痘苗病毒致炎兔皮提取物促进小胶质细胞M1向M2极化、抑制炎性因子分泌的体外研究

doi:10.3969/j.issn.1006-9771.2020.06.007

《中国康复理论与实践》 ›› 2020, Vol. 26 ›› Issue (6): 662-667.doi: 10.3969/j.issn.1006-9771.2020.06.007

痘苗病毒致炎兔皮提取物促进小胶质细胞M1向M2极化、抑制炎性因子分泌的体外研究

龚诗立^1,²,杨翠翠²,胡朝英²,王明洋²,张兰^1,²()

1.遵义医科大学基础药理教育部重点实验室,特色民族药教育部国际合作联合实验室,贵州遵义市 563000
2.首都医科大学宣武医院药学部,北京市 100053

收稿日期:2019-12-24 修回日期:2020-01-17 出版日期:2020-06-25 发布日期:2020-06-29
通讯作者: 张兰 E-mail:lanizhg@126.com
作者简介:龚诗立(1995-),女,汉族,贵州兴义市人,硕士研究生,主要研究方向：临床药理学、神经药理学。
基金资助:
1.北京市卫生系统高层次卫生技术人才项目(2014-2-014);2.首都科技领军人才培养工程项目(Z191100006119017);3.北京市博士后工作经费资助项目(2018-ZZ-105);4.中国博士后基金项目(2019M650776)

Promotion of Analgecine on Polarization of M1 Microglia to M2 and Inhibition on Secretion of Inflammatory Factors in Vitro

GONG Shi-li^1,²,YANG Cui-cui²,HU Chao-ying²,WANG Ming-yang²,ZHANG Lan^1,²()

1. Key Laboratory of Basic Pharmacology of Ministry of Education and Joint International Research Laboratory of Ethnomedicine of Ministry of Education, Zunyi Medical University, Zunyi, Guizhou 563000, China
2. Department of Pharmacy of Xuanwu Hospital Capital University, Beijing 100053, China

Received:2019-12-24 Revised:2020-01-17 Published:2020-06-25 Online:2020-06-29
Contact: ZHANG Lan E-mail:lanizhg@126.com
Supported by:
Beijing High-level Health and Technical Personal Plan(2014-2-014);Program for Training Capital Science and Technology Leading Talents(Z191100006119017);Beijing Postdoctoral Foundation(2018-ZZ-105);China Postdoctoral Science Foundation(2019M650776)

摘要/Abstract

摘要：

目的探讨痘苗病毒致炎兔皮提取物(AGC)对缺血性脑卒中细胞模型脑内炎症反应的影响及作用机制。方法采用连二亚硫酸钠(Na₂S₂O₂)无糖培养基在体外模拟缺血性脑卒中模型。将BV2细胞分为6组：对照组、对照加0.5 U/ml AGC组、氧糖剥夺再恢复(OGD/R)组,OGD/R加AGC (0.25 U/ml、0.5 U/ml、1.0 U/ml)组。OGD/R加AGC组氧糖剥夺1.5 h后换为正常培养基,同时给予不同浓度AGC,共同孵育3 h后进行细胞处理。取细胞上清液分别测定白介素-6 (IL-6)、肿瘤坏死因子-α (TNF-α)的含量。免疫荧光染色观察CD₁₆⁺+CD₃₂⁺(M1型小胶质细胞)和CD₂₀₆⁺(M2型小胶质细胞)细胞数。将BV2细胞分为7组：对照组、对照加0.5 U/ml AGC组、IL-4组、IL-4+脂多糖(LPS)+干扰素-γ(IFN-γ)组、IL-4+LPS+IFN-γ加AGC (0.25 U/m、0.5 U/m、1.0 U/ml)组。IL-4+LPS+IFN-γ加AGC 组IL-4处理24 h后,向细胞中加入LPS+IFN-γ处理18 h,加入相应浓度AGC 24 h后,流式细胞仪观察CD₁₆+CD₃₂和CD₂₀₆的表达。结果与对照组相比,OGD/R组细胞上清液中IL-6、TNF-α含量明显增加(P < 0.01),CD ₁₆⁺+CD₃₂⁺细胞数增多,CD₂₀₆⁺细胞数减少;与OGD/R组相比,AGC各剂量组IL-6、TNF-α含量均明显降低(P < 0.01),CD ₁₆⁺+CD₃₂⁺细胞数减少,CD₂₀₆⁺细胞数增多。与对照组比较,IL-4组CD₂₀₆有增多趋势,CD₁₆+CD₃₂有减少趋势;与IL-4组比较,IL-4+LPS+IFN-γ组CD₁₆+CD₃₂有增多趋势,CD₂₀₆有减少趋势;与IL-4+LPS+IFN-γ组比较,AGC各剂量组CD₁₆+CD₃₂有减少趋势,0.25 U/ml、0.5 U/ml AGC剂量组CD₂₀₆有增多趋势,1.0 U/ml AGC 剂量组CD₂₀₆表达增多(P < 0.05)。结论 AGC可通过促进小胶质细胞从M1表型向M2表型极化,抑制炎症因子的分泌。

