目的:脑出血(ICH)导致脑损伤的病理生理机制尚未完全阐明,目前缺乏有效的治疗干预措施来提高ICH患者的预后。先前的研究表明,MicroRNA-7(miR-7)可以抑制Nod样受体蛋白3(NLRP3)的表达,从而调节帕金森病发病机制中的神经炎症。然而,miR-7对ICH后NLRP3炎性体的潜在调节作用尚未确定.本研究旨在确定miR-7是否通过抑制NLRP3减轻实验性ICH后继发性脑损伤,并研究其潜在机制。
方法:将100μL自体血立体定位注入SD大鼠右侧基底节,建立ICH模型。随后,这些大鼠被分为三组:假,ICH+车辆,和ICH+miR-7,每个包含18只动物。建模后12小时,大鼠接受脑室内注射10μL生理盐水,10μL磷酸盐,和含有0.5nmolmiR-7模拟物的10μL磷酸盐缓冲盐溶液,分别。在建模后第3天评估神经功能,然后实施安乐死收集脑组织.使用干-湿重法测定脑含水量。通过免疫组织化学和Westernblot分析血肿周围脑组织中炎性细胞因子的表达。使用逆转录聚合酶链反应(RT-PCR)重新评估这些细胞因子。此外,生物信息学工具用于预测miR-7与NLRP3的结合。构建了野生型荧光素酶报告基因载体和相应的突变载体,然后将miR-7模拟物转染到HEK293T细胞中以评估荧光素酶活性。
结果:我们的研究表明,miR-7的施用可显著降低ICH后大鼠的神经功能评分并减轻脑水肿。观察到血肿附近小胶质细胞/巨噬细胞中NLRP3表达的显着上调,用miR-7模拟物治疗后显著降低。此外,这种干预改善了神经退行性改变,有效降低了促炎细胞因子的蛋白和mRNA水平,即TNF-α,IL-1β,IL-6和Caspase1在靠近血肿的脑组织中。此外,miR-7模拟物明显抑制与野生型报告基因相关的荧光素酶活性,在其突变变体中没有反映的效果。
结论:miR-7抑制NLRP3在小胶质细胞/巨噬细胞中的表达,从而减少炎性细胞因子的产生,导致大鼠ICH后进行某些神经保护。
OBJECTIVE: The pathophysiological mechanisms underlying brain injury resulting from intracerebral hemorrhage (ICH) remain incompletely elucidated, and efficacious therapeutic interventions to enhance the prognosis of ICH patients are currently lacking. Previous research indicates that MicroRNA-7 (miR-7) can suppress the expression of Nod-like receptor protein 3 (NLRP3), thereby modulating neuroinflammation in Parkinson\'s disease pathogenesis. However, the potential regulatory effects miR-7 on NLRP3 inflammasome after ICH are yet to be established. This study aims to ascertain whether miR-7 mitigates secondary brain injury following experimental ICH by inhibiting NLRP3 and to investigate the underlying mechanisms.
METHODS: An ICH model was established by stereotaxically injecting 100 μL of autologous blood into the right basal ganglia of Sprague-Dawley (SD) rats. Subsequently, these rats were allocated into three groups: sham, ICH + Vehicle, and ICH + miR-7, each comprising 18 animals. Twelve hours post-modeling, rats received intraventricular injections of 10 μL physiological saline, 10 μL phosphate, and 10 μL phosphate-buffered saline solution containing 0.5 nmol of miR-7 mimics, respectively. Neurological function was assessed on day three post-modeling, followed by euthanasia for brain tissue collection. Brain water content was determined using the dry-wet weight method. The expression of inflammatory cytokines in cerebral tissues surrounding the hematoma was analyzed through immunohistochemistry and Western blot assays. These cytokines were re-evaluated using Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Moreover, bioinformatics tools were employed to predict miR-7\'s binding to NLRP3. A wild-type luciferase reporter gene vector and a corresponding mutant vector were constructed, followed by transfection of miR-7 mimics into HEK293T cells to assess luciferase activity.
RESULTS: Our study demonstrates that the administration of miR-7 mimics markedly reduced neurological function scores and attenuated brain edema in rats following ICH. A significant upregulation of NLRP3 expression in microglia/macrophage adjacent to the hematoma was observed, substantially reduced after the treatment with miR-7 mimics. Furthermore, this intervention ameliorated neurodegenerative changes and effectively decreased the protein and mRNA levels of pro-inflammatory cytokines, namely TNF-α, IL-1β, IL-6, and Caspase1, in the cerebral tissues proximate to the hematomas. In addition, miR-7 mimics distinctly inhibited the luciferase activity associated with the wild-type reporter gene, an effect not mirrored in its mutant variant.
CONCLUSIONS: The miR-7 suppressed NLRP3 expression in microglia/macrophage to reduce the production of inflammatory cytokines, leading to conducting certain neuroprotection post-ICH in rats.