基于巨噬细胞M2表型极化胃电起搏修复Cajal间质细胞损伤的机制研究

Interstitial cells of Cajal (ICC) injury is the pathologic basis of gastrointestinal motility disorders. It has recently found that heme oxygenase-1 (HO-1) secreted by resident macrophages contributes to the repair of ICC injury. We previously found that exogenous gastric electrical pacing could enhance the expression of HO-1, repair ICC injury and improve the delayed gastric emptying in diabetic rats. However, the molecular mechanism of gastric electrical pacing to repair ICC injury is still unclear. On the basis of our preliminary work, this project is to study the effects of gastric electrical pacing on gastric emptying, gastric antrum contraction and ICC with a model of diabetic gastroparesis rats; to verify whether gastric electrical pacing promote the macrophage M2 phenotype polarization by HO-1 using laser confocal microscopy and Western blot; and further to investigate the effect of HO-1 on the downstream molecules NF-κB to resist ICC apoptosis in diabetic gastroparesis rats with ChIP-qPCR and transmission electron microscopy. To explore the mechanism of gastric electrical pacing to repair ICC damage via macrophages polarization provides a new theoretical basis for the treatment of gastrointestinal motility disorders.

Cajal间质细胞（ICC）损伤是多种胃肠动力障碍性疾病发生发展的共同病理基础。近来发现，胃肠道定居的巨噬细胞分泌的血红素氧合酶-1（HO-1）在ICC损伤修复中发挥关键作用。我们前期研究发现外源性胃电起搏促进糖尿病大鼠胃窦HO-1表达，修复ICC损伤，改善胃排空延迟。但胃电起搏参与修复ICC损伤的分子机制尚不明确。本项目拟在前期工作基础上，以糖尿病胃轻瘫大鼠为模型研究胃电起搏对胃排空、胃窦收缩及胃窦ICC的影响；应用激光共聚焦、Western blot等技术验证胃电起搏促进巨噬细胞M2表型极化上调HO-1表达的关键调控机制；利用ChIP-qPCR、透射电镜等技术探讨HO-1作用下游效应分子NF-κB而抗ICC凋亡的分子机制。阐明胃电起搏介导巨噬细胞参与ICC损伤修复的机制，为胃肠动力障碍性疾病的治疗提供理论依据。

全球糖尿病的患病率呈急剧增加的趋势，我国糖尿病的患病率超过11.0%。胃肠动力障碍是糖尿病常见的并发症。糖尿病胃轻瘫的治疗主要依赖于促动力药物，长期应用疗效欠佳。目前研究证实Cajal间质细胞（ICC）损伤是糖尿病胃轻瘫发生发展的共同病理基础。近来发现，胃肠道定居的巨噬细胞分泌的血红素氧合酶-1（HO-1）在ICC损伤修复中发挥关键作用。我们前期研究发现胃电起搏促进糖尿病大鼠胃窦HO-1表达，修复ICC损伤，改善胃排空延迟。但胃电起搏参与修复ICC损伤的分子机制尚不明确。本研究以糖尿病胃轻瘫大鼠为模型，通过酚红餐排空法及器管浴槽技术发现胃电起搏明显改善延迟的胃排空及紊乱的胃窦收缩；通过western blot技术及免疫荧光染色技术显示胃电起搏增加胃窦ICC表达，通过透射电镜技术显示胃电起搏改善损伤ICC超微结构；通过免疫荧光技术发现胃电起搏促进巨噬细胞 M2 表型极化，表现为糖尿病大鼠胃内总的巨噬细胞无明显变化，而M1型巨噬细胞明显增加M2型巨噬细胞明显减少，胃电起搏减少M1型巨噬细胞数量增加了M2型巨噬细胞的数量；通过western blot技术及免疫组化技术显示糖尿病大鼠胃窦Nrf2/HO-1表达明显减少，而给予胃电起搏后Nrf2/HO-1的表达明显增加；通过总超氧化物歧化酶测定、丙二醛测定及流式细胞术检测ROS显示，氧化应激水平在糖尿病明显升高，胃电起搏可降低糖尿病氧化应激水平；通过western blot技术及免疫组化技术显示胃电起搏促进糖尿病大鼠NF-κB p65 磷酸化、核转位，进一步通过与SCF作用减少ICC凋亡。因此，本研究明确了胃电起搏改善胃动力的机制，通过促进巨噬细胞M2型表型极化及Nrf2/HO-1通路活化，降低氧化应激水平及促进NF-κB p65 磷酸化、核转位增加ICC的表达。本研究为胃电起搏治疗糖尿病胃肠动力障碍性疾病的临床推广提供理论依据。