摘要
目的探讨Keap1-Nrf2通路在体外蛛网膜下腔出血(subarachnoid hemorrhage,SAH)细胞模型中调控炎症反应的机制。方法采用25μM氧合血红蛋白(oxyhemoglobin,OxyHb)处理神经元-小胶质细胞共培养体系24 h建立SAH模型,分为Control组、OxyHb组、OxyHb+sh-Keap1组(短发夹RNA干扰Kelch样环氧氯丙烷相关蛋白1,small hairpin RNA Kelch-1ike ECH-associated protein l,sh-Keap1)、OxyHb+sh-NC组(small hairpin RNA Negative Contros,sh-NC)。检测细胞增殖、白细胞介素6(interleukin 6,IL-6)、肿瘤坏死因子α(tumor necrosis factor,TNF-α)、白细胞介素10(interleukin 10,IL-10)、转化生长因子β(transforming growth factor-β,TGF-β)水平;检测活性氧(reactive oxygen species,ROS)变化、丙二醛(malondialdehyde,MDA)含量和谷胱甘肽(glutathione,GSH)水平;检测Kelch样环氧氯丙烷相关蛋白1(Kelch-1ike ECH-associated protein l,Keap1)、红系衍生的核因子2相关因子2(Nuclear factor erythroid 2-related factor 2,Nrf2)、重链结合蛋白(heavy-chain binding protein,BIP)、肌醇需要酶1α(Inositol-requiring enzyme 1α,IRE1α)、蛋白激酶RNA样内质网激酶(protein kinase RNA-like endoplasmic reticulum kinase,PERK)、磷酸化蛋白激酶RNA样内质网激酶(phospho-protein kinase RNA-like endoplasmic reticulum kinase,p-PERK)、激活转录因子6(activating transcription factor 6,ATF6)表达;检测M1型小胶质细胞表面标志物诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)和M2型小胶质细胞表面标志物精氨酸酶1(arginase-1,Arg1)表达。结果与对照组相比,氧合血红蛋白组细胞活力显著降低(P<0.05),氧化应激和炎症反应加剧。敲低Keap1可激活Nrf2通路,减轻氧化应激和炎症反应(P<0.05)并显著提高细胞活力(P<0.05)。相比氧合血红蛋白组,氧合血红蛋白+敲低Keap1载体组小胶质细胞向M2型极化显著增加(P<0.05)。结论Keap1-Nrf2通路通过调控内质网氧化应激及小胶质细胞极化调控SAH炎症反应,可为治疗提供新靶点和方向。
Objective To investigate the role of Kelch-like epichlorohydrin associated protein 1(Keap1)/nuclear factor erythroid derived 2 related factor 2(Nrf2)signaling pathways on inflammatory response in subarachnoid hemorrhage(SAH)cell model.Methods A 25μM oxyhemoglobin treatment was used in the neuron-microglia co-culture system for 24 hours to simulate the SAH cell model,which was further divided into four groups:Control,OxyHb,OxyHb+sh-Keap1 and OxyHb+sh-NC.Cell proliferation was measured by CCK8;ELISA was conducted to test the expressions of IL-6,TNF-α,IL-10,and TGF-β;DCFH probe was used to detect changes in ROS;the expressions of Keap1,Nrf2,BIP,IRE1α,PERK,p-PERK,and ATF6 in cells were measured by Western Blot,the levels of MDA and GSH were expressed using commercial kit respectively;real-time quantitative reverse transcription PCR(qRT-PCR)was conducted to detect mRNA levels of M1 surface marker(iNOS)and M2 surface marker(Arg1).Results Compared to the control group,cell proliferation in the OxyHb group decreased significantly due to oxidative stress and inflammation(P<0.05).Keap1 knockdown activated the Nrf2 signaling pathways,alleviating oxidative stress and inflammation(P<0.05),and significantly increased cell proliferation(P<0.05).The levels of M2 microglial polarization significantly increased in the OxyHb+sh-Keap1 group compared to the OxyHb group(P<0.05).Conclusions The Keap1-Nrf2 signaling pathway can regulate inflammation by modulating endoplasmic reticulum oxidative stress and microglial polarization in the SAH cell model,which may provide new therapeutic targets for the treatment of subarachnoid hemorrhage.
作者
玉石
黑悦
伍苛夫
龙乾发
刘卫平
YU Shi;HEI Yue;WU Kefu;LONG Qianfa;LIU Weiping(Department of Neurosurgery,The 923th Hospital of the Joint Logistics Support Force,Nanning,530000,China;Department of Oncology,Xijing Hospital of Air Force Medical University,Xi'an,710032,China;Department of Neurosurgery,Xi'an Central Hospital,Xi'an,710000,China;Department of Neurosurgery,Xi'an People’s Hospital,Xi'an,710000,China)
出处
《中华神经外科疾病研究杂志》
2025年第6期21-27,共7页
Chinese Journal of Neurosurgical Disease Research
基金
国家自然科学基金青年科学基金项目(82301553)。
