-aspartic acid receptor 2B (NR2B) pathway on type 2 DNP. METHODS: Male Sprague-Dawley rats were fed with a high-fat and high-sugar diet for 8 weeks. Then, rats were intraperitoneally injected with streptozotocin (STZ, 35 mg/kg) to induce type 2 diabetes mellitus in rats. Diabetic rats with <85% of their basic levels in mechanical withdrawal threshold and thermal withdrawal latency were classified as DNP rats on day 14 after STZ injection. DNP rats were treated with ROS scavenger N-tert-Butyl-α-phenylnitrone (PBN, 100 mg·kg–1·d–1) or TXNIP small interfering ribonucleic acid (10 μg/d) once daily for 14 days. The level of ROS, protein levels of NLRP3, TXNIP, cysteinyl aspartate-specific proteinase-1 (caspase-1), interleukin-1β (IL-1β), NR2B phosphorylation at Tyr1472 (p-NR2B), total NR2B (t-NR2B), and distribution of NLRP3 in the spinal cord were examined. In vitro experiments, BV2 cells and PC12 cells were individually cultured and cocultured in a high-glucose environment (35 mmol/L D-glucose). The level of ROS and protein levels of NLRP3, TXNIP, caspase-1, and IL-1β in BV2 cells, and p-NR2B, t-NR2B in PC12 cells were detected. The level of ROS was detected by the flow cytometry approach. The protein levels were detected by the Western blot technique. The location of NLRP3 was observed by immunofluorescent staining. The interaction between TXNIP and NLRP3 was detected by coimmunoprecipitation assay. RESULTS: The level of spinal ROS increased in DNP rats. The mechanical allodynia and thermal hyperalgesia of DNP rats were alleviated after systemic administration of PBN. This administration decreased protein levels of NLRP3, TXNIP, caspase-1, IL-1β, and p-NR2B and the coupling of TXNIP to NLRP3 in spinal cords of DNP rats. Furthermore, knockdown of spinal TXNIP alleviated nociceptive hypersensitivity and decreased protein levels of NLRP3, TXNIP, caspase-1, IL-1β, and p-NR2B in DNP rats. The level of ROS and protein levels of NLRP3, TXNIP, caspase-1, IL-1β, the coupling of TXNIP to NLRP3, and the IL-1β secretion increased in BV2 cells, and the protein expression of p-NR2B increased in cocultured PC12 cells in a high-glucose environment. All of these in vitro effects were significantly blocked after treatment of PBN. CONCLUSIONS: Our findings suggest that spinal ROS can contribute to type 2 DNP through TXNIP-NLRP3-NR2B pathway....

中文翻译：

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活性氧通过硫氧还蛋白相互作用蛋白 NOD 样受体蛋白 3-N-甲基-D-天冬氨酸受体 2B 通路导致 2 型糖尿病神经性疼痛

-2 型 DNP 上的天冬氨酸受体 2B (NR2B) 途径。方法：雄性 Sprague-Dawley 大鼠饲喂高脂高糖饮食 8 周。然后，给大鼠腹腔注射链脲佐菌素（STZ，35 mg/kg），诱导大鼠发生2型糖尿病。在 STZ 注射后第 14 天，机械戒断阈值和热戒断潜伏期低于其基本水平的 85% 的糖尿病大鼠被归类为 DNP 大鼠。DNP 大鼠每天用 ROS 清除剂 N-叔丁基-α-苯基硝酮 (PBN, 100 mg·kg–1·d–1) 或 TXNIP 小干扰核糖核酸 (10 μg/d) 处理 14 天。ROS水平、NLRP3蛋白水平、TXNIP、半胱氨酰天冬氨酸特异性蛋白酶1（caspase-1）、白细胞介素1β（IL-1β）、NR2B在Tyr1472的磷酸化（p-NR2B）、总NR2B（t-NR2B ), 检查了NLRP3在脊髓中的分布和分布。在体外实验中，BV2细胞和PC12细胞分别在高糖环境（35 mmol/L D-葡萄糖）中培养和共培养。检测BV2细胞中的ROS水平和NLRP3、TXNIP、caspase-1、IL-1β和PC12细胞中p-NR2B、t-NR2B的蛋白水平。流式细胞仪检测 ROS 水平。通过蛋白质印迹技术检测蛋白质水平。通过免疫荧光染色观察NLRP3的位置。通过免疫共沉淀法检测TXNIP和NLRP3之间的相互作用。结果：DNP大鼠脊髓ROS水平升高。全身给予PBN后，DNP大鼠的机械性异常性疼痛和热痛觉过敏得到缓解。该给药降低了 NLRP3、TXNIP、caspase-1、DNP 大鼠脊髓中 IL-1β 和 p-NR2B 以及 TXNIP 与 NLRP3 的偶联。此外，敲除脊髓 TXNIP 可减轻 DNP 大鼠的伤害性超敏反应并降低 NLRP3、TXNIP、caspase-1、IL-1β 和 p-NR2B 的蛋白质水平。BV2细胞中ROS水平和NLRP3、TXNIP、caspase-1、IL-1β、TXNIP与NLRP3偶联、IL-1β分泌增加，p-NR2B蛋白表达增加。高糖环境中的细胞。在 PBN 治疗后，所有这些体外效应均被显着阻断。结论：我们的研究结果表明，脊髓 ROS 可以通过 TXNIP-NLRP3-NR2B 通路促进 2 型 DNP。敲除脊髓 TXNIP 可减轻 DNP 大鼠的伤害性超敏反应并降低 NLRP3、TXNIP、caspase-1、IL-1β 和 p-NR2B 的蛋白质水平。BV2细胞中ROS水平和NLRP3、TXNIP、caspase-1、IL-1β、TXNIP与NLRP3偶联、IL-1β分泌增加，p-NR2B蛋白表达增加。高糖环境中的细胞。在 PBN 治疗后，所有这些体外效应均被显着阻断。结论：我们的研究结果表明，脊髓 ROS 可以通过 TXNIP-NLRP3-NR2B 通路促进 2 型 DNP。敲除脊髓 TXNIP 可减轻 DNP 大鼠的伤害性超敏反应并降低 NLRP3、TXNIP、caspase-1、IL-1β 和 p-NR2B 的蛋白质水平。BV2细胞中ROS水平和NLRP3、TXNIP、caspase-1、IL-1β、TXNIP与NLRP3偶联、IL-1β分泌增加，p-NR2B蛋白表达增加。高糖环境中的细胞。在 PBN 治疗后，所有这些体外效应均被显着阻断。结论：我们的研究结果表明，脊髓 ROS 可以通过 TXNIP-NLRP3-NR2B 通路促进 2 型 DNP。在高糖环境下共培养的 PC12 细胞中 p-NR2B 的蛋白表达增加。在 PBN 治疗后，所有这些体外效应均被显着阻断。结论：我们的研究结果表明，脊髓 ROS 可以通过 TXNIP-NLRP3-NR2B 通路促进 2 型 DNP。在高糖环境下共培养的 PC12 细胞中 p-NR2B 的蛋白表达增加。在 PBN 治疗后，所有这些体外效应均被显着阻断。结论：我们的研究结果表明，脊髓 ROS 可以通过 TXNIP-NLRP3-NR2B 通路促进 2 型 DNP。