NADPH氧化酶4在骨癌痛模型大鼠背根神经节中的作用

The role of NADPH oxidase 4 in cancer bone pain of rats

目的:研究NADPH氧化酶4(Nox4)在骨癌痛(CIBP)大鼠背根神经节(DRG)中的表达。方法:48只成年雄性SD大鼠随机分为4组:假手术组(Sham)、骨癌痛组(CIBP)、空病毒对照组(Lenti-NC)与Nox4基因沉默组(Lenti-shNox4)。通过鞘内注射方法给予大鼠重组慢病毒处理,通过胫骨骨髓腔接种Walker256细胞的方法建立大鼠骨癌痛模型,通过胫骨HE染色及行为学检测鉴定大鼠CIBP模型,应用real time RT-PCR检测大鼠DRG中Nox4 mRNA的表达,利用Western Blot和免疫组织荧光染色检测Nox4蛋白在大鼠DRG中的表达和细胞定位,利用商品化试剂盒检测大鼠DRG中H2O2的含量,应用real time RT-PCR和Western Blot检测Nox4基因沉默对大鼠DRG中神经型一氧化氮合酶(n NOS)、超氧化物歧化酶(SOD)及N-甲基-D-天冬氨酸受体2D(NMDAR2D)和γ-氨基丁酸A型γ2受体(GABAA-γ2)表达的影响。结果:大鼠接种Walker256细胞可造成胫骨破坏及肿瘤细胞生长,PWT减少和TWL缩短;在骨癌痛大鼠DRG中,Nox4的表达显著升高;免疫组织荧光染色结果表明Nox4主要表达于DRG小胶质细胞中;敲减DRG中Nox4表达能减轻骨癌痛大鼠的机械痛敏和热痛敏,降低DRG中H2O2的水平,下调nNOS和NMDAR2D表达,上调SOD和GABAA-γ2表达。结论:敲减DRG中Nox4表达可通过减轻氧化应激反应缓解大鼠骨癌痛。

Objective: To investigate the expression of NADPH oxidase 4(Nox4) in the dorsal root ganglia(DRG) of rats with bone cancer pain.Methods: Forty-eight adult male SD rats were randomly divided into four groups: sham group(sham),bone cancer pain group(CIBP),control lentivirus group(Lenti-NC),and down regulated Lenti-shNox4 group(Lenti-shNox4).The rats were treated with recombinant lentivirus by intrathecal injection.The bone cancer pain model of rats was established by injecting Walker 256 cells into the bone marrow of the tibia.Three days after intrathecal injection of lentivirus,the tibial marrow cavity of rats in CIBP group,Lenti-shNox4 group and Lenti-NC group were inoculated with walker256 cells to establish bone cancer pain model.HE staining of tibia and behavioral detection were carried out to identify the CIBP model.Nox4 mRNA expression in DRG of rats was detected by real time RT-PCR.The expression and cell localization of Nox4 protein in DRG of rats were detected by Western Blot and immunofluorescence staining.The content of H2O2 in rat DRG was detected by commercial kit.The expression of neuronal nitic oxide synthase(n NOS),super oxide dimutese(SOD),N-methyl-D-aspartate receptor 2D(NMDAR2D),and γ-aminobutyric acid type A receptor subunit γ-2(GABAA-γ2) in DRG of rats was detected by real time RT-PCR and Western Blot.Results: Walker256 cells inoculated in rats could cause tibia damage,tumor cell growth,reduced PWT and shortened TWL.Nox4 expression was significantly increased in DRG of bone cancer pain rats.Nox4 was mainly expressed in DRG microglia cells.Nox4 knockdown in DRG of rats can can alleviate the mechanical and thermal pain sensitivity of bone cancer pain rats.In addition,the levels of H2O2 in DRG were decreased,the expressions of n NOS and NMDAR2D were down-regulated,and the expressions of SOD and GABAA-γ2 were up-regulated.Conclusion: Nox4 knockdown in DRG can alleviate the pain of bone cancer by reducing oxidative stress.