基于IRE1/Beclin1通路研究芍药甘草汤调控Cajal间质细胞自噬水平改善Oddi括约肌功能障碍的作用机制

Sphincter of Oddi dysfunction (SOD) is a dynamic disorder characterized by persistent abdominal pain caused by smooth muscle spasm. Studies have confirmed that the up-regulation of IRE1/Beclin1 signaling pathway to initiate excessive autophagy and inhibit the function of interstitial cells of Cajal (ICC) in smooth muscle is an important pathological mechanism of SOD. Combining with the significant effect of Shaoyao Gancao Decoction on SOD and the working basis of its inhibiting GPR78 and Beclin1 levels, it is proposed that " Regulates IRE1/Beclin1 signaling pathway and alleviates excessive autophagy caused by endoplasmic reticulum stress in ICC, is the pharmacological mechanism of improving SOD by Shaoyao Gancao Decoction”. This study intends to carry out: (1) Reproduce SOD model of guinea pig in vivo, using IRE1 inhibitor and c-kit inhibitor as control, then separate SO myocycle and give Shaoyao Gancao Decoction to verify the efficacy again; (2) Separate primary ICC in SO in vitro, construct IRE1 overexpression/silence ICC after Shaoyao Gancao Decoction intervention.This study aimed to clarify the mechanism of Shaoyao Gancao Decoction in promoting SO relaxation and improving SOD by inhibiting excessive autophagy through down-regulating IRE1/Beclin1 pathway to promote the function of ICC, and to consolidate the foundation for the pharmacological study of traditional Chinese medicine in regulating biliary dynamics.

Oddi括约肌功能障碍（SOD）是以平滑肌痉挛引起持久腹痛为特征的动力失常性疾病。研究证实，激活IRE1/Beclin1信号启动过度自噬从而抑制Oddi括约肌（SO）内Cajal间质细胞（ICC）功能是SOD的重要病理机制。结合芍药甘草汤改善SOD腹痛效果显著及其抑制GPR78、Beclin1水平的工作基础，提出假说：芍药甘草汤抑制IRE1/Beclin1信号通路，减轻ICC内质网应激引起的过度自噬，从而改善SOD平滑肌舒张效应。拟开展：①体内复制SOD豚鼠模型，以IRE1和c-kit抑制剂为对照，芍药甘草汤干预，并分离SO肌环离体给药予以验证；②体外分离SO内原代ICC，构建IRE1过表达/沉默的ICC后予芍药甘草汤干预。本研究旨在阐明芍药甘草汤通过下调IRE1/Beclin1通路抑制ICC过度自噬，以促进ICC功能而改善SOD的机制，为推进中医药调节胆道动力学的理论研究夯实基础。

Oddi括约肌功能障碍（SOD）是一组因Oddi括约肌（SO）舒张功能异常引起的临床综合征，虽没有器质性改变证据，却会导致持久难忍的疼痛和肝功能异常；临床治疗手段有限，内镜下括约肌切开对部分患者有效。芍药甘草汤是《伤寒论》中的经典名方，前期研究表明它可有效减轻SOD患者腹痛症状，缩短胆汁排泄时间，具体作用机制尚不明确。本研究拟探讨芍药甘草汤对SO中Cajal细胞（ICC）及自噬蛋白表达的影响，为中医药治疗胆道动力疾病的理论夯实基础。. 通过吗啡注射成功建立豚鼠SOD模型，以芍药甘草浸膏液灌胃，证实模型组炎细胞浸润、纤维组织增生、环形肌紊乱，芍药甘草组和IRE1阳性对照组病理改变好转。Western blot法和RT-PCR检测提示，模型组c-kit表达降低，自噬启动基因Beclin1、自噬标记蛋白LC3B明显增高，提示SOD豚鼠自噬增加，使用芍药甘草汤干预后，这一现象有所逆转；而使用c-kit抑制剂STI571模拟ICC功能失活状态，芍药甘草汤的作用减弱，提示芍药甘草汤通过减少过度自噬、提升Cajal细胞活性，从而发挥舒张Oddi括约肌功效。离体实验同样证实芍药甘草汤药效，透射电镜证实模型组ICC内出现大量内质网和分泌颗粒，可见较多自噬小体，芍药甘草汤干预后自噬小体较模型组显著减少。体外实验构建雨蛙素诱导的AR42J细胞株自噬模型，证明芍药甘草汤显著降低了模型组Beclin1、LC3B、p62、JNK等自噬相关标记物水平。. 通过豚鼠SOD标本测序和药物质谱分析、网络药理学手段，鉴定出芍药甘草汤中32种候选化合物，筛选出SOD中247个上调基因和402个下调基因，MCODE和ClusterONE网络分析出22个交叉基因，通过RT-PCR验证SO中mRNA表达水平，证实经芍药甘草汤治疗后，Ccnb1、Cacna2d2和Chrnb4免疫和炎症调节相关基因水平显著恢复。. 本研究初步证实，芍药甘草汤提升了Cajal细胞活性、降低了细胞内过度自噬水平，发挥对SOD平滑肌的舒张效应，为芍药甘草汤的临床应用提供了坚实理论依据。