肥大细胞(MC)已被认为是中风期间炎症的效应物或炎症因子的触发因素。据报道,LJ529在缺血后通过Gi蛋白偶联腺苷A3受体(A3R)减轻炎症。这里,目的研究LJ529对蛛网膜下腔出血(SAH)大鼠模型肥大细胞相关炎症的保护作用及其机制。
实验中使用了155只Sprague-Dawley成年雄性大鼠。血管内穿孔用于SAH模型。SAH后1小时进行腹膜内LJ529。SAH后24小时测量神经系统评分。在SAH后21天评估旋转杆和莫里斯水迷宫测试。用甲苯胺蓝染色和糜酶/分型酶蛋白表达评估肥大细胞脱粒。使用IL-6,TNF-α和MCP-1蛋白表达评估肥大细胞相关的炎症。在SAH前1小时和30分钟分别腹膜内(腹膜内)给予GPR18抑制剂MRS1523和PKCε抑制剂ε-V1-2,以进行机制研究。用westernblot和免疫荧光染色检测通路相关蛋白。
A3R的表达式,PKCε在SAH后增加。LJ529治疗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的施用导致A3R的表达增加,PKCε,ALDH2蛋白与糜蛋白酶表达降低,分型酶,IL-6、TNF-α和MCP-1蛋白。MRS1523取消了LJ529对神经行为和蛋白质水平的治疗作用。ε-V1-2还通过降低PKCε下游的蛋白表达来逆转LJ529给药的结果。
SAH后,LJ529通过A3R-PKCε-ALDH2通路抑制脱颗粒,从而减轻肥大细胞相关炎症。LJ529可作为减轻SAH后脑损伤的潜在治疗策略。
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 (A3R) 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.
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.
Expression of A3R, 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 A3R, 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.
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.