LJ529 通过蛛网膜下腔出血大鼠的 AR-PKCε-ALDH2 通路减轻肥大细胞相关炎症。
LJ529 attenuates mast cell-related inflammation via AR-PKCε-ALDH2 pathway after subarachnoid hemorrhage in rats.
机构信息
Department of Neurosurgery, Xuanwu Hospital, Capital Medical University, Beijing, China.
Departments of Physiology and Pharmacology, Loma Linda University, Loma Linda, CA, USA.
出版信息
Exp Neurol. 2021 Jun;340:113686. doi: 10.1016/j.expneurol.2021.113686. Epub 2021 Mar 10.
BACKGROUND AND PURPOSE
Mast cells (MCs) has been recognized as an effector of inflammation or a trigger of inflammatory factors during stroke. LJ529 was reported to attenuate inflammation through a Gi protein-coupled Adenosine A3 receptor (AR) after ischemia. Here, we aim to study the protective effect and its mechanism of LJ529 in subarachnoid hemorrhage (SAH) rat model for mast cell-related inflammation.
METHODS
155 Sprague-Dawley adult male rats were used in experiments. Endovascular perforation was used for SAH model. Intraperitoneal LJ529 was performed 1 h after SAH. Neurological scores were measured 24 h after SAH. Rotarod and morris water maze tests were evaluated for 21 days after SAH. Mast cell degranulation was assessed with Toluidine blue staining and Chymase/Typtase protein expressions. Mast cell-related inflammation was evaluated using IL-6, TNF-α and MCP-1 protein expressions. MRS1523, inhibitor of GPR18 and ε-V1-2, inhibitor of PKCε were respectively given intraperitoneally (i.p.) 1 h and 30 min before SAH for mechanism studies. Pathway related proteins were investigated with western blot and immunofluorescence staining.
RESULTS
Expression of AR, PKCε increased after SAH. LJ529 treatment attenuated mast cell degranulation and inflammation. Meanwhile, both short-term and long-term neurological functions were improved after LJ529 treatment. Administration of LJ529 resulted in increased expressions of AR, PKCε, ALDH2 proteins and decreased expressions of Chymase, Typtase, IL-6, TNF-α and MCP-1 proteins. MRS1523 abolished the treatment effects of LJ529 on neurobehavior and protein levels. ε-V1-2 also reversed the outcomes of LJ529 administration through reduction in protein expressions downstream of PKCε.
CONCLUSIONS
LJ529 attenuated mast cell-related inflammation through inhibiting degranulation via A3R-PKCε-ALDH2 pathway after SAH. LJ529 may serve as a potential treatment strategy to relieve post-SAH brain injury.
背景与目的
肥大细胞（MCs）在中风期间被认为是炎症的效应物或炎症因子的触发物。据报道，LJ529 通过缺血后与 Gi 蛋白偶联的腺苷 A3 受体（AR）来减轻炎症。在此，我们旨在研究 LJ529 在蛛网膜下腔出血（SAH）大鼠模型中对与肥大细胞相关的炎症的保护作用及其机制。
方法
实验使用了 155 只成年雄性 Sprague-Dawley 大鼠。采用血管内穿孔法制作 SAH 模型。SAH 后 1 小时进行腹腔内 LJ529 给药。SAH 后 24 小时测量神经功能评分。SAH 后 21 天进行转棒和 Morris 水迷宫测试。甲苯胺蓝染色和糜酶/类胰蛋白酶蛋白表达评估肥大细胞脱颗粒。使用 IL-6、TNF-α 和 MCP-1 蛋白表达评估肥大细胞相关炎症。MRS1523、GPR18 抑制剂 ε-V1-2、PKCε 抑制剂分别在 SAH 前 1 小时和 30 分钟腹腔内给药，用于机制研究。Western blot 和免疫荧光染色用于研究通路相关蛋白。
结果
SAH 后 AR、PKCε 表达增加。LJ529 治疗可减轻肥大细胞脱颗粒和炎症。同时，LJ529 治疗后短期和长期神经功能均得到改善。LJ529 给药导致 AR、PKCε、ALDH2 蛋白表达增加，糜酶、类胰蛋白酶、IL-6、TNF-α 和 MCP-1 蛋白表达减少。MRS1523 消除了 LJ529 对神经行为和蛋白水平的治疗作用。ε-V1-2 通过减少 PKCε 下游蛋白的表达也逆转了 LJ529 给药的结果。
结论
LJ529 通过抑制 A3R-PKCε-ALDH2 通路抑制 SAH 后肥大细胞脱颗粒来减轻与肥大细胞相关的炎症。LJ529 可能成为缓解蛛网膜下腔出血后脑损伤的一种潜在治疗策略。
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