大鼠脑缺血再灌注损伤后极化的小胶质细胞排斥性导向分子A的表达变化
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摘要
目的探讨大鼠大脑中动脉缺血再灌注损伤后极化的小胶质细胞排斥性导向分子A(RGMa)的表达变化。方法取雄性SD大鼠30只,随机分成对照组,缺血再灌注损伤7 d、14 d模型组(I/R),采用线栓法制作大鼠大脑中动脉缺血再灌注损伤模型(t MCAO); Real-time PCR检测M1、M2型小胶质细胞标记分子白细胞介素-1β(IL-1β)、诱导性一氧化氮合酶(i NOS)、精氨酸酶1(Arg-1)及CD206 mRNA的表达;免疫荧光检测缺血区小胶质细胞RGMa的表达;体外培养小胶质细胞,分别用M1或M2型小胶质细胞的诱导物脂多糖(LPS)或IL-4诱导24 h后,利用Real-time PCR检测M1、M2型小胶质细胞标记分子IL-1β、i NOS、Arg-1、CD206 mRNA的表达; Western blotting检测M1、M2型小胶质细胞上RGMa的表达。结果缺血再灌注损伤后7 d、14 d,M1、M2型小胶质细胞的标记分子表达增加。缺血后7 d、14 d激活的小胶质细胞上RGMa大量表达。RGMa在体外培养的LPS诱导极化的M1型小胶质细胞和IL-4诱导极化的M2型小胶质细胞上表达均显著增加。结论大鼠大脑中动脉缺血再灌注损伤后,RGMa在缺血区M1型和M2型极化的小胶质细胞上均大量表达,RGMa可能在小胶质细胞激活极化过程中发挥重要作用。RGMa可能是缺血性脑卒中治疗的新靶点。
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(t MCAO) 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(i NOS),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,i NOS,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.
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(t MCAO) 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(i NOS),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,i NOS,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.
引文
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[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 longterm 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.
[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-γdifferentially 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 6FNⅢon 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 longterm 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.