Erythropoietin-overexpressed umbilical cord mesenchymal stem cells inhibit neuroapoptosis in ischemic-hypoxic SH-SY5Y and its mechanism

doi:10.12307/2024.704

Abstract

Abstract: BACKGROUND: Previous studies have successfully constructed erythropoietin-overexpressed umbilical cord mesenchymal stem cells. It was found that the apoptosis of ischemic and hypoxic human neuroblastoma cell line (SH-SY5Y) was significantly reduced by erythropoietin-overexpressed umbilical cord mesenchymal stem cells.
OBJECTIVE: To explore the possible neuroprotective mechanisms of erythropoietin-overexpressed umbilical cord mesenchymal stem cells against ischemic-hypoxic SH-SY5Y and their associated epigenetic mechanisms.
METHODS: Oxygen-glucose deprivation was applied to ischemia-hypoxia-induced SH-SY5Y cell injury, and multifactorial assays were applied to detect the expression levels of inflammatory factors in the cells before and after hypoxia and co-culture, respectively, with mesenchymal stem cells, as well as lentiviral-transfected null-loaded plasmids of the negative control mesenchymal stem cells and erythropoietin-overexpressed umbilical cord mesenchymal stem cells. The expression levels of supernatant inflammatory factors were detected by multifactor assay after co-culture. Proteomics was used to detect the differentially expressed proteins of negative control mesenchymal stem cells and erythropoietin-overexpressed umbilical cord mesenchymal stem cells. Cleavage under targets and tagmentation sequencing was applied to detect genomic H3K4me2 modification, and joint analysis was conducted with RNA-sequencing. Lentiviral vector infection was applied to construct the stable knockdown of REST in SH-SY5Y cells. qRT-PCR and western blot assay were performed to detect the expression level of REST. The apoptosis was detected by flow cytometry after co-culture of oxygen-glucose deprivation treatment with erythropoietin-overexpressed umbilical cord mesenchymal stem cells. The expression difference of H3K36me3 group proteins was detected by western blot assay, and transcriptome sequencing was performed to analyze the differentially expressed genes.
RESULTS AND CONCLUSION: (1) Compared with the control group, monocyte chemotactic protein 1, interleukin-6, interleukin-18, and interleukin-1 beta, interferon α2, and interleukin-23 levels significantly increased in the cerebrospinal fluid supernatant of patients with ischemic-hypoxic encephalopathy (P < 0.01). (2) After co-culturing SH-SY5Y cells with erythropoietin-overexpressed umbilical cord mesenchymal stem cells under ischemia and hypoxia, the expression levels of monocyte chemotactic protein 1 and interleukin-6 were significantly reduced. (3) Analysis of protein network interactions revealed significant downregulation of monocyte chemotactic protein 1, interleukin-6 related regulatory proteins CXCL1 and BGN. (4) Transcriptome sequencing analysis found that pro-inflammatory genes were down-regulated, and functional enrichment of histone modifications, and the expression of transcription factors REST and TET3 significantly up-regulated in the erythropoietin-overexpressed umbilical cord mesenchymal stem cell group compared with the negative control mesenchymal stem cell group. (5) Combined analysis of transcriptome sequencing and cleavage under targets and tagmentation revealed changes in epigenetic levels as well as significant activation of the promoter regions of transcription factors REST and TET3. (6) Stable knockdown REST in SH-SY5Y cells was successfully constructed; the transcript levels of REST mRNA and protein expression were both decreased. (7) After the REST knockdown SH-SY5Y cells were co-cultured with erythropoietin-overexpressed umbilical cord mesenchymal stem cells, apoptosis was significantly increased and H3K36me3 expression was significantly decreased. Transcriptome sequencing results showed that the expression of inflammation-related genes Aldh1l2 and Cth, as well as apoptosis-suppressor genes Mapk8ip1 and Sod2 was reduced at mRNA transcription level (P < 0.01). (8) It is concluded that erythropoietin-overexpressed umbilical cord mesenchymal stem cells activated the expression of REST and TET3 by altering the kurtosis of H3K4me2 and upregulated the modification level of H3K36me3, which in turn regulated the expression of inflammation-related genes Aldh1l2 and Cth, as well as apoptosis-suppressor genes Mapk8ip1 and Sod2, and facilitated neuronal survival.

Key words: umbilical cord mesenchymal stem cell, histone modification, ischemic-hypoxic encephalopathy, gene modification, chromosome targeted cleavage and labeling, neuron-restrictive silencer factor, monocyte chemotactic protein-1, interleukin-6

CLC Number:

Li Ruibo, Kong Ning, Sun Lei, Ma Baodong, Jin Ranran, Zhang Wenjin, Yue Han, Zhang Hui. Erythropoietin-overexpressed umbilical cord mesenchymal stem cells inhibit neuroapoptosis in ischemic-hypoxic SH-SY5Y and its mechanism[J]. Chinese Journal of Tissue Engineering Research, 2024, 28(31): 4937-4944.

