Role and mechanisms of G-protein-coupled receptor 39 in brain injury after intracerebral hemorrhage in mice

Zhongyi ZHANG; Yuguang TANG; Jinyu DAI; Zongyi XIE

doi:10.16016/j.2097-0927.202312135

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Role and mechanisms of G-protein-coupled receptor 39 in brain injury after intracerebral hemorrhage in mice

Basic Medicine | 更新时间：2026-01-09

- Role and mechanisms of G-protein-coupled receptor 39 in brain injury after intracerebral hemorrhage in mice
- Journal of Army Medical University Vol. 46, Issue 11, Pages: 1254-1264(2024)
- 作者机构：
  
  400010 重庆，重庆医科大学附属第二医院神经外科
- 作者简介：
- 基金信息：
  
  the General Project of Natural Science Foundation of Chongqing(CSTB2023NSCQ-MSX0107)
- DOI：10.16016/j.2097-0927.202312135
  CLC： R341;R363.21;R743.34
- Received：28 December 2023，
  
  Revised：2024-02-23，
  
  Published：15 June 2024
- 稿件说明：
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Zhongyi ZHANG, Yuguang TANG, Jinyu DAI, et al. Role and mechanisms of G-protein-coupled receptor 39 in brain injury after intracerebral hemorrhage in mice[J]. Journal of Army Medical University, 2024, 46(11): 1254-1264.
DOI：

Zhongyi ZHANG, Yuguang TANG, Jinyu DAI, et al. Role and mechanisms of G-protein-coupled receptor 39 in brain injury after intracerebral hemorrhage in mice[J]. Journal of Army Medical University, 2024, 46(11): 1254-1264. DOI： 10.16016/j.2097-0927.202312135.

摘要

目的

探究激活G蛋白偶联受体39(G-protein-coupled receptor 39

GPR39)对小鼠脑出血(intracerebral hemorrhage

ICH)后神经炎症和脑损伤的影响及其作用机制。

方法

176只雄性C57/BL6小鼠按随机数字表法分成8组：Sham组(

=42)、ICH组(

=34)、ICH+Vehicle组(

=32)、ICH+TC-G 1008组(

=44)、ICH+GPR39 siRNA组(

=6)、ICH+Scramble siRNA组(

=6)、ICH+TC-G 1008+666-15组(

=6)、ICH+TC-G 1008+Vehicle 2组(

=6)。通过小鼠自体血建立ICH模型。在小鼠ICH建模后1 h和25 h灌胃GPR39特异性激动剂TC-G 1008治疗。在ICH建模前24 h，通过侧脑室注射GPR39 siRNA、环磷腺苷效应元件结合蛋白(cAMP response element binding protein

CREB)抑制剂666-15分别抑制内源性的GPR39和p-CREB的表达。小鼠ICH后48 h，通过改良的加西亚实验、前肢置放实验、转角实验评估小鼠短期的神经功能缺陷，脑组织湿/干法测定脑含水量；免疫荧光检测血肿周围脑组织GPR39与神经元、小胶质细胞共定位和NOD样受体热蛋白结构域相关蛋白3(NOD-like receptor thermal protein domain associated protein 3

NLRP3)在神经元中的表达情况；ELISA检测血肿周围组织中的白介素-1β (interleukin-1β

IL-1β)，肿瘤坏死因子-α(tumor necrosis factor-α

TNF-α)和髓过氧化物酶(myeloperoxidase

MPO)的含量；TUNEL检测血肿周围凋亡神经元的数量；尼氏染色评估血肿周围神经元损伤情况；Western blot检测血肿周围脑组织中GPR39、p-CREB、CREB、NRLP3、裂解型半胱天冬酶1(Cleaved caspase-1

C-caspase-1)、gasdermin-D蛋白(GSDMD)的表达。

结果

GPR39在小鼠ICH后48 h达到表达高峰(

＜0.05)，在神经元和小胶质细胞均有表达。TC-G 1008(24 mg/kg)激活GPR39后显著改善了小鼠ICH后改良的加西亚实验评分，左前肢放置成功率和左转次数增加(

