Salidroside Provides Neuroprotection by Modulating the Expression of Immunomodulatory Protein Interleukin-4 Induced Protein 1 and Activating Microglia after Ischemic Brain Injury

doi:10.3969/j.issn.1673-5765.2022.02.006

Abstract

Abstract:

Objective To explore the effect of salidroside on the expression of interleukin 4 inducible protein 1 (IL4I1) and microglia polarization after ischemic brain injury.

Methods 38 male C57BL/6J mice were randomly divided into sham-operated group (4 mice), saline group (17 mice) and salidroside-treated group (17 mice). The model of ischemic brain injury was constructed by middle cerebral artery embolization method (reperfusion after ischemia for 60 minutes). After reperfusion for 10 minutes, saline and salidroside (10 mg/kg) were injected respectively through the tail vein at saline group and salidroside-treated group, once a day for 28 days. Collecting the serum of mice in sham-operated group and the model groups at 48 hours, 7 days and 28 days after reperfusion, the concentrations of IL4I1 were measured by ELISA. The infarct volume at 7 days was observed by NeuN immunofluorescence labeled staining. The polarization of microglia and changes in the length and number of protrusions at 7 days were observed by CD16/32, CD206 and Iba1 labeled immunofluorescent staining. To explore the relationship between IL4I1 and phenotypes of microglia in vitro cell experiment, the polarization of mouse BV2 microglia cell line were induced by LPS plus INF-γ or IL-4 plus IL-13, to transform into M1 proinflammatory type and M2 anti-inflammatory type microglia, respectively, with the blank control group as M0 microglia. The expression of IL4I1 in different phenotype BV2 cells was observed by Western blot.

Results Compared with sham-operated group, the serum concentration of IL4I1 in saline group decreased at 48 hours after reperfusion (P =0.0302); compared with saline group, it increased in salidroside-treated group at 7 days and 28 days (P =0.0229, P =0.0076). Compared with saline group, the salidroside-treated group had less cerebral infarction volume at 7 days (P =0.0389). Compared with saline group, the protrusion length and number, the number of branch points and terminal points of microglia around ischemic area increased in salidroside-treated group at 7 days (P =0.0040, P <0.001, P <0.001, P <0.001). Salidroside treatment significantly decreased the number of Iba1+CD16/32+ M1 proinflammatory type microglia (P =0.0407, P =0.0032) and increased the number of Iba1+CD206+ M2 anti-inflammatory type microglia (P =0.0206, P =0.0075) in the cortex and striatum at 7 days. In vitro cell experiment showed that the expression of IL4I1 decreased in both M1 and M2 phenotype microglia compared with that in M0 phenotype microglia (P =0.0008, P =0.0155), and the expression level was lower in M1 phenotype than that in M2 phenotype (P =0.0406).

Conclusions Salidroside may play neuroprotective action by increasing the expression of IL4I1 in serum and ischemic brain tissue, and activating microglia from M1 proinflammatory phenotype into M2 anti-inflammatory phenotype after ischemic injury, to reduce the volume of cerebral infarction.

Key words: Ischemic brain injury; Salidroside; Microglia polarization; Interleukin 4 induced protein 1

摘要：

目的探讨毛柳苷在缺血性脑损伤后对白介素4诱导蛋白1（interleukin-4 induced protein 1，IL4I1）表达及小胶质细胞极化的影响。

方法 38只雄性C57BL/6J小鼠随机分为假手术组（4只）、毛柳苷组（17只）和生理盐水组（17只）。通过线栓法制作大脑中动脉缺血再灌注模型（缺血60 min拔栓实现再灌注），再灌注10 min后，毛柳苷组与生理盐水组小鼠分别经尾静脉按照10 mg/kg剂量注射毛柳苷（1 mg/mL）与生理盐水，每天1次，持续28 d。ELISA法检测假手术组及模型组缺血再灌注后48 h、7 d、28 d血清中的IL4I1浓度。采用神经元特异核蛋白（neuronal nuclei，NeuN）抗体免疫荧光染色标记神经元，观察缺血再灌注后7 d脑梗死体积。采用CD16/32、CD206和离子化钙结合适配分子1（ionized calcium binding adapter molecule 1，Iba1）免疫荧光染色观察缺血再灌注后7 d小胶质细胞极化状态及突起的长度、数量变化。通过细胞实验探究IL4I1与小胶质细胞不同表型之间的联系。培养小鼠BV2小胶质细胞，设置M0型细胞为空白对照组，使用脂多糖（lipopolysaccharide，LPS）和INF-γ诱导BV2细胞极化为M1促炎型，使用IL-4和IL-13诱导BV2 细胞极化为M2抗炎型细胞。BV2细胞诱导48 h后提取细胞蛋白，通过免疫印迹检测BV2细胞不同表型中IL4I1的表达情况。

结果与假手术组相比，生理盐水组脑缺血再灌注后48 h血清中IL4I1浓度下降（P =0.0302）；与生理盐水组相比，毛柳苷组脑缺血再灌注后7 d和28 d血清中IL4I1浓度增加（P =0.0229，P =0.0076）。毛柳苷组脑缺血再灌注后7 d脑梗死体积较生理盐水组降低（P =0.0389）。与生理盐水组相比，毛柳苷组脑缺血再灌注后7 d缺血区周围小胶质细胞突起长度、突起数量、突起的分叉点及终末端点数量均增加（P =0.0040，P <0.001，P <0.001，P <0.001），皮层与纹状体区域M1型小胶质细胞数量减少（P =0.0407，P =0.0032），皮层与纹状体区域M2型小胶质细胞数量增多（P =0.0206，P =0.0075）。体外细胞实验BV2小胶质细胞不同表型中，M1型与M2型较M0型IL4I1表达下降（P =0.0008，P =0.0155），与 M2型相比，M1型IL4I1表达更低（P =0.0406）。

结论毛柳苷可能通过增加脑缺血再灌注损伤后血清及缺血脑组织中IL4I1的表达，并使激活的小胶质细胞向M2抗炎表型转化，减少脑梗死体积，从而发挥神经保护作用。

关键词: 缺血性脑损伤; 毛柳苷; 小胶质细胞极化; 白介素4诱导蛋白1

LIU Wenqian, ZHAO Shunying, YE Weizhen, JIANG Mingyu, CHEN Wentao, CHEN Qingfang, WEN Shaohong, DONG Wen, LIU Xiangrong. Salidroside Provides Neuroprotection by Modulating the Expression of Immunomodulatory Protein Interleukin-4 Induced Protein 1 and Activating Microglia after Ischemic Brain Injury[J]. Chinese Journal of Stroke, 2022, 17(02): 134-141.

刘文倩, 赵顺英, 叶维贞, 姜鸣钰, 陈文涛, 陈青芳, 温少红, 董雯, 刘向荣 . 毛柳苷在缺血性脑损伤中对白介素4诱导蛋白1表达和小胶质细胞激活及表型的影响[J]. 中国卒中杂志, 2022, 17(02): 134-141.

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