Tumor necrosis factor alpha-induced apoptosis in bone marrow mesenchymal stem cells

doi:10.3969/j.issn.2095-4344.2016.36.001

Abstract

Abstract:

BACKGROUND: Stem cell transplantation has achieved good results in the treatment of cerebral ischemia, and how to reduce apoptosis of transplanted cells has become the focus of the therapy.
OBJECTIVE: To investigate the injured effect of tumor necrosis factor alpha (TNF-α) on bone marrow mesenchymal stem cells and its mechanism.
METHODS: Primary cultured bone marrow mesenchymal stem cells from Sprague-Dawley rats were treated with 200 μg/L TNF-α for 6 hours. Cell vitality was assayed by MTT, and cell apoptosis was observed by Hoechst33342 staining. Apoptotic rate was detected by Annexin-V/PI double staining. Level of oxidative stress was evaluated by determination of malondialdehyde and superoxide dismutase levels. The protein expressions of phosphorylated-Akt, Akt, phosphorylated-FoxO1, FoxO1 were detected by western blot analysis.
RESULTS AND CONCLUSION: After treatment with TNF-α, the cell vitality of bone marrow mesenchymal stem cells decreased, the apoptotic rate increased, and the cells were arrested in the S phase. Moreover, the oxidative stress level was elevated, and the protein expression of phosphorylated-Akt and phosphorylated-FoxO1 was significantly reduced compared with the control group (P < 0.05). These results suggest that TNF-α at high level contributes to the S-stage arrest, responsible for the apoptosis processes of bone marrow mesenchymal stem cells via the Akt-FoxO1 pathway.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Tumor Necrosis Factor-alpha, Bone Marrow, Mesenchymal Stem Cells, Apoptosis, Tissue Engineering

CLC Number:

R394.2

Li Jie-mei, Wang Huai-gao, Deng Da-shi, Lu Fang, Zhang Chen-chen, Liu Guo-zeng, Zhou Yan-fang. Tumor necrosis factor alpha-induced apoptosis in bone marrow mesenchymal stem cells[J]. Chinese Journal of Tissue Engineering Research, 2016, 20(36): 5325-5331.

Figures/Tables 2

2.1 骨髓间充质干细胞的分离、培养及形态学观察结果原代培养48 h后首次换液，可见有少许细胞集落及已贴壁的圆形和短梭形的散在单个细胞，72 h后细胞增殖加快，呈团簇集落样生长，细胞形态呈长梭形或多角形，有较强的折光性，1周后细胞铺满瓶底达到融合状态，漩涡状生长，见图1。 2.2 肿瘤坏死因子α对骨髓间充质干细胞活力的影响 200 μg/L肿瘤坏死因子α作用6 h后，细胞存活率降低，与对照组相比，差异有显著性意义(P < 0.05)，见图2。 2.3 流式细胞术测定肿瘤坏死因子α对骨髓间充质干细胞周期的影响肿瘤坏死因子α作用后，流式细胞仪检测细胞周期发现：S期、G2期细胞比例有所下降，G1期细胞比例增加，差异有显著性意义(P < 0.05)，说明肿瘤坏死因子α能抑制骨髓间充质干细胞的增殖，将细胞阻滞在G1期(图3)。 2.4 荧光显微镜骨髓间充质干细胞凋亡的形态荧光显微镜下观察，对照组细胞核荧光均匀，实验组细胞可见致密强荧光，细胞核变小皱缩，染色质高度凝聚，边缘化，可见核裂解为碎块的凋亡小体(图4)。 2.5 流式细胞仪检测肿瘤坏死因子α对骨髓间充质干细胞凋亡的影响肿瘤坏死因子α作用后，细胞凋亡率为8.6%，较对照组细胞凋亡率0.4%有明显增高，差异有显著性意义(P < 0.05)，说明缺血微环境中肿瘤坏死因子α可促进骨髓间充质干细胞凋亡(图5)。 2.6 肿瘤坏死因子α对p-Akt的及p-FoxO1的下调作用 Western blot结果表示，正常骨髓间充质干细胞内有一定水平p-Akt及p-FoxO1的表达。200 μg/L肿瘤坏死因子α处理骨髓间充质干细胞6 h可明显下调p-Akt及其下游p-FoxO1表达水平，与对照组比较，差异有显著性意义(P < 0.05，P < 0.01)，见图6。 2.7 肿瘤坏死因子α引起骨髓间充质干细胞内超氧化物歧化酶生成减少，丙二醛生成增加 200 μg/L肿瘤坏死因子α处理骨髓间充质干细胞6 h后，胞内超氧化物歧化酶水平明显降低(P < 0.01)，丙二醛水平明显升高(P < 0.05)，与对照组比较，差异有显著性意义。实验结果说明，缺血微环境中肿瘤坏死因子α能促进细胞的氧化应激水平，增强细胞损伤(表1)。"

