丁酸梭菌通过调节结肠微生物结构和短链脂肪酸含量影响小鼠肌纤维类型

doi:10.3969/j.issn.1004-1524.20240197

浙江农业学报 ›› 2025, Vol. 37 ›› Issue (3): 568-578.DOI: 10.3969/j.issn.1004-1524.20240197

丁酸梭菌通过调节结肠微生物结构和短链脂肪酸含量影响小鼠肌纤维类型

吴兴凤¹^,²(), 肖英平², 吕文涛², 马灵燕², 陈渠², 温洋², 徐娥¹^,^*()

1.贵州大学动物科学学院,高原山地动物遗传育种与繁殖教育部重点实验室,贵州贵阳 550025
2.浙江省农业科学院农产品质量安全与营养研究所,省部共建农产品质量安全与危害因子防控国家重点实验室,浙江杭州 310021

收稿日期:2024-02-29 出版日期:2025-03-25 发布日期:2025-04-02
作者简介:吴兴凤(1998—),女,布依族,贵州都匀人,硕士研究生,研究方向为动物营养与饲料科学。E-mail:wxf18826@163.com
通讯作者: * 徐娥,E-mail:exu@gzu.edu.cn
基金资助:
国家自然科学基金(32160798)

Clostridium butyricum affects muscle fiber type in mice by modulating colonic microbial structure and short-chain fatty acids content

WU Xingfeng¹^,²(), XIAO Yingping², LYU Wentao², MA Lingyan², CHEN Qu², WEN Yang², XU E¹^,^*()

1. Key Laboratory of Genetics, Breeding and Reproduction of Plateau Mountain Animals, Ministry of Education, School of Animal Science, Guizhou University, Guiyang 550025, China
2. State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-Products, Institute of Agro-Product Safety and Nutrition, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China

Received:2024-02-29 Online:2025-03-25 Published:2025-04-02

摘要/Abstract

摘要：

为探究丁酸梭菌对小鼠肌纤维类型、结肠微生物结构和短链脂肪酸含量的影响,选取14只C57BL/6J雄性小鼠分为CB组和NC组,每组7只,CB组小鼠连续8周灌胃200 μL丁酸梭菌悬液(10⁸ CFU·mL^-1),NC组小鼠则给予等量生理盐水。分别采用实时荧光定量PCR、16S rRNA基因测序和气相色谱法测定小鼠腓肠肌肌纤维类型基因表达、肠道菌群和短链脂肪酸含量的变化。结果显示,NC组和CB组小鼠的肌纤维直径差异不显著,但CB组腓肠肌肌纤维的横截面面积较NC组显著(P<0.05)增加。CB组小鼠腓肠肌MyHC I和MyHC IIa的mRNA水平较NC组显著升高,MyHC IIb的mRNA水平显著降低。相较于NC组,CB组小鼠肠道中拟杆菌门(Bacteroidota)和变形菌门(Proteobacteria)的丰度显著升高,厚壁菌门(Firmicutes)的丰度显著降低;罗氏菌属(Roseburia)、拟杆菌属(Bacteroides)、副拟杆菌属(Parabacteroides)的丰度显著升高。CB组小鼠肠道菌群间的互作关系比NC组更为密切。CB组小鼠肠道菌群的柠檬酸循环、泛酸和辅酶A生物合成、脂肪酸生物合成、脂肪酸代谢等代谢通路较NC组被显著激活。CB组小鼠结肠中的总短链脂肪酸、乙酸和丁酸含量显著高于NC组。综上,丁酸梭菌可能通过调节肠道菌群和短链脂肪酸影响小鼠肌纤维类型相关基因的表达,促使肌纤维类型发生转化。

关键词: 丁酸梭菌, 肌纤维类型, 肠道菌群, 短链脂肪酸

Abstract:

