补阳还五汤调控小胶质细胞/巨噬细胞极化抑制大鼠脑缺血后炎症反应研究
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摘要
[目的]研究补阳还五汤(Buyang Huanwu Decoction, BYHWD)对大鼠脑缺血后小胶质细胞/巨噬细胞M1/M2极化及神经炎症的影响。[方法]采用线栓法建立大鼠大脑中动脉阻塞(middle cerebral artery occlusion, MCAO)模型,缺血90 min后再灌注。将大鼠随机分为假手术组、模型组和BYHWD组,BYHWD组缺血后24h开始予BYHWD(13g·kg-1)灌胃,连续给药14d。缺血后第14天处死大鼠,分别采用Iba1/CD16/32、Iba1/CD206免疫荧光双标染色检测缺血区M1型和M2型小胶质细胞/巨噬细胞表型,qRT-PCR检测M1型小胶质细胞/巨噬细胞表面标记物CD86、诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)及促炎因子肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-1β(interleukin-1β,IL-1β)、白细胞介素-6(interleukin-6,IL-6)的m RNA表达;M2型小胶质细胞/巨噬细胞表面标记物CD206、精氨酸酶-1(arginase-1,Arg-1)及抗炎因子白细胞介素-10(interleukin-10, IL-10)、转化生长因子-β(transforming growth factor-β,TGF-β)的mRNA表达。[结果]免疫荧光双标染色结果表明,与模型组比较,BYHWD显著减少缺血区M1型小胶质细胞/巨噬细胞(CD16/32+)数量(P<0.01),增加M2型小胶质细胞(CD206+)数量(P<0.05)。qRT-PCR结果表明,与模型组比较,BYHWD显著下调M1型小胶质细胞/巨噬细胞表面标记物CD86、iNOS和促炎因子TNF-α、IL-1β、IL-6 mRNA表达(P<0.01),上调M2型小胶质细胞/巨噬细胞表面标记物CD206、Arg-1和抗炎因子IL-10、TGF-βmRNA表达(P<0.01)。[结论] BYHWD可能通过促进激活的小胶质细胞/巨噬细胞从M1型向M2型转换,从而抑制大鼠脑缺血后炎症反应。
[Objective] To investigate the effects of Buyang Huanwu Decoction(BYHWD) on microglia/macrophage M1/M2 polarization and neuro-inflammation after cerebral ischemia in rats. [Methods] Focal cerebral ischemia was induced by occlusion of the right middle cerebral artery for 90 minutes using the intraluminal filament model. The rats were randomly divided into sham operation group, model group and BYHWD group. BYHWD(13g·kg-1) was administered intragastricly in BYHWD group 24 h after ischemia and once daily for 14 days. Microglia/macrophage M1/M2 phenotype was determined by double immune-fluorescence labeling. The expression of M1/M2 microglia/macrophage markers were detected by qRT-PCR. [Results]Compared with model group, double immune-fluorescence labeling showed that BYHWD significantly reduced the expression of M1 marker CD16/32( P<0.01) and enhanced the expression of M2 marker CD206 in microglia/macrophage after cerebral ischemia( P<0.05). q RT-PCR results showed that BYHWD significantly down-regulated the expression of M1 microglia/macrophage surface markers CD86, inducible nitric oxide synthase(iNOS), and pro-inflammatory cytokines tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β), interleukin-6(IL-6) mRNA(P<0.01). In contrast, BYHWD up-regulated the expression of M2 microglia/macrophage surface markers CD206, arginase-1(Arg-1), and anti-inflammatory cytokines interleukin-10(IL-10) and transforming growth factor beta(TGF-β) mRNA(P<0.01). [Conclusion] These findings suggest that BYHWD inhibits inflammation after cerebral ischemia in rats, which may be related to promoting the polarization of the microglia/macrophage from M1 to M2 phenotype in the ischemic brain.
