肝郁脾虚证大鼠胃肠钠尿肽信号通路的变化及疏肝健脾法干预机制

Clinical observation showed that The common symptoms of patient suffering from stagnation of liver-QI with deficiency of the spleen is gastrointestinal motility dysfunction. Research of combining traditional Chinese and Western medicine indicated that stagnation of liver-QI with deficiency of the spleen-induced gastrointestinal motility dysfunction was closely related to inappropriate secretion of neurotransmitter and hormone. Natriuretic peptide is polypeptide hormone existing both in the nervous system and the gastrointestinal tract. As for the relationship between NP and stagnation of liver-QI with deficiency of the spleen-induced gastrointestinal motility has not been definitely concluded at present. Our findings demonstrated what NP inhibited gastric motility by NP-cGMP-PKG pathway.Expression of Natriuretic peptide receptor expression ascend in stomach. Gastric motility was remarkably weakened in stagnation of liver-QI with deficiency of the spleen rat and sensitivity of gastric motility to NP-induced inhibition was increased. However, the arepresentative prescription of soothing Liver and strengthening spleen, Xiaoyaosan,significantly improve stagnation of liver-QI with deficiency of the spleen-induced gastrointestinal motility dysfunction (see our work). Based on the early works, Our study plan to choose gastric antrum, upper part of jejunum and descending colon as research object and investigate the change of NP signal pathway(ligand,receptor,enzyme and ion channel) in stagnation of liver-QI with deficiency of the spleen rat and the intervention effect of Xiaoyao san.The aim of of this study was to clarify material base of stagnation of liver-QI with deficiency of the spleen-induced gastrointestinal motility dysfunction from tissue to cellular level, from gene to protein level. At the same time, it will elucidate the probable intervention poin of Xiaoyao san on improving stagnation of liver-QI with deficiency of the spleen-induced gastrointestinal motility dysfunction in NP signal pathway. The purpose of the present study is to explain connotation of theory of spleen and stomach of spleen and stomach . Present study provide the theory basis for further developement and application of Xiaoyao san.

临床观察显示肝郁脾虚患者存在明显的胃肠运动障碍。研究表明肝郁脾虚证胃肠运动障碍与神经递质、激素和细胞因子分泌异常密切相关。钠尿肽（NP）是共存于神经系统和胃肠道的肽类激素，与胃肠运动密切相关，但目前尚无NP与肝郁脾虚证胃肠运动障碍间关系的报道。我们研究发现NP通过NP-cGMP-PKG通路抑制胃动力；肝郁脾虚大鼠胃NP受体表达增加，胃动力明显减弱并对NP抑制反应敏感性增加，而疏肝健脾法代表方剂-逍遥散对其有明显改善作用（见工作基础）。在此基础上本研究拟以大鼠胃窦、空肠上段和降结肠为研究对象，观察肝郁脾虚证大鼠胃肠NP信号通路关键环节（配体、受体、酶及离子通道）变化及逍遥散的干预作用。从组织到细胞水平，从基因到蛋白水平，揭示肝郁脾虚证胃肠运动障碍的物质基础，阐明逍遥散改善肝郁脾虚证胃肠运动障碍在NP信号通路上的可能干预位点。进一步阐释脾胃学说的内涵，为逍遥散的进一步开发应用提供理论依据。

临床观察显示肝郁脾虚证患者存在明显的胃肠运动障碍，研究表明肝郁脾虚证胃肠运动障碍与神经递质、激素以及细胞因子的分泌异常以及胃肠起搏细胞——Cajal间质细胞（Interstitial cells of Cajal，ICC）的功能密切相关。钠尿肽（Natriuretic Peptides, NPs）是一类共存于神经系统和胃肠道的肽类激素，许多研究表明钠尿肽及其受体广泛分布于胃肠道，并通过NPs-NPRs-pGC-cGMP-PKG信号通路对胃肠平滑肌收缩活动起抑制作用。钠尿肽信号通路是否参与肝郁脾虚证引起的胃肠运动障碍，以及疏肝健脾法的代表方剂——逍遥散对肝郁脾虚证胃肠运动障碍的改善是否与钠尿肽信号通路有关尚不清楚。本研究选用慢性温和不可预知性刺激结合孤养的方法建立肝郁脾虚证大鼠模型，以大鼠胃Cajal间质细胞、胃窦、空肠上段和降结肠为研究对象，研究钠尿肽信号通路在肝郁脾虚证大鼠模型中的变化情况。研究结果表明慢性温和不可预知性刺激结合孤养的方法可以成功制备肝郁脾虚证动物模型；NPs-NPRs-pGC-cGMP信号通路上调可能是肝郁脾虚证大鼠胃肠运动障碍的原因之一，米氮平及逍遥散剂量依赖性地下调该信号通路发挥治疗效果；肝郁脾虚证胃肠运动障碍可能与胃泌素、抑胃肽含量变化有关，逍遥散可能通过促进胃泌素，抑制抑胃肽分泌发挥治疗作用；在肝郁脾虚证大鼠胃中钠尿肽信号通路表达上调的同时，SCF/c-kit信号通路表达下调， CNP/NPR-A,B/cGMP信号通路引起SCF表达下调， cANF/NPR-C信号通路抑制胃平滑肌细胞的增殖，推测钠尿肽信号通路上调可能通过降低SCF的表达间接参与Cajal细胞的丢失。CNP对胃底及胃窦环形肌的自发性收缩起抑制作用，在肝郁脾虚证大鼠中这种抑制作用更为明显，CNP的抑制作用可被钾通道抑制剂TEA减弱，推测CNP可能是通过钾离子通道来抑制胃底及胃窦环形肌的自发性收缩。