自噬抑制剂3-MA对脑缺血再灌注损伤大鼠脑内炎症反应的影响

doi:10.3969/j.issn.1006-7795.2024.06.010

摘要/Abstract

摘要： 目的研究自噬抑制剂3-甲基腺嘌呤（3-methyladenine, 3-MA）对局灶性脑缺血再灌注大鼠缺血侧脑组织趋化因子受体2（C-X-C chemokine receptor type 2, CXCR2）、肿瘤坏死因子α（tumor necrosis factor-α，TNF-α）、白细胞介素-1β（interleukin-1β, IL-1β）、环氧化酶２（cyclooxygenase-2，COX2）的影响，探究脑缺血再灌注损伤过程中自噬对炎症的作用。方法将雄性SD大鼠采用随机数字表法分为：假手术（Sham）组、大脑中动脉梗死（middle cerebral artery occlusion，MCAO）组和MCAO再灌注24 h+3-MA（3-MA）组，每组5只。按照改良线栓法制作MCAO大鼠模型，模拟缺血90 min。3-MA组大鼠于 MCAO 前腹腔注射10 mg/kg 3-MA。所有组别大鼠均再灌注24 h。达到再灌注时间后将大鼠处死，迅速断头、取脑，应用Western blotting 法检测大鼠缺血侧脑组织CXCR2 蛋白水平，免疫荧光染色法检测脑组织冰冻切片缺血半暗带区炎症因子TNF-α、 IL-1β及 COX2 阳性细胞数量，并分别对IL-1β、COX2进行细胞定位。结果 ①MCAO 组大鼠再灌注24 h缺血脑组织CXCR2蛋白水平比 Sham 组明显升高（P ＜ 0.05）。3-MA组大鼠缺血侧脑组织 CXCR2 蛋白水平比 MCAO 组显著减少（P ＜ 0.05）。 ② Sham组大鼠脑内偶见TNF-α、IL-1β和 COX2阳性细胞，而MCAO组大鼠脑缺血半暗带区TNF-α、IL-1β和COX2阳性细胞数量均比Sham 组显著升高（P ＜ 0.05）。相较于MCAO组，给予自噬抑制剂3-MA的缺血大鼠脑组织半暗带区中TNF-α、IL-1β和COX2阳性细胞数量均显著降低（P ＜ 0.05）。③ IL-1β和COX2在脑缺血大鼠缺血半暗带区内分别与神经元标志物NeuN共定位。结论自噬抑制剂 3-MA降低脑缺血再灌注大鼠缺血侧脑组织中CXCR2表达以及炎症因子TNF-α、 IL-1β和COX2的阳性细胞数量，从而减轻脑缺血后的炎症反应，说明自噬是促进脑缺血后炎症反应的因素之一。

