G6PD缺乏诱导P2X3受体可塑性变化在糖尿病神经病理性疼痛中的作用及机制研究

Painful diabetic peripheral neuropathy is common in diabetic patients, which affects quality of life severely, and the cause of the pain has not been fully elucidated. It is well known that oxidative stress is one of the most important mechanisms underlying diabetic neuropathy and that G6PD deficiency is a major cause of oxidative stress. However, it is not clear whether G6PD takes part in the development of diabetic pain. Recently, we found that the level of G6PD and NADPH were decreased in hind-paw specific DRGs in diabetic rats, and that G6PD was co-localized with P2X3 receptors in DiI labeled hind-paw specific DRG neurons. In addition, neuronal excitability was increased and the ATP evoked currents were enhanced. More importantly, purinergic P2X3 receptor expression in hind-paw specific DRGs was increased in diabetic rats while miR 31-5p expression was reduced which may be involved in the regulation of P2X3 expression. Based on these exciting preliminary data, we hypothesize that (1). Reductase G6PD deficiency plays a crucial role in hind-paw hyperalgesia by up-regulating expression and function of P2X3 receptors and increasing excitability of hind-paw specific DRG neurons in diabetic rats, and (2). Oxidative stress due to reductase G6PD deficiency regulates expression of P2X3 receptors through epigenetic mechanism. To test these hypotheses, we will investigate the following specific aims: Specific Aim 1: To determine the role of G6PD in hind-paw hyperalgesia in diabetic rats; Specific Aim 2: To investigate the epigenetic mechanisms of P2X3 up-regulation due to G6PD deficiency in diabetic rats. VFF and thermal stimulator will be used to record painful behavior in normal and diabetic rats. Patch clamp recordings will be performed in vitro on single hind-paw-specific primary sensory neurons acutely isolated from these rats labeled with DiI. Expression of key nociceptive gene (i.e., G6PD, P2X3, miR31-5P) will be examined with immunocytochemistry, western blotting and (or) RT-PCR analyses. We expect that our studies may clarify mechanisms by which oxidative stress induced epigenetic regulation of key nociceptive genes in the diabetic neuropathy. This added knowledge would provide new strategies for treatment for peripheral neuropathic pain in patients with diabetes.

糖尿病痛性神经病变对患者生活质量影响大，机制尚不明确。还原酶G6PD功能减退是引起氧化应激的重要原因，预实验显示：1.糖尿病大鼠后肢特异性脊髓背根节（L4-6 DRG）中G6PD表达下调，NADPH降低，G6PD与P2X3共定位于上述神经元中；2.上述 DRG中ATP配体门控通道P2X3表达上调，神经元ATP电流增加、兴奋性增加；3.可能参与调控P2X3表达的miR31-5p水平降低。由此假设：高糖状态下，G6PD缺乏引起神经元氧化应激，通过降低miR31-5p水平，上调P2X3的表达及功能，增加神经元兴奋性，导致疼痛。本课题将通过在体动物模型和离体研究，探讨：1. 糖尿病神经病理性疼痛模型中是否存在G6PD表达与功能缺乏，后者能否激活P2X3引起疼痛。2.G6PD缺乏参与调控P2X3可塑性（表达与功能）的具体机制。从分子水平解释糖尿病痛性神经病变的形成机制，为治疗提供理论依据。

糖尿病痛性神经病变对患者生活质量影响大，机制尚不明确。结合前期研究结果，本项目拟探讨：1.糖尿病大鼠是否存在G6PD缺乏，后者能否激活P2X3引起疼痛。2.G6PD缺乏是否通过调控miRNA31-5p影响P2X3可塑性变化。后续实验发现，在体过表达G6PD后能降低糖尿病大鼠L4-6 DRG中P2X3的表达，但对miRNA31-5p表达没有明显影响，提示G6PD缺失并不是通过调控miRNA来影响P2X3的表达。结合更多的实验结果，调整研究内容为：1. P2X3启动子区DNA甲基化状态及其与转录因子NF-κB的相互作用。2. G6PD缺乏对TLR4/NF-κB信号通路的调控作用。方法：应用VFF机械刺激及热刺激技术，测定大鼠缩爪阈值；应用膜片钳技术检测后肢特异性DRG神经元ATP电流密度；应用Western blotting及RT-PCR技术检测表达水平；应用免疫荧光技术研究分子的定位及表达；应用MS-PCR及BS-PCR技术测定P2X3启动子区甲基化状态；应用CHIP技术检测NF-κB与P2X3启动子区的结合度。结果显示：1. 鞘内注射P2X受体抑制剂Suramin及P2X3选择性抑制剂A-317491均能明显缓解糖尿病大鼠后肢痛觉过敏。2. 糖尿病大鼠L4-6 DRG神经元ATP电流密度明显升高，P2X3表达明显高于对照组。3. 糖尿病大鼠L4-6 DRG中p2x3基因启动子区DNA甲基化水平明显降低，且NF-kB与p2x3基因启动子区的结合度明显增加。4. 糖尿病大鼠L4-6 DRG中NF-κB表达明显升高，腹腔注射NF-κB抑制剂PDTC能明显缓解糖尿病大鼠后肢痛觉过敏、明显降低P2X3表达。5. 给予NF-κB siRNA慢病毒鞘内注射后，糖尿病大鼠L4-6 DRG中P2X3表达明显下调。结果还显示：1. 糖尿病大鼠L4-6 DRG中G6PD表达水平显著低于对照大鼠，而TLR4表达水平显著高于对照大鼠。2. 免疫荧光结果显示G6PD及TLR4均广泛表达于DRG神经元中，并且广泛共定位。3. 鞘内注射G6PD过表达腺病毒能够明显缓解糖尿病大鼠后肢痛觉过敏，且能显著降低L4-6 DRG中TLR4的表达。4. 鞘内注射TLR4选择性抑制剂CLI095能够明显缓解糖尿病大鼠后肢痛觉过敏。预期通过本项目的研究初步揭示糖尿病后肢痛觉过敏的形成机理，为治疗提供理论依据。

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