Department of Pediatric Surgery, Xin Hua Hospital, Affiliated to Shanghai JiaoTong University School of Medicine, 1665 Kong Jiang Road, 200092 Shanghai, India; Department of Physiology, Shanghai Jiaotong University School of Medicine, Shanghai 200240, China.
Department of Physiology, Shanghai Jiaotong University School of Medicine, Shanghai 200240, China.
Eur J Pharmacol. 2019 May 15;851:151-160. doi: 10.1016/j.ejphar.2019.02.033. Epub 2019 Feb 21.
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 ([Ca]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 [Ca]i increase in Hek293 cells with heterologous expression of P2X7R (Hek293-P2X7R cells) and the same dose of ATP-induced [Ca]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.
一般认为肠神经病是糖尿病性胃轻瘫的原因之一。我们之前的研究表明，糖尿病小鼠存在一氧化氮合酶神经元的丢失。然而，其潜在机制尚不清楚。本研究旨在阐明神经元 P2X7R 与 NOS 神经元损伤之间的关系。通过定量 RT-PCR、免疫荧光、western blot、等长力记录、细胞内钙([Ca]i)测量和全细胞膜片钳技术，研究 P2X7R 对糖尿病诱导的胃 NOS 神经元损伤的影响及其机制。免疫组织化学和 western blot 结果表明，糖尿病小鼠中 nNOS 表达明显下调，同时，电刺激诱导的 NOS 敏感松弛明显受到抑制。肌间神经节神经元表达 P2X7R 和 pannexin1，糖尿病小鼠中 P2X7R 和 pannexin1 的 mRNA 和蛋白水平上调。P2X7R 激活剂 BzATP 可引起异源表达 P2X7R 的 Hek293 细胞([Ca]i 增加，相同剂量的 ATP 诱导的[Ca]i 在 Hek293-P2X7R 细胞中比在 Hek293 细胞中更明显。BzATP 的应用以剂量依赖的方式激活 Hek293-P2X7R 的内向电流。Hek293-P2X7R 而非未转染的 Hek293 细胞可摄取 YO-PRO-1。此外，P2X7 拮抗剂 oxATP 和 pannexin1 抑制剂 CBX 可阻断 Hek293-P2X7R 对 YO-PRO-1 的摄取。结果表明，肠神经元的 P2X7R 可能通过与 pannexin-1 结合形成跨膜孔，导致大分子物质和钙进入细胞，从而参与糖尿病诱导的 NOS 神经元损伤。
