Effect and mechanism of chronic oxidative stress induced by hydrogen peroxide on microglial celluar senescence_West China Medical Journal

Authors：

LI Hongyan ¹ , MAO Anqiong ¹ , LIU Ting ² , PENG Minghui ¹ , ZHANG Min ¹ ,  ZHANG Ying ¹

1. Department of Anesthesiology, the Affiliated Traditional Chinese Medicine Hospital of Southwest Medical University, Luzhou, Sichuan 646000, P. R. China;
2. Department of Anesthesiology, Zigong first people’s hospital, Zigong, Sichuan 643000, P. R. China;

Corresponding author：

ZHANG Ying, Email: zhangying021210@163.com

Keywords：

Aging; microglia; hydrogen peroxide; neurodegenerative diseases; celluar senescence

DOI：

10.7507/1002-0179.202302081

Video：

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Objective To explore the role of hydrogen peroxide (H₂O₂) in inducing chronic oxidative stress in microglia aging. Methods BV2 microglia purchased from ATCC in less than 10 generations were treated with 0, 50, 100, 200 μmol/L H₂O₂ at different concentrations. According to the concentration of H₂O₂ used, the BV2 microglia were divided into a control group and H₂O₂ -50 μmol/L Group, H₂O₂ -100 μmol/L Group, H₂O₂ -200 μmol/L Group. Cell proliferation was measured by CCK8 cell proliferation assay. Age-related β-galactosidase (SA-β-gal) staining assay, and expression of age-related cyclin molecules p16, p21, p53 and senescence sssociated secretory phenotype interleukin 1 beta (IL-1β), transforming growth factor-β (TGF-β) and matrix metalloprotein 9 (MMP9) detected by quantitative real-time polymerase chain reaction were used to measure celluar senescence. Results During the induction process, H₂O₂-200 μmol/L caused significant damage to BV2 microglia, therefore no subsequent testing was conducted. Finally, the control group, H₂O₂-50 μmol/L group and H₂O₂-100 μmol/L group cells were collected. The differences in cell survival rate (F=46.176, P<0.001) and positive rate of SA-β-gal staining (F=553.1, P<0.001) among the three groups were statistically significant. The cell survival rate of H₂O₂-50 μmol/L group had no significant change (P>0.05), while the cell survival rate of H₂O₂-100 μmol/L group decreased significantly (P<0.001). The positive rate of SA-β-gal staining in H₂O₂-50 μmol/L group and H₂O₂-100 μmol/L group was increased (P<0.001), and the positive rate of SA-β-gal staining in H₂O₂-100 μmol/L group was higher than that in H₂O₂-50 μmol/L group (P<0.001). The mRNA levels of senescence related cyclin molecules p16, p21 and p53 were up-regulated under the induction of 50, 100 μmol/L H₂O₂ (P<0.05), and the expressions of IL-1β, TGF-β and MMP9 of senescence associated secretory phenotype were increased (P<0.05). The increase of H₂O₂-50 μmol/L group was more obvious (P<0.05). Conclusion The aging model of BV2 microglia can be successfully established by inducing 8 d with 100 μmol/L H₂O₂, and the mechanism may be related to promoting the secretion of p16, p21, p53, IL-1β, TGF-β and MMP9.

Citation： LI Hongyan, MAO Anqiong, LIU Ting, PENG Minghui, ZHANG Min, ZHANG Ying. Effect and mechanism of chronic oxidative stress induced by hydrogen peroxide on microglial celluar senescence. West China Medical Journal, 2023, 38(8): 1188-1194. doi: 10.7507/1002-0179.202302081 Copy