关键词: 脑缺血再灌注损伤, 炎症, 自噬

Abstract: Objective To investigate the effects of the autophagy inhibitor 3-methyladenine (3-MA) on C-X-C chemokine receptor type 2 (CXCR2), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and cyclooxygenase-2 (COX2) in the ischemic brain tissue of rats subjected to transient focal cerebral ischemia/reperfusion, in order to explore the role of autophagy in inflammation during cerebral ischemia/reperfusion injury. Methods The male Sprague Dawley rats were randomized into: sham group, middle cerebral artery occlusion (MCAO) group and MCAO+3-MA (3-MA) group, with n=5 for each group. A model of MCAO was induced by using the intraluminal suture method. The rats underwent 90 minutes of ischemia. Rats in 3-MA group were intraperitoneally injected with 10 mg/kg of 3-MA before MCAO. All groups of rats underwent reperfusion for 24 h. After reperfusion time, the rats were quickly decapitated, and their brains were removed. The levels of CXCR2 protein in the ischemic brain tissue were detected by Western blotting. The number of positive cells of TNF-α, IL-1β, and COX2 were detected by immunofluorescence staining in the ischemic penumbra, and the cellular localization of IL-1β and COX2 was evaluated respectively. Results ①Compared with sham group, the level of CXCR2 protein was upregulated obviously in the ischemic brain tissue of MCAO rats after 24 h reperfusion (P < 0.05). The level of CXCR2 protein decreased significantly in the ischemic brain tissue of 3-MA group compared with that of MCAO group (P < 0.05). ②There were few TNF-α, IL-1β, and COX2 positive cells in the sham group. Compared with sham group, the number of TNF-α, IL-1β, and COX2 positive cells in the ischemic penumbra of MCAO group rats obviously increased after 24 h of reperfusion (P < 0.05). Compared with MCAO group, the number of positive cells of TNF-α, IL-1β and COX2 decreased significantly in the ischemic penumbra of the MCAO+3-MA group rats (P < 0.05). ③The IL-1β and COX2 positive cells were colocalized with NeuN, a general neuronal marker, in the ischemic penumbra of cerebral ischemia/reperfusion rats. Conclusions The autophagy inhibitor 3-MA may attenuate inflammation by inhibiting the expression levels of chemokine receptor CXCR2, as well as the number of positive cells of TNF-α, IL-1β, and COX2 inflammatory factors in ischemic brain tissue of MCAO rats after reperfusion, indicating that autophagy is one of the factors that promote the inflammatory response after ischemia.

Key words: cerebral ischemia/reperfusion injury, inflammation, autophagy

中图分类号:

R743.3

安琪, 杨楠, 朱玥荃, 师文娟, 黄敏琪, 赵咏梅. 自噬抑制剂3-MA对脑缺血再灌注损伤大鼠脑内炎症反应的影响[J]. 首都医科大学学报, 2024, 45(6): 1008-1015.

An Qi, Yang Nan, Zhu Yuequan, Shi Wenjuan, Huang Minqi, Zhao Yongmei. Effect of autophagy inhibitor 3-MA on inflammatory response in the brain of rats with cerebral ischemia/reperfusion injury[J]. Journal of Capital Medical University, 2024, 45(6): 1008-1015.

