兰州大学机构库 >药学院
三种病生理条件下大/小鼠体内胆酸盐的变化及机制研究
Alternative TitleExploring the alterations of bile acids and their mechanisms under three different pathophysiological conditions in rats and mice
张帆
Thesis Advisor武新安
2019-05-28
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name博士
Degree Discipline药物化学生物学
Keyword胆酸盐 葡萄糖 核受体 代谢酶 转运体 信号通路 胆囊摘除 雌激素 17α-炔雌醇 肝内胆汁淤积 2型糖尿病
Abstract目的  胆酸盐作为体内重要的内源性物质,具有非常广泛的生物学活性,参与体内多种生理和病理过程,其作为信号分子与核受体包括法尼醇X受体(FXR)、G蛋白偶联胆酸盐受体(TGR5)、孕烷X受体(PXR)、组成型雄甾烷受体(CAR)等结合,在转录水平对下游的代谢酶和转运体等功能蛋白的基因表达进行调控,且相互之间形成信号通路网络,以维持体内物质代谢平衡及机体内稳态。现研究表明,胆酸盐对其自身、葡萄糖和脂类等的代谢平衡均发挥核心调控作用,其体内代谢紊乱是导致机体生理环境改变乃至诱发肝内胆汁淤积、糖尿病尤其是2型糖尿病、肥胖、非酒精性脂肪肝、动脉粥样硬化等疾病的关键因素。此外,胆酸盐在不同的生理条件和病理进程中,其浓度和组成存在明显差异,利用此特点,有望实现对相关疾病的早期诊断和分型。然而,目前有关胆酸盐与相关疾病之间的关联性尚未完全阐明,仍需要进行多方面的研究。为此,本论文以大/小鼠为研究对象,基于胆酸盐及其相关信号通路,探究胆囊摘除后的生理条件改变情况下,以及雌激素紊乱型肝内胆汁淤积和2型糖尿病的疾病条件下,机体内胆酸盐的变化及其机制,并以此为基础,探讨胆酸盐代谢紊乱与上述疾病发生和转归之间的关系,以期为寻找相关疾病防治的新靶点和新措施提供思路。方法  本论文涉及3部分实验方法和内容:(1)小鼠胆囊摘除后体内胆酸盐及其昼夜节律的变化与机制   昆明种雄性小鼠,分别实施胆囊摘除手术和假手术,手术后恢复2周,收集血清、肝脏、回肠、胆囊、小肠(含内容物)及粪便样本。采用紫外法测定上述各样本中的总胆酸盐浓度和胆酸池含量,并考察其昼夜节律性;观察肝脏和回肠的病理学变化;采用HPLC/MS/MS方法定量分析血清、肝脏组织、回肠组织和粪便中各胆酸盐的浓度和比例,并考察其昼夜节律性;使用实时荧光定量PCR(RT-qPCR)和Western Blotting技术,分别检测肝脏和回肠中胆酸盐相关核受体、代谢酶和转运体的mRNA表达量和蛋白表达量,并分析相关功能蛋白mRNA水平的昼夜节律性。 (2)基于胆酸盐“肠-肝轴”和“肝-肾轴”阐释雌激素诱导肝内胆汁淤积的致病、代偿与失代偿机制   以Wistar雄性大鼠及人类HepaRG肝细胞为研究对象。首先,以常用雌激素17α-炔雌醇(EE)为模型诱导药物,于大鼠颈部皮下分别注射EE丙二醇溶液7、14和28天,诱导致病期、代偿期和失代偿期三个不同病程阶段的雌激素紊乱型肝内胆汁淤积模型。其次,于诱导结束后,分别采集大鼠血清、肝脏、回肠、肾脏、胆汁、粪便和尿液样本,使用相应方法分别测定并分析:血清转氨酶和胆红素水平;上述各样本中总胆酸盐的浓度以及胆酸池含量;肝脏病理学变化;血清和胆汁样本以及肝脏和回肠组织样本中各胆酸盐的浓度和比例;肝脏、回肠和肾脏部位胆酸盐相关核受体、代谢酶和转运体的mRNA表达量。最后,采用计算机模拟分子对接技术及HepaRG细胞实验对所得结论进行验证。 (3)2型糖尿病模型大鼠体内胆酸盐的变化与机制及其对葡萄糖代谢紊乱的影响初探     Wistar雄性大鼠,采用高脂饲料致肥胖后,联合小剂量链脲佐菌素诱导2型糖尿病模型,即:高脂饲料喂养大鼠7周诱导肥胖后,一次性腹腔注射小剂量链脲佐菌素(30 mg/kg)后,再用高脂饲料喂养1周,以制备2型糖尿病大鼠模型。模型诱导成功后,分别采集大鼠血清、肝脏、回肠、结肠、胰腺、小肠(包含内容物)和粪便样本,采用相应方法分别测定并分析:血清中的转氨酶、葡萄糖、胆固醇和甘油三酯的水平;血清、肝脏、小肠(包含内容物)和粪便样本中总胆酸盐的浓度以及胆酸池的含量;肝脏病理学变化;血清、肝脏、回肠、结肠和胰腺组织样本中各胆酸盐的浓度和比例;肝脏、回肠、结肠和胰腺部位胆酸盐及与葡萄糖代谢相关的核受体、代谢酶和转运体的mRNA表达量。结果     小鼠胆囊摘除后:体内总胆酸池显著减小,其与回肠Na+依赖性胆酸盐转运体(Asbt)蛋白表达量降低导致的胆酸盐回肠吸收障碍及大量胆酸盐经粪便流失有关;胆囊收缩节律消失导致胆酸盐回肠重吸收的昼夜节律消失,并最终造成总胆酸池的节律性消失;胆酸盐合成限速酶Cyp7a1基因表达的昼夜节律消失,导致初级胆酸盐肝脏合成的节律性明显消失;回肠Fxr及其调控下游的功能蛋白-促进成纤维生长因子(Fgf15)基因表达的昼夜节律发生了明显改变,且此通路决定了肝脏Cyp7a1 mRNA水平的昼夜节律特征;肝药酶Cyp3a11 mRNA和蛋白表达量均显著下调,同时Cyp3a11以及肝脏药物转运体Oatp2和Mrp2 mRNA水平的昼夜节律性明显消失。      EE诱导的大鼠肝内胆汁淤积:动物实验、分子对接实验和HepaRG细胞实验结果均表明,EE通过抑制Fxr后,在转录水平下调肝细胞胆管侧膜的胆酸盐输出泵(Bsep)的表达,从而造成胆酸盐经胆汁排泄障碍,最终导致肝内胆汁淤积的发生;在代偿期,模型大鼠回肠和肝脏组织中胆酸盐浓度和比例发生了差异性变化,进而通过影响Fxr“肠-肝轴”,上调肝脏胆酸盐合成酶Cyp7a1与Cyp27a1的基因表达并下调 Cyp8b1的基因表达,从而增加了肝组织中鹅去氧胆酸(CDCAs)的水平,CDCAs作为Fxr的强效激动剂逆转了EE对Fxr-Bsep通路的抑制作用,从而促使胆酸盐经胆汁外排能力恢复并增强,以发挥代偿效应;胆酸盐“肝-肾轴”适应性改变,包括肝脏Pxr-硫酸化转移酶2a1(Sult2a1)/多药耐药相关蛋白3(Mrp3)通路的激活、肾脏外排转运体Mrp4表达的上调以及肾脏重吸收转运体Asbt和有机溶质转运体β(Ostβ)表达的下调,促使胆酸盐经尿液排泄显著增加,从而发挥代偿作用;失代偿期的模型大鼠,肝脏病理变化加重,肝细胞死亡增多,肝脏中多数功能蛋白的基因表达下调,与此同时,肝组织中具较高毒性的胆酸盐CDCAs的水平升高,同时Fxr拮抗剂β-鼠胆酸(β-MCAs)比例的增加,可能是造成其向失代偿期发展的原因;分子对接实验显示,内源性雌二醇与EE分子对Fxr存在非常接近的结合模式和结合力。       