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17.	Chen H, Gu X, Liu Y, et al. PDGF signalling controls age-dependent proliferation in pancreatic β-cells. Nature, 2011, 478(7369): 349-355.
18.	He S, Sharpless NE. Senescence in health and disease. Cell, 2017, 169(6): 1000-1011.
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21.	刘洋, 吴晓冰, 荆永光, 等. 过氧化氢诱导间充质干细胞衰老模型的建立. 军事医学, 2015, 39(5): 329-333.
22.	潘长伟, 赵峰, 郎宏鑫, 等. 过氧化氢诱导人皮肤成纤维细胞衰老模型的建立. 解剖科学进展, 2019, 25(2): 193-195, 199.
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31.	Lopes-Paciencia S, Saint-Germain E, Rowell MC, et al. The senescence-associated secretory phenotype and its regulation. Cytokine, 2019, 117: 15-22.
32.	Huang Z, Lan J, Gao X. Feprazone mitigates IL-1β-induced cellular senescence in chondrocytes. ACS Omega, 2021, 6(14): 9442-9448.
33.	付美艳, 杨镜以, 温欣, 等. 青藤碱通过阻止 NF-κB 通路激活抑制 IL-1β诱导的椎间盘终板软骨细胞炎症及退变. 中国老年学杂志, 2022, 42(16): 4045-4049.
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35.	叶洪, 金建生, 陈晓春, 等. 人参皂苷 Rg1 通过下调 TGFβ₁ 及βigh3 表达延缓拟衰老小鼠肾脏纤维化. 中国老年学杂志, 2014, 34(21): 6143-6146.
36.	Lynn SA, Soubigou F, Dewing JM, et al. An exploratory study provides insights into MMP9 and Aβ levels in the vitreous and blood across different ages and in a subset of AMD patients. Int J Mol Sci, 2022, 23(23): 14603.
37.	Pan J, Ma N, Yu B, et al. Transcriptomic profiling of microglia and astrocytes throughout aging. J Neuroinflammation, 2020, 17(1): 97.