参考文献

［1］ Zhou H Q, Zhang L M, Li X, et al. Crosstalk between autophagy and inflammation in chronic cerebral Ischaemia［J］. Cell Mol Neurobiol, 2023, 43(6): 2557－2566.
［2］ Tao J H, Shen C, Sun Y C, et al. Neuroprotective effects of pinocembrin on ischemia/reperfusion-induced brain injury by inhibiting autophagy［J］. Biomed Pharmacother, 2018, 106: 1003－1010.
［3］ Xing S H, Zhang Y S, Li J J, et al. Beclin 1 knockdown inhibits autophagic activation and prevents the secondary neurodegenerative damage in the ipsilateral thalamus following focal cerebral infarction［J］. Autophagy, 2012, 8(1): 63－76.
［4］房亚兰, 黄语悠, 赵咏梅, 等. 大黄酚对局灶性脑缺血再灌注小鼠Beclin1及Bax的作用［J］. 首都医科大学学报, 2019, 40(5): 703－708.
［5］ Lukasik A, Brzozowska I, Zielenkiewicz U, et al. Detection of plant miRNAs abundance in human breast milk［J］. Int J Mol Sci, 2017, 19(1): 37.
［6］ Miao M S, Yan X L, Guo L, et al. Effects of the Rabdosia rubescens total flavonoids on focal cerebral ischemia reperfusion model in rats［J］. Saudi Pharm J, 2017, 25(4): 607－614.
［7］房亚兰, 黄语悠, 赵咏梅, 等. 大黄酚对局灶性脑缺血再灌注小鼠缺血半暗带区环氧化酶2和基质金属蛋白酶-9表达的影响［J］. 首都医科大学学报, 2017, 38(1): 47－52.
［8］ Ceccariglia S, Cargnoni A, Silini A R, et al. Autophagy: a potential key contributor to the therapeutic action of mesenchymal stem cells［J］. Autophagy, 2020, 16(1): 28－37.
［9］ Cosin-Roger J, Simmen S, Melhem H, et al. Hypoxia ameliorates intestinal inflammation through NLRP3/mTOR downregulation and autophagy activation［J］. Nat Commun, 2017, 8(1): 98.
［10］ Lee Y R, Kuo S H, Lin C Y, et al. Dengue virus-induced ER stress is required for autophagy activation, viral replication, and pathogenesis both in vitro and in vivo［J］. Sci Rep, 2018, 8(1): 489.
［11］ Schlemmer F, Boyer L, Soumagne T, et al. Beclin1 circulating levels and accelerated aging markers in COPD［J］. Cell Death Dis, 2018, 9(2): 156.
［12］ Sorbara M T, Ellison L K, Ramjeet M, et al. The protein ATG16L1 suppresses inflammatory cytokines induced by the intracellular sensors Nod1 and Nod2 in an autophagy-independent manner［J］. Immunity, 2013, 39(5): 858－873.
［13］ Tang D F, Yao R Y, Zhao D D, et al. Trichostatin a reverses the chemoresistance of lung cancer with high IGFBP2 expression through enhancing autophagy［J］. Sci Rep, 2018, 8(1): 3917.
［14］ Bussi C, Peralta Ramos J M, Arroyo D S, et al. Autophagy down regulates pro-inflammatory mediators in BV2 microglial cells and rescues both LPS and alpha-synuclein induced neuronal cell death［J］. Sci Rep, 2017, 7: 43153.
［15］ Luo J, Chen J L, Yang C H, et al. 6-Gingerol protects against cerebral ischemia/reperfusion injury by inhibiting NLRP3 inflammasome and apoptosis via TRPV1/FAF1 complex dissociation-mediated autophagy［J］. Int Immunopharmacol, 2021, 100: 108146.
［16］ Yang Z, Zhong L N, Zhong S C, et al. Hypoxia induces microglia autophagy and neural inflammation injury in focal cerebral ischemia model［J］. Exp Mol Pathol, 2015, 98(2): 219－224.
［17］ Xu S C, Huang P, Yang J H, et al. Calycosin alleviates cerebral ischemia/reperfusion injury by repressing autophagy via STAT3/FOXO3a signaling pathway［J］. Phytomedicine, 2023, 115: 154845.
［18］ Wang L, Jin Z C, Wang J K, et al. Detrimental effect of Hypoxia-inducible factor-1α-induced autophagy on multiterritory perforator flap survival in rats［J］. Sci Rep, 2017, 7(1): 11791.
［19］ Li W, Yang X Q, Ding M, et al. Zinc accumulation aggravates cerebral ischemia/reperfusion injury by promoting inflammation［J］. Front Cell Neurosci, 2023, 17: 1065873.
［20］ Zhang W F, Jin Y C, Li X M, et al. Protective effects of leptin against cerebral ischemia/reperfusion injury［J］. Exp Ther Med, 2019, 17(5): 3282－3290.
