Effect of directive differentiation of microglia by SN50 on hypoxia-caused neurons injury in mice_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

 HAN Fangfang ^1,2 , LIU Chunlong ² , YANG Changqing ³ , SUN Yuanhua ³

1. Medical Treatment Department, Luohe Medical College, Luohe Henan, 462002, P.R.China;
2. Clinical Medical College of Acupuncture Moxibustion and Rehabilitation, Guangzhou University of Chinese Medicine, Guangzhou Guangdong, 510006, P.R.China;
3. Department of Pediatrics, the Third Affiliated Hospital, Luohe Medical College, Luohe Henan, 462002, P.R.China;

Corresponding author：

HAN Fangfang, Email: hanfangfanglh@163.com

Keywords：

Microglia; neuron; nuclear factor κB; cell differentiation; hypoxic injury; mouse

DOI：

10.7507/1002-1892.201905131

Video：

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Objective To explore the effect and mechanism of directive differentiation of microglia by SN50 on hypoxia-caused neurons injury in mice.Methods The microglia were isolated and purified from brain tissue of new-born BALB/c mice through differential velocity adherent and vibration technique. The quantity of the microglia was identified by immunofluorescence staining of inducible nitric oxide synthetase (iNOS) and ionized calcium binding adapter molecule 1 (Iba1) and real-time fluorescence quantitative PCR (qRT-PCR) for special expression genes [iNOS, CD32, and interlenkin 10 (IL-10)]. Then the microglia were cultured with SN50, and the expressions of nuclear factor κB (NF-κB), differentiation-related genes (iNOS, CD11b, IL-10, and CD206), and apoptosis were detected by Western blot, qRT-PCR, and flow cytometry, respectively. The hypoxia model of neuron was established, and the cell apoptosis was evaluated by MTT after 0, 2, 6, 12, 24, and 48 hours of anoxic treatment. The apoptosis related markers (Bcl-2 and Caspase-3) were measured by Western blot and flow cytometry. In addition, the neurons after anoxic treatment were co-cultured with SN50 treated microglia (experimental group) and normal microglia (control group) for 24 hours. And the cell viability and apoptosis related markers (Bcl-2 and Caspase-3) were also measured.Results Immunofluorescence staining and qRT-PCR analysis showed that the cells expressed the specific proteins and genes of microglia. Compared with the normal microglia, the relative expressions of NF-κB protein and iNOS and CD11b mRNAs in the microglia treated with SN50 significantly decreased (P<0.05), the relative expressions of IL-10 and CD206 mRNAs significantly increased (P<0.05), and the cell apoptosis rate had no significant change (P>0.05). Compared with the normal neurons, the cell viability, the relative expressions of Bcl-2 and Caspase-3 proteins after anoxic treatment significantly decreased (P<0.05), while the relative expressions of cleaved-Caspase-3 protein and cell apoptosis rate of neurons significantly increased (P<0.05). In the co-culture system, the cell viability, the relative expressions of Bcl-2 and Caspase-3 proteins were significantly higher in experimental group than those in control group (P<0.05), while the relative expressions of cleaved-Caspase-3 protein and cell apoptosis rate were significantly lower in experimental group than those in control group (P<0.05).Conclusion SN50 can induce the microglia differentiation into M2 type through NF-κB pathway. The SN50-induced microglia can protect neurons from hypoxic injury.

Citation： HAN Fangfang, LIU Chunlong, YANG Changqing, SUN Yuanhua. Effect of directive differentiation of microglia by SN50 on hypoxia-caused neurons injury in mice. Chinese Journal of Reparative and Reconstructive Surgery, 2020, 34(4): 509-517. doi: 10.7507/1002-1892.201905131 Copy

