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：

Export PDF Favorites Scan Get Citation

Abstract Full text Figures/Tables Video References Cited by

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.	泰文嬌, 葉旋, 鮑秀琦, 等. 小膠質細胞與腦缺血關系的研究進展. 藥學學報, 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.	任憲盛, 丁巍, 楊小玉. 蝦青素對大鼠脊髓損傷后細胞凋亡的影響. 中國修復重建外科雜志, 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.	劉靜, 劉婷, 徐睿玲, 等. SN50 聯合 5-氟尿嘧啶通過調節 NF-κB 信號通路抑制人胃癌裸鼠移植瘤生長的研究. 胃腸病學和肝病學雜志, 2016, 25(1): 5-8.
20.	習亮, 梁偉, 張永峰. NF-κB 信號通路抑制劑對 P 物質作用后脊髓星形膠質細胞 GFAP、炎性因子表達的影響. 山東醫藥, 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.	鐘澤祥, 周亦楠, 馮思思, 等. 慢病毒介導小干擾 RNA 干擾促分裂原和應激激活的蛋白激酶 1 對大鼠脊髓損傷的修復作用. 中國修復重建外科雜志, 2018, 32(7): 941-950.
26.	龔飛宇, 魏在榮, 金文虎, 等. 人羊膜間充質干細胞來源的雪旺細胞樣細胞移植在皮瓣神經再生中的作用. 中國修復重建外科雜志, 2018, 32(1): 80-90.
27.	徐明均, 李曉國, 馬晨, 等. 人胎盤 MSCs 移植對急性肺損傷小鼠肺血管內皮通透性及肺組織損傷修復的影響. 中國修復重建外科雜志, 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. 泰文嬌, 葉旋, 鮑秀琦, 等. 小膠質細胞與腦缺血關系的研究進展. 藥學學報, 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. 任憲盛, 丁巍, 楊小玉. 蝦青素對大鼠脊髓損傷后細胞凋亡的影響. 中國修復重建外科雜志, 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. 劉靜, 劉婷, 徐睿玲, 等. SN50 聯合 5-氟尿嘧啶通過調節 NF-κB 信號通路抑制人胃癌裸鼠移植瘤生長的研究. 胃腸病學和肝病學雜志, 2016, 25(1): 5-8.
20. 習亮, 梁偉, 張永峰. NF-κB 信號通路抑制劑對 P 物質作用后脊髓星形膠質細胞 GFAP、炎性因子表達的影響. 山東醫藥, 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. 鐘澤祥, 周亦楠, 馮思思, 等. 慢病毒介導小干擾 RNA 干擾促分裂原和應激激活的蛋白激酶 1 對大鼠脊髓損傷的修復作用. 中國修復重建外科雜志, 2018, 32(7): 941-950.
26. 龔飛宇, 魏在榮, 金文虎, 等. 人羊膜間充質干細胞來源的雪旺細胞樣細胞移植在皮瓣神經再生中的作用. 中國修復重建外科雜志, 2018, 32(1): 80-90.
27. 徐明均, 李曉國, 馬晨, 等. 人胎盤 MSCs 移植對急性肺損傷小鼠肺血管內皮通透性及肺組織損傷修復的影響. 中國修復重建外科雜志, 2020, 34(3): 387-392.

Previous Article
Clinical study of concealed penis correction surgery based on principle of midline symmetry
Next Article
Advance of diagnosis and treatment of Haglund syndrome

Chinese Journal of Reparative and Reconstructive Surgery

Effect of directive differentiation of microglia by SN50 on hypoxia-caused neurons injury in mice

Abstract Full text Figures/Tables Video References Cited by

Previous Article

Next Article

Format

Content