活性氧簇（NOX2）诱导的氧化应激通过蛋白激酶 C ε（PKCε）质膜易位导致大鼠背根神经节神经元神经病理性疼痛。

Oxidative stress induced by NOX2 contributes to neuropathic pain via plasma membrane translocation of PKCε in rat dorsal root ganglion neurons.

机构信息

Center for Experimental Medicine, the First Affiliated Hospital of Nanchang University, Nanchang, 330006, Jiangxi, China.

Department of Pediatrics, the First Affiliated Hospital of Nanchang University, Nanchang, 330006, Jiangxi, China.

出版信息

J Neuroinflammation. 2021 May 6;18(1):106. doi: 10.1186/s12974-021-02155-6.

BACKGROUND

Nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2)-induced oxidative stress, including the production of reactive oxygen species (ROS) and hydrogen peroxide, plays a pivotal role in neuropathic pain. Although the activation and plasma membrane translocation of protein kinase C (PKC) isoforms in dorsal root ganglion (DRG) neurons have been implicated in multiple pain models, the interactions between NOX2-induced oxidative stress and PKC remain unknown.

METHODS

A spared nerve injury (SNI) model was established in adult male rats. Pharmacologic intervention and AAV-shRNA were applied locally to DRGs. Pain behavior was evaluated by Von Frey tests. Western blotting and immunohistochemistry were performed to examine the underlying mechanisms. The excitability of DRG neurons was recorded by whole-cell patch clamping.

RESULTS

SNI induced persistent NOX2 upregulation in DRGs for up to 2 weeks and increased the excitability of DRG neurons, and these effects were suppressed by local application of gp91-tat (a NOX2-blocking peptide) or NOX2-shRNA to DRGs. Of note, the SNI-induced upregulated expression of PKCε but not PKC was decreased by gp91-tat in DRGs. Mechanical allodynia and DRG excitability were increased by ψεRACK (a PKCε activator) and reduced by εV1-2 (a PKCε-specific inhibitor). Importantly, εV1-2 failed to inhibit SNI-induced NOX2 upregulation. Moreover, the SNI-induced increase in PKCε protein expression in both the plasma membrane and cytosol in DRGs was attenuated by gp91-tat pretreatment, and the enhanced translocation of PKCε was recapitulated by HO administration. SNI-induced upregulation of PKCε was blunted by phenyl-N-tert-butylnitrone (PBN, an ROS scavenger) and the hydrogen peroxide catalyst catalase. Furthermore, εV1-2 attenuated the mechanical allodynia induced by HO CONCLUSIONS: NOX2-induced oxidative stress promotes the sensitization of DRGs and persistent pain by increasing the plasma membrane translocation of PKCε.

背景

烟酰胺腺嘌呤二核苷酸磷酸氧化酶 2（NOX2）诱导的氧化应激，包括活性氧（ROS）和过氧化氢的产生，在神经病理性疼痛中起关键作用。尽管蛋白激酶 C（PKC）同工型在背根神经节（DRG）神经元中的激活和质膜易位已被牵连到多种疼痛模型中，但 NOX2 诱导的氧化应激与 PKC 之间的相互作用尚不清楚。

方法

在成年雄性大鼠中建立 spared nerve injury（SNI）模型。局部应用药理学干预和 AAV-shRNA 至 DRG。通过 Von Frey 测试评估疼痛行为。通过全细胞膜片钳记录 DRG 神经元的兴奋性。

结果

SNI 诱导 DRG 中长达 2 周的持续 NOX2 上调，并增加 DRG 神经元的兴奋性，这些作用被 DRG 中 gp91-tat（NOX2 阻断肽）或 NOX2-shRNA 的局部应用所抑制。值得注意的是，SNI 诱导的 PKCε表达上调，但不是 PKC，被 gp91-tat 在 DRG 中减少。ψεRACK（PKCε 激活剂）增加机械性痛觉过敏和 DRG 兴奋性，而 εV1-2（PKCε 特异性抑制剂）减少。重要的是，εV1-2 未能抑制 SNI 诱导的 NOX2 上调。此外，gp91-tat 预处理减弱了 SNI 诱导的 DRG 中 PKCε 蛋白在质膜和细胞质中的表达增加，而 HO 给药再现了 PKCε 的增强易位。SNI 诱导的 PKCε 表达上调被苯-N-叔丁基硝酮（PBN，ROS 清除剂）和过氧化氢催化剂过氧化氢酶减弱。此外，εV1-2 减弱了 HO 诱导的机械性痛觉过敏。