[Objective] To investigate the effects of Buyang Huanwu Decoction(BYHWD) on microglia/macrophage M1/M2 polarization and neuro-inflammation after cerebral ischemia in rats. [Methods] Focal cerebral ischemia was induced by occlusion of the right middle cerebral artery for 90 minutes using the intraluminal filament model. The rats were randomly divided into sham operation group, model group and BYHWD group. BYHWD(13g·kg-1) was administered intragastricly in BYHWD group 24 h after ischemia and once daily for 14 days. Microglia/macrophage M1/M2 phenotype was determined by double immune-fluorescence labeling. The expression of M1/M2 microglia/macrophage markers were detected by qRT-PCR. [Results]Compared with model group, double immune-fluorescence labeling showed that BYHWD significantly reduced the expression of M1 marker CD16/32( P<0.01) and enhanced the expression of M2 marker CD206 in microglia/macrophage after cerebral ischemia( P<0.05). q RT-PCR results showed that BYHWD significantly down-regulated the expression of M1 microglia/macrophage surface markers CD86, inducible nitric oxide synthase(iNOS), and pro-inflammatory cytokines tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β), interleukin-6(IL-6) mRNA(P<0.01). In contrast, BYHWD up-regulated the expression of M2 microglia/macrophage surface markers CD206, arginase-1(Arg-1), and anti-inflammatory cytokines interleukin-10(IL-10) and transforming growth factor beta(TGF-β) mRNA(P<0.01). [Conclusion] These findings suggest that BYHWD inhibits inflammation after cerebral ischemia in rats, which may be related to promoting the polarization of the microglia/macrophage from M1 to M2 phenotype in the ischemic brain.
引文
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[11]王清任.医林改错[M].北京:人民卫生出版社,2005:22.WANG Qingren.Corrections on the Errors of Medical Works[M].Beijing:People’s Medical Publishing House,2005:22.
[12] Longa E Z, Weinstein P R, Carlson S, et al. Reversible middle cerebral artery occlusion without craniectomy in rats[J]. Stroke, 1989, 20(1):84-91.
[13] Kumar A, Alvarez-Croda D M, Stoica B A, et al. Microglial/macrophage polarization dynamics following traumatic brain injury[J]. J Neurotrauma, 2016, 33(19):1732-1750.
[14] Hu X, Leak R K, Shi Y, et al. Microglial and macrophage polarization-new prospects for brain repair[J].Nat Rev Neurol, 2015, 11(1):56-64.
[15] Liu Z, Ran Y, Huang S, et al. Curcumin protects against ischemic stroke by titrating microglia/macrophage polarization[J]. Front Aging Neurosci,2017,9:233.
[16] Liu X, Wen S, Yan F, et al. Salidroside provides neuroprotection by modulating microglial polarization after cerebral ischemia[J].J Neuroinflammation,2018,15(1):39.
[17] 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.
[18] Moretti R, Leger P L, Besson V C, et al. Sildenafil, a cyclic GMP phosphodiesterase inhibitor, induces microglial modulation after focal ischemia in the neonatal mouse brain[J]. J Neuroinflammation, 2016, 13(1):95.
[19] Ekdahl C T, Claasen J H, Bonde S, et al. Inflammation is detrimental for neurogenesis in adult brain[J]. Proc Natl Acad Sci U S A, 2003, 100(23):13632-13637.
[20] Xiong X Y, Liu L, Yang Q W. Functions and mechanisms of microglia/macrophages in neuroinflammation and neurogenesis after stroke[J].Prog Neurobiol,2016,142:23-44.
[21] Liu Z, Fan Y, Won S J, et al. Chronic treatment with minocycline preserves adult new neurons and reduces functional impairment after focal cerebral ischemia[J].Stroke, 2007, 38(1):146-152.
[22] Zhang J C, Xu H, Yuan Y, et al. Delayed treatment with green tea polyphenol EGCG promotes neurogenesis after ischemic stroke in adult mice[J].Mol Neurobiol,2017,54(5):3652-3664.
[2] Chamorroá, Dirnagl U, Urra X, et al. Neuroprotection in acute stroke:targeting excitotoxicity, oxidative and nitrosative stress, and inflammation[J]. Lancet Neurol, 2016,15(8):869-881.
[3] Jiménez Fernández D, Lamkanfi M. Inflammatory caspases:key regulators of inflammation and cell death[J].Biol Chem, 2015, 396(3):193-203.
