茵陈蒿汤阻断毒性胆汁酸对SCF/c-kit通路的影响治疗胆石症的研究

Studies have demonstrated that inhibition of SCF/c-kit pathway in the gallbladder can cause the reduction of the number of ICC and cause the dysfunction of gallbladder contraction and led to the formation of gallstones. Based on the regulatory role of YINCHENHAO DECOCTION on bile acid transport system and its significant clinical curative effects on cholelithiasis, combined with the previous findings that increased toxic bile acids concentration is the manifestations of the bile lithogenicity, this research proposes the scientific hypothesis that by blocking the effects of toxic bile acids on SCF/c-kit pathway, avoiding the decrease in the number of ICC, recoving the function of gallbladder contraction, and inhibiting the gallstone formation are the possible mechanism of YINCHENHAO DECOCTION for treating cholelithiasis.This study will collect clinical samples of bile and serum from the patients with cholelithiasis, and use Ultra Pressure Liquid Chromatography-Triple Quadrupole Mass Spectrometry to detect the composition and concentration of bile acids so as to construct the bile acid profile of patients with cholelithiasis. Through the application of animal model of cholelithiasis and gallbladder muscle strip cultured in vitro,this study will explore the mechanism of YINCHENHAO DECOCTION about blocking the effects of toxic bile acid on SCF/c-kit signaling pathway. This experiment will clarify the characteristics of bile acid of cholelithiasis and the mechanism about the effects of toxic bile acids on SCF/c-kit pathway, and the mechanism of YINCHENHAO DECOCTION on the treatment of cholelithiasis by regulating the toxic bile acids. This study will provide new ideas and methods on Traditional Chinese Medicine therapy for the prevention and treatment of cholelithiasis.

研究已证实胆囊中SCF/c-kit通路抑制可致ICC减少引起胆囊收缩功能障碍进而导致胆结石形成，基于茵陈蒿汤对胆汁酸转运系统的调控作用及对胆石症的显著临床疗效，结合前期研究发现毒性胆汁酸浓度升高是胆汁致石性的表现，本研究提出“茵陈蒿汤治疗胆石症的机制可能与其阻断毒性胆汁酸对SCF/c-kit通路的影响，避免了ICC数量的减少，恢复了胆囊收缩功能进而抑制胆石形成”的科学假说。本研究从临床采集胆石症患者胆汁及血清样本，应用超高效液相色谱三重四级杆质谱检测胆汁酸成分及浓度变化，构建胆石症患者胆汁酸谱特征。同时应用胆石症动物模型及胆囊肌条离体培养研究茵陈蒿汤阻断毒性胆汁酸对SCF/c-kit通路影响的相关机制。通过此研究将明确胆石症患者胆汁酸谱变化特征，阐明毒性胆汁酸对SCF/c-kit通路的影响机制及茵陈蒿汤调控毒性胆汁酸而治疗胆石症的作用机制，为临床中医药防治胆石症提供新的思路与方法。

背景：茵陈蒿汤(YCHD)具有清热祛湿、利胆退黄等功效，目前临床上对茵陈蒿汤治疗胆石症的作用机制尚不明确，观察茵陈蒿汤对致石饮食所致小鼠胆囊胆固醇结石模型的影响，并探讨其可能的作用机制，为临床运用茵陈蒿汤防治胆囊胆固醇结石提供科学实验依据。.主要研究内容：实验一:YCHD 抑制小鼠胆囊胆固醇结石作用的研究，即研究对小鼠胆囊胆固醇结石的预防作用。致石饮食建立胆固醇结石小鼠模型，采用雄性 C57BL/6 小鼠随机分为:正常对照组(N)、模型组(M)、茵陈蒿汤低剂量组(YCHD-L)、茵陈蒿汤高剂量组(YCHD-H)和牛磺熊去氧胆酸组(TUDCA)。造模的同时灌胃给药，每天一次，分别持续4周和8周。.实验二: YCHD 治疗干预小鼠胆囊胆固醇结石发展的研究，即研究 YCHD 对小鼠胆囊胆固醇结石的治疗作用。致石饮食建立胆固醇结石小鼠模型，采用雄性 C57BL/6 小鼠随机分为:N、M、YCHD-L、YCHD-H 和 TUDCA 组。造模4周后开始灌胃给药，每天一次，分别持续4周和8周。.观察项目：(1)观察小鼠一般情况及胆囊内结石形成情况，双盲法判定胆结石的等级；(2)酶比色法测定血清肝功能和血脂、肝组织脂质含量；(3)肝组织HE染色；(4)酶比色法测定胆汁成分，计算胆固醇饱和指数(CSI)；(5)RT-qPCR、Western blot法观察肝脏和小肠胆固醇代谢通路相关基因表达水平的变化。 .重要结果：茵陈蒿汤可以有效干预致石饮食诱导所致胆囊胆固醇结石模型的发生与发展，改善血清及肝脏生化异常，存在一定的量效关系。茵陈蒿汤通过调节胆汁中胆固醇、磷脂和胆汁酸三者之间的相对浓度平衡以改善胆固醇过饱和，其作用机制可能是通过调控胆固醇代谢，即抑制小肠胆固醇吸收，影响肝脏胆固醇的转运，从而起到防治胆囊胆固醇结石的作用。