Study on the Brain Protection of Granulocyte Colony-Stimulating Factor in Stroke after Alcohol Use Disorder

doi:10.3969/j.issn.1673-5765.2024.10.010

Abstract

Abstract: Objective To examine the effects of granulocyte colony-stimulating factor (G-CSF) treatment on the expression of transforming growth factor-α (TGF-α) in microglia and its neuroprotective effects in stroke after alcohol use disorder (AUD) in rats.
Methods Male Sprague-Dawley rats were used to establish an AUD model using the double bottle method. Based on the AUD model, the focal cerebral ischemia model of middle cerebral artery occlusion (MCAO) was established using the thread embolism method. TGF-α small interfering RNA (siRNA) or negative control siRNA was injected into the lateral ventricle
24 h before MCAO. G-CSF was injected intraperitoneally l h after ischemia-reperfusion injury. Rats were randomly divided into the AUD group (18 rats), the AUD+MCAO group (18 rats), the AUD+MCAO+G-CSF group (18 rats), the AUD+MCAO+G-CSF+TGF-α siRNA group (18 rats), and the AUD+MCAO+G-CSF+control siRNA group (6 rats). Alcohol intake, changes in alcohol preference, and neurological function scores of the rats were recorded. The volume of cerebral infarction 24 h after MCAO was measured by 2,3,5-triphenyltetrazolium chloride staining. The number of positive cells of transmembrane protein 119 (TMEM119), a specific marker of microglia in the cerebral cortex of the ischemic side, and the G-CSF receptor (G-CSFR), and the protein expression of TGF-α were observed by immunofluorescence staining. The expression levels of TMEM119 and TGF-α protein in brain tissue of the ischemic side were detected by Western blot.
Results Alcohol intake and alcohol preference of rats gradually increased with the increase of drinking time. G-CSFR was significantly expressed in microglia. Compared with the AUD group, the fluorescence intensity of TMEM119 protein and TGF-α protein in the AUD+MCAO group was significantly enhanced, and the number of positive cells increased. Compared with the AUD+MCAO group, the volume of infarction in the AUD+MCAO+G-CSF group was significantly reduced, and the symptoms of neurological impairment improved. The fluorescence intensity of TMEM119 protein was significantly weakened, and the number of positive cells decreased. The fluorescence intensity of TGF-α protein was significantly enhanced, and the number of positive cells increased. Volume of cerebral infarction increased and neurological deficits aggravated in the rats after lateral ventricle injection of TGF-α siRNA.
Conclusions G-CSF plays an important brain protective effect in stroke after AUD by increasing the expression of TGF-α in microglia.

Key words: Alcohol use disorder; Stroke; Microglia; Transforming growth factor-α; Granulocyte colony-stimulating factor

摘要： 目的探讨大鼠酒精使用障碍（alcohol use disorder，AUD）后卒中应用粒细胞集落刺激因子（granulocyte colony-stimulating factor，G-CSF）治疗对小胶质细胞中转化生长因子α（transforming growth factor-α，TGF-α）表达的影响及其神经保护作用。
方法选择雄性Sprague-Dawley大鼠，应用双瓶法构建AUD模型，在AUD模型基础上应用线栓法制作大脑中动脉栓塞（middle cerebral artery occlusion，MCAO）局部脑缺血模型。MCAO前24 h侧脑室注射TGF-α小干扰RNA（small interfering RNA，siRNA）或阴性对照siRNA。缺血再灌注损伤1 h后腹腔注射G-CSF。大鼠随机分为AUD组（18只）、AUD+MCAO组（18只）、AUD+MCAO+G-CSF组（18只）、AUD+MCAO+G-CSF+TGF-α siRNA组（18只）及AUD+MCAO+G-CSF+阴性对照siRNA组（6只）。记录大鼠酒精摄入量、酒精偏好变化及神经功能评分。采用2，3，5-氯化三苯基四氮唑染色法测定MCAO后24 h脑梗死体积。采用免疫荧光染色观察缺血侧大脑皮质小胶质细胞特异性标志物跨膜蛋白119（transmembrane protein 119，TMEM119）阳性细胞数量、G-CSF受体（G-CSF receptor，G-CSFR）及TGF-α蛋白表达的变化。采用免疫印迹法检测缺血侧脑组织TMEM119及TGF-α蛋白表达水平。
结果大鼠酒精摄入量和酒精偏好随饮酒时间的增加而逐渐增加。G-CSFR在小胶质细胞中有明显的表达。与AUD组相比，AUD+MCAO组TMEM119和TGF-a蛋白荧光强度明显增强，阳性细胞数量增加。与AUD+MCAO组相比，AUD+MCAO+G-CSF组治疗后梗死体积明显缩小，大鼠神经功能缺损症状改善，TMEM119蛋白荧光强度明显减弱，阳性细胞数量减少；TGF-α蛋白荧光强度明显增强，阳性细胞数量增加。侧脑室注射TGF-α siRNA后大鼠脑梗死体积增加，神经功能缺损加重。
结论 G-CSF通过增加小胶质细胞中TGF-α的表达，在AUD后卒中中发挥重要的脑保护作用。

