Bax/Bcl-2 protein and apoptosis during adipose-derived stromal cell differentiation into astrocytes

doi:10.12307/2022.777

Abstract

Abstract: BACKGROUND: Neurodegenerative diseases are a large class of major neurological diseases that can lead to the loss of patients’ ability to work and live. At present, there is no effective treatment for this disease. Adipose-derived stromal cells can induce differentiation into nerve cells in vitro, which is expected to provide a new method for the treatment of this kind of diseases.
OBJECTIVE: To explore the expression changes of Bax/Bcl-2 protein during the differentiation of adipose-derived stromal cells into astrocytes, and to further investigate whether there is mitochondrial pathway-mediated apoptosis during the induction.
METHODS: Immunocytochemistry and western blot assay were used to analyze the expression of glial fibrillary acidic protein, Bax, Bcl-2, cytochrome C, and casepase-3 in the process of adipose-derived stromal cell differentiation into astrocytes when induction for 48 hours and 7, 14, and 21 days. Transmission electron microscopy was used to observe the ultrastructure of cells and mitochondria.
RESULTS AND CONCLUSION: (1) After being induced for 48 hours and 7, 14, and 21 days, the expression of glial fibrillary acidic protein reached the peak on day 7 (P < 0.05). There was no significant difference in glial fibrillary acidic protein expression between 14 days and 7 days after induction (P > 0.05). (2) The expression of Bax, cytochrome C, and casepase-3 increased gradually with the induction time extending and reached the peak on day 14 (P < 0.05). The expression of Bcl-2 was decreased gradually and reached the lowest level on day 14 after induction (P < 0.05). (3) Typical apoptotic bodies and mitochondrial damage were observed using transmission electron microscopy. (4) Adipose-derived stromal cells were in the best survival and differentiation state at the 7th to 14th day of differentiation. During adipose-derived stromal cell differentiation into astrocytes, the inhibitory effect of Bcl-2 on apoptosis was significantly weakened, and the pro apoptotic effect of Bax was gradually enhanced. Cytochrome C was released into the cytoplasm by damaged mitochondria, which led to apoptosis in mitochondrial pathway.

Key words: adipose-derived stromal cell, induction, differentiation, astrocyte, mitochondria, apoptosis, protein

CLC Number:

Zhang Lili, Ou Ya, Yuan Xiaodong, Zhang Pingshu. Bax/Bcl-2 protein and apoptosis during adipose-derived stromal cell differentiation into astrocytes[J]. Chinese Journal of Tissue Engineering Research, 2022, 26(31): 4982-4987.