In order to investigate the effects of Clostridium butyricum on muscle fiber types, colonic microbial structure and short-chain fatty acids content in mice, fourteen C57BL/6J male mice were selected and divided into CB and NC groups randomly with 7 mice in each group. The mice in CB group were treated by oral gavage with 200 μL C. butyricum suspension (10⁸ CFU·mL^-1) for 8 weeks, while the mice in the NC group were given an equal amount of saline. Real-time fluorescence quantitative PCR (RT-qPCR), 16S rRNA gene sequencing and gas chromatography were used to determine the changes of muscle fiber type gene expression, intestinal microflora structure and short-chain fatty acids content in gastrocnemius muscle of mice. It was shown that the difference in muscle fiber diameter between the NC and CB groups was not significant, yet the cross-sectional area of gastrocnemius muscle fibers was significantly (P<0.05) increased in the CB group than that in the NC group. Compared with the NC group, mRNA levels of MyHC I and MyHC IIa were significantly increased in the CB group, yet the mRNA level of MyHC IIb was significantly decreased. The abundance of Bacteroidota and Proteobacteria in the intestine of the CB group was significantly increased than that of the NC group, yet the abundance of Firmicutes in the CB group was significantly decreased. The abundance of Roseburia, Bacteroides, Parabacteroides were significantly increased in the CB group than that of the NC group. The interactions between the intestinal microflora of mice in the CB group were more closely related than those in the NC group. Metabolic pathways such as citrate cycle, pantothenate and coenzyme A (CoA) biosynthesis, fatty acid biosynthesis, and fatty acid metabolism were significantly activated in the intestinal microflora of the CB group as compared with the NC group. The contents of total short-chain fatty acids, acetic acid and butyric acid in the colon of CB group were significantly higher than those in the NC group. In conclusion, Clostridium butyricum treatment affected the expression of genes related to muscle fiber types in mice by regulating the intestinal microflora and short-chain fatty acids produced by the bacteria, hence promoted the transformation of muscle fiber types.

Key words: Clostridium butyricum, muscle fiber type, intestinal microflora, short-chain fatty acid

中图分类号:

S852.2

吴兴凤, 肖英平, 吕文涛, 马灵燕, 陈渠, 温洋, 徐娥. 丁酸梭菌通过调节结肠微生物结构和短链脂肪酸含量影响小鼠肌纤维类型[J]. 浙江农业学报, 2025, 37(3): 568-578.

WU Xingfeng, XIAO Yingping, LYU Wentao, MA Lingyan, CHEN Qu, WEN Yang, XU E. Clostridium butyricum affects muscle fiber type in mice by modulating colonic microbial structure and short-chain fatty acids content[J]. Acta Agriculturae Zhejiangensis, 2025, 37(3): 568-578.

图/表 9

表1 用于实时荧光定量PCR的引物序列

Table 1 Primers sequences used for real-time fluorescent quantitative PCR

基因名Gene name	正向引物序列Sequence of forward primer	反向引物序列Sequence of reverse primer
MyHC I	5'-ATATACAGCCCCTGAGACCAG-3'	5'-GAACCTGGAGCCCCTTATCC-3'
MyHC IIa	5'-GAACATGGAGCAGACCGTGA-3'	5'-GCGCATGACCAAAGGTTTCA-3'
MyHC IIx	5'-GAAGTTGCATCCCTAAAGGCAG-3'	5'-CCCCTGCATTTTGCCAGAAG-3'
MyHC IIb	5'-TCACCTACCAGACCGAGGAG-3'	5'-TCTCCTGTCACCTCTCAACAGA-3'
GAPDH	5'-TTCACCACCATGGAGAAGGC-3'	5'-TGAAGTCGCAGGAGACAACC-3'

表2 小鼠体重和腓肠肌质量

Table 2 Body weight and gastrocnemius muscle weight of mice

处理 Treatment	初始体重 Initial body weight/g	结束体重 Final body weight/g	腓肠肌质量 Gastrocnemius muscle weight/g	腓肠肌质量分数 Mass fraction of gastrocnemius muscle/%
NC	22.01	25.25	0.36	1.42
CB	21.94	24.99	0.37	1.49

图1 小鼠腓肠肌的HE染色切片(A)与肌纤维直径(B)和横截面面积(C) “*”表示差异显著(P<0.05)。下同。

Fig.1 HE staining sections (A), fiber diameter (B) and cross-sectional area (c) of gastrocnemius muscle in mice “*” indicates significant difference at P<0.05. The same as below.