2型糖尿病模型大鼠体内:血清中胆酸盐的浓度和组成与对照组存在明显差异,其中以12α-OH/non-12α-OH胆酸盐比例的升高为主要特征,且其与肝脏介导12α-OH胆酸盐合成的Cyp8b1基因表达比例升高有关,此外,模型大鼠体内次级胆酸盐水平明显降低;在模型大鼠的结肠、胰腺和肝脏组织中,Fxr和Tgr5激动剂型胆酸盐的水平下降,同时,在Fxr和Tgr5介导调控葡萄糖代谢的相关信号通路上,部分功能蛋白的mRNA表达水平也下调,这种改变可能将进一步加重体内葡萄糖的代谢紊乱并促进2型糖尿病的发展。 结论     胆囊摘除、雌激素紊乱型肝内胆汁淤积和2型糖尿病条件下,机体内胆酸盐出现了明显的代谢紊乱,这与相关核受体-代谢酶-转运体组成的信号通路的变化有关。与此同时,胆酸盐及其信号通路的改变与胆囊摘除后相关疾病、雌激素紊乱型肝内胆汁淤积和2型糖尿病的发生与转归关联。实验所得结论,为胆酸盐代谢紊乱与相关疾病之间关系的阐明提供了新的依据。
Other AbstractObjective        Bile acids (BAs), the important endogenous substances, have extensive biological activities and play an important role in various physiological and pathological processes in vivo. BAs as the pivotal signaling molecules, combined with the nuclear receptors, farnesoid X receptor (Fxr), pregnane X receptor (Pxr), constitutive androstane receptor (Car) and G protein-coupled bile acid receptor (Tgr5), regulate the gene expressions of the target enzymes, transporters and the others relevant functional proteins at transcriptional level, which altogether form the signaling pathways networks to control and maintain the substances metabolic balance and the physiological homeostasis. It has been reported that BAs play a key role in regulating the homeostasis of BAs, glucose and lipids in vivo, and BAs metabolic disorder can induce the physiologic derangement even diseases including intrahepatic cholestasis, diabetes especially the type 2 diabetes mellitus (T2DM), obesity, nonalcoholic fatty liver disease, atherosclerosis, etc.. In addition, the alterations of the concentrations and compositions of BAs in different pathophysiological conditions exhibit significantly distinctive characters, which can be employed to diagnose and classify the relevant diseases at an early stage. It is, however, that, the associations between BAs and the relevant diseases still have not been identified clearly, and more comprehensive and systematic studies are needed. As a result, in the present study, rats and mice were employed respectively as our experimental animals. We primarily elucidated the alterations of BAs and their mechanisms in the context of cholecystectomy which is the disease resulting in the physiologic derangement, and under the pathological conditions including estrogen-induced intrahepatic cholestasis and T2DM. Thereafter, we explored the relevance between BAs metabolic disorder and the occurrence and the development of the diseases mentioned above. The findings from this study are expected to provide valuable cues for identifying novel and effective therapeutic targets for amelioration or treatment of the relevant diseases.Methods    There are three parts of the experiments in the current study:   (1) The alterations