MRS2578通过P2Y6-ROCK-MILC信号通路抑制海马CA3区域小胶质细胞吞噬作用并加重小鼠癫痫发作

MRS2578 Aggravates Seizure via Blocking the P2Y6-ROKC-MLC Signaling Pathway and Inhibits Microglial Phagocytosis in Hippocampal CA3 of Mice

许多研究表明,癫痫过程中P2Y6受体表达增加。MRS2578作为P2Y6受体的不可逆抑制剂,其在癫痫中作用尚不明确。本研究旨在阐明MRS2578在癫痫中的作用及其潜在的分子机制。研究发现,小鼠脑室注射海人酸诱导癫痫模型,同时注射MRS2578能加重癫痫发作,此作用呈现浓度依赖效应;MRS2578增加海马CA3区域神经元丢失(FJB+神经元由71.67±8.88增加至98.45±9.97个/视野);同时抑制小胶质细胞的激活以及转变为阿米巴样细胞(小胶质细胞密度由66.18±5.86下降至55.22±10.85个/视野),小胶质细胞CD68表达也相应降低;共聚焦显微镜检测发现,MRS2578处理后,CA3区域小胶质细胞吞噬细胞所形成的"吞噬杯"结构减少;Western印迹检测证实,MRS2578抑制小胶质细胞P2Y6、Rho相关激酶(ROCK)、p-MLC-Ser19的分子表达。上述结果提示,MRS2578加重海人酸诱导的癫痫发作,伴随小胶质细胞激活的抑制和细胞吞噬功能下调,此过程依赖P2Y6-ROCK-MLC信号途径。

Numerous studies have shown that P2 Y6 expression increased in epilepsy.As an irreversible inhibitor of the P2 Y6 receptor,the role of MRS2578 in epilepsy is not clear.This study aims to clarify the effect of MRS2578 in epilepsy and its potential molecular mechanism.C57 BL/7 mice intracerebroventricular injection of KA(kainic acid)was used as an epilepsy model.MRS2578 aggravated seizure in a dose-dependent manner.MRS2578 increased neuron loss in the hippocampal CA3 region(KA:FJB+neuron 71.67±8.88 per frame,KA+MRS2578:FJB+neuron 98.45±9.97 per frame);the activation of microglia and ameboid microglia percentage was inhibited by MRS2578(microglia density from KA was 66.18±5.86 per frame,and it decreased to55.22±10.85 per frame in KA+MRS2578 group).Meanwhile,CD68 expression in microglia decreased after MRS2578 treatment.Confocal microscopy observation showed that MRS2578 treatment attenuated the"phagocytotic cup"structure of microglia during its phagocytosis.Western blotting results revealed that P2 Y6,ROCK and p-MCL-Ser19 expression decreased in the KA+MRS2578 group compared to the KA group.These results suggest that MRS2578 could efficiently aggravate seizure in the ICV-epilepsy model,and decrease microglial activation and phagocytosis in the hippocampal CA3 region,which was dependent on inhibition of the P2 Y6-ROCK-MLC signaling pathway.