1.	Orihuela R, McPherson CA, Harry GJ. Microglial M1/M2 polarization and metabolic states. Br J Pharmacol, 2016, 173(4): 649-665.
2.	Orczykowski ME, Calderazzo SM, Shobin E, et al. Cell based therapy reduces secondary damage and increases extent of microglial activation following cortical injury. Brain Research, 2019, 1717: 147-159.
3.	泰文娇, 叶旋, 鲍秀琦, 等. 小胶质细胞与脑缺血关系的研究进展. 药学学报, 2012, 47(3): 346-353.
4.	Ma D, Zhao Y, Huang L, et al. A novel hydrogel-based treatment for complete transection spinal cord injury repair is driven by microglia/macrophages repopulation. Biomaterials, 2020, 237: 119830.
5.	Pronin A, Pham D, An W, et al. Inflammasome activation induces pyroptosis in the retina exposed to ocular hypertension injury. Front Mol Neurosci, 2019, 12: 36.
6.	Wei W, Lan XB, Liu N, et al. Echinacoside alleviates hypoxic-ischemic brain injury in neonatal rat by enhancing antioxidant capacity and inhibiting apoptosis. Neurochem Res, 2019, 44(7): 1582-1592.
7.	任宪盛, 丁巍, 杨小玉. 虾青素对大鼠脊髓损伤后细胞凋亡的影响. 中国修复重建外科杂志, 2018, 32(5): 41-46.
8.	Frank-Cannon TC, Alto LT, McAlpine FE, et al. Does neuroinflammation fan the flame in neurodegenerative diseases? Mol Neurodegener, 2009, 4: 47.
9.	Chian CF, Chiang CH, Chuang CH, et al. SN50, a cell-permeable-inhibitor of nuclear factor-κB, attenuates ventilator-induced lung injury in an isolated and perfused rat lung model. Shock, 2016, 46(2): 194-201.
10.	Mizuma A, Kim JY, Kacimi R, et al. Microglial calcium release-activated calcium (CRAC) channel inhibition improves outcome from experimental traumatic brain injury and microglia-induced neuronal death. J Neurotrauma, 2019, 36(7): 996-1007.
11.	Zhang W, Potrovita I, Tarabin V, et al. Neuronal activation of NF-kappaB contributes to cell death in cerebral ischemia. J Cereb Blood Flow Metab, 2005, 25(1): 30-40.
12.	Kim EJ, Kwon KJ, Park JY, et al. Effects of peroxisome proliferator-activated receptor agonists on LPS-induced neuronal death in mixed cortical neurons: associated with iNOS and COX-2. Brain Res, 2002, 941(1-2): 1-10.
13.	Qin H, Xu HZ, Gong YQ. Mechanism of NF-κB signaling pathway and autophagy in the regulation of osteoblast differentiation. Mol Membr Biol, 2016, 33(6-8): 138-144.