and the circadian rhythms of BAs and their underlying mechanisms in mice with cholecystectomy     The cholecystectomy and sham operations were executed in Kunming male mice, respectively. After the surgery, the mice recovered for 2 weeks, and then the specimens including serum, liver, ileum, gallbladder, small intestine containing contents and feces were collected. The ultraviolet spectrophotometry was used to determine the total BAs (TBAs) concentrations in all of the specimens and the BAs pool size. The pathology of liver and ileum was investigated applying the histopathological method. The concentrations and compositions of individual BAs in serum, liver tissue, ileum tissue, ileum contents and feces were determined by HPLC/M/MS. The Real Time-quantification PCR (RT-qPCR) and Western Blotting were respectively used to determine the mRNA and protein expressions of the relevant nuclear receptors, enzymes and transporters. Furtermore, the circadian rhythms of BAs and the mRNA expressions of the functional genes were evaluated.   (2) Exploring the pathogenic, compensatory and decompensatory mechanisms of estrogen-induced intrahepatic cholestasis via BAs and the “intestine-liver axis” and the “liver-kidney axis” in rats     In this work, we employed Wistar male rats and HepaRG cells as our experimental objects. Firstly, the pathogenic, compensatory and decompensatory stages of intrahepatic cholestasis model were respectively induced by the subcutaneously administration with 17α-ethynylestradiol (EE) in rats for 7, 14 and 28 days. Secondary, after EE administration, the specimens of serum, liver, ileum, kidney, bile, feces and urine were collected and analyzed. The analyses included the levels of transaminases and bilirubin in serum, the TBAs concentrations in all of the specimens, BAs pool size, the liver pathology, the individual BAs concentrations and compositions in serum, bile, liver tissue and ileum tissue, and the mRNA expressions of the relevant nuclear receptors, enzymes and transporters in liver, ileum and kidney. Furthermore, the molecular docking and HepaRG cells studies were employed for verification.      (3) Exploring the mechanisms about BAs alterations and preliminarily investigating the effects of the altered BAs on glucose metabolic disorder in rats with T2DM         T2DM model was induced in Wistar male rats by high-fat diet combined with the low dose of streptozotocin (STZ). Briefly, rats were fed with a high-fat diet for 7 weeks to induce obesity, and then a low dose (30 mg/kg) of streptozotocin was intraperitoneally injected into rats, followed by another one-week high-fat diet to induce T2DM. Following the intervention scheme, the specimens of serum, liver, ileum, colon, pancreas, small intestine with the contents