MOTS-C通过调控线粒体动力学与氧化应激抑制IPEC-J2细胞凋亡并增强能量代谢

doi:10.3864/j.issn.0578-1752.2026.14.016

摘要/Abstract

摘要：

【目的】通过获取猪MOTS-C基因序列并分析其在猪小肠上皮细胞（IPEC-J2）中对线粒体动力学与抗氧化能力影响，检测其对细胞增殖及肠道屏障的作用，为利用MOTS-C改善仔猪肠道健康提供了新的分子靶点和理论依据。【方法】扩增并系统分析猪MOTS-C基因，构建过表达载体或外源性MOTS-C肽处理IPEC-J2细胞，评估其对细胞增殖、屏障、线粒体动力学、氧化应激及凋亡的影响。【结果】猪MOTS-C由线粒体基因组基因编码高度保守的含21个氨基酸的肽。在IPEC-J2细胞中，MOTS-C过表达显著下调了线粒体动力学相关基因DRP1（P<0.01）和Fis1（P<0.05）的mRNA水平，上调了OPA1的mRNA水平（P<0.01）和MFN2的蛋白水平（P<0.01）。过表达MOTS-C降低了ROS的产生，提高了CAT和T-SOD的活性（P<0.01），降低了MDA含量（P<0.01），并上调抗氧化相关基因，包括SOD3和CAT的mRNA水平（P<0.05）以及SOD2和GCLM的蛋白表达（P<0.05）。MOTS-C促进紧密连接相关基因Claudin和Occludin以及增殖相关基因CDK2、CCNDBP1的mRNA水平（P<0.05），提高细胞周期蛋白CCND1的蛋白表达（P<0.05）。此外，MOTS-C抑制促凋亡基因BAX和CASP3的mRNA水平（P<0.05），上调抗凋亡基因BCL2的蛋白水平（P<0.05）。外源MOTS-C肽处理显著下调DRP1（P<0.05）以及FIS1（P<0.01）的mRNA水平，同时显著上调OPA1的mRNA水平（P<0.05）。Western blot 结果显示MFN2蛋白呈上升趋势，但差异不显著。外源MOTS-C肽处理显著提高CAT酶活性水平（P<0.05），显著降低MDA含量（P<0.05），且显著上调SOD3和HO-1的mRNA表达水平（P<0.05），Western blot分析结果进一步表明，MOTS-C肽处理后，HO-1的mRNA与蛋白均显著升高（P<0.05）。与对照组相比，MOTS-C肽显著降低BAX/BCL2的mRNA（P<0.05）和蛋白水平（P<0.05）。【结论】MOTS-C增强IPEC-J2细胞线粒体动力学和抗氧化能力，促进细胞增殖，增强肠屏障完整性，减少氧化应激和细胞凋亡。

关键词: MOTS-C, 细胞凋亡, 氧化应激, 线粒体动力学, IPEC-J2

Abstract:

【Objective】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.【Method】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.【Result】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).【Conclusion】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.

Key words: MOTS-C, apoptosis, oxidative stress, mitochondrial dynamics, IPEC-J2

霍乐乐, 田婉茹, 王彬彬, 曹涵洋, 李梦轩, 刘峻莹, 罗刚, 沈曼曼, 孙刘妹, 刘吉英. MOTS-C通过调控线粒体动力学与氧化应激抑制IPEC-J2细胞凋亡并增强能量代谢[J]. 中国农业科学, 2026, 59(14): 3220-3236.

HUO LeLe, TIAN WanRu, WANG BinBin, CAO HanYang, LI MengXuan, LIU JunYing, LUO Gang, SHEN ManMan, SUN LiuMei, LIU JiYing. MOTS-C Regulates Mitochondrial Dynamics and Oxidative Stress to Inhibit Apoptosis and Enhance Energy Metabolism in IPEC-J2 Cells[J]. Scientia Agricultura Sinica, 2026, 59(14): 3220-3236.

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基因 Gene	引物序列 Primer sequence	引物长度 Primer length	产物长度 Product length
MOTS-C （载体构建 Vector construction）	F:GATCCATGGGTTGGAAAGAAATGGGCTACA TTTTCTACATAAGAATACCCACCATACGAAAGTTTTTATGA R:CTCGATCATAAAAACTTTCGTATGGTGGGT ATTCTTATGTGAAAAATGTAGCCCATTTCTTTCCAACCCAT	71 71	151
MOTS-C （qRT-PCR）	F:ATGGGTTGGAAAGAAATG R:TCATAAAAACTTTCGTATGGTG	18 22	135
GAPDH	F:TCGGAGTGAACGGATTTGGC R:TGCCGTGGGTGGAATCATAC	20 20	147
DRP1	F:TCTGAATCTGGTGGGCATGATTGC R:CTCCGCAGTAAAGGACTCGAAGTG	24 24	91
FIS1	F:CTACCCAAAGGGAGCAAAGAG R:GTCCAATGAGTCCAGCCAGTC	21 21	250
MFF	F:GACCTCCTCCAACCCCTCAAAATG R:GACTAGCTGTCCATTCTGGCGAAC	24 24	104
OPA1	F:ACAGAGGATGGTGCTTGTTGAC R:CAGTATGATGGCGTTGGGATT	22 21	130
MFN1	F:AGAAAGCACAAAGCACAGGGGATG R:CACTGCTGACTGCGAGATACACTC	24 24	126
MFN2	F:GCCACACCACCAACTGCTTCC R:TCTTGACGCTCCTCTTCTCCTCTG	21 24	96
SOD-1	F:ATTCTGTGATCGCCCTCT R:AGCATTTCCCGTCTTTGT	18 18	119
SOD-2	F:TCTGGACAAATCTGAGCCCTAA R:TGGACGCCGACGGATACA	22 18	127
SOD-3	F:TGACACCCCTTCAAGAACCC R:AGAAGCGGAGAGTGTGTCTG	21 20	145
CAT	F:CGAAGGCGAAGGTGTTTG R:CAAACCCACGAGGGTCAC	18 18	114
HO-1	F:GCTAGCCTGGTGCAAGATACT R:AAGCTGAGAGTGAGGACCCA	21 20	110
CDK2	F:TGCATCTTTGCTGAGATGGTGA R:TTGCGATAACAAGCTCCGTC	22 20	225
CDK4	F:ATGGCTACCTCCCGGTATGA R:CTCCAGCACCTCCTCCATTG	20 20	139
PCNA	F:TGCAGATGTACCCCTTGTTGT R:CATCTTCGATCTTGGGAGCCA	21 21	83
CCNDBP1	F:CACCAACACCGACGTGGATT R:GAAGGAGAGAGCCGATTGGAC	20 21	206
CCNE2	F:GGGTCTGGCGAGGTGTCA R:TGAGGGGAATCCGTCTGACT	18 20	104
Claudin	F:CTAGTGATGAGGCAGATGAA R:AGATAGGTCCGAAGCAGAT	20 19	250
Occludin	F:GAGTGATTCGGATTCTGTCT R:TAGCCATAACCATAGCCATAG	20 21	181
ZO-1	F:TTGATAGTGGCGTTGACA R:CCTCATCTTCATCATCTTCTAC	18 22	126
BAX	F:GCCGAAATGTTTGCTGAC R:GCCGATCTCGAAGGAAGT	18 18	154
BCL2	F:TCAGGGATGGGGTGAACT R:TCAGAGACAGCCAGGAGAAAT	18 21	240
Caspase3	F:TTGGACTGTGGGATTGAGACG R:CGCTGCACAAAGTGACTGGA	21 20	165