14.	Lin L, Hu K. Tissue-type plasminogen activator modulates macrophage M2 to M1 phenotypic change through annexin A2-mediated NF-κB pathway. Oncotarget, 2017, 8(50): 88094-88103.
15.	Conti P, Lauritano D, Caraffa A, et al. Microglia and mast cells generate proinflammatory cytokines in the brain and worsen inflammatory state: Suppressor effect of IL-37. Eur J Pharmacol, 2020.
16.	Ganbold T, Bao Q, Zandan J, et al. Modulation of microglia polarization through Silencing of NF-kB p65 by functionalized curdlan nanoparticle mediated RNAi. ACS Applied Materials & Interfaces, 2020. doi: 10.1021/acsami.9b23004.
17.	Yan S, Xuan Z, Yang M, et al. CSB6B prevents β-amyloid-associated neuroinflammation and cognitive impairments via inhibiting NF-κB and NLRP3 in microglia cells. Int Immunopharmacol, 2020, 81: 106263.
18.	Chian CF, Chiang CH, Chuang CH, et al. Inhibitor of nuclear factor-κB, SN50, attenuates lipopolysaccharide-induced lung injury in an isolated and perfused rat lung model. Transl Res, 2014, 163(3): 211-220.
19.	刘静, 刘婷, 徐睿玲, 等. SN50 联合 5-氟尿嘧啶通过调节 NF-κB 信号通路抑制人胃癌裸鼠移植瘤生长的研究. 胃肠病学和肝病学杂志, 2016, 25(1): 5-8.
20.	习亮, 梁伟, 张永峰. NF-κB 信号通路抑制剂对 P 物质作用后脊髓星形胶质细胞 GFAP、炎性因子表达的影响. 山东医药, 2017, 57(44): 51-53.
21.	Sun YX, Dai DK, Liu R, et al. Therapeutic effect of SN50, an inhibitor of nuclear factor-κB, in treatment of TBI in mice. Neurol Sci, 2013, 34(3): 345-355.
22.	Wilcock DM. Neuroinflammatory phenotypes and their roles in Alzheimer’s disease. Neurodegener Dis, 2013, 13(2-3): 183-185.
23.	Tang Y, Le W. Differential roles of M1 and M2 microglia in neurodegenerative diseases. Mol Neurobiol, 2016, 53(2): 1181-1194.
24.	Polazzi E, Monti B. Microglia and neuroprotection: from in vitro studies to therapeutic applications. Prog Neurobiol, 2010, 92(3): 293-315.
25.	钟泽祥, 周亦楠, 冯思思, 等. 慢病毒介导小干扰 RNA 干扰促分裂原和应激激活的蛋白激酶 1 对大鼠脊髓损伤的修复作用. 中国修复重建外科杂志, 2018, 32(7): 941-950.
26.	龚飞宇, 魏在荣, 金文虎, 等. 人羊膜间充质干细胞来源的雪旺细胞样细胞移植在皮瓣神经再生中的作用. 中国修复重建外科杂志, 2018, 32(1): 80-90.
27.	徐明均, 李晓国, 马晨, 等. 人胎盘 MSCs 移植对急性肺损伤小鼠肺血管内皮通透性及肺组织损伤修复的影响. 中国修复重建外科杂志, 2020, 34(3): 387-392.

