By obtaining the genetic sequence of MOTS-C and analyzing its effects on mitochondrial dynamics and antioxidant capacity in porcine intestinal epithelial cells (IPEC-J2), its effects on cell proliferation and intestinal barrier were detected, so as to provide a new molecular target and theoretical basis for the use of MOTS-C to improve piglet intestinal health.

Overexpression vectors or exogenous MOTS-C peptides were used to treat IPEC-J2 cells, and their effects on cell proliferation, barrier function, mitochondrial dynamics, oxidative stress, and apoptosis were evaluated.

Pig MOTS-C was encoded by a highly conserved 21-amino acid peptide from the mitochondrial genome. In IPEC-J2 cells, MOTS-C overexpression significantly downregulated the mitochondrial dynamics-related genes mRNA levels of DRP1 (P<0.01) and Fis1 (P<0.05), while upregulating the fusion-promoting protein OPA1 (P<0.01) and the protein expression of MFN2 (P<0.01). MOTS-C reduced the production of ROS, increased the activities of CAT and T-SOD (P<0.01), lowered MDA content (P<0.01), and upregulated antioxidative genes, including superoxide SOD3 and CAT (P<0.05) at the mRNA level, as well as SOD2 and GCLM at the protein level (P<0.05). MOTS-C also enhanced the expression of tight junction-related genes, including Claudin and Occludin (P<0.05), and proliferation-associated genes, such as CDK2 and CCNDBP1 (P<0.05), and increased CCND1 protein expression (P<0.05). In addition, MOTS-C suppressed the mRNA expression of pro-apoptotic genes BAX and CASP3 (P<0.05), while elevating the protein level of the anti-apoptotic gene BCL2 (P<0.05). MOTS-C peptide significantly downregulated the mRNA levels of DRP1 (P<0.05) and FIS1 (P<0.01), while significantly upregulating OPA1 (P<0.05). Western blot results showed that the expression of MFN2 protein was increased (P>0.05). MOTS-C peptide also significantly increased CAT enzyme activity levels (P<0.05) and significantly reduced intracellular MDA content (P<0.05). Moreover, it significantly increased SOD3 and HO-1 mRNA levels (P<0.05). Further Western blot analysis indicated MOTS-C peptide significantly increased HO-1 protein level, and it consistent with the trend in mRNA levels (P<0.05). In addition, the MOTS-C peptide downregulated the mRNA expression levels of pro-apoptotic genes BAX (P > 0.05), and the BAX/BCL2 was significantly downregulated (P<0.05). Compared with the control group, MOTS-C peptide significantly reduced the mRNA and protein levels of BAX/BCL2 (P<0.05).

MOTS-C enhanced mitochondrial dynamics and antioxidative capacity in IPEC-J2 cells, promoted cell proliferation, strengthend intestinal barrier integrity, and reduced oxidative stress and apoptosis.