共生微生物对仔猪肠道发育、代谢和线粒体功能的影响

doi:10.13560/j.cnki.biotech.bull.1985.2023-0492

生物技术通报 ›› 2024, Vol. 40 ›› Issue (1): 332-343.doi: 10.13560/j.cnki.biotech.bull.1985.2023-0492

共生微生物对仔猪肠道发育、代谢和线粒体功能的影响

张进威¹^,²^,³(), 吴元霞¹^,⁴, 孙静¹^,²^,³, 李晓开¹^,³^,⁵, 陆路⁵, 李周权⁴, 葛良鹏¹^,²^,³()

1.重庆市畜牧科学院，重庆402460
2.农业农村部养猪科学重点实验室养猪科学重庆市重点实验室，重庆402460
3.国家生猪技术创新中心，重庆 402460
4.西南大学动物科学技术学院生物饲料与分子营养实验室，重庆400715
5.四川农业大学动物科技学院，成都611130

收稿日期:2023-05-21 出版日期:2024-01-26 发布日期:2024-02-06
通讯作者: 葛良鹏，男，博士，研究员，研究方向：实验猪资源创新开发与利用；E-mail: geliangpeng1982@163.com
作者简介:张进威，男，博士，助理研究员，研究方向：SPF猪培育与利用；E-mail: jinweizhang50@163.com
基金资助:
国家自然科学基金项目(32072687);国家自然科学基金项目(32302712);国家生猪技术创新中心先导科技项目(NCTIP-XD/B13);重庆市自然科学基金面上项目(cstc2021jcyj-msxmX0630);四川省科技厅区域创新合作项目(2022YFQ0022)

Effects of Commensal Microbiota on Intestinal Development, Metabolism, and Mitochondrial Function in Piglets

ZHANG Jin-wei¹^,²^,³(), WU Yuan-xia¹^,⁴, SUN Jing¹^,²^,³, LI Xiao-kai¹^,³^,⁵, LU Lu⁵, LI Zhou-quan⁴, GE Liang-peng¹^,²^,³()

1. Chongqing Academy of Animal Sciences, Chongqing 402460
2. Ministry of Agriculture Key Laboratory of Pig Sciences, Chongqing Key Laboratory of Pig Sciences, Chongqing 402460
3. National Center of Technology Innovation for Pigs, Chongqing 402460
4. Key Laboratory for Bio-Feed and Molecular Nutrition, College of Animal Science and Technology, Southwest University, Chongqing 400715
5. College of Animal Science and Technology, Sichuan Agricultural University, Chengdu 611130

Received:2023-05-21 Published:2024-01-26 Online:2024-02-06

摘要/Abstract

摘要：

【目的】以猪为动物模型研究共生微生物对宿主肠道发育代谢的调控作用。【方法】经无菌剖腹产、无菌饲养等技术培育无菌（germ-free, GF）仔猪和无特定病原（specific pathogen-free, SPF）仔猪，通过形态学观察、液相色谱分析、RNA-seq等方法研究共生微生物对仔猪肠道形态、代谢、基因表达及线粒体功能的影响。【结果】共生微生物对仔猪肠道的形态结构、短链脂肪酸含量、氨基酸代谢和肠道细胞线粒体含量等方面产生不同程度的影响；共生微生物影响仔猪肝脏、回肠和结肠组织的整体基因表达，调控线粒体氧化磷酸化、氨基酸代谢、脂质代谢等生物学过程相关基因表达，导致线粒体功能发生改变，从而影响仔猪肠道营养物质的吸收与代谢。【结论】共生微生物通过调控肠道细胞线粒体从而影响仔猪肠道发育和代谢，为“微生物-宿主互作”调控仔猪肠道健康相关研究奠定理论基础。

关键词: 无菌猪, 无特定病原猪, 共生微生物, 能量代谢, 线粒体功能

Abstract:

【Objective】Using pig as animal model to study the regulatory effects of commensal microbiota on host intestinal development and metabolism.【Method】Germ free (GF) piglets and specific pathogen free (SPF) piglets were cultivated through techniques such as aseptic cesarean section and aseptic feeding, and used to study the effects of commensal microbiota on intestinal morphology, metabolism, gene expression and mitochondrial function of piglets through morphological observation, liquid chromatography analysis, RNA seq, and other methods.【Result】Commensal microbiota affected the intestinal morphology and structure, short-chain fatty acid content, amino acid metabolism and mitochondrial content of intestinal cells. Commensal microbiota affected the overall gene expression of the liver, ileum and colon of piglets, and regulated the expression of genes related to biological processes such as mitochondrial oxidation and phosphorylation, amino acid metabolism and lipid metabolism, resulting in the changes in mitochondrial function, thus affected the absorption and metabolism of intestinal nutrients in piglets. 【Conclusion】Commensal microbiota affected the development and metabolism of piglet intestines by regulating the mitochondria of intestinal cells, which provided a theoretical foundation for the study on the regulation of "microbe-host interaction" in piglet’s intestinal health.