关键词: 酒精使用障碍; 卒中; 小胶质细胞; 转化生长因子α; 粒细胞集落刺激因子

CLC Number:

HAO Dandan, WANG Danyang, LI Li. Study on the Brain Protection of Granulocyte Colony-Stimulating Factor in Stroke after Alcohol Use Disorder[J]. Chinese Journal of Stroke, 2024, 19(10): 1170-1179.

郝丹丹, 王丹阳, 李丽. 粒细胞集落刺激因子在酒精使用障碍后卒中中的脑保护作用研究[J]. 中国卒中杂志, 2024, 19(10): 1170-1179.

References

[1] FEIGIN V L，BRAININ M，NORRVING B，et al. World Stroke Organization（WSO）：global stroke fact sheet[J]. Int J Stroke，2022，17（1）：18-29.
[2] 唐春花，郭露，李琼，等. 2022年全球卒中数据报告解读[J]. 诊断学理论与实践，2023，22（3）：238-246.
TANG C H，GUO L，LI Q，et al. Interpretation on the report of global stroke data 2022[J]. J Diagn Concepts Pract，2023，22（3）：238-246.
[3] THAYABARANATHAN T，KIM J，CADILHAC D A，et al. Global stroke statistics[J]. Int J Stroke，2022，17（9）：946-956.
[4] 王拥军，李子孝，谷鸿秋，等. 中国卒中报告2020（中文版）（1）[J]. 中国卒中杂志，2022，17（5）：433-447.
WANG Y J，LI Z X，GU H Q，et al. China stroke statistics 2020（1）[J]. Chin J Stroke，2022，17（5）：433-447.
[5] QU X，LIU M，KE C R，et al. Burden of alcohol use disorders in China and the regions with different income levels over the world[J/OL]. J Glob Health，2021，11：08011[2024-05-09]. https://doi.org/10.7189/jogh.
11.08011.
[6] VAFAEI MASTANABAD M，NOORAEI A，HASSAN ZADEH TABATABAEI M S，et al. Granulocyte-colony stimulating factor（G-CSF）：an emerging therapeutic approach for amyotrophic lateral sclerosis（ALS）[J]. Acta Neurol Belg，2023，123（3）：763-771.
[7] SARKALA H B，JAHANSHAHI M，DOLATABADI L K，et al. G-CSF improved the memory and dendritic morphology impairments in the hippocampal CA1 pyramidal neurons after brain ischemia in the male rats[J]. Metab Brain Dis，2023，38（8）：2573-2581.
[8] RAHI V，JAMWAL S，KUMAR P. Neuroprotection through G-CSF：recent advances and future viewpoints[J]. Pharmacol Rep，2021，73（2）：372-385.
[9] HASHEMIZADEH S，HOSSEINDOOST S，OMIDI A，et al. Novel therapeutic approach to slow down the inflammatory cascade in acute/subacute spinal cord injury：early immune therapy with lipopolysaccharide enhanced neuroprotective effect of combinational therapy of granulocyte colony-stimulating factor and bone-marrow mesenchymal stem cell in spinal cord injury[J/OL]. Front Cell Neurosci，2022，16：993019[2024-05-09]. https://doi.org/10.3389/fncel.2022.993019.
[10] 吕振，龚志刚. 酒精使用障碍动物模型构建的研究进展[J]. 中国实验动物学报，2023，31（3）：382-389.
LYU Z，GONG Z G. Advances in establishment of alcohol use disorder models[J]. Acta Lab Anim Sci Sin，2023，31（3）：382-389.
[11] LI X X，YANG T，WANG N，et al. 7，8-
dihydroxyflavone attenuates alcohol-related behavior in rat models of lcohol consumption via TrkB in the ventral tegmental area[J/OL]. Front Neurosci，2020，14：467[2024-05-09]. https://doi.org/10.3389/fnins.2020.00467.
[12] MOORE C F，LYNCH W J. Alcohol preferring（P）rats as a model for examining sex differences in alcohol use disorder and its treatment[J/OL]. Pharmacol Biochem Behav，2015，132：1-9[2024-05-09]. https://doi.org/10.1016/j.pbb.2015.02.014.
[13] SHI Y Y，HAN L J，ZHANG X X，et al. Selenium alleviates cerebral ischemia/reperfusion injury by regulating oxidative stress，mitochondrial fusion and ferroptosis[J]. Neurochem Res，2022，47（10）：2992-3002.