1. Niccoli T, Partridge L. Ageing as a risk factor for disease. Curr Biol, 2012, 22(17): R741-R752.
2. Hou Y, Dan X, Babbar M, et al. Ageing as a risk factor for neurodegenerative disease. Nat Rev Neurol, 2019, 15(10): 565-581.
3. Mertens J, Reid D, Lau S, et al. Aging in a dish: iPSC-derived and directly induced neurons for studying brain aging and age-related neurodegenerative diseases. Annu Rev Genet, 2018, 52: 271-293.
4. Kritsilis M, V Rizou S, Koutsoudaki PN, et al. Ageing, cellular senescence and neurodegenerative disease. Int J Mol Sci, 2018, 19(10): 2937.
5. Mecca C, Giambanco I, Donato R, et al. Microglia and aging: the role of the TREM2-DAP12 and CX3CL1-CX3CR1 axes. Int J Mol Sci, 2018, 19(1): 318.
6. Aguzzi A, Barres BA, Bennett ML. Microglia: scapegoat, saboteur, or something else?. Science, 2013, 339(6116): 156-161.
7. Hua K, Schindler M K, McQuail JA, et al. Regionally distinct responses of microglia and glial progenitor cells to whole brain irradiation in adult and aging rats. PLoS One, 2012, 7(12): e52728.
8. Lee JD, Kamaruzaman NA, Fung JN, et al. Dysregulation of the complement cascade in the hSOD1G93A transgenic mouse model of amyotrophic lateral sclerosis. J Neuroinflammation, 2013, 10: 119.
9. Kwon HS, Koh SH. Neuroinflammation in neurodegenerative disorders: the roles of microglia and astrocytes. Transl Neurodegener, 2020, 9(1): 42.
10. HAYFLICK L. The limited in vitro lifetime of human diploid cell strains. Exp Cell Res, 1965, 37: 614-636.
11. 张凌云, 刘缨. 细胞衰老检测指标及衰老模型研究进展. 生命科学研究, 2022, 26(3): 264-270.
12. 张宇, 柏云, 魏璐, 等. 构建肾小管上皮细胞衰老模型的研究. 中华老年医学杂志, 2021, 40(10): 1255-1259.
13. 牛海名, 李建伟, 陈妙莲, 等. 外源性硫化氢改善人脐静脉内皮细胞早熟性衰老与 eNOS 表达的关系. 中国处方药, 2022, 20(1): 7-11.
14. Muñoz-Espín D, Serrano M. Cellular senescence: from physiology to pathology. Nat Rev Mol Cell Biol, 2014, 15(7): 482-496.
15. Campisi J. Aging, cellular senescence, and cancer. Annu Rev Physiol, 2013, 75: 685-705.
16. Chang J, Wang Y, Shao L, et al. Clearance of senescent cells by ABT263 rejuvenates aged hematopoietic stem cells in mice. Nat Med, 2016, 22(1): 78-83.
17. Chen H, Gu X, Liu Y, et al. PDGF signalling controls age-dependent proliferation in pancreatic β-cells. Nature, 2011, 478(7369): 349-355.
18. He S, Sharpless NE. Senescence in health and disease. Cell, 2017, 169(6): 1000-1011.
19. Mohamad Kamal NS, Safuan S, Shamsuddin S, et al. Aging of the cells: insight into cellular senescence and detection methods. Eur J Cell Biol, 2020, 99(6): 151108.
20. Toussaint O, Royer V, Salmon M, et al. Stress-induced premature senescence and tissue ageing. Biochem Pharmacol, 2002, 64(5/6): 1007-1009.
21. 刘洋, 吴晓冰, 荆永光, 等. 过氧化氢诱导间充质干细胞衰老模型的建立. 军事医学, 2015, 39(5): 329-333.
22. 潘长伟, 赵峰, 郎宏鑫, 等. 过氧化氢诱导人皮肤成纤维细胞衰老模型的建立. 解剖科学进展, 2019, 25(2): 193-195, 199.
23. González-Gualda E, Baker AG, Fruk L, et al. A guide to assessing cellular senescence in vitro and in vivo. FEBS J, 2021, 288(1): 56-80.
24. Dimri GP, Lee X, Basile G, et al. A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci U S A, 1995, 92(20): 9363-9367.
25. Lewis-McDougall FC, Ruchaya PJ, Domenjo-Vila E, et al. Aged-senescent cells contribute to impaired heart regeneration. Aging Cell, 2019, 18(3): e12931.
26. Dang Y, An Y, He J, et al. Berberine ameliorates cellular senescence and extends the lifespan of mice via regulating p16 and cyclin protein expression. Aging Cell, 2020, 19(1): e13060.
27. Jiang Y, Dong G, Song Y. Nucleus pulposus cell senescence is alleviated by resveratrol through regulating the ROS/NF-κB pathway under high-magnitude compression. Biosci Rep, 2018, 38(4): BSR20180670.
28. Beauséjour CM, Krtolica A, Galimi F, et al. Reversal of human cellular senescence: roles of the p53 and p16 pathways. EMBO J, 2003, 22(16): 4212-4222.
29. McHugh D, Gil J. Senescence and aging: causes, consequences, and therapeutic avenues. J Cell Biol, 2018, 217(1): 65-77.
30. Birch J, Gil J. Senescence and the SASP: many therapeutic avenues. Genes Dev, 2020, 34(23/24): 1565-1576.
31. Lopes-Paciencia S, Saint-Germain E, Rowell MC, et al. The senescence-associated secretory phenotype and its regulation. Cytokine, 2019, 117: 15-22.
32. Huang Z, Lan J, Gao X. Feprazone mitigates IL-1β-induced cellular senescence in chondrocytes. ACS Omega, 2021, 6(14): 9442-9448.
33. 付美艳, 杨镜以, 温欣, 等. 青藤碱通过阻止 NF-κB 通路激活抑制 IL-1β诱导的椎间盘终板软骨细胞炎症及退变. 中国老年学杂志, 2022, 42(16): 4045-4049.
34. Morikawa M, Derynck R, Miyazono K. TGF-β and the TGF-β family: context-dependent roles in cell and tissue physiology. Cold Spring Harb Perspect Biol, 2016, 8(5): a021873.
35. 叶洪, 金建生, 陈晓春, 等. 人参皂苷 Rg1 通过下调 TGFβ₁ 及βigh3 表达延缓拟衰老小鼠肾脏纤维化. 中国老年学杂志, 2014, 34(21): 6143-6146.
36. Lynn SA, Soubigou F, Dewing JM, et al. An exploratory study provides insights into MMP9 and Aβ levels in the vitreous and blood across different ages and in a subset of AMD patients. Int J Mol Sci, 2022, 23(23): 14603.
37. Pan J, Ma N, Yu B, et al. Transcriptomic profiling of microglia and astrocytes throughout aging. J Neuroinflammation, 2020, 17(1): 97.

West China Medical Journal

Effect and mechanism of chronic oxidative stress induced by hydrogen peroxide on microglial celluar senescence

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