[4] Danton G H, Dietrich W D. Inflammatory mechanisms after ischemia and stroke[J].J Neuropathol Exp Neurol,2003, 62(2):127-136.
[5] Tang Y, Le W D. Differential roles of M1 and M2 microglia in neurodegenerative diseases[J].Mol Neurobiol,2016,53(2):1181-1194.
[6] Ma Y, Wang J, Wang Y, et al. The biphasic function of microglia in ischemic stroke[J].Prog Neurobiol,2017,157:247-272.
[7] 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.
[8]姚广明,刘朋飞,李鹏亮,等.补阳还五汤对脑缺血治疗作用机制[J].中国老年学杂志,2016,36(6):1533-1535.YAO Guangming,LIU Pengfei, LI Pengliang, et al. Therapeutic mechanism of Buyang Huanwu Decoction on cerebral ischemia[J]. Chinese Journal of Gerontology,2016,36(6):1533-1535.
[9]李琳,刘志婷,储利胜,等.补阳还五汤诱导脑缺血后血管生成促进侧脑室下区神经母细胞迁移[J].中国中药杂志,2015,40(2):298-302.LI Lin, LIU Zhiting, CHU Lisheng, et al. Buyang Huanwu Decoction promotes neuroblast migration from subventricular zone via inducing angiogenesis after ischemia[J].China Journal of Chinese Materia Medica,2015,40(2):298-302.
[10]曲铁兵,俞天虹,刘志婷,等.补阳还五汤及其拆方对大鼠脑缺血后神经发生的影响[J].中国中西医结合杂志,2014,34(3):342-347.QU Tiebing, YU Tianhong, LIU Zhiting, et al. Effect of Buyang Huanwu Decoction and its disassembled recipes on rats’ neurogenesis after focal cerebral ischemia[J].Chin J Integr Trad West Med,2014,34(3):342-347.
[11]王清任.医林改错[M].北京:人民卫生出版社,2005:22.WANG Qingren.Corrections on the Errors of Medical Works[M].Beijing:People’s Medical Publishing House,2005:22.
[12] Longa E Z, Weinstein P R, Carlson S, et al. Reversible middle cerebral artery occlusion without craniectomy in rats[J]. Stroke, 1989, 20(1):84-91.
[13] Kumar A, Alvarez-Croda D M, Stoica B A, et al. Microglial/macrophage polarization dynamics following traumatic brain injury[J]. J Neurotrauma, 2016, 33(19):1732-1750.
[14] Hu X, Leak R K, Shi Y, et al. Microglial and macrophage polarization-new prospects for brain repair[J].Nat Rev Neurol, 2015, 11(1):56-64.
[15] Liu Z, Ran Y, Huang S, et al. Curcumin protects against ischemic stroke by titrating microglia/macrophage polarization[J]. Front Aging Neurosci,2017,9:233.
[16] Liu X, Wen S, Yan F, et al. Salidroside provides neuroprotection by modulating microglial polarization after cerebral ischemia[J].J Neuroinflammation,2018,15(1):39.
[17] 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.
[18] Moretti R, Leger P L, Besson V C, et al. Sildenafil, a cyclic GMP phosphodiesterase inhibitor, induces microglial modulation after focal ischemia in the neonatal mouse brain[J]. J Neuroinflammation, 2016, 13(1):95.
[19] Ekdahl C T, Claasen J H, Bonde S, et al. Inflammation is detrimental for neurogenesis in adult brain[J]. Proc Natl Acad Sci U S A, 2003, 100(23):13632-13637.
[20] Xiong X Y, Liu L, Yang Q W. Functions and mechanisms of microglia/macrophages in neuroinflammation and neurogenesis after stroke[J].Prog Neurobiol,2016,142:23-44.
[21] Liu Z, Fan Y, Won S J, et al. Chronic treatment with minocycline preserves adult new neurons and reduces functional impairment after focal cerebral ischemia[J].Stroke, 2007, 38(1):146-152.
[22] Zhang J C, Xu H, Yuan Y, et al. Delayed treatment with green tea polyphenol EGCG promotes neurogenesis after ischemic stroke in adult mice[J].Mol Neurobiol,2017,54(5):3652-3664.