［21］ Hong Y, Liu Y J, Zhang G, et al. Progesterone suppresses Aβ42-induced neuroinflammation by enhancing autophagy in astrocytes［J］. Int Immunopharmacol, 2018, 54: 336－343.
［22］ Barisas D A G, Choi K. Extramedullary hematopoiesis in cancer［J］. Exp Mol Med, 2024, 56(3): 549－558.
［23］ Mohamud Yusuf A, Hagemann N, Ludewig P, et al. Roles of polymorphonuclear neutrophils in ischemic brain injury and Post-Ischemic brain remodeling［J］. Front Immunol, 2021, 12: 825572.
［24］ Jia P, Xu S J, Ren T, et al. LncRNA IRAR regulates chemokines production in tubular epithelial cells thus promoting kidney ischemia-reperfusion injury［J］. Cell Death Dis, 2022, 13(6): 562.
［25］ Ullah A, Zhao J, Singla R K, et al. Pathophysiological impact of CXC and CX3CL1 chemokines in preeclampsia and gestational diabetes mellitus［J］. Front Cell Dev Biol, 2023, 11: 1272536.
［26］ Rong M M, Yan X Y, Zhang H Y, et al. Dysfunction of decidual macrophages is a potential risk factor in the occurrence of preeclampsia［J］. Front Immunol, 2021, 12: 655655.
［27］ Yang N, Yang X Q, Fang Y L, et al. Nitric oxide promotes cerebral ischemia/reperfusion injury through upregulating hypoxia-inducible factor1-α-associated inflammation and apoptosis in rats［J］. Neurosci Lett, 2023, 795: 137034.
［28］ Jiang Y J, Zhu J H, Wu L, et al. Tetracycline inhibits local inflammation induced by cerebral ischemia via modulating autophagy［J］. PLoS One, 2012, 7(11): e48672.
［29］ Zhou Y F, Dong W T, Wang L K, et al. Cystatin C attenuates perihematomal secondary brain injury by inhibiting the cathepsin B/NLRP3 signaling pathway in a rat model of intracerebral hemorrhage［J］. Mol Neurobiol, 2024, 61(11): 9646－9662.
［30］ Xie L, Wu H, He Q P, et al. A slow-releasing donor of hydrogen sulfide inhibits neuronal cell death via anti-PANoptosis in rats with spinal cord ischemia-reperfusion injury［J］. Cell Commun Signal, 2024, 22(1): 33.
［31］ Lv S Y, Liu H Y, Wang H G. The interplay between autophagy and NLRP3 inflammasome in ischemia/reperfusion injury［J］. Int J Mol Sci, 2021, 22(16): 8773.
［32］ Wang X H, Chi J H, Huang D, et al. α-synuclein promotes progression of parkinson's disease by upregulating autophagy signaling pathway to activate NLRP3 inflammasome［J］. Exp Ther Med, 2020, 19(2): 931－938.
［33］ Zhou Y R, Zhao Y, Bao B H, et al. SND-117, a sinomenine bivalent alleviates type II collagen-induced arthritis in mice［J］. Int Immunopharmacol, 2015, 26(2):423－431.
［34］ Liu J, Jin Y, Ye Y, et al. The neuroprotective effect of short chain fatty acids against sepsis-associated encephalopathy in mice［J］. Front Immunol, 2021, 12:626894.
［35］ Akarasereenont P, Thiemermann C. The induction of cyclo-oxygenase-2 in human pulmonary epithelial cell culture (A549) activated by IL-1beta is inhibited by tyrosine kinase inhibitors［J］. Biochem Biophys Res Commun, 1996, 220(1): 181－185.
［36］ Lin C H, Sheu S Y, Lee H M, et al. Involvement of protein kinase C-gamma in IL-1beta-induced cyclooxygenase-2 expression in human pulmonary epithelial cells［J］. Mol Pharmacol, 2000, 57(1): 36－43.
［37］ Herseth J I, Refsnes M, Låg M, et al. Role of IL-1 beta and COX2 in silica-induced IL-6 release and loss of pneumocytes in co-cultures［J］. Toxicol In Vitro, 2009, 23(7): 1342－1353.
［38］ Varinthra P, Ganesan K, Huang S P, et al. The 4-(phenylsulfanyl) butan-2-one improves impaired fear memory retrieval and reduces excessive inflammatory response in triple transgenic alzheimer's disease mice［J］. Front Aging Neurosci, 2021, 13: 615079.

[1]	孙婧, 辛灵恩, 冯贤珍, 丁雨璇, 张玉彪, 王佳. 白芍总苷对脂多糖诱导的小鼠急性肺损伤的保护作用及机制[J]. 首都医科大学学报, 2024, 45(2): 302-311.
[2]	魏星月, 王连双, 王媛媛, 高孟泽, 何琼, 张瑶, 罗建文. 基于多模态超声成像数据的慢性肝病肝纤维化、炎症和脂肪变性的智能分级诊断[J]. 首都医科大学学报, 2023, 44(6): 928-935.
[3]	周妍, 吴巍. 萝卜硫素干扰微管与线粒体动力学抗肿瘤机制[J]. 首都医科大学学报, 2023, 44(3): 363-369.