＜0.05)。同侧基底节(basal ganglia

BG)和皮层(cortex

CX)的脑水肿显著减轻(

＜0.05)。血肿周围凋亡和受损的神经元数量显著减少(

＜0.05)。焦亡相关分子NLRP3、C-caspase-1、GS

DMD的表达和炎症相关因子IL-1β、TNF-α、MPO的水平降低(

＜0.05)。敲低GPR39和下调p-CREB的表达显著增加了小鼠ICH后血肿周围脑组织焦亡相关分子的表达和炎症相关因子的含量(

＜0.05)。

结论

GPR39激活可能部分通过CREB信号通路抑制小鼠ICH后的神经炎症和脑损伤，GPR39可能是ICH后减轻神经炎症和脑损伤潜在的治疗靶点。

Abstract

Objective

To investigate the effect and underlying mechanisms of G-protein-coupled receptor 39 (GPR39) activation on neuroinflammation and brain injury after experimental intracerebral hemorrhage in mice.

Methods

Mouse model of intracerebral hemorrhage (ICH) was established by intracerebral injection of autologous blood. A total of 176 male C57/BL6 mice were randomly divided into 8 groups: Sham group (

=42)

ICH group (

=34)

ICH+Vehicle group (

=32)

ICH+TC-G 1008 group (

=44)

ICH+GPR39 siRNA group (

=6)

ICH+Scramble siRNA group (

=6)

ICH+TC-G 1008+666-15 group (

=6)

and ICH+TC-G 1008+Vehicle 2 group (

=6). GPR39-specific agonist TC-G 1008 was administered

via

oral gavage at 1 and 25 h post-ICH modeling. Additionally

GPR39 siRNA and cAMP response element binding protein (CREB) inhibitor 666-15 were intracerebroventricularly injected 24 h before induction of ICH to inhibit the expression levels of GPR39 and p-CREB. At 48 h after ICH

modified Garcia test

forelimb placement test and corner turn test were used to evaluate the short-term neurological deficits in mice. Brain water content was determined by wet/dry method. Immunofluorescence assay was performed to detect the co-localization of GPR39 in neurons and microglia in the brain tissue surrounding the hematoma

as well as the expression of NOD-like receptor thermal protein domain associated protein 3 (NLRP3) in neurons. ELISA was employed to measure IL-1β

TNF-α and myeloperoxidase (MPO) levels in peri-hematoma tissue. TUNE

L staining was performed to quantify apoptotic neurons around the hematoma. Nissl staining was utilized to evaluate neuronal damage. Western blotting was conducted to detect the expression of GPR39

p-CREB

CREB

NLRP3

Cleaved caspase-1 (C-caspase-1)

and gasdermin-D protein (GSDMD) in peri-hematoma brain tissue.

Results

GPR39 expression peaked at 48 h post-ICH in mice (

0.05)

and it was expressed in both neurons and microglia. Activation of GPR39 by TC-G 1008 (24 mg/kg) significantly improved the modified Garcia score

and increased success rate of left forelimb placement and the number of left turns (

0.05). Brain edema in the ipsilateral basal ganglia (BG) and cortex (CX) was significantly reduced (

0.05). The numbers of apoptotic and damaged neurons around the hematoma were obviously decreased (

0.05). The expression of pyroptosis-related molecules

including NLRP3

C-caspase-1 and GSDMD and the levels of inflammation-related factors

including IL-1β

TNF-α and MPO were notably decreased (

0.05). However

knockdown of GPR39 and downregulation of p-CREB expression significantly increased the expression of pyroptosis related molecules and inflammatory-related factors in peri-hematoma brain tissue post-ICH in mice (

0.05).

Conclusion

GPR39 activation may inhibit neuroinflammation and brain injury after ICH in mice partly through the CREB signaling pathway. Therefore

GPR39 may be a potential therapeutic target for mitigating neuroinflammation and brain damage after ICH.

关键词

Keywords

references

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