Key words: germ-free piglets, specific pathogen-free piglets, commensal microbiota, energy metabolism, mitochondrial function

张进威, 吴元霞, 孙静, 李晓开, 陆路, 李周权, 葛良鹏. 共生微生物对仔猪肠道发育、代谢和线粒体功能的影响[J]. 生物技术通报, 2024, 40(1): 332-343.

ZHANG Jin-wei, WU Yuan-xia, SUN Jing, LI Xiao-kai, LU Lu, LI Zhou-quan, GE Liang-peng. Effects of Commensal Microbiota on Intestinal Development, Metabolism, and Mitochondrial Function in Piglets[J]. Biotechnology Bulletin, 2024, 40(1): 332-343.

图/表 9

表1 RT-qPCR引物序列

Table 1 Primer sequences used for RT-qPCR

目的基因 Target gene	引物序列Primer sequence（5'-3'）	登录号Accession No.	产物长度Product length/bp
p53	Forward: ATTTCACCCTCCAGATCCGTG	NM_213824.3	153
p53	Reverse: AGGGAGACTGCCCCTTCTTA	NM_213824.3	153
PIG3	Forward: GCGGACTTACTCCAGAGACAA	XM_003125363.6	194
PIG3	Reverse: AGGAACCCTTCAGGGACAGT	XM_003125363.6	194
Cyt C	Forward: GCCAACAAGAACAAAGGCATC	NM_001129970.1	113
Cyt C	Reverse: CTCCCTTCTTCTTAATGCCAGC	NM_001129970.1	113
Apaf-1	Forward: TACCCTGTTGGCGACTGGAGATG	XM_021093026.1	87
Apaf-1	Reverse: ACTGGAGCACACGAATGAAGAAGC	XM_021093026.1	87
NRF1	Forward: GCCAGTGAGATGAAGAGAAACG	AK393002.1	166
NRF1	Reverse: CTACAGCAGGGACCAAAGTTCAC	AK393002.1	166
TFAM	Forward: GCTCTCCGTTCAGTTTTGCG	NM_001130211.1	187
TFAM	Reverse: GGAAGTTCCCTCCACAGCTC	NM_001130211.1	187
PGC-1α	Forward: TGGACTGACATCGAGTGTGCT	NM_213963.2	127
PGC-1α	Reverse: TGAGTCCACCCAGAAAGCTG	NM_213963.2	127
HO-1	Forward: TCCTGCTCAACATTCAGCTGTT	NM_001004027.1	135
HO-1	Reverse: TTGTCACGGGAGTGGAGTCT	NM_001004027.1	135
CPT-1a	Forward: CAAGATGGGCATGAACGCTG	NM_001129805.1	145
CPT-1a	Reverse: TGGAATGTTGGGGTTGGTGT	NM_001129805.1	145
ACC1	Forward: CTGGAGGTGTATGTGCGAAG	XM_021066238.1	177
ACC1	Reverse: GTGGTTGAGGTTGGAGGAGA	XM_021066238.1	177
GAPDH	Forward: ACATGGCCTCCAAGGAGTAAGA	NM_001206359.1	106
GAPDH	Reverse: GATCGAGTTGGGGCTGTGACT	NM_001206359.1	106

图1 共生微生物对肠道绒毛高度、隐窝深度及黏膜层厚度的影响 A：回肠绒毛高度；B：回肠隐窝深度；C：回肠黏膜层厚度；D：结肠绒毛高度；E：结肠隐窝深度；F：结肠黏膜层厚度。*表示P < 0.05，**表示P < 0.01，***表示P < 0.001。下同

Fig. 1 Effects of commensal microbiota on intestinal villus height, crypt depth, and mucosal thickness A: Ileal villus height; B: ileal crypt depth; C: ileal mucosal thickness; D: colonic villus height; E: colonic crypt depth; F: colonic mucosal thickness. * indicates P < 0.05, ** P < 0.01, and *** P < 0.001. The same below

