摘要
探讨神经元一氧化氮合酶(nNOS)通过Akt S-亚硝基化促进出血性脑损伤的机制。建立SD大鼠脑出血模型,Western blot和RT-qPCR检测nNOS表达,生物素置换法结合Western blot检测Akt S-亚硝基化水平。使用nNOS抑制剂1400W和NO清除剂c-PTIO进行干预,评估Akt S-亚硝基化程度、神经元凋亡(TUNEL染色)和脑水肿(干湿重法)。在原代培养神经元中过表达或敲低nNOS,验证其对Akt S-亚硝基化的直接影响。脑出血后nNOS表达显著上调(P<0.05),Akt S-亚硝基化水平同步升高(P<0.05)。1400W和c-PTIO处理均可降低Akt S-亚硝基化(均P<0.05),减少神经元凋亡(均P<0.05)和脑水肿(均P<0.05)。体外实验证实nNOS过表达直接导致Akt S-亚硝基化增加(P<0.05),而nNOS敲低则降低Akt S-亚硝基化水平(P<0.05)。nNOS通过诱导Akt S-亚硝基化促进出血性脑损伤,为脑出血治疗提供新的潜在靶点。
This study aimed to explore the mechanism by which neuronal nitric oxide synthase(nNOS)promotes hemorrhagic brain injury via Akt S-nitrosylation.A rat model of intracerebral hemorrhage(ICH)was established using Sprague-Dawley(SD)rats.Western blotting and RT-qPCR were performed to measure the expression of nNOS.The biotin switch assay coupled with Western blotting was employed to detect the levels of Akt S-nitrosylation.The nNOS-specific inhibitor 1400W and the nitric oxide(NO)scavenger c-PTIO were administered to intervene in vivo,and their effects on Akt S-nitrosylation,neuronal apoptosis(assessed by TUNEL staining),and brain edema(measured by the wet-dry weight method)were evaluated.Furthermore,primary cultured neurons were used in vitro to overexpress or knockdown nNOS,verifying its direct role in Akt S-nitrosylation.After ICH,the expression of nNOS was significantly increased compared to controls(P<0.05),and a corresponding elevation in Akt S-nitrosylation was observed simultaneously(P<0.05).Treatment with either 1400W or c-PTIO effectively reduced Akt S-nitrosylation(both P<0.05),significantly decreased neuronal apoptosis(both P<0.05),and alleviated brain edema(both P<0.05).Additionally,in vitro experiments provided direct evidence that overexpression of nNOS markedly elevated Akt S-nitrosylation levels(P<0.O5),whereas knockdown of nNOS significantly reduced Akt S-nitrosylation(P<0.O5).These findings clearly indicate a causal relationship between nNOS activity and Akt S-nitrosylation levels.The authors'findings suggest that nNOS exacerbates hemorrhagic brain injury by inducing Akt S-nitrosylation,highlighting a critical pathway involved in neuronal injury post-ICH.Thus,modulation of nNOS and the Akt S-nitrosylation signaling pathway emerges as a promising therapeutic target for mitigating secondary brain injury following intracerebral hemorrhage.Further investigation into this signaling axis could potentially lead to novel strategies in clinical management,improving outcomes for patients suffering from hemorrhagic stroke.
作者
张继东
纪子腾
赵赛赛
刘玲
王春晴
ZHANG Jidong;JI Ziteng;ZHAO Saisai;LIU Ling;WANG Chunqing(Department of Neurosurgery,Xuzhou Municipal Hospital Affiliated to Xuzhou Medical University,Xuzhou 221000,China;Department of Neurosurgery,Xuzhou First People's Hospital,Xuzhou 221000,China;Department of General Practice,Affiliated Hospital of Xuzhou Medical University,Xuzhou 221000,China)
出处
《药物生物技术》
2025年第6期737-742,共6页
Pharmaceutical Biotechnology
基金
徐州市科学技术局资助项目(No.KC23162)。
