Role of NADPH oxidase1 in the development of inflammatory pain has been demonstrated by gene knockout studies. Nevertheless, pharmacological inhibition of NOX1 is a requisite approach for therapeutic utility. Recently, we have reported the anti-nociceptive effect of newly identified NOX1 specific inhibitor ML171 (2-acetylphenothiazine). Inhibition of NOX1 resulted in attenuation of nociceptive sensitization during acute inflammatory pain via inhibition of ROS generation and its downstream ERK1/2 activation. However, glial activation accompanying inflammation is closely related to the initiation and maintenance of pain. Peripheral nociceptive inputs activate the primary afferents via release of various chemical mediators which are potentially capable of mediating signals from neuron to glia in DRG and subsequently in spinal cord dorsal horn. The subsequent interactions between neuron and glia contribute to pain hypersensitivity. Thus, the present study was focused to investigate the effect of ML171 on ERK1/2 signaling, glial activation, and crosstalk between neuron and glia in a mouse model of formalin induced acute nociception. Thus, the present study was focused to investigate the effect of ML171 on ERK1/2 signaling, glial activation, and crosstalk between neuron and glia in DRG and dorsal horn of the spinal cord of lumbar region (L3-L5) in a mouse model of formalin induced acute nociception. Intraperitoneal administration of ML171 decreased nociceptive behavioral responses, i.e. the flinch and lick counts, in formalin induced nociceptive mice. Immunofluorescence and Western blot analysis demonstrated decreased levels of nociceptive mediators like p-ERK1/2, p-NFκB p65, Iba1 and GFAP in DRG as well as in spinal cord dorsal horn; supporting anti-nociceptive potential of ML171. Further, co-localization studies showed the neuron-glia crosstalk in tissue dependent manner. ERK1/2 was found to be activated in glia and NFκB in neurons in DRG; whereas in case of spinal cord ERK1/2 was activated in neurons and NFκB in astrocytes. Decrease in nociceptive behavioral response and activation of nociceptive mediators after intraperitoneal administration of ML171 strongly advocate anti-nociceptive potential of ML171. This is the first report demonstrating modulation of ERK1/2-NFκB signaling pathway, glial activation and regulation of neuron-glia crosstalk by NADPH oxidase1 inhibition towards its anti-nociceptive action.

中文翻译：

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NADPH氧化酶1抑制通过减弱ERK1 /2-NFκB信号传导和神经胶质激活而导致福尔马林诱导的急性伤害感受中枢中枢敏化程度的降低。

基因敲除研究已证明NADPH氧化酶1在炎性疼痛发生中的作用。然而，NOX1的药理抑制是治疗实用性的必要方法。最近，我们报道了新发现的NOX1特异性抑制剂ML171（2-乙酰吩噻嗪）的抗伤害作用。抑制NOX1可通过抑制ROS的产生及其下游ERK1 / 2的活化来减弱急性炎症性疼痛中的伤害性敏化作用。然而，伴随炎症的神经胶质激活与疼痛的发生和维持密切相关。周围的伤害感受输入通过释放各种化学介质来激活初级传入，这些化学介质可能能够介导DRG中神经元到神经胶质的信号，并随后在脊髓背角中介导神经胶质。神经元和胶质细胞之间的后续相互作用导致疼痛超敏反应。因此，本研究的重点是在福尔马林诱导的急性伤害性小鼠模型中研究ML171对ERK1 / 2信号传导，神经胶质激活以及神经元与神经胶质之间的串扰的影响。因此，本研究的重点是研究ML171对ERK1 / 2信号传导，神经胶质活化以及DRG和腰区脊髓背角（L3-L5）的神经元和神经胶质之间的串扰的影响。福尔马林诱导的急性伤害感受。在福尔马林诱导的小鼠中，腹腔注射ML171可降低其伤害性行为反应，即退缩和舔食计数。免疫荧光和蛋白质印迹分析表明，伤害性介质如p-ERK1 / 2，p-NFκBp65，DRG以及脊髓背角中的Iba1和GFAP；支持ML171的抗伤害感受能力。此外，共定位研究表明神经元-神经胶质细胞串扰以组织依赖性方式发生。发现DRG的神经元中的胶质细胞和NFκB激活了ERK1 / 2。而在脊髓中，神经元激活ERK1 / 2，星形胶质细胞激活NFκB。腹膜内给予ML171后，伤害感受行为反应的减少和伤害感受介质的激活强烈提倡ML171的抗伤害感受能力。这是第一个证明NADPH氧化酶1抑制ERK1 /2-NFκB信号传导途径，调节神经胶质激活和调节神经胶质细胞串扰的抗伤害感受作用的报告。共定位研究表明神经元-神经胶质细胞串扰以组织依赖性方式发生。发现DRG的神经元中的胶质细胞和NFκB激活了ERK1 / 2。而在脊髓中，神经元激活ERK1 / 2，星形胶质细胞激活NFκB。腹膜内给予ML171后，伤害感受行为反应的减少和伤害感受介质的激活强烈提倡ML171的抗伤害感受能力。这是第一个证明NADPH氧化酶1抑制ERK1 /2-NFκB信号传导途径，调节神经胶质激活和调节神经胶质细胞串扰的抗伤害感受作用的报告。共定位研究表明神经元-神经胶质细胞串扰以组织依赖性方式发生。发现DRG中神经元的胶质细胞和NFκB激活了ERK1 / 2。而在脊髓中，神经元激活ERK1 / 2，星形胶质细胞激活NFκB。腹膜内给予ML171后，伤害感受行为反应的减少和伤害感受介质的激活强烈提倡ML171的抗伤害感受能力。这是第一个证明NADPH氧化酶1抑制ERK1 /2-NFκB信号传导途径，调节神经胶质激活和调节神经胶质细胞串扰的抗伤害感受作用的报告。而在脊髓中，神经元激活ERK1 / 2，星形胶质细胞激活NFκB。腹膜内给予ML171后，伤害感受行为反应的减少和伤害感受介质的激活强烈提倡ML171的抗伤害感受能力。这是第一个证明NADPH氧化酶1抑制ERK1 /2-NFκB信号传导途径，调节神经胶质激活和调节神经胶质细胞串扰的抗伤害感受作用的报告。而在脊髓中，神经元激活ERK1 / 2，星形胶质细胞激活NFκB。腹腔注射ML171后伤害感受行为反应的减少和伤害感受介质的激活强烈提倡ML171的抗伤害感受能力。这是第一份证明NADPH氧化酶1抑制ERK1 /2-NFκB信号传导途径，调节神经胶质细胞激活和调节神经胶质细胞串扰的抗伤害感受作用的报告。