FGF21增强线粒体功能抑制猪卵巢颗粒细胞凋亡

doi:10.11843/j.issn.0366-6964.2023.03.017

摘要/Abstract

摘要： 旨在探究成纤维细胞生长因子21(fibroblast growth factor 21, FGF21)对猪(Sus scrofa)卵巢颗粒细胞凋亡的作用及其调控机制。本研究采集大白×长白二元杂交母猪((8±1)月龄)新鲜卵巢组织抽取卵泡液分离颗粒细胞作为研究对象，通过RNAi技术和添加不同浓度的FGF21重组蛋白(0、0.1、1、10 ng · mL^-1)处理颗粒细胞24 h。24 h后通过流式细胞术检测细胞凋亡情况，利用Western blot、qRT-PCR等技术探究颗粒细胞BAX和BCL2的表达水平。为了进一步明确FGF21影响颗粒细胞凋亡与线粒体之间的关系，本试验检测了不同处理后颗粒细胞线粒体复合物的蛋白水平、细胞中线粒体数量、ATP浓度、总抗氧化能力、活性氧水平以及线粒体生成相关基因的表达水平。研究发现，利用RNAi技术敲低颗粒细胞中FGF21表达后，处于晚期凋亡的细胞比例显著上升(P<0.05)，而活细胞比例显著降低(P<0.05)。同时，FGF21敲低组颗粒细胞促凋亡基因BAX的mRNA和蛋白水平显著升高(P<0.05)，而抑制细胞凋亡的基因BCL2的表达显著降低(P<0.05)。相反，重组蛋白FGF21的添加有效阻止了颗粒细胞凋亡，并且上调抗凋亡基因BCL2的表达(P<0.05)，抑制促凋亡基因BAX的表达(P<0.05)。进一步研究发现，颗粒细胞中敲低FGF21的表达后，线粒体氧化磷酸化复合物II琥珀酸脱氢酶(succinate dehydrogenase complex iron sulfur subunit B，SDHB)表达显著降低(P<0.05)，同时，线粒体DNA拷贝数极显著降低(P<0.001)；此外，检测发现FGF21敲低后，细胞三磷酸腺苷(adenosine triphosphate，ATP)浓度、总抗氧化能力(total antioxidant capacity，T-AOC)极显著下降(P<0.001)，线粒体生成相关基因的表达显著被抑制(P<0.05)，细胞活性氧(ROS)水平显著升高(P<0.05)。于此相反，FGF21重组蛋白处理极显著增加了SDHB表达(P<0.01)以及细胞线粒体数目(P<0.05)，同时显著增加了线粒体DNA拷贝数(P<0.01)和ATP浓度(P<0.05)、T-AOC活性(P<0.05)和线粒体生成相关基因的表达，而ROS水平显著降低(P<0.05)。以上结果说明，FGF21促进了颗粒细胞线粒体生物合成以及维持了细胞内氧化-抗氧化系统的平衡从而抑制了颗粒细胞凋亡的发生。本研究为进一步揭示家畜卵泡发育潜在的调控机制提供理论依据。

关键词: 卵巢颗粒细胞, FGF21, 凋亡, 线粒体功能, 母猪

Abstract: The purpose of this study was to investigate the effect of fibroblast growth factor 21 (FGF21) on the apoptosis of porcine (Sus scrofa) ovarian granulosa cells and its regulatory mechanism. In this study, follicular fluid was collected from fresh ovarian tissue of Large white×Landrace sows ((8±1) months old) to separate granulosa cells as the research object. Granulosa cells were treated by RNAi technology and added with different concentrations of FGF21 recombinant protein (0, 0.1, 1, 10 ng·mL^-1) for 24 h. After 24 h, the cell apoptosis was detected by flow cytometry, and the expression levels of BAX and BCL2 in granulosa cells were investigated by Western blot, qRT-PCR. In order to further clarify the relationship between the effect of FGF21 on apoptosis of granulosa cells and mitochondria, the protein levels of mitochondrial complexes in granulosa cells after different treatments, the number of mitochondria in cells, ATP concentration, total antioxidant activity, reactive oxygen species levels and the expression levels of mitochondrial production-related genes were detected. The study results showed that after knockdown of FGF21 expression in granulosa cells by RNAi technology, the proportion of cells in late apoptosis was significantly increased (P<0.05) and the proportion of viable cells was significantly decreased (P<0.05). At the same time, the mRNA and protein levels of the pro-apoptotic gene BAX in granulosa cells in the FGF21 knockdown group were significantly increased (P<0.05), while the expression of the apoptosis-inhibiting gene BCL2 was significantly decreased (P<0.05). On the contrary, the addition of recombinant protein FGF21 effectively prevented the apoptosis of granulosa cells, and up-regulated the expression of anti-apoptotic gene BCL2 (P<0.05) and inhibited the expression of pro-apoptotic gene BAX (P<0.05). Further study found that after knockdown of FGF21 expression in granulosa cells, the expression of mitochondrial oxidative phosphorylation complex II succinate dehydrogenase complex iron sulfur subunit B (SDHB) was significantly decreased (P<0.05). At the same time, the copy number of mitochondrial DNA was significantly decreased (P<0.001); In addition, it was found that, after FGF21 knockdown, the concentration of adenosine triphosphate (ATP) and the total antioxidant capacity (T-AOC) significantly decreased (P<0.001), the expression of mitochondrial production-related genes was significantly inhibited (P<0.05), and the level of cellular reactive oxygen species (ROS) was significantly increased (P<0.05). In contrast, FGF21 recombinant protein treatment significantly increased SDHB expression (P<0.01) and mitochondrial number (P<0.05), and significantly increased mitochondrial DNA copy number (P<0.01) and ATP concentration (P<0.05), T-AOC activity (P<0.05) and expression of mitochondrial production-related genes, while ROS levels were significantly decreased (P<0.05). The above results indicated that FGF21 promoted mitochondrial biosynthesis in granulosa cells and maintained the balance of intracellular oxidation-antioxidant system, thereby inhibiting the occurrence of apoptosis of granulosa cells. This study provides a theoretical basis for further revealing the potential regulatory mechanism of follicular development in livestock.