图2 腓肠肌中肌球蛋白重链基因mRNA表达水平

Fig.2 The mRNA expression level of myosin heavy chain gene in gastrocnemius muscle

表3 肠道菌群的α多样性

Table 3 Alpha diversity of intestinal microflora

处理 Treatment	ASV数量 ASV number	香农指数 Shannon index	辛普森指数 Simpson index	Chao1指数 Chao1 index
NC	430±20	4.29±0.11	0.04±0.01	431.0±19.7
CB	432±23	4.47±0.05	0.03±0.01	433.3±22.2

图3 小鼠肠道菌群的主坐标分析(A)和结构组成(B) Firmicutes,厚壁菌门;Bacteroidetes,拟杆菌门;Proteobacteria,变形菌门;Deferribacteres,脱铁杆菌门;Cyanobacteria,蓝藻门;Others,其他。Bacteroidales S24-7 group_norank,拟杆菌门S24-7群未分类属;Erysipelotrichaceae_uncultured,丹毒丝菌科未纯培养菌株;Lactobacillus,乳杆菌属;Prevotellaceae UCG-001,普雷沃氏菌科UCG-001;Lachnospiraceae_uncultured,毛螺菌科未纯化菌株;Lachnospiraceae NK4A136 group,毛螺菌科NK4A136群;Erysipelotrichaceae_unclassified,丹毒丝菌科未知分类属;Parabacteroides,副拟杆菌属;Roseburia,罗氏菌属;Bacteroides,拟杆菌属。图4同。

Fig.3 Principal coordinate analysis (A) and structural composition (B) of intestinal microflora in mice

图4 小鼠肠道菌群的线性判别分析(A)、功能预测(B)与网络互作分析(C) Faecalibaculum,肠杆菌属;Turicibacter,苏黎世杆菌属;Coriobacteriaceae UCG_002,红蝽菌科UCG_002;Adlercreutzia,阿德勒克伊茨菌属;Paraprevotella,普雷沃氏菌属;Acetitomaculum,聚乙酸菌属;Rhodospirillaceae_uncultured,红螺菌科未纯培养菌株;Anaerotruncus,厌氧棍状菌属;Clostridiales vadinBB60 group_norank,梭菌目vadinBB60群未知分类;Oscillibacter,颤杆菌克属;Rikenellaceae RC9 gut group,理研菌科RC9群;Streptococcus,链球菌属。网络图中节点的大小与肠道菌群丰度比例呈正相关;菌群间的连接线越多,表示菌群间的联系关系越密切。

Fig.4 Linear discriminant analysis effect size (LEfSe) analysis (A), functional prediction (B) and network interaction analysis (C) of mouse intestinal microflora The size of the nodes in the figure is positively correlated with the proportion of intestinal microflora abundance. The more the connection lines between the microflora, the closer the relationship between the microflora.

表4 小鼠结肠的短链脂肪酸含量

Table 4 Short chain fatty acids content in colon of mice mg·g-1

处理 Treatment	乙酸含量 Acetic acid content	丙酸含量 Propionic acid content	异丁酸含量 Isobutyric acid content	丁酸含量 Butyric acid content	异戊酸含量 Isovaleric acid content	戊酸含量 Valeric acid content	总短链脂肪酸含量 Total short chain fatty acids content
NC	2.23±0.32	0.41±0.17	0.08±0.07	0.62±0.11	0.05±0.01	0.07±0.01	3.47±0.49
CB	3.35±0.35^*	0.56±0.24	0.46±0.19	1.15±0.14^*	0.11±0.03^*	0.14±0.03^*	5.77±0.60^*

图5 小鼠肠道差异菌属、SCFA含量和肌纤维类型相关基因的关联分析 “*”和“**”分别表示显著(P<0.05)与极显著(P<0.01)相关。

Fig.5 Correlation analysis within intestinal differential microflora, SCFA content and muscle fiber types of mice “*” and “**”indicate significant correlation at P<0.05 and P<0.01 level, respectively.

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丁酸梭菌通过调节结肠微生物结构和短链脂肪酸含量影响小鼠肌纤维类型

Clostridium butyricum affects muscle fiber type in mice by modulating colonic microbial structure and short-chain fatty acids content

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