Figures/Tables 5

References

[1] CHI H, CHANG HY, SANG TK. Neuronal Cell Death Mechanisms in Major Neurodegenerative Diseases. Int J Mol Sci. 2018;19(10):3082.
[2] DE GIOIA R, BIELLA F, CITTERIO G, et al. Neural Stem Cell Transplantation for Neurodegenerative Diseases. Int J Mol Sci. 2020; 21(9):3103.
[3] YE CQ, YUAN XD, LIU H, et al. Ultrastructure of neuronal-like cells differentiated from adult adipose-derived stromal cells. Neural Regen Res. 2010;5(19):1456-1463.
[4] OU Y, YUAN XD, CAI YN, et al. A novel ethanol-based method to induce differentiation of adipose-derived stromal cells into astrocytes. Neural Regen Res. 2011;6(10):738-743.
[5] YUAN XD, CAI YN, OU Y, et al. Adult adipose-derived stromal cells differentiate into neurons with normal electrophysiological functions. Neural Regen Res. 2011;6(34):2681-2686.
[6] 蔡亚楠,元小冬,欧亚,等.成人脂肪基质细胞体外诱导分化为神经前体细胞的数量达峰时间研究[J].中华行为医学与脑科学杂志, 2011,20(4):302-305.
[7] OU Y, YUAN XD, CAI YN, et al. Ultrastructure and electrophysiology of astrocytes differentiated from adult adipose-derived stromal cells. Chin Med J (Engl). 2011;124(17):2656-2660.
[8] LU YH, YUAN XD, OU Y, et al. Autophagy and apoptosis during about adult adipose-derived stromal cells differentiation into neuron-like cells in vitro. Neural Regen Res. 2012;7(16):1205-1212.
[9] LIN Z, XIE F, TRIVIÑO M, et al. Ectopic Expression of a Self-Incompatibility Module Triggers Growth Arrest and Cell Death in Vegetative Cells. Plant Physiol. 2020;183(4):1765-1779.
[10] WON M, LUO Y, LEE DH, et al. BAX is an essential key mediator of AP5M1-induced apoptosis in cervical carcinoma cells. Biochem Biophys Res Commun. 2019;518(2):368-373.
[11] HU H, TIAN M, DING C, et al. The C/EBP Homologous Protein (CHOP) Transcription Factor Functions in Endoplasmic Reticulum Stress-Induced Apoptosis and Microbial Infection. Front Immunol. 2019;9:3083.
[12] TAN BL, NORHAIZAN ME, CHAN LC. ROS-Mediated Mitochondrial Pathway is Required for Manilkara Zapota (L.) P. Royen Leaf Methanol Extract Inducing Apoptosis in the Modulation of Caspase Activation and EGFR/NF-κB Activities of HeLa Human Cervical Cancer Cells. Evid Based Complement Alternat Med. 2018;2018:6578648.
[13] VEZZANI B, CARINCI M, PATERGNANI S, et al. The Dichotomous Role of Inflammation in the CNS: A Mitochondrial Point of View. Biomolecules. 2020;10(10):1437.
[14] 郑凯,杨梅桂,闫朝君,等.线粒体动力学与细胞凋亡[J].中国细胞生物学学报,2019,41(8):1465-1476.
[15] MARTINIS P, GRANCARA S, KANAMORI Y, et al. Involvement of the biogenic active amine agmatine in mitochondrial membrane permeabilization and release of pro-apoptotic factors. Amino Acids. 2020;52(2):161-169.
[16] MACCHIONI L, PETRICCIUOLO M, DAVIDESCU M, et al. Palmitate lipotoxicity in enteric glial cells: Lipid remodeling and mitochondrial ROS are responsible for cyt c release outside mitochondria. Biochim Biophys Acta Mol Cell Biol Lipids. 2018;1863(8):895-908.
[17] WANG Q, ZHANG L, YUAN X, et al. The Relationship between the Bcl-2/Bax Proteins and the Mitochondria-Mediated Apoptosis Pathway in the Differentiation of Adipose-Derived Stromal Cells into Neurons. PLoS One. 2016;11(10):e0163327.
[18] ANTONSSON B. Bax and other pro-apoptotic Bcl-2 family “killer-proteins” and their victim the mitochondrion. Cell Tissue Res. 2001; 306(3):347-361.
[19] 冯健愉,朱玉山,陈佺,等.Bcl-2家族蛋白的生理功能及结构基础[J].中国细胞生物学学报,2019,41(8):1477-1489.
[20] RENAULT TT, DEJEAN LM, MANON S. A brewing understanding of the regulation of Bax function by Bcl-xL and Bcl-2. Mech Ageing Dev. 2017;161(Pt B):201-210.
[21] MAHDAVI S, KHODARAHMI P, ROODBARI NH. Effects of cadmium on Bcl-2/ Bax expression ratio in rat cortex brain and hippocampus. Hum Exp Toxicol. 2018;37(3):321-328.
[22] BERGANDI L, MUNGO E, MORONE R, et al. Hyperglycemia Promotes Chemoresistance Through the Reduction of the Mitochondrial DNA Damage, the Bax/Bcl-2 and Bax/Bcl-XL Ratio, and the Cells in Sub-G1 Phase Due to Antitumoral Drugs Induced-Cytotoxicity in Human Colon Adenocarcinoma Cells. Front Pharmacol. 2018;9:866.
[23] HOPE JM, LOPEZ-CAVESTANY M, WANG W, et al. Activation of Piezo1 sensitizes cells to TRAIL-mediated apoptosis through mitochondrial outer membrane permeability. Cell Death Dis. 2019;10(11):837.
[24] YANG Z, WANG WE, ZHANG Q. CIAPIN1 siRNA inhibits proliferation, migration and promotes apoptosis of VSMCs by regulating Bcl-2 and Bax. Curr Neurovasc Res. 2013;10(1):4-10.