图2 共生微生物对仔猪短链脂肪酸含量的影响 A：回肠短链脂肪酸含量；B：结肠短链脂肪酸含量；C：肝脏短链脂肪酸含量；D：血清短链脂肪酸含量。Acetic acid：乙酸；Propionic acid：丙酸；Isobutyric acid：异丁酸；Butyric acid：丁酸；Pentanoic acid：戊酸；Isopentoic acid：异戊酸；Caproic acid：己酸

Fig. 2 Effects of commensal microbiota on the contents of short-chain fatty acids in piglets A: Ileum short-chain fatty acid content; B: colonic short-chain fatty acid content; C: liver short-chain fatty acid content; D: serum short-chain fatty acid content

图3 共生微生物对仔猪氨基酸含量的影响 A-B:回肠氨基酸含量；C-D：结肠氨基酸含量；E-F：肝脏氨基酸含量；G-F：血清氨基酸含量。Ala：丙氨酸；Arg：精氨酸；Asn：天冬酰胺；Asp：天冬氨酸；Cys：半胱氨酸；Gln：谷氨酰胺；Glu：谷氨酸；Gly：甘氨酸；His：组氨酸；Ile：异亮氨酸；Leu：亮氨酸；Lys：赖氨酸；Met：甲硫氨酸（蛋氨酸）；Phe：苯丙氨酸；Pro：脯氨酸；Ser：丝氨酸；Thr：苏氨酸；Trp：色氨酸；Tyr：酪氨酸；Val：缬氨酸

Fig. 3 Effects of commensal microbiota on the amino acid contents in piglets A-B: Ileum amino acids content; C-D: colonic amino acids content; E-F: liver amino acids content; G-F: serum amino acids content. Ala: alanine; Arg: arginine; Asn: asparagine; Asp: asparticacid; Cys: cysteine; Gln: glutamine; Glu: glutamicacid; Gly: glycine; His: histidine; Ile: isoleucine; Leu: leucine; Lys: lysine; Met: methionine; Phe: phenylalanine; Pro: proline; Ser: serine; Thr: threonine; Trp: tryptophan; Tyr: tyrosine; Val: valine

图4 共生微生物对仔猪回肠和结肠线粒体含量的影响左边图为线粒体免疫组化染色图片。A：回肠线粒体含量；B：结肠线粒体含量

Fig. 4 Effects of commensal microbiota on the mitochondrial contents in the ileum and colon of piglets Left picture shows mitochondria immunohistochemical staining pictures. A: Ileal mitochondrial content; B: colonic mitochondrial content

图5 共生微生物对回肠、结肠和肝脏基因表达量的影响 A：基因表达相关性分析；B：基于所有样本中mRNA整体表达的聚类分析；C：肝脏、回肠和结肠中共有和特有的差异mRNA；D-G：共生微生物对肝脏、回肠和结肠基因表达的影响

Fig. 5 Effects of commensal microbiota on the gene expressions in ileum, colon, and liver A: Gene expression correlation analysis. B: Cluster analysis based on overall gene expression in all samples. C: The common and unique differential mRNA in the liver, ileum, and colon. D-G: Commensal microbiota induced the differential gene expression in liver, ileum, and colon, respectively

图6 回肠、结肠和肝脏差异基因功能富集分析

Fig. 6 Results of functional enrichment analysis of differential genes in the ileum, colon, and liver

图7 共生微生物对线粒体功能相关基因表达的影响红色为上调，蓝色为下调，★表示P < 0.05

Fig. 7 Effect of commensal microbiota on mitochondrial function-related gene expression Red indicates upregulation, blue indicates downregulation, and ★ indicates P<0.05

图8 共生微生物对线粒体功能相关基因表达的影响 A-C：肝脏脂质代谢、细胞凋亡和氧化应激过程相关基因表达量；D-F：回肠脂质代谢、细胞凋亡和氧化应激过程相关基因表达量；G-I：结肠脂质代谢、细胞凋亡和氧化应激过程相关基因表达量

Fig. 8 Effects of commensal microbiota on the expressions of mitochondrial function-related genes A-C: The expressions of genes related to lipid metabolism, apoptosis, and oxidative stress in liver. D-F: The expression of genes related to lipid metabolism, apoptosis, and oxidative stress in ileal. G-I: The expression of genes related to lipid metabolism, apoptosis and oxidative stress in colon

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共生微生物对仔猪肠道发育、代谢和线粒体功能的影响

Effects of Commensal Microbiota on Intestinal Development, Metabolism, and Mitochondrial Function in Piglets

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