大柴胡汤加减对胆囊胆固醇结石湿热证小鼠FXR/FGF15/FGFR4信号通路的影响  

Effect of Modified Da Chaihutang on FXR/FGF15/FGFR4 Signaling Pathway in Mice with Cholesterol Gallstone Differentiated into Damp-Heat Syndrome

作　　者：王素英 谢玉春 闵莉[1] WANG Su-ying;XIE Yu-chun;MIN Li(Fujian University of Traditional Chinese Medicine(TCM),China Molecular Biology Laboratory of TCM Syndrome Research Base,Top Level Laboratory of the State Administration of TCM,Fuzhou 350122,China)

机构地区：[1]福建中医药大学中医证研究基地分子生物实验室,国家中医药管理局三级科研实验室,福州350122

出　　处：《中国实验方剂学杂志》2022年第3期15-24,共10页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金项目(81603541);福建省自然科学基金项目(2021J01131533)。

摘　　要：目的:基于法尼酯衍生物X受体(FXR)/成纤维细胞生长因子15(FGF15)/成纤维细胞生长因子受体4(FGFR4)通路观察大柴胡汤加减对胆囊胆固醇结石(CS)湿热证模型小鼠的影响,以从方证对应的角度,探讨CS湿热证的分子生物学机制。方法:将48只6周龄小鼠随机分为空白组、模型组、大柴胡汤加减组(23.4 g·kg^(-1))和熊去氧胆酸组(0.12 g·kg^(-1)),每组12只;空白组作为对照,其他3组采用“内湿+外湿+高胆固醇致石饲料”的模式造模12周建立CS湿热证小鼠模型,大柴胡汤加减组和熊去氧胆酸组给予相应的药物灌胃,模型组和空白组给予等量的生理盐水灌胃,共干预4周。造模前后每组小鼠均要进行旷场行为学实验判断其活动度和精神状态,每周观察记录各组小鼠体质量、饮食量、皮毛、二便等一般情况变化以评判其湿热证候表现;干预结束后,取各组小鼠肝脏、胆囊组织进行苏木素-伊红(HE)染色,对血清中γ-谷氨酰基转移酶(GGT),碱性磷酸酶(ALP),总胆红素(TBIL)指标进行生化检测;酶联免疫吸附测定法(ELISA)检测胆汁中总胆固醇(TC),总胆汁酸(TBA)含量;实时荧光定量聚合酶链式反应(Real-time PCR)及蛋白免疫印迹法(Western blot)检测各组小鼠回肠FXR,FGF15,以及肝脏FGFR4,胆固醇7α-羟化酶(CYP7A1)mRNA与蛋白表达水平。结果:与空白组比较,模型组小鼠胆囊增大,胆汁呈褐色浑浊状态,肉眼可见絮状沉淀;湿热证候表现明显;肝脏组织有明显的脂肪样变性,胆囊壁毛糙、增厚;血清肝功能指标ALP,GGT,TBIL水平均显著升高(P<0.01);胆汁内TC含量显著增多(P<0.01),TBA含量显著减少(P<0.01);回肠组织的FXR mRNA与蛋白表达均显著升高(P<0.01),FGF15 mRNA和蛋白表达均明显升高(P<0.05,P<0.01),肝脏FGFR4 mRNA和蛋白表达量均明显增多(P<0.05),CYP7A1 mRNA表达显著降低(P<0.01),蛋白表达量呈降低趋势但差异无统计学意义。与模型组比较,两用药组小鼠胆�Objective:To observe the effect of modified Da Chaihutang on cholesterol gallstone(CS)in mice due to damp-heat based on the farnesoid X receptor(FXR)/fibroblast growth factor 15(FGF15)/fibroblast growth factor receptor 4(FGFR4)pathway and explore the molecular biological mechanisms of CS differentiated into damp-heat syndrome from the perspective of correspondence between prescription and syndrome.Method:Forty-eight six-week-old mice were randomly divided into the blank group,model group,modified Da Chaihutang(23.4 g·kg^(-1))group,and ursodeoxycholic acid(0.12 g·kg^(-1))group,with 12 mice in each group.The ones in the latter three groups were exposed to"internal dampness+external dampness+highcholesterol diet"for 12 weeks for inducing CS due to damp-heat.Mice in the modified Da Chaihutang group and ursodeoxycholic acid group were gavaged with the corresponding drugs,while those in the model and blank groups with the same amount of normal saline for a total of four weeks.Before and after modeling,mice in each group were subjected to open field tests for determining their activities and mental states.Such general conditions as body mass,food intake,fur,and urine and stool of mice in each group were observed and recorded weekly for judging the damp-heat syndrome.After the intervention,the sampled liver and gallbladder tissues of mice in each group were stained with hematoxylin-eosin(HE)staining,and the serumγ-glutamyltransferase(GGT),alkaline phosphatase(ALP),and total bilirubin(TBIL)were determined.The total cholesterol(TC)and total bile acid(TBA)contents in bile were measured by enzyme-linked immunosorbent assay(ELISA).The mRNA and protein expression levels of FXR,FGF15,FGFR4,and cholesterol 7α-hydroxylase gene(CYP7A1)were assayed by real-time fluorescence quantitative polynucleotide chain reaction(Real-time PCR)and Western blot.Result:Compared with the blank group,the model group exhibited enlarged gallbladder,brown turbid bile with flocculent precipitation visible to the naked eye,obvious damp-heat syndrome,

关 键 词：胆囊胆固醇结石 湿热证 大柴胡汤加减 胆固醇-胆汁酸代谢 法尼酯衍生物X受体(FXR)/成纤维细胞生长因子15(FGF15)/成纤维细胞生长因子受体4(FGFR4)通路 

分 类 号：R2-0[医药卫生—中医学] R22