Figures/Tables 10

References

[1] 王文广,王斌,胡颖杰,等.MRI与CT对新生儿缺血缺氧性脑病诊断价值的分析[J].中国妇幼保健,2015,30(4):639-641.
[2] WANG W, JIA L. Regulatory Mechanism of MicroRNA-30b on Neonatal Hypoxic-Ischemic Encephalopathy (HIE). J Stroke Cerebrovasc Dis. 2021;30(3):105553.
[3] LIU CY, AL-WARD H, NGAFFO MEKONTSO F, et al. Experimental Study on the Correlation between miRNA-373 and HIF-1α, MMP-9, and VEGF in the Development of HIE. Biomed Res Int. 2021;2021:5553486.
[4] EDMONDS CJ, HELPS SK, HART D, et al. Minor neurological signs and behavioural function at age 2 years in neonatal hypoxic ischaemic encephalopathy (HIE). Eur J Paediatr Neurol. 2020;27:78-85.
[5] LI JB, WU WS, DU B, et al. Impact of mild hypothermia therapy on hemodynamics during the induction stage in neonates with moderate to severe hypoxic-ischemic encephalopathy. Zhongguo Dang Dai Er Ke Za Zhi. 2021;23(2):133-137.
[6] YE L, FENG Z, DOYCHEVA D, et al. CpG-ODN exerts a neuroprotective effect via the TLR9/pAMPK signaling pathway by activation of autophagy in a neonatal HIE rat model. Exp Neurol. 2018;301(Pt A):70-80.
[7] HU X, LI S, DOYCHEVA DM, et al. Rh-CSF1 Attenuates Oxidative Stress and Neuronal Apoptosis via the CSF1R/PLCG2/PKA/UCP2 Signaling Pathway in a Rat Model of Neonatal HIE. Oxid Med Cell Longev. 2020;2020:6801587.
[8] MASSARO AN, MURTHY K, ZANILETTI I, et al. Short-term outcomes after perinatal hypoxic ischemic encephalopathy: a report from the Children’s Hospitals Neonatal Consortium HIE focus group. J Perinatol. 2015;35(4):290-296.
[9] 余静,薛伟,李亚萍.亚低温联合促红细胞生成素对HIE患儿脑部生理和神经系统发育的改善作用[J].临床研究,2023,31(5):65-68.
[10] LI F, ZHANG K, LIU H, et al. The neuroprotective effect of mesenchymal stem cells is mediated through inhibition of apoptosis in hypoxic ischemic injury. World J Pediatr. 2020;16(2):193-200.
[11] LI T, LIU Y, YU L, et al. Human Umbilical Cord Mesenchymal Stem Cells Protect Against SCA3 by Modulating the Level of 70 kD Heat Shock Protein. Cell Mol Neurobiol. 2018;38(3):641-655.
[12] FRENETTE PS, PINHO S, LUCAS D, et al. Mesenchymal stem cell: keystone of the hematopoietic stem cell niche and a stepping-stone for regenerative medicine. Annu Rev Immunol. 2013;31:285-316.
[13] PARK WS, SUNG SI, AHN SY, et al. Hypothermia augments neuroprotective activity of mesenchymal stem cells for neonatal hypoxic-ischemic encephalopathy. PLoS One. 2015;10(3):e0120893.
[14] PARK M, KIM HM, SHIN HA, et al. Human Pluripotent Stem Cell-Derived Neural Progenitor Cells Promote Retinal Ganglion Cell Survival and Axon Recovery in an Optic Nerve Compression Animal Model. Int J Mol Sci. 2021;22(22):12529.
[15] AHMAD KA, BENNETT MM, JUUL SE, et al. Utilization of Erythropoietin within the United States Neonatal Intensive Care Units from 2008 to 2017. Am J Perinatol. 2021;38(7):734-740.
[16] ZHOU H, HE Y, XIONG W, et al. MSC based gene delivery methods and strategies improve the therapeutic efficacy of neurological diseases. Bioact Mater. 2022;23: 409-437.
[17] BELL KF, BENT RJ, MEESE-TAMURI S, et al. Calmodulin kinase IV-dependent CREB activation is required for neuroprotection via NMDA receptor-PSD95 disruption. J Neurochem. 2013;126(2):274-287.
[18] 班跃耀,孙蕾,马保东,等.促红细胞生成素基因修饰的人脐带间充质干细胞对神经元周期和凋亡的调控作用及机制[J].新乡医学院学报,2023,40(10): 909-916.
[19] HWANG JY, ZUKIN RS. REST, a master transcriptional regulator in neurodegenerative disease. Curr Opin Neurobiol. 2018;48:193-200.
[20] DAY JJ. Genetic and epigenetic editing in nervous system. Dialogues Clin Neurosci. 2019;21(4):359-368.
[21] ALY H, KHASHABA MT, EL-AYOUTY M, et al. IL-1beta, IL-6 and TNF-alpha and outcomes of neonatal hypoxic ischemic encephalopathy. Brain Dev. 2006;28(3): 178-182.
[22] 邹良,何秋红,陈秋杨,等. IL-17/IL-23炎症轴在新生儿缺氧缺血性脑病发病中的作用机制探究[J].河北医科大学学报,2023,44(3):305-309.