Key words: ovarian granulosa cells, FGF21, apoptosis, mitochondrial function, sows

中图分类号:

S828.3

胡亚美, 宋湘容, 黄亮, 张璐通, 高磊, 庞卫军, 杨公社, 褚瑰燕. FGF21增强线粒体功能抑制猪卵巢颗粒细胞凋亡[J]. 畜牧兽医学报, 2023, 54(3): 1034-1045.

HU Yamei, SONG Xiangrong, HUANG Liang, ZHANG Lutong, GAO Lei, PANG Weijun, YANG Gongshe, CHU Guiyan. FGF21 Enhances Mitochondrial Function and Inhibits Apoptosis of Porcine Ovarian Granulosa Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(3): 1034-1045.

参考文献 37

[1]	DISTL O.Mechanisms of regulation of litter size in pigs on the genome level[J].Reprod Domest Anim,2007,42(Suppl 2):10-16.
[2]	VINET A,DROUILHET L,BODIN L,et al.Genetic control of multiple births in low ovulating mammalian species[J].Mamm Genome,2012,23(11-12):727-740.
[3]	TANTASUPARUK W,TECHAKUMPHU M,DORNIN S.Relationships between ovulation rate and litter size in purebred Landrace and Yorkshire gilts[J].Theriogenology,2005,63(4):1142-1148.
[4]	DUMESIC D A,MELDRUM D R,KATZ-JAFFE M G,et al.Oocyte environment:follicular fluid and cumulus cells are critical for oocyte health[J].Fertil Steril,2015,103(2):303-316.
[5]	MANABE N,MATSUDA-MINEHATA F,GOTO Y,et al.Role of cell death ligand and receptor system on regulation of follicular atresia in pig ovaries[J].Reprod Domest Anim,2008,43(Suppl 2):268-272.
[6]	ZHANG K,HANSEN P J,EALY A D.Fibroblast growth factor 10 enhances bovine oocyte maturation and developmental competence in vitro[J].Reproduction,2010,140(6):815-826.
[7]	KHARITONENKOV A,ADAMS A C.Inventing new medicines:The FGF21 story[J].Mol Metab,2014,3(3):221-229.
[8]	FLIPPO K H,POTTHOFF M J.Metabolic messengers:FGF21[J].Nat Metab,2021,3(3):309-317.
[9]	FISHER F M,KLEINER S,DOURIS N,et al.FGF21 regulates PGC-1α and browning of white adipose tissues in adaptive thermogenesis[J].Genes Dev,2012,26(3):271-281.
[10]	JUSTESEN S,HAUGEGAARD K V,HANSEN J B,et al.The autocrine role of FGF21 in cultured adipocytes[J].Biochem J,2020,477(13):2477-2487.
[11]	ABU-ODEH M,ZHANG Y,REILLY S M,et al.FGF21 promotes thermogenic gene expression as an autocrine factor in adipocytes[J].Cell Rep,2021,35(13):109331.
[12]	LAN T,MORGAN D A,RAHMOUNI K,et al.FGF19,FGF21,and an FGFR1/β-klotho-activating antibody act on the nervous system to regulate body weight and glycemia[J].Cell Metab,2017,26(5):709-718.e3.
[13]	FON TACER K,BOOKOUT A L,DING X S,et al.Research resource:Comprehensive expression atlas of the fibroblast growth factor system in adult mouse[J].Mol Endocrinol,2010,24(10):2050-2064.
[14]	LIANG Q N,ZHONG L,ZHANG J L,et al.FGF21 maintains glucose homeostasis by mediating the cross talk between liver and brain during prolonged fasting[J].Diabetes,2014,63(12):4064-4075.
[15]	KIM E H,TAWEECHAIPAISANKUL A,RIDLO M R,et al.Effect of Klotho protein during porcine oocyte maturation via Wnt signaling[J].Aging (Albany NY),2020,12(23):23808-23821.
[16]	OWEN B M,BOOKOUT A L,DING X S,et al.FGF21 contributes to neuroendocrine control of female reproduction[J].Nat Med,2013,19(9):1153-1156.
[17]	SINGHAL G,DOURIS N,FISH A J,et al.Fibroblast growth factor 21 has no direct role in regulating fertility in female mice[J].Mol Metab,2016,5(8):690-698.
[18]	XU C,MESSINA A,SOMM E,et al.KLB,encoding β-Klotho,is mutated in patients with congenital hypogonadotropic hypogonadism[J].EMBO Mol Med,2017,9(10):1379-1397.