[25] ZHOU P, XIE W, LUO Y, et al. Inhibitory Effects of Ginsenoside Rb1 on Early Atherosclerosis in ApoE-/- Mice via Inhibition of Apoptosis and Enhancing Autophagy. Molecules. 2018;23(11):2912.
[26] LIU LS, BAI XQ, GAO Y, et al. PCSK9 Promotes oxLDL-Induced PC12 Cell Apoptosis Through the Bcl-2/Bax-Caspase 9/3 Signaling Pathway. J Alzheimers Dis. 2017;57(3):723-734.
[27] RAK M, BÉNIT P, CHRÉTIEN D, et al. Mitochondrial cytochrome c oxidase deficiency. Clin Sci (Lond). 2016;130(6):393-407.
[28] CLEETER MW, COOPER JM, DARLEY-USMAR VM, et al. Reversible inhibition of cytochrome c oxidase, the terminal enzyme of the mitochondrial respiratory chain, by nitric oxide. Implications for neurodegenerative diseases. FEBS Lett. 1994;345(1):50-54.
[29] TRUMPOWER BL. The protonmotive Q cycle. Energy transduction by coupling of proton translocation to electron transfer by the cytochrome bc1 complex. J Biol Chem. 1990;265(20):11409-11412.
[30] MARTINEZ LYONS A, ARDISSONE A, REYES A, et al. COA7 (C1orf163/RESA1) mutations associated with mitochondrial leukoencephalopathy and cytochrome c oxidase deficiency. J Med Genet. 2016;53(12):846-849.
[31] OSHIMA Y, VERHOEVEN N, CARTIER E, et al. The OMM-severed and IMM-ubiquitinated mitochondria are intermediates of mitochondrial proteotoxicity-induced autophagy in PRKN/parkin-deficient cells. Autophagy. 2021;17(11):3884-3886.
[32] GU J, CHEN Y, TONG L, et al. Astaxanthin-loaded polymer-lipid hybrid nanoparticles (ATX-LPN): assessment of potential otoprotective effects. J Nanobiotechnology. 2020;18(1):53.
[33] KISELEVSKY DB. Granzymes and Mitochondria. Biochemistry (Mosc). 2020;85(2):131-139.
[34] LAI CI, CHU YL, HO CT, et al. Antcin K, an active triterpenoid from the fruiting bodies of basswood cultivated Antrodia cinnamomea, induces mitochondria and endoplasmic reticulum stress-mediated apoptosis in human hepatoma cells. J Tradit Complement Med. 2015;6(1):48-56.
[35] HUANG H, LIU C, FU X, et al. Microcystin-LR Induced Apoptosis in Rat Sertoli Cells via the Mitochondrial Caspase-Dependent Pathway: Role of Reactive Oxygen Species. Front Physiol. 2016;7:397.
[36] 王艳杰,邓雯,张鹏飞.细胞色素C与细胞凋亡研究进展[J].动物医学进展,2012,33(7):89-92.
[37] BENDER CE, FITZGERALD P, TAIT SW, et al. Mitochondrial pathway of apoptosis is ancestral in metazoans. Proc Natl Acad Sci U S A. 2012; 109(13):4904-4909.
[38] 王晓庚,刘林,左健,等.沉默FoxM1通过促进线粒体释放细胞色素C诱导口腔鳞癌细胞凋亡[J].中国病理生理杂志,2019,35(3): 430-435.
[39] JEONG SY, SEOL DW. The role of mitochondria in apoptosis. BMB Rep. 2008;41(1):11-22.
[40] 朱玉山,卢铁元,王蕊,等.Bcl-2家族蛋白调控线粒体膜通透性和细胞色素C释放的新机制[J].生命科学,2011,23(11):1076-1080.
[41] SALIMI A, BAHREINI F, JAMALI Z, et al. Mesalazine Induces Oxidative Stress and Cytochrome c Release in Isolated Rat Heart Mitochondria: An Analysis of Cardiotoxic Effects. Int J Toxicol. 2020;39(3):241-247.
[42] WANG L, MA G, ZHANG Y, et al. Effect of mitochondrial cytochrome c release and its redox state on the mitochondrial-dependent apoptotic cascade reaction and tenderization of yak meat during postmortem aging. Food Res Int. 2018;111:488-497.
[43] LI L, WANG F, ZHANG J, et al. Typical phthalic acid esters induce apoptosis by regulating the PI3K/Akt/Bcl-2 signaling pathway in rat insulinoma cells. Ecotoxicol Environ Saf. 2021;208:111461.
[44] NOMURA M, SHIMIZU S, SUGIYAMA T, et al. 14-3-3 Interacts directly with and negatively regulates pro-apoptotic Bax. J Biol Chem. 2003; 278(3):2058-2065.
[45] JEZEK J, CHANG KT, JOSHI AM, et al. Mitochondrial translocation of cyclin C stimulates intrinsic apoptosis through Bax recruitment. EMBO Rep. 2019;20(9):e47425.
[46] GIULIANO M, LAURICELLA M, CALVARUSO G, et al. The apoptotic effects and synergistic interaction of sodium butyrate and MG132 in human retinoblastoma Y79 cells. Cancer Res. 1999;59(21):5586-5595.
[47] LI H, TONG J, BAO J, et al. Hematoporphyrin monomethyl ether combined with He-Ne laser irradiation-induced apoptosis in canine breast cancer cells through the mitochondrial pathway. J Vet Sci. 2016; 17(2):235-242.
[48] GOGVADZE V, ORRENIUS S, ZHIVOTOVSKY B. Multiple pathways of cytochrome c release from mitochondria in apoptosis. Biochim Biophys Acta. 2006;1757(5-6):639-647.
[49] WANG X. Molecular mechanism of cytochrome c release from mitochondria during apoptosis. Biochimica et Biophysica Acta (BBA) - Bioenergetics. 2016;1857:e7.