References

[1] Ding Y, Clark JC. Cerebrovascular injury in stroke. Neurol Res. 2006;28(1):3-10.
[2] Schippers EF, van't Veer C, van Voorden S, et al. TNF-alpha promoter, Nod2 and toll-like receptor-4 polymorphisms and the in vivo and ex vivo response to endotoxin. Cytokine. 2004;26(1):16-24.
[3] El-Obeid A, Hassib A, Pontén F, et al. Effect of herbal melanin on IL-8: a possible role of Toll-like receptor 4 (TLR4). Biochem Biophys Res Commun. 2006;344(4): 1200-1206.
[4] Jin Z, Liang J, Wang J, et al. MCP-induced protein 1 mediates the minocycline-induced neuroprotection against cerebral ischemia/reperfusion injury in vitro and in vivo. J Neuroinflammation. 2015;12:39.
[5] Hong SQ, Zhang HT, You J, et al. Comparison of transdifferentiated and untransdifferentiated human umbilical mesenchymal stem cells in rats after traumatic brain injury. Neurochem Res. 2011;36(12): 2391-2400.
[6] Jeong CH, Kim SM, Lim JY, et al. Mesenchymal stem cells expressing brain-derived neurotrophic factor enhance endogenous neurogenesis in an ischemic stroke model. Biomed Res Int. 2014;2014:129145.
[7] Mahmood A, Lu D, Qu C, et al. Treatment of traumatic brain injury with a combination therapy of marrow stromal cells and atorvastatin in rats. Neurosurgery. 2007;60(3):546-553.
[8] Rao RR, Peterson AW, Ceccarelli J, et al. Matrix composition regulates three-dimensional network formation by endothelial cells and mesenchymal stem cells in collagen/fibrin materials. Angiogenesis. 2012; 15(2):253-264.
[9] Sheikh AM, Nagai A, Wakabayashi K, et al. Mesenchymal stem cell transplantation modulates neuroinflammation in focal cerebral ischemia: contribution of fractalkine and IL-5. Neurobiol Dis. 2011;41(3):717-724.
[10] Lee EJ, Park HW, Jeon HJ, et al. Potentiated therapeutic angiogenesis by primed human mesenchymal stem cells in a mouse model of hindlimb ischemia. Regen Med. 2013;8(3):283-293.
[11] Wang H, Nagai A, Sheikh AM, et al. Human mesenchymal stem cell transplantation changes proinflammatory gene expression through a nuclear factor-κB-dependent pathway in a rat focal cerebral ischemic model. J Neurosci Res. 2013;91(11): 1440-1449.
[12] Xue Q, Luan XY, Gu YZ, et al. The negative co-signaling molecule b7-h4 is expressed by human bone marrow-derived mesenchymal stem cells and mediates its T-cell modulatory activity. Stem Cells Dev. 2010;19(1):27-38.
[13] Parr AM, Tator CH, Keating A. Bone marrow-derived mesenchymal stromal cells for the repair of central nervous system injury. Bone Marrow Transplant. 2007; 40(7):609-619.
[14] Merson TD, Bourne JA. Endogenous neurogenesis following ischaemic brain injury: insights for therapeutic strategies. Int J Biochem Cell Biol. 2014;56:4-19.
[15] Nadri S, Soleimani M, Mobarra Z, et al. Expression of dopamine-associated genes on conjunctiva stromal-derived human mesenchymal stem cells. Biochem Biophys Res Commun. 2008;377(2):423-428.
[16] Xu YX, Chen L, Hou WK, et al. Mesenchymal stem cells treated with rat pancreatic extract secrete cytokines that improve the glycometabolism of diabetic rats. Transplant Proc. 2009;41(5):1878-1884.
[17] Han EY, Chun MH, Kim ST, et al. Injection time-dependent effect of adult human bone marrow stromal cell transplantation in a rat model of severe traumatic brain injury. Curr Stem Cell Res Ther. 2013; 8(2):172-181.
[18] Walker PA, Shah SK, Jimenez F, et al. Bone marrow-derived stromal cell therapy for traumatic brain injury is neuroprotective via stimulation of non-neurologic organ systems. Surgery. 2012;152(5): 790-793.
