外源性瘦素通过上调星形胶质细胞GLT-1和GLAST的表达减轻小鼠脑缺血再灌注引起的谷氨酸兴奋毒性损伤

doi:10.12122/j.issn.1673-4254.2024.06.08

南方医科大学学报 ›› 2024, Vol. 44 ›› Issue (6): 1079-1087.doi: 10.12122/j.issn.1673-4254.2024.06.08

外源性瘦素通过上调星形胶质细胞GLT-1和GLAST的表达减轻小鼠脑缺血再灌注引起的谷氨酸兴奋毒性损伤

陈洁¹^,²(), 刘晨旭¹^,², 王春²^,³, 李丽¹^,², 陶伟婷¹^,², 徐婧茹⁴, 唐红辉⁴, 黄丽¹^,²()

^1.蚌埠医科大学，病理生理学教研室，安徽蚌埠 233000
^2.蚌埠医科大学，心脑血管疾病基础与临床重点实验室，安徽蚌埠 233000
^4.蚌埠医科大学，临床医学院，安徽蚌埠 233000
^3.蚌埠医科大学第二附属医院全科医学科，安徽蚌埠 233000

收稿日期:2023-07-14 出版日期:2024-06-20 发布日期:2024-07-01
通讯作者: 黄丽 E-mail:cj332022@163.com;zi05@163.com
作者简介:陈洁，在读硕士研究生，E-mail: cj332022@163.com
基金资助:
安徽省教育厅自然科学重点研究项目(KJ2021A0696);蚌埠医学院“512人才培育计划”(by51202204);安徽省教育厅自然科学重大研究项目(KJ2021ZD0091);安徽省研究生创新创业实践项目(2022cxcysj169);蚌埠医学院研究生科研创新计划项目(Byycx22008);国家级大学生创新项目(202210367060)

Exogenous leptin improves cerebral ischemia-reperfusion-induced glutamate excitotoxic injury in mice by up-regulating GLT-1 and GLAST expression in astrocytes

Jie CHEN¹^,²(), Chenxu LIU¹^,², Chun WANG²^,³, Li LI¹^,², Weiting TAO¹^,², Jingru XUN⁴, Honghui TANG⁴, Li HUANG¹^,²()

^1.Department of Pathophysiology, Bengbu 233000, China
^2.Preclinical and Clinical Key Laboratory of Cardiovascular and Cerebrovascular Diseases, Bengbu 233000, China
^4.School of Clinical Medicine, Bengbu Medical University, Bengbu 233000, China
^3.Department of General Medicine, Second Affiliated Hospital of Bengbu Medical University, Bengbu 233000, China

Received:2023-07-14 Online:2024-06-20 Published:2024-07-01
Contact: Li HUANG E-mail:cj332022@163.com;zi05@163.com

摘要/Abstract

摘要：

目的探究外源性瘦素（Leptin）对小鼠局灶性脑缺血再灌注损伤的保护作用及其内在机制。方法将100只C57BL/6小鼠随机分为5组：假手术（Sham）组、脑缺血/再灌注模型组（I/R组）、瘦素低剂量组（Leptin-L组，0.5 mg/kg）、瘦素中剂量组（Leptin-M组，1 mg/kg）和瘦素高剂量组（Leptin-H组，2 mg/kg），20只/组。采用改良线栓法复制小鼠局灶性脑缺血/再灌注模型，缺血1.5 h再灌注24 h后进行神经功能评分评估神经功能缺损程度，TTC染色测定脑梗死面积，HE染色观察皮层脑组织病理变化，退化神经元染色观察皮层神经元变性损伤程度，免疫组织化学染色测定皮层脑组织胶原酸性纤维蛋白的表达和形态变化，Western blot测定皮层脑组织胶原酸性纤维蛋白蛋白表达。在此基础上，另将45只C57BL/6小鼠随机分为Sham组、I/R组和Leptin组（1 mg/kg），15只/组。谷氨酸检测试剂盒检测皮层脑组织谷氨酸含量，免疫组织化学染色测定皮层谷氨酸-天冬氨酸转运体（GLAST）和谷氨酸转运体1（GLT-1）表达，Western blotting测定GLAST、GLT-1蛋白表达。结果与I/R组比较，瘦素明显降低小鼠神经功能缺损评分（P<0.0001），缩小脑梗死面积（P<0.001），减轻皮层脑组织病理损伤程度，降低皮层神经元损伤程度（P<0.0001），维持星形胶质细胞正常形态及降低星形胶质细胞的过度增生和表达（P<0.01），使GLT-1、GLAST表达上调（P<0.01、P<0.05），脑组织谷氨酸含量降低（P<0.01）。结论外源性瘦素对小鼠脑缺血再灌注损伤具有明显的神经保护作用，其机制可能与调节星形胶质细胞过度增生和上调GLT-1、GLAST的表达从而降低谷氨酸的兴奋毒性损伤有关。

关键词: 瘦素, 脑缺血再灌注损伤, 星形胶质细胞, GLT-1, GLAST, 兴奋性毒性

Abstract:

