新型吩噻嗪衍生物DA414在大鼠脑出血中的作用及机制研究  

Effects and underlying mechanisms of novel phenothiazine derivative DA414 in rats with intracerebral hemorrhage

作　　者：马渝凡 王柄乔 林森 杨清武 MA Yufan;WANG Bingqiao;LIN Sen;YANG Qingwu(Department of Neurology,Second Affiliated Hospital,Army Medical University(Third Military Medical University),Chongqing,China)

机构地区：[1]陆军军医大学(第三军医大学)第二附属医院神经内科,重庆

出　　处：《陆军军医大学学报》2025年第9期922-934,共13页Journal of Army Medical University

基　　金：重庆市自然科学基金面上项目(CSTB2024NSCQ-MSX0909);陆军军医大学科技创新能力提升专项青年培育项目(2023XQN42)。

摘　　要：目的 探讨新型吩噻嗪衍生物DA414对脑出血(intracerebral hemorrhage, ICH)大鼠的疗效及作用机制。方法 雄性SD大鼠(8~10周龄,体质量250~300 g)按随机数字表法分为假手术组、模型组、5 mg/(kg·d) DA414组和10 mg/(kg·d)DA414组,每组20只。采用磁共振成像(magnetic resonance imaging, MRI)及组织切片定量分析ICH区域的大小及损伤程度;神经功能通过行为学进行评估;Western blot探讨DA414对铁死亡调节因子谷胱甘肽过氧化物酶4(glutathione peroxidase 4, GPX4)的表达影响;多重荧光免疫组化对血肿周围组织小胶质细胞活化状态定量分析;RT-qPCR检测炎症小体通路(NLRP3、Caspase-1、IL-1β)及促炎(IL-18、TLR4、IL-6、TNF-α)和抗炎(IL-4、IL-10)细胞因子的mRNA表达水平。通过伊文思蓝染色评估血脑屏障(blood-brain barrier,BBB)的完整性,HE染色和临床生化检验评估DA414对肝脏、肾脏和心脏的安全性。结果 DA414显著促进了ICH后血肿的吸收,减少神经损伤,并显著改善神经功能评分。与模型组相比,DA414处理能够显著上调铁死亡调节因子GPX4的水平(P<0.01);且显著抑制了ICH后小胶质活化(P<0.001);RT-qPCR进一步证实其下调炎症小体通路NLRP3(P<0.01)、Caspase-1(P<0.01)、IL-1β(P<0.01)及促炎因子IL-18(P<0.01)、TLR4(P<0.05)、IL-6(P<0.05)、TNF-α(P<0.01),同时上调抗炎细胞因子IL-4、IL-10 mRNA表达(P<0.05),提示其神经保护作用可能通过调节铁死亡及炎症双重机制实现。安全性评估进一步表明DA414干预对大鼠BBB无明显影响,且其对肝脏、肾脏、心脏也无明显损伤。结论 DA414通过靶向抑制铁死亡并调节炎症反应,在ICH模型中表现出显著神经保护效应,为ICH治疗提供实验基础。Objective To explore the efficacy and action mechanism of a novel phenothiazine derivative, DA414, in rats with intracerebral hemorrhage(ICH). Methods Male Sprague-Dawley(SD) rats(aged 8~10 weeks, weighing 250~300 g) were randomly divided into sham operation, model, and low and high dose DA414 groups [5 and 10 mg/(kg·d)], with 20 animals per group. The size and extent of injury in the ICH area were measured by magnetic resonance imaging(MRI) and histopathological slices. Neurological function was evaluated with a behavioral grading system. Western blotting was used to detect the expression of ferroptosis-related factor, glutathione peroxidase 4(GPX4). Multiplex immunohistochemistry was employed to quantitatively evaluate microglial activation in perihematomal tissue. RT-qPCR was applied to measure the mRNA expression levels of NLRP3 inflammasome components(NLRP3, Caspase-1, IL-1β), pro-inflammatory(IL-18, TLR4, IL-6, TNF-α) and anti-inflammatory cytokines(IL-4, IL-10). The integrity of the blood-brain barrier(BBB) was assessed by Evans blue staining, and the biosafety of DA414 for the liver, kidneys and heart was assessed by HE staining and clinical biochemical tests. Results DA414 significantly promoted the absorption of hematoma, reduced neuronal injury, and improved neurological function scores. DA414significantly up-regulated the ferroptosis regulatory factor GPX4(P<0. 01), and also significantly inhibited the activation of microglia after ICH(P<0. 001). RT-qPCR indicated that DA414 treatment resulted in downregulation of mRNA expression in the inflammasome pathway(NLRP3, Caspase-1, IL-1β, all P<0. 01) and proinflammatory cytokines(TLR4, IL-6, both P<0. 05;IL-18, TNF-α, both P<0. 01) and up-regulation of antiinflammatory cytokines(IL-4, IL-10, both P<0. 05), suggesting that DA414 exerts its neuroprotective effect probably by regulating ferroptosis and inflammation. Safety assessment revealed that DA414 had no significant effect on BBB integrity or damage to the liver, kidneys, and heart in rats. Conclusi

关 键 词：新型吩噻嗪衍生物 脑出血 铁死亡 炎症 

分 类 号：R743.34[医药卫生—神经病学与精神病学] R966[医药卫生—临床医学] R976