Expression of repulsive guidance molecule A in polarized microglia after rat focal cerebral ischemia/reperfusion injury

doi:10.16098/j.issn.0529-1356.2019.04.001

Abstract

Abstract:

Objective To investigate the expression of repulsive guidance molecule A (RGMa) in polarized microglia after rat focal cerebral ischemia/reperfusion injury. Methods Adult male Sprague-Dawley (SD) rats were randomly divided into control group, 7 days cerebral ischemia/reperfusion injury group (I/R), and 14 days I/R group. The transient middle cerebral occlusion (tMCAO) model was induced by ligation with nylon monofilament. Real-time PCR was used to test the mRNA expression of M1 and M2 microglia marker interleukin-1β(IL-1β), inducible nitric oxide synthase (iNOS), arginase 1 (Arg-1) and CD206 in ipsilateral cortex at day 7 and day 14.Double immunofluorescence staining was performed to investigate the co-expression of RGMa and Iba1 in microglia. The microglia was incubated with lipopolysaccharides (LPS)or IL-4 in vitro. The mRNA expression of M1 and M2 microglia marker (IL-1β, iNOS, Arg-1, CD206) was tested by Real-time PCR. Western blotting was used to assess the proteins level of RGMa in M1 and M2 microglia. Results M1 and M2 microglia marker mRNA level were increased in ipsilateral cortex at day 7 and day 14. RGMa was strongly expressed in reactive microglia at day 7 and day 14. LPS or IL-4 treatment polarized microglia to M1 or M2 in vitro. The expression of RGMa protein in M1 and M2 microglia was increased. Conclusion RGMa was strongly expressed in reactive M1 and M2 microglia after ischemia/reperfusion injury. RGMa may play an important role in the process of microglia activation and polarization. RGMa may be a novel therapeutic target for stroke.

Key words: Repulsive guidance molecule A, Brain ischemia, Reperfusion injury, Microglia polarization, Western blotting, Real-time PCR, Rat

WU Yan-ping QIN Xin-yue ZHANG Rong-rong HUANG Si-yuan ZHANG Lei. Expression of repulsive guidance molecule A in polarized microglia after rat focal cerebral ischemia/reperfusion injury[J]. Acta Anatomica Sinica, 2019, 50(4): 405-410.

References

［1］ Moskowitz MA, Lo EH, Iadecola C. The science of stroke: mechanisms in search of treatments ［J］. Neuron, 2010, 67(2):181-198.

［2］ Ponomarev ED, Maresz K, Tan Y, et al. CNS-derived interleukin-4 is essential for the regulation of autoimmune inflammation and induces a state of alternative activation in microglial cells［J］. J Neurosci, 2007, 27(40):10714-10721.

［3］ Butovsky O, Ziv Y, Schwartz A, et al. Microglia activated by IL-4 or IFN-γ differentially induce neurogenesis and oligodendrogenesis from adult stem/progenitor cells［J］. Mol Cell Neurosci, 2006, 31(1):149-160.

［4］ Lah GJ, Key B. Novel roles of the chemorepellent axon guidance molecule RGMa in cell migration and adhesion［J］. Mol Cell Biol, 2012, 32(5):968-980.

［5］ Wang T, Wu X, Yin C, et al. CRMP-2 is involved in axon growth inhibition induced by RGMa in vitro and in vivo.［J］. Mol Neurobiol, 2013, 47(3):903-913.

［6］ Feng J, Wang T, Li Q, et al. RNA interference against repulsive guidance molecule A improves axon sprout and neural function recovery of rats after MCAO/reperfusion［J］. Exp Neurol, 2012, 238(2):235-242.

［7］ Xie F,Qin XY, Zhang ＲR, et al. Effect of repulsive guidance molecule a specific inhibitor 6FNⅢ on cortex neuron autophagy in rats after cerebral ischemia/reperfusion injury ［J］.Acta Anatomica Sinica,2017, 48(4):381-386. (in Chinese)

谢扉,秦新月,张融融,等. 排斥性导向分子A抑制剂6FNⅢ对大鼠大脑皮质神经元缺血再灌注后自噬的作用［J］. 解剖学报, 2017, 48(4):381-386.

［8］ Zhang G, Wang R, Cheng K, et al. Repulsive guidance molecule a inhibits angiogenesis by downregulating VEGF and phosphorylated focal adhesion kinase in vitro［J］. Front Neurol, 2017, 8: 504.

［9］ Wang Y, Zhang R, Xing X, et al. Repulsive guidance molecule a suppresses angiogenesis after ischemia/reperfusion injury of middle cerebral artery occlusion in rats［J］. Neurosci Lett, 2018, 662:318-323.

［10］Zhang R, Wu Y, Xei F, et al. RGMa mediates reactive astrogliosis and glial scar formation through TGFβ1/Smad2/3 signaling after stroke［J］. Cell Death Differ, 2018, 25(8):1503-1516.

［11］Longa EZ, Weinstein PR, Carlson S, et al. Reversible middle cerebral artery occlusion without craniectomy in rats［J］. Stroke, 1989, 20(1):84-91.

［12］Zhang X. The modulation of atp on microglia polarization in neuropathic pain［D］.Shanghai Jiao Tong University,2015. (in Chinese)

张昕. ATP调控小胶质细胞极化状态进而参与神经病理性疼痛的研究［D］.上海交通大学,2015.

［13］Sun D, Yu Z, Fang X, et al. LncRNA GAS5 inhibits microglial M2 polarization and exacerbates demyelination［J］. EMBO Rep, 2017, 18(10):1801-1816.

［14］Hu X, Li P, Guo Y, et al. Microglia/macrophage polarization dynamics reveal novel mechanism of injury expansion after focal cerebral ischemia［J］. Stroke, 2012, 43(11):3063-3070.

［15］Liu X, Liu J, Zhao S, et al. Interleukin-4 is essential for microglia/macrophage M2 polarization and long-term recovery after cerebral ischemia［J］. Stroke, 2016, 47(2):498-504.

［16］Luo XQ, Li A, Yang X, et al. Paeoniflorin exerts neuroprotective effects by modulating the M1/M2 subset polarization of microglia/macrophages in the hippocampal CA1 region of vascular dementia rats via cannabinoid receptor 2［J］. Chin Med, 2018, 13(1):14-30.

［17］Suenaga J, Hu X, Pu H, et al. White matter injury and microglia/macrophage polarization are strongly linked with age-related long-term deficits in neurological function after stroke［J］. Exp Neurol, 2015, 272:109-119.

［18］Shao Y, Deng T, Zhang T, et al. FAM19A3, a novel secreted protein, modulates the microglia/macrophage polarization dynamics and ameliorates cerebral ischemia.［J］. FEBS Lett, 2016, 589(4):467-475.

［19］Jin Q, Cheng J, Liu Y, et al. Improvement of functional recovery by chronic metformin treatment is associated with enhanced alternative activation of microglia/macrophages and increased angiogenesis and neurogenesis following experimental stroke［J］. Brain Behav Immun, 2014, 40:131-142.

［20］Liu HC, Zheng MH, Du YL, et al. N9 microglial cells polarized by LPS and IL4 show differential responses to secondary environmental stimuli［J］. Cell Immunol, 2012, 278(1-2):84-90.

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