基因组DNA提取、16SrDNA特异引物PCR扩增、扩增产物纯化、DNA测序及序列比对等步骤，是能够对细菌进行种属鉴定快速获得细菌种属信息的方法。

背景：龟鹿二仙胶由龟甲胶、鹿角胶、枸杞子和人参组成。在早期的临床观察中，发现使用龟鹿二仙胶治疗肝肾不足型膝骨关节炎患者能有效缓解膝关节疼痛、增加运动范围、提高行走能力。然而，口服龟鹿二仙胶能够对膝关节产生局部治疗作用的确切机制尚不清楚。
目的：研究龟鹿二仙胶对膝骨关节炎大鼠肠道菌群的影响，并利用16SrDNA测序和机器学习分析评估其作用机制。
方法：将18只雄性SD大鼠随机分为3组：空白组、模型组、龟鹿二仙胶组，每组6只。模型组和龟鹿二仙胶组采用内侧半月板失稳手术制备膝骨关节炎模型。造模成功后，龟鹿二仙胶组灌胃龟鹿二仙胶水煎剂，空白组和模型组则给予等量的蒸馏水。持续干预28 d后，使用高效液相色谱技术检测龟鹿二仙胶有效成分；MRI成像观察大鼠膝关节软骨情况采集各组大鼠粪便样本，使用试剂盒提取DNA，通过PCR扩增及纯化，构建Illumina测序文库，利用Illumina MiSeq平台进行高通量测序，生成原始序列数据。得到原始数据后，使用QIIME2软件对数据进行处理，使用线性判别分析效应值和随机森林算法对微生物数据进行差异性物种筛选，使用KEGG和MetaCyc功能通路分析探索关键菌群与实验组间的关联，使用线性判别分析效应值和随机森林算法筛选差异性物种，使用关联网络分析微生物群落间的相互作用，结合机器学习方法分析肠道菌群组成和变化。
结果与结论：①龟鹿二仙胶中药复方进行LC-MS成分鉴定，共指出7种有效成分。②MRI成像显示，龟鹿二仙胶组大鼠高密度影的滑膜炎症范围减少，且股骨头内侧髁软骨较模型组退化较少。③16SrDNA测序显示模型组大鼠表现出明显的菌群失衡，在门水平上厚壁菌门和拟杆菌门的丰度显著降低，而变形菌门的比例显著增加(P < 0.05)。龟鹿二仙胶组大鼠的肠道菌群结构有了显著改善，其厚壁菌门和拟杆菌门的比例回升，恢复了更为多样的菌群组成，接近空白组(P < 0.05)。④KEGG和MetaCyc功能通路分析显示，龟鹿二仙胶组显著激活了多条代谢通路，包括氨基酸代谢、脂质代谢和生物素合成通路(P < 0.05)。⑤上述结果提示，龟鹿二仙胶含有7种有效成分，并利用16SrDNA测序分析膝骨关节炎大鼠肠道菌群变化；龟鹿二仙胶能显著改善肠道菌群失衡，恢复有益菌丰度，对肠道菌群组成有显著影响，为龟鹿二仙胶的药效及机制提供了科学依据。
https://orcid.org/0009-0005-9197-1084(顾富城)；https://orcid.org/0009-0008-0863-0783(李楠)

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

关键词: 龟鹿二仙胶, 膝骨关节炎, 肠道菌群, 16SrDNA测序, 机器学习, α多样性, β多样性, 差异通路

Abstract: BACKGROUND: The Guilu Erxian Glue consists of Testudinis Plastrum, Cornu Cervi, Lycii Fructus, and Ginseng Radix. In earlier clinical observations, it is discovered that using Guilu Erxian Glue to treat patients with liver-kidney deficiency type knee osteoarthritis effectively alleviated knee pain, increased the range of motion, and improved walking ability. However, the exact mechanism by which oral administration of Guilu Erxian Glue can produce local therapeutic effects on the knee joint is still unclear.  
OBJECTIVE: To investigate the effects of Guilu Erxian Glue on gut microbiota in rats with knee osteoarthritis and to evaluate its mechanism using 16S rDNA sequencing and machine learning analysis. 
METHODS: Totally 18 female SD rats were randomly divided into three groups: blank group, model group, and Guilu Erxian Glue group, with 6 rats in each group. A knee osteoarthritis model was prepared using the destabilization of the medial meniscus surgical method. After successful modeling, the Guilu Erxian Glue group was given a decoction of Guilu Erxian Glue by gavage, while the blank and model groups were given an equal amount of distilled water. After 28 days of continuous intervention, high performance liquid chromatography was used to detect the active ingredients of Guilu Erxian Glue. MRI imaging was used to observe the condition of rat knee articular cartilage. Fecal samples were collected; DNA was extracted using a kit, amplified and purified by PCR, and an Illumina sequencing library was constructed. The Illumina MiSeq platform was used for high-throughput sequencing to generate raw sequence data. After obtaining the raw data, QIIME2 software was used to process the data. Linear Discriminant Analysis Effect Size analysis and random forest algorithm were used to screen for differential species in microbial data. KEGG and MetaCyc functional pathway analyses were used to explore the association between key microbial communities and experimental groups. Linear discriminant analysis effect values and random forest algorithm were used to screen for differential species. Association networks were used to analyze the interactions between microbial communities, and machine learning methods were used to analyze the composition and changes of gut microbiota.
RESULTS AND CONCLUSION: (1) LC-MS component identification was conducted on the traditional Chinese medicine formula of Guilu Erxian Glue, and a total of 7 effective ingredients were identified. (2) MRI imaging showed that synovitis scope of high-density shadows in rats of the Guilu Erxian Glue group was reduced, and the degeneration of medial femoral condyle cartilage was less than that in the model group. (3) 16S rDNA sequencing showed that the model group rats exhibited significant microbial imbalance, with a significant decrease in the abundance of Firmicutes and Bacteroidetes at the phylum level, while the proportion of Proteobacteria increased significantly (P < 0.05). The gut microbiota structure of rats in the Guilu Erxian Glue group was significantly improved, and the proportion of Firmicutes and Bacteroidetes increased, restoring a more diverse microbiota composition, approaching that of the blank group (P < 0.05). (4) KEGG and MetaCyc functional pathway analysis showed that the Guilu Erxian Glue group significantly activated multiple metabolic pathways, including amino acid metabolism, lipid metabolism, and biotin synthesis pathways (P < 0.05). (5) The results indicate that Guilu Erxian Glue contains seven active ingredients, and the changes in gut microbiota of knee osteoarthritis rats were analyzed using 16S rDNA sequencing. Guilu Erxian Glue can significantly improve the imbalance of gut microbiota, restore the abundance of beneficial bacteria, and have a significant impact on the composition of gut microbiota, providing scientific basis for the efficacy and mechanism of Guilu Erxian Glue. 

Key words: Guilu Erxian Glue, knee osteoarthritis, gut microbiota, 16S rDNA sequencing, machine learning, alpha diversity, beta diversity, differential pathway

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