关键词: 缺血性脑卒中, 痘苗病毒致炎兔皮提取物, 小胶质细胞极化, 炎症

Abstract:

Objective To investigate the effect of analgecine (AGC) on inflammatory response in the cell model of ischemic stroke and its mechanism.Methods Sodium hydrosulfite (Na₂S₂O₂) combined with sugar-free culture-medium was used to stimulate the model of ischemic stroke in vitro. BV2 cells were divided into six groups: control group, control with 0.5 U/ml AGC group, oxygen deprivation and recovery (OGD/R) group, OGD/R with AGC (0.25 U/ml, 0.5 U/ml, 1 U/ml) groups. After oxygen and glucose deprivation for 1.5 hours, they were changed to normal medium and given different concentrations of AGC in OGD/R with AGC groups. After co-incubation for three hours, the cells were treated. The content of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in the supernatant was detected. The expression of M1-type microglia marker CD₁₆+CD₃₂ and M2-type microglia marker CD₂₀₆ were detected with immunofluorescent staining. BV2 cells were divided into seven groups: control group, control with 0.5 U/ml AGC group, IL-4 group, IL-4 + lipopolysaccharide (LPS) + interferon (IFN)-γ group, IL-4 + LPS + IFN-γ with AGC (0.25 U/ml, 0.5 U/ml, 1 U/ml) groups. After 24 hours of IL-4 treatment, LPS + IFN-γ were added for 18 hours, they were changed to normal medium and given different concentrations of AGC for 24 hours, the expression of CD₁₆+CD₃₂ and CD₂₀₆ were observed by flow cytometry.Results Compared with the control group, the IL-6 and TNF-α level increased (P< 0.01), the number of CD₁₆⁺+CD₃₂⁺ increased and the number of CD₂₀₆⁺ decreased in OGD/R group. Compared with the OGD/R group, the IL-6 and TNF-α level decreased (P< 0.01), the number of CD₁₆⁺+CD₃₂⁺ decreased and the number of CD₂₀₆⁺ increased in AGC groups. Compared with the control group, the number of CD₂₀₆ tended to increase, and the number of CD₁₆+CD₃₂ tended to decrease in IL-4 group; compared with IL-4 group, the number of CD₁₆+CD₃₂ tended to increase, and the number of CD₂₀₆ tended to decrease in IL-4 + LPS + IFN-γ group; compared with IL-4 + LPS + IFN-γ group, the number of CD₁₆+CD₃₂ tended to decrease, and the number of CD₂₀₆ tended to increase in IL-4 + LPS + IFN-γ + 0.25 U/ml AGC group and IL-4 + LPS + IFN-γ + 0.5 U/ml AGC group, while the number of CD₂₀₆ increased in IL-4 + LPS + IFN-γ + 1.0 U/ml AGC group (P < 0.05). Conclusion AGC could inhibit the secretion of inflammation factors by promoting the polarization of microglia from M1 phenotype to M2 phenotype.

Key words: ischemic stroke, analgecine, microglia polarization, inflammation

中图分类号:

R743.3

龚诗立,杨翠翠,胡朝英,王明洋,张兰. 痘苗病毒致炎兔皮提取物促进小胶质细胞M1向M2极化、抑制炎性因子分泌的体外研究[J]. 《中国康复理论与实践》, 2020, 26(6): 662-667.