[23] KUMAR H, KAWAI T, AKIRA S. Pathogen recognition by the innate immune system. Int Rev Immunol. 2011;30(1):16-34.
[24] SINGH S, ANSHITA D, RAVICHANDIRAN V. MCP-1: Function, regulation, and involvement in disease. Int Immunopharmacol. 2021;101(Pt B):107598.
[25] 许云鹤,刘永刚,赵小妹,等.血清SAA、RBP4、MCP-1与缺血性脑卒中脑损伤及梗死程度的关系研究[J].临床和实验医学杂志,2018,17(3):255-258.
[26] 张艳华,孔慧霞,张嘉雯,等.血清及脑脊液IL-1β、PCT、MCP-1水平变化在化脓性脑膜炎患儿病情评估及预后判断中的应用价值[J].中国临床实用医学,2017,8(3):45-47.
[27] GROH J, HEINL K, KOHL B, et al. Attenuation of MCP-1/CCL2 expression ameliorates neuropathy in a mouse model for Charcot-Marie-Tooth 1X. Hum Mol Genet. 2010;19(18):3530-3543.
[28] KOLATTUKUDY PE, NIU J. Inflammation, endoplasmic reticulum stress, autophagy, and the monocyte chemoattractant protein-1/CCR2 pathway. Circ Res. 2012; 110(1):174-189.
[29] HUANG XY, HU QP, SHI HY, et al. Everolimus inhibits PI3K/Akt/mTOR and NF-kB/IL-6 signaling and protects seizure-induced brain injury in rats. J Chem Neuroanat. 2021;114:101960.
[30] HOU SM, CHEN PC, LIN CM, et al. CXCL1 contributes to IL-6 expression in osteoarthritis and rheumatoid arthritis synovial fibroblasts by CXCR2, c-Raf, MAPK, and AP-1 pathway. Arthritis Res Ther. 2020;22(1):251.
[31] ZHANG X, ZHANG F, YAO F, et al. Bergenin has neuroprotective effects in mice with ischemic stroke through antioxidative stress and anti-inflammation via regulating Sirt1/FOXO3a/NF-κB signaling. Neuroreport. 2022;33(13):549-560.
[32] ZHONG Q, ZOU Y, LIU H, et al. Toll-like receptor 4 deficiency ameliorates β2-microglobulin induced age-related cognition decline due to neuroinflammation in mice. Mol Brain. 2020;13(1):20.
[33] CHENG Y, WANG G, ZHAO L, et al. Periplocymarin Induced Colorectal Cancer Cells Apoptosis Via Impairing PI3K/AKT Pathway. Front Oncol. 2021;11:753598.
[34] TIAN Q, GUO Y, FENG S, et al. Inhibition of CCR2 attenuates neuroinflammation and neuronal apoptosis after subarachnoid hemorrhage through the PI3K/Akt pathway. J Neuroinflammation. 2022;19(1):312.
[35] NIE Y, SHU C, SUN X. Cooperative binding of transcription factors in the human genome. Genomics. 2020;112(5):3427-3434.
[36] WANG S, MEYER DH, SCHUMACHER B. H3K4me2 regulates the recovery of protein biosynthesis and homeostasis following DNA damage. Nat Struct Mol Biol. 2020;27(12):1165-1177.
[37] SOLLINGER C, LILLIS J, MALIK J, et al. Erythropoietin Signaling Regulates Key Epigenetic and Transcription Networks in Fetal Neural Progenitor Cells. Sci Rep. 2017;7(1):14381.
[38] KAYA-OKUR HS, WU SJ, CODOMO CA, et al. CUT&Tag for efficient epigenomic profiling of small samples and single cells. Nat Commun. 2019;10(1):1930.
[39] RYAN BJ, BENGOA-VERGNIORY N, WILLIAMSON M, et al. REST Protects Dopaminergic Neurons from Mitochondrial and α-Synuclein Oligomer Pathology in an Alpha Synuclein Overexpressing BAC-Transgenic Mouse Model. J Neurosci. 2021;41(16):3731-3746.
[40] LAM XJ, MANIAM S, CHEAH PS, et al. REST in the Road Map of Brain Development. Cell Mol Neurobiol. 2023;43(7):3417-3433.
[41] PERERA A, EISEN D, WAGNER M, et al. TET3 is recruited by REST for context-specific hydroxymethylation and induction of gene expression. Cell Rep. 2015; 11(2):283-294.
[42] ABDERRAHMANI A, STEINMANN M, PLAISANCE V, et al. The transcriptional repressor REST determines the cell-specific expression of the human MAPK8IP1 gene encoding IB1 (JIP-1). Mol Cell Biol. 2001;21(21):7256-7267.
[43] MARTIN D, ALLAGNAT F, GESINA E, et al. Specific silencing of the REST target genes in insulin-secreting cells uncovers their participation in beta cell survival. PLoS One. 2012;7(9):e45844.
[44] KINUGAWA S, WANG Z, KAMINSKI PM, et al. Limited exercise capacity in heterozygous manganese superoxide dismutase gene-knockout mice: roles of superoxide anion and nitric oxide. Circulation. 2005;111(12):1480-1486.