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

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

Abstract

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

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.

Figures/Tables 9

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

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

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

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

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

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

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

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

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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