Objective To investigate the protective effect of exogenous leptin against focal cerebral ischemia-reperfusion (I/R) injury in mice and explore the underlying mechanism. Methods A total of 100 C57BL/6 mice were randomly divided into 5 groups, including a sham-operated group, cerebral I/R model group, and 3 leptin treatment groups with intraperitoneal injections of 0.5, 1.0 or 2.0 leptin immediately after occlusion of the internal carotid artery. At 24 h after reperfusion, neurological function scores of the mice were assessed, and TTC staining was used to determine the area of cerebral infarction. The pathological changes in the cortical brain tissue of the mice were observed using HE staining, and degenerative damage of the cortical neurons were assessed with Fluoro-Jade C staining. The expression of glial fibrillary acidic protein in cortical brain tissues was detected using immunohistochemistry and Western blotting. In another 45 C57BL/6 mice with sham operation, I/R modeling, or leptin (1 mg/kg) treatment, glutamic acid in the cortical brain tissue was detected using glutamate assay, and cortical glutamate-aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1) protein expressions were detected using immunohistochemistry. Results Compared with the I/R model mice, the leptin-treated mice had significantly lower neurological deficit scores, smaller cerebral infarct area, milder pathologies in the cortical brain tissue, and lessened cortical neuronal damage with normal morphology and less excessive proliferation of the astrocytes. Leptin treatment significantly up-regulated the expressions of GLT-1 and GLAST and lowered the content of glutamic acid in the brain tissue of the I/R mice. Conclusion Exogenous leptin has obvious neuroprotective effect against cerebral I/R injury in mice, mediated probably by controlling excessive astrocyte proliferation and up-regulating cortical GLT-1 and GLAST expressions to reduce glutamate-mediated excitotoxic injury of the astrocytes.

Key words: leptin, cerebral ischemia-reperfusion injury, astrocytes, glutamate transporter-1, glutamate-aspartate transporter, excitotoxicity

陈洁, 刘晨旭, 王春, 李丽, 陶伟婷, 徐婧茹, 唐红辉, 黄丽. 外源性瘦素通过上调星形胶质细胞GLT-1和GLAST的表达减轻小鼠脑缺血再灌注引起的谷氨酸兴奋毒性损伤[J]. 南方医科大学学报, 2024, 44(6): 1079-1087.

Jie CHEN, Chenxu LIU, Chun WANG, Li LI, Weiting TAO, Jingru XUN, Honghui TANG, Li HUANG. Exogenous leptin improves cerebral ischemia-reperfusion-induced glutamate excitotoxic injury in mice by up-regulating GLT-1 and GLAST expression in astrocytes[J]. Journal of Southern Medical University, 2024, 44(6): 1079-1087.

图/表 8

图1 镜下小鼠右侧CCA及其分支

Fig.1 Microscopic observation of mouse right common carotid artery (CCA) and its branches.

图2 脑I/R后实验鼠体态(A)和神经功能评分(B)

Fig.2 Posture of the mice following brain I/R modeling (A) and neurological deficit scores of the mice in different groups (B), n=9. ****P<0.0001 vs Sham group; ####P<0.0001 vs I/R group.

图3 TTC染色测定脑梗死面积的变化

Fig.3 TTC staining for determining the size of cerebral infarct area of the mice. A: TTC staining of the brain tissue sections. B: Quantitative analysis of brain infarct area (n=3). ****P<0.0001 vs Sham group; ###P<0.001, ####P<0.0001 vs I/R group.

图4 HE染色观察皮层脑组织病理学变化

Fig.4 HE staining for examining histopathological changes in the cortical brain tissues of the mice (scale bar=20 μm).

图5 FJC 染色观察皮层神经元变性损伤

Fig.5 FJC staining for assessing cortical neuronal degenerative damage. A: FJC staining iamges (scale bar=20 μm). B: Positive rate of the degenerated neurons. ****P<0.0001 vs Sham group; ####P<0.0001 vs I/R group.

图6 GFAP蛋白在小鼠皮层脑组织中的表达情况

Fig.6 Expression of GFAP protein in mouse cortical brain tissue. A: Expression of GFAP detected by immunohistochemistry (Original magnification: ×40). B: Expression of GFAP detected by Western blotting (n=3). C: Quantitative analysis of GFAP expression. ***P<0.001 vs Sham group; ##P<0.01 vs I/R group.

图7 GLT-1、GLAST蛋白在小鼠皮层脑组织中的表达情况

Fig.7 Expression of GLT-1 and GLAST proteins in mouse cortical brain tissue (×40).

图8 小鼠皮层脑组织GLT-1、GLAST蛋白表达情况和Glu的含量的变化

Fig.8 Changes in the expression of GLT-1 and GLAST proteins and Glu content in mouse cortical brain tissue. A: Expression of GLT-1 and GLAST detected by Western blotting (n=3). B: Quantitative analysis of GLT-1 expression. C: Quantitative analysis of GLAST expression; D: Content of Glu (n=3). ***P<0.001, ****P<0.0001 vs Sham group; #P<0.01, ###P<0.001 vs I/R group.

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外源性瘦素通过上调星形胶质细胞GLT-1和GLAST的表达减轻小鼠脑缺血再灌注引起的谷氨酸兴奋毒性损伤

Exogenous leptin improves cerebral ischemia-reperfusion-induced glutamate excitotoxic injury in mice by up-regulating GLT-1 and GLAST expression in astrocytes

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