[19]	ZHUO Y,HUA L,FENG B,et al.Fibroblast growth factor 21 coordinates adiponectin to mediate the beneficial effects of low-protein diet on primordial follicle reserve[J].EBioMedicine,2019,41:623-635.
[20]	LEE M S.Role of mitochondrial function in cell death and body metabolism[J].Front Biosci (Landmark Ed),2016,21(6): 1233-1244.
[21]	JI K Q,ZHENG J F,LV J W,et al.Skeletal muscle increases FGF21 expression in mitochondrial disorders to compensate for energy metabolic insufficiency by activating the mTOR-YY1-PGC1α pathway[J].Free Radic Biol Med,2015,84:161-170.
[22]	RESTELLI L M,OETTINGHAUS B,HALLIDAY M,et al.Neuronal mitochondrial dysfunction activates the integrated stress response to induce fibroblast growth factor 21[J].Cell Rep,2018,24(6):1407-1414.
[23]	SA-NGUANMOO P,TANAJAK P,KERDPHOO S,et al.FGF21 improves cognition by restored synaptic plasticity,dendritic spine density,brain mitochondrial function and cell apoptosis in obese-insulin resistant male rats[J].Horm Behav,2016,85:86-95.
[24]	WU G,LI C Y,TAO J L,et al.FSH mediates estradiol synthesis in hypoxic granulosa cells by activating glycolytic metabolism through the HIF-1α-AMPK-GLUT1 signaling pathway[J].J Biol Chem,2022,298(5):101830.
[25]	TUZLAK S,KAUFMANN T,VILLUNGER A.Interrogating the relevance of mitochondrial apoptosis for vertebrate development and postnatal tissue homeostasis[J].Genes Dev,2016,30(19):2133-2151.
[26]	BOCK F J,TAIT S W G.Mitochondria as multifaceted regulators of cell death[J].Nat Rev Mol Cell Biol,2020,21(2):85-100.
[27]	COSTERMANS N G J,TEERDS K J,KEIJER J,et al.Follicular development of sows at weaning in relation to estimated breeding value for within-litter variation in piglet birth weight[J].Animal,2019,13(3):554-563.
[28]	SINGH R,LETAI A,SAROSIEK K.Regulation of apoptosis in health and disease:the balancing act of BCL-2 family proteins[J].Nat Rev Mol Cell Biol,2019,20(3):175-193.
[29]	YOULE R J,STRASSER A.The BCL-2 protein family:opposing activities that mediate cell death[J].Nat Rev Mol Cell Biol,2008,9(1):47-59.
[30]	SHAO C S,ZHOU X H,MIAO Y H,et al.In situ observation of mitochondrial biogenesis as the early event of apoptosis[J]. iScience, 2021,24(9):103038.
[31]	BURKE P J.Mitochondria,bioenergetics and apoptosis in cancer[J].Trends Cancer,2017,3(12):857-870.
[32]	CAO C H,YU H Y,WU F,et al.Antibiotic anisomycin induces cell cycle arrest and apoptosis through inhibiting mitochondrial biogenesis in osteosarcoma[J].J Bioenerg Biomembr,2017,49(6):437-443.
[33]	XU G F,LIU S,HUANG M Q,et al.Cadmium induces apoptosis of human granulosa cell line KGN via mitochondrial dysfunction-mediated pathways[J].Ecotoxicol Environ Saf,2021,220:112341.
[34]	SCHIEBER M,CHANDEL N S.ROS function in redox signaling and oxidative stress[J].Curr Biol,2014,24(10):R453-R462.
[35]	LI X,HONG Y M,HE H W,et al.FGF21 mediates mesenchymal stem cell senescence via regulation of mitochondrial dynamics[J]. Oxid Med Cell Longev,2019,2019:4915149.
[36]	ABATE M,FESTA A,FALCO M,et al.Mitochondria as playmakers of apoptosis,autophagy and senescence[J].Semin Cell Dev Biol,2020,98:139-153.
[37]	LIU Y,PANG Y,ZHU B Q,et al.Therapeutic targeting of SDHB-mutated pheochromocytoma/paraganglioma with pharmacologic ascorbic acid[J].Clin Cancer Res,2020,26(14):3868-3880.