自噬诱导剂亚精胺改善脑出血后脑损伤  

Autophagy inducer spermidine improves brain injury after intracerebral hemorrhage

作　　者：祁凌霄 闫改丽 薛孟周 QI Lingxiao;YAN Gaili;XUE Mengzhou(Department of Cerebrovascular Diseases,Second Affiliated Hospital of Zhengzhou University,Zhengzhou University,Zhengzhou,Henan,China;Academy of Medical Sciences,Zhengzhou University,Zhengzhou,Henan,China)

机构地区：[1]郑州大学第二附属医院脑血管病科,河南郑州 [2]郑州大学医学科学院,河南郑州

出　　处：《陆军军医大学学报》2025年第7期639-648,共10页Journal of Army Medical University

基　　金：国家自然科学基金面上项目(82071331);国家重点研发计划(2018YFC1312200)。

摘　　要：目的探讨亚精胺(spermidine,SPD)在脑出血(intracerebral hemorrhage,ICH)中的作用及其潜在机制。方法选用8~10周龄的雄性C57BL/6小鼠构建胶原酶诱导的ICH模型。将108只小鼠随机分为3组,即假手术组、ICH组和亚精胺治疗组,每组36只。亚精胺治疗组小鼠在术后接受连续3 d的15 mg/kg亚精胺腹腔注射。在ICH后第3天,通过神经行为学测试(改良Garcia神经功能评分、前肢放置试验)评估小鼠神经功能缺损、HE染色观察脑组织病理损伤、免疫荧光染色检测小胶质细胞/巨噬细胞与星形胶质细胞活化,以及通过Western blot分析自噬标志物Beclin-1、P62及炎症因子MMP-9、NLRP3、COX-2的表达。以血红素刺激HT-22细胞模拟体外ICH,3-甲基腺嘌呤(3-methyladenine,3-MA)作为自噬抑制剂。将细胞随机分为4组(n=7):对照组、血红素组、血红素+亚精胺组和血红素+亚精胺+3-MA组。在血红素处理24 h后,通过CCK-8法检测各组细胞存活率、Western blot检测自噬相关蛋白LC3-II、P62的表达,通过测定超氧化物歧化酶(superoxide dismutase,SOD)活性与丙二醛(malondialdehyde,MDA)含量以评估氧化应激水平。结果在ICH造模后第3天,与ICH组相比,亚精胺显著减少脑损伤面积(P<0.05),促进神经功能恢复(P<0.05),激活自噬Beclin-1表达升高,P62表达降低(P<0.05),抑制小胶质细胞/巨噬细胞和星形胶质细胞活化(P<0.01),降低MMP-9、NLRP3及COX-2表达,同时提高SOD活性并减少MDA含量(P<0.05)。在细胞实验中,亚精胺提高HT-22细胞存活率(P<0.05)、增强SOD活性并降低MDA水平(P<0.01),而自噬抑制剂3-MA有效抑制了自噬,使LC3-II表达降低、P62表达升高(P<0.05),并完全逆转了上述保护效应(P<0.05)。结论亚精胺在ICH后激活自噬并通过抑制神经炎症和氧化应激改善ICH后脑损伤。Objective To investigate the role and underlying mechanism of spermidine(SPD)in intracerebral hemorrhage(ICH).Methods Male C57BL/6 mice were subjected to establish a collagenaseinduced ICH model.The 108 mice were randomly divided into Sham group,ICH group and ICH+SPD group(intraperitoneal injection of 15 mg/kg SPD for 3 consecutive days after modeling),with 36 mice in each group.On the 3rd day after ICH,neurological deficits were evaluated using modified Garcia scoring and forelimb placing test;brain pathological damage was assessed with HE staining;activation of microglia/macrophages(Iba-1)and astrocytes(GFAP)was detected by immunofluorescence assay;expression of autophagy markers(Beclin-1,P62)and inflammatory factors(MMP-9,NLRP3,COX-2)was measured with Western blotting.In in vitro experiments,hemin was used to stimulate HT-22 cells to mimic ICH.The HT-22 cells were randomly divided into Control group,Hemin group,Hemin+SPD group,and Hemin+SPD+3-methyladenine(3-MA,an autophagy inhibitor)group(n=7).After 24 h of hemin treatment,cell viability was detected with CCK-8 assay,the expression of autophagy-related proteins(LC3-II and P62)were detected with Western blotting,and oxidative stress was determined by measuring superoxide dismutase(SOD)activity and malondialdehyde(MDA)content.Results On day 3 post-ICH,SPD significantly reduced the area of brain damage(P<0.05),improved neurological recovery(P<0.05),activated autophagy with up-regulation of Beclin-1 while down-regulation of P62(P<0.05),suppressed the activation of microglia/macrophage and astrocytes(P<0.01),reduced the expression of MMP-9,NLRP3 and COX-2,and enhanced SOD activity and decreased MDA content(P<0.05)when compared with the ICH group.SPD increased the viability of HT-22 cells(P<0.05),improved SOD activity and reduced MDA content(P<0.01).Autophagy inhibitor 3-MA effectively blocked the down-regulation of LC3-II and up-regulation of P62,and completely reversed above protective effects caused by SPD(P<0.05).Conclusion SPD activates autophagy after ICH and

关 键 词：脑出血 亚精胺 自噬 神经炎症 氧化应激 

分 类 号：R743.34[医药卫生—神经病学与精神病学] R914.4[医药卫生—临床医学] R963