1. Orihuela R, McPherson CA, Harry GJ. Microglial M1/M2 polarization and metabolic states. Br J Pharmacol, 2016, 173(4): 649-665.
2. Orczykowski ME, Calderazzo SM, Shobin E, et al. Cell based therapy reduces secondary damage and increases extent of microglial activation following cortical injury. Brain Research, 2019, 1717: 147-159.
3. 泰文娇, 叶旋, 鲍秀琦, 等. 小胶质细胞与脑缺血关系的研究进展. 药学学报, 2012, 47(3): 346-353.
4. Ma D, Zhao Y, Huang L, et al. A novel hydrogel-based treatment for complete transection spinal cord injury repair is driven by microglia/macrophages repopulation. Biomaterials, 2020, 237: 119830.
5. Pronin A, Pham D, An W, et al. Inflammasome activation induces pyroptosis in the retina exposed to ocular hypertension injury. Front Mol Neurosci, 2019, 12: 36.
6. Wei W, Lan XB, Liu N, et al. Echinacoside alleviates hypoxic-ischemic brain injury in neonatal rat by enhancing antioxidant capacity and inhibiting apoptosis. Neurochem Res, 2019, 44(7): 1582-1592.
7. 任宪盛, 丁巍, 杨小玉. 虾青素对大鼠脊髓损伤后细胞凋亡的影响. 中国修复重建外科杂志, 2018, 32(5): 41-46.
8. Frank-Cannon TC, Alto LT, McAlpine FE, et al. Does neuroinflammation fan the flame in neurodegenerative diseases? Mol Neurodegener, 2009, 4: 47.
9. Chian CF, Chiang CH, Chuang CH, et al. SN50, a cell-permeable-inhibitor of nuclear factor-κB, attenuates ventilator-induced lung injury in an isolated and perfused rat lung model. Shock, 2016, 46(2): 194-201.
10. Mizuma A, Kim JY, Kacimi R, et al. Microglial calcium release-activated calcium (CRAC) channel inhibition improves outcome from experimental traumatic brain injury and microglia-induced neuronal death. J Neurotrauma, 2019, 36(7): 996-1007.
11. Zhang W, Potrovita I, Tarabin V, et al. Neuronal activation of NF-kappaB contributes to cell death in cerebral ischemia. J Cereb Blood Flow Metab, 2005, 25(1): 30-40.
12. Kim EJ, Kwon KJ, Park JY, et al. Effects of peroxisome proliferator-activated receptor agonists on LPS-induced neuronal death in mixed cortical neurons: associated with iNOS and COX-2. Brain Res, 2002, 941(1-2): 1-10.
13. Qin H, Xu HZ, Gong YQ. Mechanism of NF-κB signaling pathway and autophagy in the regulation of osteoblast differentiation. Mol Membr Biol, 2016, 33(6-8): 138-144.
14. Lin L, Hu K. Tissue-type plasminogen activator modulates macrophage M2 to M1 phenotypic change through annexin A2-mediated NF-κB pathway. Oncotarget, 2017, 8(50): 88094-88103.
15. Conti P, Lauritano D, Caraffa A, et al. Microglia and mast cells generate proinflammatory cytokines in the brain and worsen inflammatory state: Suppressor effect of IL-37. Eur J Pharmacol, 2020.
16. Ganbold T, Bao Q, Zandan J, et al. Modulation of microglia polarization through Silencing of NF-kB p65 by functionalized curdlan nanoparticle mediated RNAi. ACS Applied Materials & Interfaces, 2020. doi: 10.1021/acsami.9b23004.
17. Yan S, Xuan Z, Yang M, et al. CSB6B prevents β-amyloid-associated neuroinflammation and cognitive impairments via inhibiting NF-κB and NLRP3 in microglia cells. Int Immunopharmacol, 2020, 81: 106263.
18. Chian CF, Chiang CH, Chuang CH, et al. Inhibitor of nuclear factor-κB, SN50, attenuates lipopolysaccharide-induced lung injury in an isolated and perfused rat lung model. Transl Res, 2014, 163(3): 211-220.
19. 刘静, 刘婷, 徐睿玲, 等. SN50 联合 5-氟尿嘧啶通过调节 NF-κB 信号通路抑制人胃癌裸鼠移植瘤生长的研究. 胃肠病学和肝病学杂志, 2016, 25(1): 5-8.
20. 习亮, 梁伟, 张永峰. NF-κB 信号通路抑制剂对 P 物质作用后脊髓星形胶质细胞 GFAP、炎性因子表达的影响. 山东医药, 2017, 57(44): 51-53.
21. Sun YX, Dai DK, Liu R, et al. Therapeutic effect of SN50, an inhibitor of nuclear factor-κB, in treatment of TBI in mice. Neurol Sci, 2013, 34(3): 345-355.
22. Wilcock DM. Neuroinflammatory phenotypes and their roles in Alzheimer’s disease. Neurodegener Dis, 2013, 13(2-3): 183-185.
23. Tang Y, Le W. Differential roles of M1 and M2 microglia in neurodegenerative diseases. Mol Neurobiol, 2016, 53(2): 1181-1194.
24. Polazzi E, Monti B. Microglia and neuroprotection: from in vitro studies to therapeutic applications. Prog Neurobiol, 2010, 92(3): 293-315.
25. 钟泽祥, 周亦楠, 冯思思, 等. 慢病毒介导小干扰 RNA 干扰促分裂原和应激激活的蛋白激酶 1 对大鼠脊髓损伤的修复作用. 中国修复重建外科杂志, 2018, 32(7): 941-950.
26. 龚飞宇, 魏在荣, 金文虎, 等. 人羊膜间充质干细胞来源的雪旺细胞样细胞移植在皮瓣神经再生中的作用. 中国修复重建外科杂志, 2018, 32(1): 80-90.
27. 徐明均, 李晓国, 马晨, 等. 人胎盘 MSCs 移植对急性肺损伤小鼠肺血管内皮通透性及肺组织损伤修复的影响. 中国修复重建外科杂志, 2020, 34(3): 387-392.

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