内质网应激通路PERK-LC3/IRE1-Beclin1介导自噬调控ICC及疏肝理气法对FD效应机制

Functional dyspepsia (FD) is a common frequently-occurring disease, the treatment curative effect of dynamics-promoting medicine combined with antidepressant is poor and it often repeats. The function of ICC self-discipline pacemaker could adjust the integrity of ENS-ICC-SMC system and maintain normal gastric dynamics. The latest researches show that endoplasmic reticulum stress - ICC autophagy takes part in forming the gastric slow wave. Our hypothesis: that PERK/IRE1 pathway of endoplasmic reticulum stress starts the ICC autophagy and causes obstacle of ENS - ICC - SMC system is the basis of pathogenesis of FD. Our previous studies have confirmed that the method of Soothing Liver and Regulating Qi has the effect of promoting gastric dynamics on FD. In this study, FD animal model was made by cliped tail stress, gastric emptying and ultrastructures like ICC form, autophagy body were observed with laser confocal microscope and transmission electron microscope before and after intervention of Chaihu Shugan Powder. The changes of gene transcription and protein expression on PERK、eIF2α、Atg12、LC3、IRE1、TRAF2、JNK、Beclin1 were detected by RT-PCR, immunohistochemistry and Western Blot method etc.. It would provide an objective basis for TCM FD prevention with the method of soothing liver and regulating qi to explore the mechanisms of ICC autophagy mediated by PERK/IRE1 pathway of the endoplasmic reticulum stress on pathogenesis of FD and the intervention effects of Chaihu Shugan Powder.

功能性消化不良(FD)是常见多发病，促动力药甚至与抗抑郁药联合治疗时常疗效欠佳，病情反复。ICC自律起搏功能调节ENS-ICC-SMC系统的完整性保持正常胃动力。最新研究表明内质网应激-ICC自噬参与胃慢波形成。我们假说：内质网应激PERK/IRE1通路启动ICC自噬，导致“ENS-ICC-SMC”系统障碍是FD的发病基础。我们前期研究证实疏肝理气法对FD的促胃动力效应。本项目通过制备FD动物模型，运用柴胡疏肝散干预，观察干预前后胃排空、激光共聚焦显微镜、电镜观察ICC形态、自噬体等超微结构，RT-PCR、免疫组化、Western Blot等方法检测PERK、eIF2α、Atg12、LC3、IRE1、TRAF2、JNK、Beclin1基因转录及蛋白的表达变化；探讨内质网应激PERK/IRE1通路介导ICC自噬在FD发病中的机制及柴胡疏肝散的干预效应，为中医疏肝理气法防治FD提供客观依据。

背景：功能性消化不良（FD）是临床常见多发病，严重影响患者生活质量，疏肝理气法是治疗FD的大法，但是相关的机理尚未明确。胃肠Cajal间质细胞（ICC）在胃肠运动中发挥重要作用，FD的发病机理可能与ICC内质网应激-自噬有关，而疏肝理气法发挥疗效的机理可能与干预内质网应激PERK/IRE1通路介导ICC自噬有关。.研究内容：本课题按照研究目的分别从动物实验、细胞实验进行研究，证实疏肝理气法促胃动力作用，深入探讨内质网应激PERK/IRE1通路介导ICC自噬在FD发病中的机制及柴胡疏肝散的干预效应。.关键数据：与正常对照组比较，模型组大鼠胃排空率降低（P<0.05）；与模型组比较，柴胡疏肝散低、中、高剂量组及枳实组胃排空率升高（P<0.05）。与正常组比较，ERS组ICCs内质网肿胀、扩张、呈囊泡化、相互离散，粗面型内质网脱颗粒明显，可见大量自噬体；枳实组ICCs的内质网管腔扩张不明显，粗面型内质网少量脱颗粒，可见少量自噬体。与正常组比较，模型组ICC内Beclin1、LC3B荧光强度升高（P=0.00）；与模型组比较，柴胡疏肝散高、中剂量组内ICC内Beclin1、LC3B荧光强度下降（P=0.00）。与正常组比较，模型组PERK、p-eIF2α蛋白表达增高（P<0.05）；与模型组比较，柴胡疏肝散组PERK、p-eIF2α蛋白表达降低（P<0.05）。与正常组比较，模型组的IRE1、TRAF2、GRP78、JNK蛋白及mRNA的表达升高（P<0.05）；与模型组比较，柴胡疏肝散组IRE1、TRAF2、GRP78、JNK蛋白及mRNA表达降低（P<0.05）。与正常组比较，模型组Bax、CHOP mRNA表达升高（P<0.05）；与模型组比较，枳实低、中、高剂量组Bax、CHOP mRNA表达降低，Bcl-2、PERK mRNA表达升高（P<0.05）。.科学意义：FD胃肠动力障碍的机制之一可能是胃ICC内质网应激导致过度自噬，柴胡疏肝散通过下调GRP78的表达、抑制PERK/LC3B途径及IRE1/Beclin1途径，减轻ICC内质网应激-自噬损伤，从而促进胃动力。内质网应激及自噬与细胞启动程序性凋亡密切相关，枳实通过上调Bcl-2、PERK mRNA，下调Bax、CHOP mRNA来调节ICC内质网应激途径的细胞凋亡，促进ICC细胞存活，改善胃动力障碍。