GONG Shi-li,YANG Cui-cui,HU Chao-ying,WANG Ming-yang,ZHANG Lan. Promotion of Analgecine on Polarization of M1 Microglia to M2 and Inhibition on Secretion of Inflammatory Factors in Vitro[J]. 《Chinese Journal of Rehabilitation Theory and Practice》, 2020, 26(6): 662-667.

图/表 5

表1

图1

图2

图3

表2

参考文献 26

[1]	Thion M S, Ginhoux F, Garel S. Microglia and early brain development: an intimate journey[J]. Science, 2018, 362(6411):185-189. doi: 10.1126/science.aat0474
[2]	Chhor V, Le Charpentier T, Lebon S, et al. Characterization of phenotype markers and neuronotoxic potential of polarized primary microglia in vitro[J]. Brain Behave Immun, 2013, 32:70-85.
[3]	Hu X M, Li P Y, Guo Y L, et al. Microglia/macrophage polarization dynamics reveal novel mechanism of injury expansion after focal cerebral ischemia[J]. Stroke, 2012, 43(11):3063-3070. doi: 10.1161/STROKEAHA.112.659656
[4]	Cherry J D, Olschowka J A, O'Banion M K. Neuroinflammation and M2 microglia: the good, the bad, and the inflamed[J]. J Neuroinflammation, 2014, 11:98. doi: 10.1186/1742-2094-11-98
[5]	Tang Y, Le W D. Differential Roles of M1 and M2 Microglia in neurodegenerative diseases[J]. Mol Neurobiol, 2016, 53(2):1181-1194. doi: 10.1007/s12035-014-9070-5 pmid: 25598354
[6]	Carlo P, Stefano F, Maria-Grazia D S. Temporal pattern of expression and colocalization of microglia/macrophage phenotype markers following brain ischemic injury in mice[J]. J Neuroinflammation, 2011, 8:174. doi: 10.1186/1742-2094-8-174
[7]	Yang X M, He R G. Effect of extracts from rabbit skin inflamed by vaccinia virus in the management of postherpetic neuralgia and on serum interleukin-6 level in aged patients[J]. Nan Fang Yi Ke Da Xue Xue Bao, 2007, 27(12):1941-1943.
[8]	费秋惠. 恩再适治疗带状疱疹后遗神经痛的可行性和安全性[J]. 医学理论与实践, 2019, 32(4):557-559.
[9]	陈和星. 恩再适在治疗带状疱疹后遗神经痛中的可行性及对脑源性神经营养因子的表达意义[J]. 江西医药, 2014, 49(11):1293-1295.
[10]	Dong J, Tu H P, Ding W Y, et al. Analgecine, the extracts of Vaccinia-inoculated rabbit skin, effectively alleviates the chronic low back pain with little side effect: a randomized multi-center double-blind placebo-controlled phase 3 clinical trial[J]. Contemp Clin Trials Commun, 2016, 2:16-24. doi: 10.1016/j.conctc.2015.11.002
[11]	Poomarin S, Passakorn S, Nattayaporn A, et al. Role of microglia under cardiac and cerebral ischemia/reperfusion (I/R) injury[J]. Metab Brain Dis, 2018, 33(4):1019-1030. doi: 10.1007/s11011-018-0232-4
[12]	Hu L, He D, Bai Y. Microglia-mediated inflammation and neurodegenerative disease[J]. Mol Neurobiol, 2016, 53(10):6709-6715. doi: 10.1007/s12035-015-9593-4
[13]	Hu J J, Qin L J, Liu Z Y, et al. MiR-15a regulates oxygen glucose deprivation/ reperfusion (OGD/R)-induced neuronal injury by targeting BDNF[J]. Kaohsiung J Med Sci, 2020, 36(1):27-34. doi: 10.1002/kjm2.v36.1
[14]	Li H, Ma J W, Fang Q, et al. Botch protects neurons from ischemic insult by antagonizing Notch-mediated neuroinflammation[J]. Exp Neurol, 2019, 321:113028. doi: 10.1016/j.expneurol.2019.113028
[15]	Dugan L L, Kim-Han J S. Astrocyte mitochondria in in vitro models of ischemia[J]. J Bioenerg Biomembr, 2004, 36(4):317-321. doi: 10.1023/B:JOBB.0000041761.61554.44
[16]	Ryou M G, Mallet R T. An in vitro oxygen-glucose deprivation model for studying ischemia-reperfusion injury of neuronal cells[J]. Methods Mol Biol, 2018, 1717:229-235.
[17]	Kroner A, Greenhalgh A D, Zarruk J G, et al. TNF and increased intracellular iron alter macrophage polarization to a detrimental M1 phenotype in the injured spinal cord[J]. Neuron, 2014, 83(5):1098-1116. doi: 10.1016/j.neuron.2014.07.027 pmid: 25132469
[18]	Bernstein J E, Savla P, Dong F, et al. Inflammatory markers and severity of intracerebral hemorrhage[J]. Cureus, 2018, 10(10):e3529.
[19]	Kettenmann H, Hanisch U K, Noda M, et al. Physiology of microglia[J]. Physiol Rev, 2011, 91(2):461-553. doi: 10.1152/physrev.00011.2010 pmid: 21527731
[20]	Ghosh S, Castillo E, Frias E S, et al. Bioenergetic regulation of microglia[J]. Glia, 2018, 66(6):1200-1212. doi: 10.1002/glia.v66.6
[21]	Varnum M M, Ikezu T. The classification of microglial activation phenotypes on neurodegeneration and regeneration in Alzheimer's disease brain[J]. Arch Immunol Ther Exp (Warsz), 2012, 60(4):252-266.
[22]	Li C W, Bian Y Q, Feng Y, et al. Neuroprotective effects of BHDPC, a novel neuroprotectant, on experimental stroke by modulating microglia polarization[J]. ACS Chem Neurisci, 2019, 10(5):2434-2449.
[23]	Ma Y Y, Wang J X, Wang Y T, et al. The biphasic function of microglia in ischemic stroke[J]. Prog Neurobiol, 2017, 157:247-272. doi: 10.1016/j.pneurobio.2016.01.005
[24]	Colonna M, Butovsky O. Microglia function in the central nervous system during health and neurodegeneration[J]. Annu Rev Immunol, 2017, 35:441-468. doi: 10.1146/annurev-immunol-051116-052358 pmid: 28226226
[25]	Orihuela R, McPherson C A, Harry G J. Microglia M1/M2 polarization and metabolic states[J]. Br J Pharmacol, 2016, 173(4):649-665. doi: 10.1111/bph.v173.4
[26]	Kigerl K A, Gensel J C, Ankeny D P, et al. Identification of two distinct macrophage subsets with divergent effectscausing either neurotoxicity or regeneration in the injured mouse spinal cord[J]. J Neurosci, 2009, 29(43):13435-13444. doi: 10.1523/JNEUROSCI.3257-09.2009

