青蒿琥酯调控NLRP3/ASC/Caspase-1信号通路减轻脑出血小鼠炎症及保护神经功能的作用研究

Mechanism of Artesunate Regulating NLRP3/ASC/Caspase-1 Signaling Pathway to Reduce Inflammation and Protect Neurological Function in Mice with Intracerebral Hemorrhage

背景炎症反应是脑出血(ICH)病情进展的主要因素。青蒿琥酯(ART)具有抗菌和抗炎的药理活性,ART在脑内浓度较高,但对脑出血损伤的神经保护作用尚不清楚。目的观察ICH后ART对炎症反应的影响并探讨其机制。方法2022年3月—2023年2月选取108只8~10周雄性SPF级C57BL/6小鼠,随机分为假手术组(Sham组,n=36)、ICH对照组(ICH+Vehicle组,n=36)、ART治疗组(ICH+ART组,n=36),建立ICH模型,ICH+ART组在造模后2 h腹腔注射ART溶液150 mg·kg^(-1)·d^(-1),ICH+Vehicle组腹腔注射5%碳酸氢钠溶液,连续注射3 d。观察小鼠行为学指标,HE染色观察各组小鼠脑组织损伤情况,免疫组织化学染色检测白介素(IL)6、IL-1β、髓过氧化物酶(MPO)单位面积阳性细胞数,兔抗小胶质标志物(IBA1)免疫荧光染色观察小胶质细胞/巨噬细胞的激活情况,TUNEL/NeuN免疫荧光双染色观察神经元死亡情况,蛋白质印迹法比较小鼠MPO、IL-1β、肿瘤坏死因子α(TNF-α)、NOD样受体蛋白3(NLRP3)、凋亡相关颗粒样蛋白(ASC)和半胱氨酸天冬氨酸蛋白酶1(Caspase-1)水平。结果ICH+Vehicle组改良神经系统严重程度评分、右转弯的百分比高于Sham组,ICH+ART组低于ICH+Vehicle组(P<0.05)。HE染色结果示Sham组纹状体周围有少量血液,水肿可忽略不计,ICH+Vehicle组脑组织严重损伤,细胞间隙增加、血管周围血肿、炎性细胞浸润增加;ICH+ART组上述症状均有明显改善。ICH+Vehicle组IL-6、IL-1β、MPO单位面积阳性细胞数均高于Sham组,ICH+ART组IL-6、IL-1β、MPO单位面积阳性细胞均低于ICH+Vehicle组(P<0.05)。ICH+Vehicle组小胶质细胞/巨噬细胞激活数高于Sham组,ICH+ART组低于ICH+Vehicle组(P<0.05)。3组小鼠神经元死亡数量比较,差异有统计学意义(P<0.05),其中ICH+Vehicle组高于Sham组,ICH+ART组低于ICH+Vehicle组(P<0.05)。ICH+Vehicle组MPO、IL-1β、TNF-α、NLRP3、ASC和Caspase-1水平均高于Sham组,ICH+ART组MPO、IL-1β、TNF-α、NLRP3、ASC和Caspase-1水平均低于ICH+Vehicle组(P<0.05)。结论ICH后ART治疗可通过靶向调节NLRP3/ASC/Caspase-1信号通路减轻小鼠纹状体炎症反应,减少小胶质细胞激活,最终减轻纹状体神经元凋亡并改善脑水肿。

Background The inflammatory response is a major factor in the progression of intracerebral hemorrhage(ICH).Artesunate(ART)has antibacterial and anti-inflammatory pharmacological activity with high concentrations in the brain,but its neuroprotective effect on cerebral hemorrhage injury remains unclear.Objective To observe the effect of ART on inflammatory response after intracerebral hemorrhage and explore its mechanism.Methods From March 2022 to February 2023,108 male C57BL/6 mice aged 8 to 10 weeks were selected and randomly divided into the sham-operated group(Sham group,n=36),ICH control group(ICH+Vehicle group,n=36)and ART treatment group(ICH+ART group,n=36).ICH model was established.The mice in the ICH+ART group were intraperitoneally injected with 150 mg·kg^(-1)·d^(-1) 2h after modeling,and the mice in the ICH+Vehicle group were intraperitoneally injected with 5%sodium bicarbonate solution for 3 consecutive days.The behavioral indicators of mice were observed.The brain tissue damage of mice in each group was observed by HE staining;the number of positive cells per unit area of interleukin(IL)6,IL-1βand myeloperoxidase(MPO)were detected by immunohistochemical staining;the activation of microglia/macrophages was observed by IBA1 immunofluorescence staining.TUNEL/NeuN immunofluorescence double staining was performed to observe neuronal death.The levels of MPO,IL-1β,tumor necrosis factorα(TNF-α),Nod-like receptor protein 3(NLRP3),apoptosis-related granuloid protein(ASC)and cysteine aspartic protease 1(Caspase-1)were compared by western blotting.Results The modified neurological severity scores and percentages of right turn of mice were higher in the ICH+Vehicle group than the Sham group,and lower in the ICH+ART group than the ICH+Vehicle group(P<0.05).HE staining results showed that there was a small amount of blood around the striatum in the Sham group and negligible edema.In the ICH+Vehicle group,brain tissue was seriously damaged,with increased intercellular space,perivascular hematoma and inflammatory cell infiltration.All of the above symptoms were significantly improved in the ICH+ART group.The numbers of IL-6,IL-1βand MPO positive cells in the ICH+Vehicle group were higher than the Sham group,the numbers of IL-6,IL-1βand MPO positive cells in the ICH+ART group were lower than the ICH+Vehicle group(P<0.05).The number of activated microglia/macrophages in the ICH+Vehicle group was higher than the Sham group,the number of activated microglia/macrophages in the ICH+ART group was lower than the ICH+Vehicle group(P<0.05).The number of neuron death in the ICH+Vehicle group was higher than the Sham group,and lower in the ICH+ART group than the ICH+Vehicle group(P<0.05).The levels of MPO,IL-1β,TNF-α,NLRP3,ASC and Caspase-1 in the ICH+Vehicle group were higher than the Sham group,the levels of MPO,IL-1β,TNF-α,NLRP3,ASC and Caspase-1 in the ICH+ART group were lower than the ICH+Vehicle group(P<0.05).Conclusion ART therapy after ICH attenuates the inflammatory response of striatum and the activation of microglia in mice through targeted regulation of NLRP3/ASC/Caspase-1 signaling pathway,and ultimately reduces striatal neuronal apoptosis and improves brain edema.