基于"脾主肌肉"理论探讨糖尿病胃轻瘫胃窦平滑肌RhoA/RocK信号通路的响应及红芪多糖的干预机制

Diabetic gastroparesis (DGP) is a complication which seriously influences the life quality of the diabetic patients and has a relatively high morbidity. Traditional Chinese medicine holds “spleen governing muscles”. The main pathogenesis of DGP is due to spleen qi-deficiency and muscular flaccidity which is closely related to the malfunction of the smooth muscle. Studies show that RhoA/RocK signal is the significant molecular mechanism mediating gastric antrum smooth muscle impairment. Our previous experiments demonstrated that hedysarum polybotrys saccharide (HPS) played a role on the protection of the gastric mucosa of the experimental rat with spleen deficiency, but the molecular mechanism of its intervention in DGP smooth muscle cells is still unclear. Combining the theory of“spleen governing muscles”and previous research foundation, we establish the hypothesis that the effective mechanism of HPS in the treatment of DGP is closely related to its intervention in the RhoA/RocK signaling pathway in smooth muscles. In this study,we will set up DGP rat models, interven in them with HPS, then use transmission electron microscope, RT-PCR and western blotting technology to detect the ultrastructure of gastric antrum smooth muscle cells in DGP rats and the mRNA and protein expression of correlation factors in RhoA/RocK signaling pathway. Finally, we will elucidate the molecular mechanism of HPS in the protection and restoration of DGP gastric antrum smooth muscle cells, make an explanation of the theory“spleen governing muscles”and provide an experimental and theoretical support for the development of superior Chinese medicine for prevention and treatment of DGP.

糖尿病胃轻瘫是发病率较高且严重影响糖尿病患者生活质量的并发症。中医理论认为“脾主肌肉”，糖尿病胃轻瘫发生的主要病机是脾气虚弱，肌肉松弛，而肌肉松弛与平滑肌功能失调密切相关。研究表明，RhoA/RocK信号是介导胃窦平滑肌受损的重要分子机制。前期实验证实，红芪多糖对实验性脾虚大鼠胃黏膜有一定保护作用，但其干预糖尿病胃轻瘫平滑肌细胞受损的分子机制尚不明确。研究结合“脾主肌肉”理论和前期研究基础，建立“红芪多糖治疗糖尿病胃轻瘫的疗效机制与其干预胃窦平滑肌RhoA/RocK信号通路密切相关”的假说，复制糖尿病胃轻瘫大鼠模型，以红芪多糖干预，采用透射电镜、RT-PCR、免疫印迹等技术检测大鼠胃窦平滑肌细胞超微结构、RhoA/RocK信号通路相关因子的mRNA和蛋白表达情况，探讨红芪多糖保护和修复胃窦平滑肌细胞的分子机制，阐释“脾主肌肉”理论的科学内涵，为开发防治该病的优势中药提供实验依据和理论支撑。

项目背景：.糖尿病胃轻瘫(diabetic Gastroparesis,DGP)是糖尿病(diabetes mellitus,DM)众多慢性消化道并发症之一，发病率极高且其发病机制十分复杂，现代研究表明，DGP 的胃动力低下主要是因胃平滑肌细胞受损所致，因此，提高胃动力促进胃排空的关键是修复胃平滑肌功能。RhoA/Rock信号通路是介导DGP发生的重要环节。中医理论认为“脾主肌肉”，糖尿病胃轻瘫发生的主要病机是脾气虚弱，肌肉松弛，而肌肉松弛与平滑肌功能失调密切相关。前期实验证实，红芪多糖(Hedysarum polybotrys saccharide,HPS)对实验性脾虚大鼠胃黏膜有一定保护作用，但其干预糖尿病胃轻瘫平滑肌细胞受损的分子机制尚不明确。研究结合“脾主肌肉”理论和前期研究基础，建立“红芪多糖治疗糖尿病胃轻瘫的疗效机制与其干预胃窦平滑肌RhoA/RocK信号通路密切相关”的假说，复制糖尿病胃轻瘫大鼠模型，以红芪多糖干预，开展本课题。.主要研究内容：以GK大鼠为研究对象，制备DGP模型，观察红芪多糖对DGP大鼠血糖值、胃排空率，胃黏膜超微结构，MTL、GAS、SP、Ach、DA、5-HT的含量以及对胃窦组织RhoA/RocK信号通路相关因子RhoA、Rock1、Rock2、MYPT1、p-MYPT1蛋白定性或半定量表达的影响等，以探究HPS促进糖尿病胃轻瘫大鼠胃动力的作用机制。.重要结果：.1.红芪多糖对大鼠一般情况具有较好恢复效果，能显著降低DGP大鼠血糖，提升胃排空率，对胃黏膜层损伤有不同程度的修复，提升胃激素含量，增加胃窦组织RhoA、Rock1、Rock2、MYPT1、p-MYPT1蛋白及mRNA表达量。.关键数据：.1.透射电镜结果：与模型组比较，红芪多糖高剂量组胃黏膜超微结构无明显的线粒体肿胀和细胞核变性以及染色质增多等现象。.2.与红芪多糖低剂量组比较，红芪多糖高剂量组RhoA、Rock1、Rock2、MYPT1、p-MYPT1蛋白表达显著升高，且阳性药组与红芪多糖高剂量组之间蛋白表达量无显著差异。.科学意义：.采用补气之品红芪的提取物红芪多糖，能够改善DGP大鼠一般情况，降低血糖，提升胃激素含量，促进胃排空率，对胃黏膜修复有一定促进作用，说明红芪多糖针对DGP疗效可靠，可以为后期药物研发提供实验依据。