基于PLC-IP3-Ca2+/NO-cGMP-PKG信号通路的半夏泻心汤防治糖尿病胃轻瘫的机理研究

Banxia xiexin Tang is a classic prescription for the treatment of stomach disorders,there is considerable evidence that Banxia xiexin Tang is effective on gastric motility disorders. Gastric smooth muscle is the structural basis of gastrointestinal motility, gastric smooth muscle contraction function, and diastolic dysfunction is an important factor in the pathogenesis of diabetic gastroparesis. Smooth muscle contraction of the stomach caused by motilin downstream signal transduction pathways(PLC-IP3-Ca2+), causing calcium ion concentration to exert contractile regulation of gastric motility; gastric smooth muscle relaxation is to play the role of nitric oxide by adjusting the downstream signaling pathways(NO-cGMP-PKG). Traditional medicines primarily for a single contract or relaxation, and can not be adjusted for systolic and diastolic overall, Banxia xiexin Tang may have multiple targets, the onset of the advantages of multi-channel. We found that after Banxia xiexin Tang treatment of gastric smooth muscle strips of rat activity increased, serum motilin and nitric oxide levels were higher. To verify and investigate the possibility of the overall regulation of diabetic gastroparesis disease network through multiple signaling pathways impact on gastric smooth muscle contract and relaxation effect of Banxia xiexin Tang, in vivo and in vitro experiments are combined to find the signaling pathways (PLC-IP3-Ca2/ NO-cGMP-PKG), and thereby clarify the mechanism of Banxia xiexin Tang.

半夏泻心汤是《伤寒论》治疗痞证的经典方剂，临床上有大量证据表明其对胃动力障碍疾病有确实疗效。胃平滑肌是胃运动的结构基础，胃平滑肌的收缩舒张功能下降是糖尿病胃轻瘫发病的重要因素。胃平滑肌收缩是通过胃动素引起下游信号通路(PLC-IP3-Ca2+)的传导，引起钙离子浓度升高，来发挥作用；胃平滑肌舒张是通过一氧化氮调节下游信号通路(NO-cGMP-PKG)发挥作用。传统药物主要针对单一收缩或舒张信号通路起作用，无法对收缩舒张整体进行调节，半夏泻心汤可能具有多靶点、多通路起效的优势。我们研究发现半夏泻心汤治疗后大鼠胃平滑肌肌条活动度增加，血清胃动素、一氧化氮含量明显增高。为了验证半夏泻心汤对大鼠胃平滑肌收缩舒张效应的影响并探讨其是否可通过多信号途径整体调控糖尿病胃轻瘫疾病网络，实验采用体内与体外实验相结合的方法研究相关信号通路的变化，来阐明半夏泻心汤防治糖尿病胃轻瘫的机理。

半夏泻心汤是《伤寒论》治疗上腹部饱胀综合征的经典方剂。大量的证据表明半夏泻心汤可以缓解胃运动障碍，提示半夏泻心汤通过双信号途径调节胃平滑肌功能，促进胃运动，预防和治疗糖尿病性胃轻瘫（DGP）。由Sprague–Dawley（SD）大鼠制成的糖尿病胃轻瘫模型大鼠最初在腹腔内注射40 mg/kg剂量的链脲佐菌素（STZ），72小时后，监测空腹血糖（FBG），确定FBG≥16.7mol/L的大鼠为糖尿病（DM）模型。2个月后，通过记录胃排空、肠转运速度和胃肌电活动，证实了DGP模型。DGP模型大鼠随机分为6组：正常组、DGP组、高、中、低剂量半夏泻心汤组和多潘立酮组，各组分别用相应的药物治疗4周，然后通过逆转录聚合酶链反应检测磷脂酶C（plc）、肌醇三磷酸（ip3）、神经元一氧化氮合酶（nNOS）和环鸟苷一磷酸（cGMP）依赖性蛋白激酶G（pkg）信使核糖核酸（mRNA）的表达。用Western blot法检测plc、ip3、nNOS和pkg蛋白的表达，酶联免疫吸附法检测一氧化氮（no）和cGMP。大鼠胃组织取自对照组和模型组，用于原代细胞培养制备。测定半夏泻心汤治疗前后模型组胃平滑肌细胞长度及细胞内Ca2+浓度（[Ca2+]i）变化，将半夏泻心汤组与模型组的体内、体外数据进行比较，提出并分析了促进胃运动的机制。半夏泻心汤能剂量依赖性地增加plc、ip3、no、nNOS、cGMP和pkg的表达（P<0.01），促进胃平滑肌细胞的收缩（P<0.01），增加[Ca2+]i（P<0.01）。本研究结果表明，半夏泻心汤对糖尿病胃轻瘫大鼠的治疗作用可能与plc-ip3-ca2+/no-cGMP-pkg信号通路有关。