Abstract：Methionine enkephalin (Met-Enk) is an opioid neuropeptide with regulatory effects on cell proliferation and metabolism. In order to investigate the effect of Met-Enk on apoptosis, mitochondrial structure and function of porcine (Sus scrofa) precursor adipocytes, in this study, Met-Enk was used to treat porcine primary precursor adipocytes, and detected apoptosis by using cell counting kit 8 (CCK-8) and annexin V/propidium iodide (AV/PI) staining, and observed mitochondrial structural changes by means of transmission electron microscope (TEM) and atomic force microscope (AFM) and detected the changes of mitochondrial function by JC-1 fluorescent probe and other methods. The results showed that Met-Enk treatment resulted in decreased activity and increased apoptosis rate of porcine precursor adipocytes, causing changes in the expression levels of apoptosis markers Caspase 3 and Caspase 9, etc. The TEM results showed that the number of mitochondria in the precursor adipocytes was reduced, the volume was swollen, and mitochondrial cristae were reduced. The AFM results showed that the mitochondria were deformed, and the surface of the membrane appeared to be concave and the roughness was increased; Immunoblot analysis and qPCR revealed that revealed that Met-Enk treatment led to impaired mitochondrial function and promoted apoptotic factors such as Bax/Bak through opioid growth factor receptor (Ogfr), which prompted the release of cytochrome C from mitochondria and promoted apoptosis. This study deeply investigated the mechanism of Met-Enk inducing apoptosis in porcine precursor adipocytes by affecting mitochondrial structure and function, so as to provide a reference for the use of Met-Enk to improve the development of adipose tissues in livestock and poultry.
[1] 柏雪丽. 2022. 蛋氨酸脑啡肽(MENK)通过阿片受体(OGFr)抑制人表皮鳞癌及其相关机制的研究[D]. 博士学位论文, 中国医科大学, 导师: 单风平, pp. 33-37. (Bai X L.Methionine enkephalin (MENK) inhibits human epidermal squamous cell carcinoma through opioid receptor (OGFR) and its related mechanism[D]. Thesis for Ph.D., China Medical University, Supervisor: Shan F P, pp. 33-37.) [2] 柴立昂, 孙法壮, 蔡和乐, 等. 2020. 甲硫脑啡肽对脂多糖作用下奶牛子宫内膜上皮细胞增殖的影响[J]. 中国兽医学报, 40(04): 772-779. (Chai L A, Sun F Z, Cai H L, et al.2020. Effect of met-enkephalin on lipopolysaccharide-stimulated proliferation of bovine endometrial epithelial cells[J]. Chinese Journal of Veterinary Science, 40(04): 772-779.) [3] 郭万松, 杨波. 2016. 阿片生长因子及其受体抗肿瘤机制研究进展[J]. 中华临床医师杂志(电子版), 10(19): 2950-2953. (Guo W S, Yang B.2016. Research progress of opioid growth factor and its receptor on anti-tumor[J]. Chinese Journal of Clinicians (Electronic Edition), 10(19): 2950-2953.) [4] 高歌, 汪志鹏, 段春礼, 等. 2016. 原子力显微镜检测α-突触核蛋白转基因鼠线粒体微观结构的变化[J]. 首都医科大学学报, 37(06): 806-811. (Gao G, Wang Z P, Duan C L, et al.2016. Topographic images analysis of mitochondria from α-synuclein transgenic mice utilizing atomic force microscopy[J]. Journal of Capital Medical University, 37(06): 806-811.) [5] 黄冬爱, 李平风, 李刚. 2006. 甲硫氨酸脑啡肽和抗δ阿片受体抗体对小鼠骨髓瘤细胞增殖的影响[J]. 海南医学院学报, 12(2): 103-105. (Huang D A, Li P F, Li G.2006. The role of methionine enkephalin and anti-delta opioid receptor antibodies on the proliferation of mouse myeloma cells[J]. Journal of Hainan Medical University, 12(2): 103-105.) [6] 刘霏霏, 刘泽源. 2012. 阿片生长因子甲硫氨酸脑啡肽抗肿瘤作用研究进展[J]. 药学服务与研究, 12(05): 387-391. (Liu F F, Liu Z Y.2012. Advances in research on the anti-tumore ffect of opioid growth factor methionine enkephalin[J]. Pharmaceutical Care and Research, 12(05): 387-391.) [7] 申振通. 2022. MetEnk对小鼠白脂棕色化和脂肪细胞线粒体功能的调节机制研究[D]. 西北农林科技大学, 硕士学位论文, 西北农林科技大学, 导师: 孙超, pp. 38-45. (Sheng Z T.2022. The mechanism research on MetEnk regulating browning of white adipose tissue and mitochondrial function of adipocyte in mice[D]. Thesis for M. S., Northwest A&F University, Supervisor: Sun C, pp. 38-45.) [8] 汪志鹏, 高歌, 段春礼, 等. 2018. α-突触核蛋白对线粒体膜造成孔道样损伤[J].中国生物化学与分子生物学报, 34(09): 1013-1020. (Wang Z P, Gao G, Duan C L, et al.2018. α-synuclein induces pore-like impairments of mitochondrial membrane[J]. Chinese Journal of Biochemistry and Molecular Biology, 34(09): 1013-1020.) [9] 汪蒙蒙, 韩岚, 刘竹青, 等. 2020. 细胞凋亡、细胞焦亡、坏死性凋亡异同与交互[J].生命的化学, 40(11): 1932-1940. (Wang M M, Han L, Liu Z Q, et al.2020. The similarities, differences and crosstalks of apoptosis, pyroptosis and necroptosis[J]. Chemistry of Life, 40(11): 1932-1940.) [10] 杨珺, 陈晓宇, 宋先兵, 等. 2019. 