Diabetes-induced damage of gastric nitric oxide neurons mediated by P2X7R in diabetic mice
Diabetes-induced damage of gastric nitric oxide neurons mediated by P2X7R in diabetic mice
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P2X7R 介导的糖尿病小鼠胃一氧化氮神经元损伤
DOI：
10.1016/j.ejphar.2019.02.033
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发表时间：
2019
影响因子：
5
通讯作者：
Xu Wen-Xie
中科院分区：
文献类型：
--
作者：
Zhang Chun-Mei;Huang Xu;Lu Hong-Li;Meng Xiang-Min;Song Ni-Na;Chen Lu;Kim Young-Chul;Chen Jie;Xu Wen-Xie
一般认为肠神经病变是糖尿病胃轻瘫的致病因素之一。我们之前的研究表明，糖尿病小鼠的 NOS 神经元出现了损失。然而，其根本机制仍不清楚。本研究旨在阐明神经元 P2X7R 与 NOS 神经元损伤之间的关系。采用定量RT-PCR、免疫荧光、蛋白质印迹、等长力记录、细胞内钙([Ca2+]i)测量和全细胞膜片钳技术研究P2X7R对糖尿病引起的胃NOS神经元损伤的影响及其机制。免疫组化和Western blot结果显示，糖尿病小鼠中nNOS表达显着下调，同时电场刺激诱导的NOS敏感性舒张也显着受到抑制。肌间神经元表达P2X7R和pannexin1，并且糖尿病小鼠中P2X7R和pannexin1的mRNA和蛋白水平上调。 P2X7R 激活剂 BzATP 会引起异源表达 P2X7R 的 Hek293 细胞（Hek293-P2X7R 细胞）中 [Ca2+]i 增加，且相同剂量的 ATP 诱导的 [Ca2+]i 在 Hek293-P2X7R 细胞中比 Hek293 细胞更明显。 BzATP 的应用以剂量依赖性方式激活 Hek293-P2X7R 的内向电流。 Hek293-P2X7R 但未转染的 Hek293 细胞可以摄取 YO-PRO-1。此外，Hek293-P2X7R对YO-PRO-1的摄取被P2X7拮抗剂oxATP和pannexin1抑制剂CBX阻断。结果提示，肠神经元的P2X7R可能通过与pannexin-1结合形成跨膜孔，诱导大分子物质和蛋白的产生，从而参与糖尿病诱导的NOS神经元损伤。 钙进入细胞。
It is generally considered that enteric neuropathy is one of the causative factors in diabetic gastroparesis. Our previous study demonstrated that there is a loss of NOS neurons in diabetic mice. However, the underlying mechanism remains unclear. The present study was designed to clarify the relationship between neuronal P2X7R and NOS neuron damage. The effect of P2X7R on diabetes-induced gastric NOS neurons damage and its mechanism were investigated by using quantitative RT-PCR,immunofluorescence, western blot, isometric force recording, intracellular calcium ([Ca2+]i) measurement and whole-cell patch clamp techniques. The immunohistochemistry and western blot results showed that nNOS expression was significantly down-regulated in diabetic mice, meanwhile, electric field stimulation-induced NOS sensitive relaxation was significantly suppressed. Myenteric neurons expressed P2X7R and pannexin1, and the mRNA and protein level of P2X7R and pannexin1 were up-regulated in diabetic mice. BzATP, a P2X7R activator, evoked [Ca2+]i increase in Hek293 cells with heterologous expression of P2X7R (Hek293-P2X7R cells) and the same dose of ATP-induced [Ca2+]i was more obvious in Hek293-P2X7R cells than in Hek293 cells. Application of BzATP activated an inward current of Hek293-P2X7R in a dose dependent manner. Hek293-P2X7R but not untransfected Hek293 cells could take up of YO-PRO-1. In addition, the uptake of YO-PRO-1 by Hek293-P2X7R was blocked by oxATP, a P2X7 antagonist and CBX, a pannexin1 inhibitor.The results suggest that the P2X7R of enteric neurons may be involved in diabetes-induced NOS neuron damage via combining with pannexin-1 to form transmembrane pores which induce macromolecular substances and calcium into the cells.