[19] Kawai T, Akira S. TLR signaling. Semin Immunol. 2007; 19(1):24-32.
[20] Xu JW, Ikeda K, Yamori Y. Inhibitory effect of polyphenol cyanidin on TNF-alpha-induced apoptosis through multiple signaling pathways in endothelial cells. Atherosclerosis. 2007;193(2):299-308.
[21] 余科科,汪思应.TNF-α诱导肝干细胞凋亡及信号转导途径的改变[J].世界华人消化杂志,2010,18(7):707-710.
[22] Qiao L, Yacoub A, Studer E, et al. Inhibition of the MAPK and PI3K pathways enhances UDCA-induced apoptosis in primary rodent hepatocytes. Hepatology. 2002;35(4):779-789.
[23] Basak S, Hoffmann A. Crosstalk via the NF-kappaB signaling system. Cytokine Growth Factor Rev. 2008; 19(3-4):187-197.
[24] Wang L, Zhao Y, Liu Y, et al. IFN-γ and TNF-α synergistically induce mesenchymal stem cell impairment and tumorigenesis via NFκB signaling. Stem Cells. 2013;31(7):1383-1395.
[25] 张玉龙. TNF-α抑制骨髓间充质干细胞参与糖尿病创面愈合的机制研究[D].重庆:第三军医大学,2013.
[26] Erceg S, Ronaghi M, Oria M, et al. Transplanted oligodendrocytes and motoneuron progenitors generated from human embryonic stem cells promote locomotor recovery after spinal cord transection. Stem Cells. 2010;28(9):1541-1549.
[27] Zheng Z, Yenari MA. Post-ischemic inflammation: molecular mechanisms and therapeutic implications. Neurol Res. 2004;26(8):884-892.
[28] Xian YF, Lin ZX, Mao QQ, et al. Isorhynchophylline Protects PC12 Cells Against Beta-Amyloid-Induced Apoptosis via PI3K/Akt Signaling Pathway. Evid Based Complement Alternat Med. 2013;2013:163057.
[29] Kops GJ, Dansen TB, Polderman PE, et al. Forkhead transcription factor FOXO3a protects quiescent cells from oxidative stress. Nature. 2002;419(6904): 316-321.
[30] Furukawa-Hibi Y, Yoshida-Araki K, Ohta T, et al. FOXO forkhead transcription factors induce G(2)-M checkpoint in response to oxidative stress. J Biol Chem. 2002;277(30):26729-26732.
[31] Medema RH, Kops GJ, Bos JL, et al. AFX-like Forkhead transcription factors mediate cell-cycle regulation by Ras and PKB through p27kip1. Nature. 2000;404(6779):782-787.
[32] Fukunaga K, Ishigami T, Kawano T. Transcriptional regulation of neuronal genes and its effect on neural functions: expression and function of forkhead transcription factors in neurons. J Pharmacol Sci. 2005;98(3):205-211.
[33] Okada T, Liew CW, Hu J, et al. Insulin receptors in beta-cells are critical for islet compensatory growth response to insulin resistance. Proc Natl Acad Sci U S A. 2007;104(21):8977-8982.
[34] Nakae J, Kitamura T, Kitamura Y, et al. The forkhead transcription factor Foxo1 regulates adipocyte differentiation. Dev Cell. 2003;4(1):119-129.
[35] Nemoto S, Finkel T. Redox regulation of forkhead proteins through a p66shc-dependent signaling pathway. Science. 2002;295(5564):2450-2452.
[36] Storz P. Forkhead homeobox type O transcription factors in the responses to oxidative stress. Antioxid Redox Signal. 2011;14(4):593-605.
[37] Biggs WH 3rd, Meisenhelder J, Hunter T, et al. Protein kinase B/Akt-mediated phosphorylation promotes nuclear exclusion of the winged helix transcription factor FKHR1. Proc Natl Acad Sci U S A. 1999;96(13): 7421-7426.
[38] Medema RH, Kops GJ, Bos JL, et al. AFX-like Forkhead transcription factors mediate cell-cycle regulation by Ras and PKB through p27kip1. Nature. 2000;404(6779):782-787.
[39] Huang H, Tindall DJ. Dynamic FoxO transcription factors. J Cell Sci. 2007;120(Pt 15):2479-2487.
[40] Katayama K, Nakamura A, Sugimoto Y, et al. FOXO transcription factor-dependent p15(INK4b) and p19(INK4d) expression. Oncogene. 2008;27(12): 1677-1686.