[14] LI L，MCBRIDE D W，DOYCHEVA D，et al. G-CSF attenuates neuroinflammation and stabilizes the blood-brain barrier via the PI3K/Akt/GSK-3β signaling pathway following neonatal hypoxia-ischemia in rats[J/OL]. Exp Neurol，2015，272：135-144[2024-05-09]. https://doi.org/10.1016/j.expneurol.2014.12.020.
[15] GARCIA J H，WAGNER S，LIU K F，et al. Neurological deficit and extent of neuronal necrosis attributable to middle cerebral artery occlusion in rats[J]. Stroke，1995，26（4）：627-635.
[16] LI L，KLEBE D，DOYCHEVA D，et al. G-CSF ameliorates neuronal apoptosis through GSK-3β inhibition in neonatal hypoxia-ischemia in rats[J/OL]. Exp Neurol，2015，263：141-149[2024-05-09]. https://doi.org/10.1016/j.expneurol.2014.10.004.
[17] MENDELSON S J，PRABHAKARAN S. Diagnosis and management of transient ischemic attack and acute ischemic stroke：a review[J]. JAMA，2021，325（11）：1088-1098.
[18] FESKE S K. Ischemic stroke[J]. Am J Med，2021，134（12）：1457-1464.
[19] PATEL D，GANDHI Z，DESAI R，et al. Impact of alcohol use disorder on stroke risk in geriatric patients with prediabetes：a nationwide analysis[J/OL]. Int J Clin Pract，2021，75（9）：e14477[2021-06-24]. https://doi.org/10.1111/ijcp.14477.
[20] YU H，WANG P C，LU H B，et al. Effects of G-CSF on hPDLSC proliferation and osteogenic differentiation in the LPS-induced inflammatory microenvironment[J/OL]. BMC Oral Health，2023，23（1）：422[2024-05-08]. https://doi.org/10.1186/s12903-023-03040-9.
[21] VON LEWINSKI D，SELVANAYAGAM J B，SCHATZ R A，et al.“Protocol for a phase 2，randomized，double-blind，placebo-controlled，safety and efficacy study of dutogliptin in combination with filgrastim in early recovery post-myocardial infarction”：study protocol for a randomized controlled trial[J/OL]. Trials，2020，21：744[2024-05-08]. https://doi.org/10.1186/s13063-020-04652-0.
[22] SARKALA H B，JAHANSHAHI M，DOLATABADI L K，et al. G-CSF improved the memory and dendritic morphology impairments in the hippocampal CA1 pyramidal neurons after brain ischemia in the male rats[J]. Metab Brain Dis，2023，38（8）：2573-2581.
[23] PÀMIES A，LLOP D，IBARRETXE D，et al. Angiopoietin-2，vascular endothelial growth factor family，and heparin binding endothelial growth factor are associated with subclinical atherosclerosis in rheumatoid arthritis[J/OL]. Comput Struct Biotechnol J，2024，23：1680-1688[2024-05-08]. https://doi.org/10.1016/j. csbj. 2024.04.042.
[24] KARKI P，JOHNSON J，Jr，SON D S，et al. Transcriptional regulation of human transforming growth factor-α in astrocytes[J]. Mol Neurobiol，2017，54（2）：964-976.
[25] KIMURA M，MOTEKI H，OGIHARA M. Role of hepatocyte growth regulators in liver regeneration
[J/OL]. Cells，2023，12（2）：208[2024-05-08]. https://doi.org/10.3390/cells12020208.
[26] AMUNDADOTTIR L T，NASS S J，BERCHEM G J，et al. Cooperation of TGF alpha and c-Myc in mouse mammary tumorigenesis：coordinated stimulation of growth and suppression of apoptosis[J]. Oncogene，1996，13（4）：757-765.
[27] LIDDELOW S A，BARRES B A. Reactive astrocytes：production，function，and therapeutic potential[J]. Immunity，2017，46（6）：957-967.
[28] PRINZ M，PRILLER J. Microglia and brain macrophages in the molecular age：from origin to neuropsychiatric disease[J/OL]. Nat Rev Neurosci，2014，15：300-312[2024-05-08]. https://doi.org/10.1038/nrn3722.
[29] ROTHHAMMER V，BORUCKI D M，TJON E C，et al. Microglial control of astrocytes in response to microbial metabolites[J]. Nature，2018，557（7707）：724-728.