透射电镜下观察白藜芦醇对佐剂性关节炎大鼠滑膜细胞凋亡的影响[J]. 泰山医学院学报, 40(07): 548-550. (Yang J, Chen X Y, Song X B, et al.Effect of resveratrol on synovial cell apoptosis in rats with adjuvant arthritis observed by transmission electron microscopy[J]. Journal of Shandong First Medical University, 40(07): 548-550.) [11] Barron B A, Oakford L X, Gaugl J F, et al.1995. Methionine-enkephalin-Arg-Phe immunoreactivity in heart tissue[J]. Peptides, 16(7): 1221-1227. [12] Fan C, Mclaughlin P J, Verderame M F, et al.2008. The OGF-OGFr axis utilizes the p21 pathway to restrict progression of human pancreatic cancer[J]. Molecular Cancer, 7(1): 5-5. [13] Fernández-Galilea M, Pérez-Matute P, Prieto-Hontoria P L, et al.2015. α-Lipoic acid treatment increases mitochondrial biogenesis and promotes beige adipose features in subcutaneous adipocytes from overweight/obese subjects[J]. Biochimica Et Biophysica Acta-Biomembranes, 1851(3): 273-281. [14] Hamada H, Kishioka S, Yamoto M, et al.1995.[3H]naloxone binding sites in porcine ovarian follicles and corpora lutea during the ovarian cycle[J]. European Journal of Endocrinology, 132(5): 622-626. [15] Hauser K F, Houdi A A, Turbek C S, et al.2000. Opioids intrinsically inhibit the genesis of mouse cerebellar granule neuron precursors in vitro: Differential impact of mu and delta receptor activation on proliferation and neurite elongation[J]. The European Journal of Neuroscience, 12(4): 1281-1293. [16] Kumar S, Behl T, Sachdeva M, et al.2021. Implicating the effect of ketogenic diet as a preventive measure to obesity and diabetes mellitus[J]. Life Sciences, 264: 118661. [17] Li C, Chen Q, Liu Y, et al.2023. Methionine enkephalin promotes white fat browning through cAMP/PKA pathway[J]. Life Sciences, 312: 121189. [18] Li W I.1992. Methionine-enkephalin in a porcine endometrial cell line and its responsiveness to potassium depolarization[J]. Life Sciences, 51(25): 1981-1990. [19] Qu N, Wang R, Meng Y, et al.2022. Methionine enkephalin inhibited cervical carcinoma via apoptosis promotion and reduction of myeloid derived suppressor cell infiltrated in tumor[J]. International Immunopharmacology, 110: 108933. [20] Sogi C, Takeshita N, Jiang W, et al.2020. Methionine enkephalin suppresses osteocyte apoptosis induced by compressive force through regulation of nuclear translocation of NFATc1[J]. JBMR Plus, 4(7): e10369. [21] Spalding K L, Arner E, Westermark P O, et al.2008. Dynamics of fat cell turnover in humans[J]. Nature, 453(7196): 783-787. [22] Tian J, Fu W, Xie Z, et al.2024. Methionine enkephalin (MENK) protected macrophages from ferroptosis by downregulating HMOX1 and ferritin[J]. Proteome Science, 22(1): 2. [23] Tuo Y, Tian C, Lu L, et al.2020. The paradoxical role of methionine enkephalin in tumor responses[J]. European Journal of Pharmacology, 882(Suppl.1): 173253. [24] Wang R, Zhang Y, Shan F.2019. Interaction of opioid growth factor (OGF) and opioid antagonist and their significance in cancer therapy[J]. International Immunopharmacology, 75: 105785. [25] Wang X, Jiao X, Meng Y, et al.2018. Methionine enkephalin (MENK) inhibits human gastric cancer through regulating tumor associated macrophages (TAMs) and PI3K/AKT/mTOR signaling pathway inside cancer cells[J]. International Immunopharmacology, 65: 312-322. [26] Wilson-Fritch L, Burkart A, Bell G, et al.2003. Mitochondrial biogenesis and remodeling during adipogenesis and in response to the insulin sensitizer rosiglitazone[J]. Molecular and Cellular Biology, 23(3): 1085-1094. [27] Ye K, Meng W X, Sun H, et al.2020. Characterization of an alternative BAK-binding site for BH3 peptides[J]. Nature Communications, 11(1): 3301. [28] Zagon I S, Ruth T B, McLaughlin P J.2005. Nucleocytoplasmic distribution of opioid growth factor and its receptor in tongue epithelium[J]. The Anatomical Record. Part A, Discoveries in Molecular, Cellular, and Evolutionary Biology, 282(1): 24-37. [29] Zhang S, Huang H, Handley M, et al.2021a. A novel mechanism of lung cancer inhibition by methionine enkephalin through remodeling the immune status of the tumor microenvironment[J]. International Immunopharmacology, 99: 107999. [30] Zhang S, Liu N, Ma M, et al.2021b. Methionine enkephalin (MENK) suppresses lung cancer by regulating the Bcl-2/Bax/caspase-3 signaling pathway and enhancing natural killer cell-driven tumor immunity[J]. International Immunopharmacology, 98: 107837.