组别	n	TNF-α	IL-6
对照1组	3	48.70±1.637	5.093±0.5215
AGC1组	3	25.73±5.660	1.863±0.635
OGD/R组	3	342.3±22.070^a	31.13±3.003^b
L1组	3	212.3±14.560^c	7.148±1.924^c
M1组	3	138.4±14.280^c	6.127±1.966^c
H1组	3	74.85±9.630^d	3.793±0.463^d
F值		83.600	40.840
P值		<0.0001	<0.0001

组别	n	CD₁₆+CD₃₂	CD₂₀₆
对照2组	4	46.60±5.796	5.093±0.5215
AGC2组	4	26.60±8.821	1.863±0.6350
IL-4组	4	39.00±8.256	7.163±2.115
IFN-γ组	4	55.00±9.906	2.567±0.9602
L2组	4	39.65±10.98	4.437±1.865
M2组	4	39.40±11.120	7.353±2.969
H2组	4	40.97±11.180	6.037±0.3755^a
F值		0.8271	0.917
P值		0.5582	0.508

痘苗病毒致炎兔皮提取物促进小胶质细胞M1向M2极化、抑制炎性因子分泌的体外研究

Promotion of Analgecine on Polarization of M1 Microglia to M2 and Inhibition on Secretion of Inflammatory Factors in Vitro

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 5

参考文献 26

相关文章 15

Metrics

本文评价

推荐阅读 0

[1]	邓婷, 陈敬绵, 刘小蒙, 姚晓华, 刘芦姗, 何威, 张通, 芦海涛. 轻中度急性缺血性脑卒中并发卒中相关性肺炎的危险因素分析[J]. 《中国康复理论与实践》, 2023, 29(6): 708-713.
[2]	张春龙, 刘福亮, 商娜, 李芳, 刘慧珍. 血清脂联素、超敏C-反应蛋白与急性缺血性脑卒中短期预后的相关性[J]. 《中国康复理论与实践》, 2023, 29(10): 1221-1226.
[3]	周小珏,冯婧,庞日朝,刘捷,张安仁. 隔日限食减轻脊髓损伤大鼠炎症反应的芳香烃受体/细胞因子信号传导抑制因子2/核转录因子-κB信号通路机制[J]. 《中国康复理论与实践》, 2022, 28(5): 544-551.
[4]	赖海芳,顾琳,纵亚,牛传欣,谢青. 采用多层感知器神经网络构建亚急性期缺血性脑卒中患者短期预后的预测模型[J]. 《中国康复理论与实践》, 2022, 28(3): 335-339.
[5]	朱慧,夏有兵,巩尊科,王世雁,马柯,闫金秋. 高频重复经颅磁刺激对缺血性脑卒中后中枢性面瘫的效果[J]. 《中国康复理论与实践》, 2022, 28(2): 199-203.
[6]	蓝婉婷,钟救根,沈莹莹,龚嘉恒,邹智,侯晓晖. 运动减轻孤独症神经炎症的可视化分析[J]. 《中国康复理论与实践》, 2022, 28(10): 1190-1197.
[7]	王娜,李培兰,刘芦姗,王丰容. 不同评分系统预测急性缺血性脑卒中患者的短期和长期死亡风险比较[J]. 《中国康复理论与实践》, 2021, 27(3): 256-260.
[8]	张馨予,杜小正,王金海,何文洁,王娜娜,王若州,井维尧,杨尚伟. Netrin-1对脑梗死损伤的保护和修复作用研究进展[J]. 《中国康复理论与实践》, 2021, 27(3): 316-319.
[9]	杨肇宇,李培君,李健,刘晓丹,吴卫兵. 运动对慢性阻塞性肺疾病系统性炎症和骨骼肌功能障碍干预效果的系统综述[J]. 《中国康复理论与实践》, 2021, 27(12): 1443-1449.
[10]	王娜娜, 杜小正, 何文洁, 张馨予, 郑欣, 李梦莘. 针刺治疗缺血性脑卒中的Notch信号通路研究进展[J]. 《中国康复理论与实践》, 2021, 27(1): 67-70.
[11]	陈小平,王明洋,马登磊,龚诗立,张丽,李雅莉,李林,胡朝英,张兰. 痘苗病毒致炎兔皮提取物对脑缺血再灌注脑损伤大鼠凋亡和神经炎症的影响[J]. 《中国康复理论与实践》, 2020, 26(9): 1038-1044.
[12]	刘建华,魏清川,胡秀茹,叶赛青,闫志宇,高强. 弥散张量成像各向异性分数与缺血性脑卒中患者上肢运动功能的相关性[J]. 《中国康复理论与实践》, 2020, 26(7): 749-752.
[13]	魏海萍,郭佳,王欢,葛朝明. 迷走神经刺激对脑缺血再灌注大鼠磷酸腺苷活化蛋白激酶-沉默信息调节因子2相关酶1自噬通路的影响[J]. 《中国康复理论与实践》, 2020, 26(7): 775-779.
[14]	刘慧珍,郭树彬,商娜,李芳,刘芦姗,李培兰,陈敬绵,王丰容,李俊玉. 血清25羟基-维生素D对急性缺血性脑卒中急诊患者结局的预测价值[J]. 《中国康复理论与实践》, 2020, 26(7): 830-835.
[15]	王传杰,吴毅,陶峰,杨雷. 丰富环境对缺血性脑卒中小鼠海马区Bcl-2和Bax蛋白表达和认知功能的效果[J]. 《中国康复理论与实践》, 2020, 26(5): 539-543.