and feces were collected and evaluated. The evaluation includes the levels of transaminases, glucose, cholesterol and triglyceride in serum; the TBAs concentrations in serum, liver tissue, small intestine with the contents and feces, BAs pool size, the liver pathology, the individual BAs concentrations and compositions in serum and tissues specimens of liver, ileum, colon and pancreas, and the mRNA expressions of the relevant nuclear receptors, enzymes and transporters in liver, ileum, colon and pancreas.      Results     The following results were found after the circumstance of cholecystectomy in mice. The BAs pool size in vivo reduced significantly, which could be ascribed to the down-regulated protein expression of ileum apical sodium-dependent bile acid transporter (Asbt), and the down-regulated Asbt resulted in the BAs ileal malabsorption and an abundance of BAs losing via feces. The disappearance of constriction circadian rhythms of gallbladder attenuated the circadian rhythms of BAs ileum reabsorption and resulted in the circadian rhythmicity of BAs pool flatting. The circadian rhythmicity of the Cyp7a1 gene expression flattened, which triggered the disappearance of the hepatic biosynthesis rhythmicity of the BAs. The circadian rhythms of the mRNA expressions of Fxr and its downstream gene fibroblast growth factor (Fgf15) in ileum altered significantly which eventually determined the rhythmicity of hepatic Cyp7a1. In addition, the mRNA and protein expressions of hepatic Cyp3a11 down-regulated remarkably, and the circadian rhythms of the mRNA expressions of Cyp3a11, Oatp2 and Mrp2 in liver attenuated remarkably.Under the condition of EE-induced intrahepatic cholestasis in rats, the following results can be found. The results from the animal experiment, molecular docking study and HepaRG cells experiment all indicated that EE inhibited Fxr to down regulate the gene expression of Bsep in the liver, which resulted in biliary excretion of BAs decreasing profoundly to induced the intrahepatic cholestasis. At the compensatory stage, the distinct alterations of BAs concentrations and compositions in ileum and liver tissue contributed to the adaptive regulations of Fxr-mediated “intestine-liver axis”, which increased the hepatic biosynthesis of CDCAs via up-regulating mRNA expressions of Cyp7a1 and Cyp27a1, and down-regulating the mRNA expression of Cyp8b1 in liver, and the increased hepatic CDCAs level blocked the antagonistic effect of EE on Fxr-Bsep pathway, and then promoted BAs biliary excretion to alleviate the EE-induced cholestasis. The adaptive alterations of “liver-kidney axis”, including the activation of hepatic Pxr-sulfotransferase family 2a member 1 (Sult2a1)/multidrug resistance protein 3 (Mrp3) pathway, the up-regulation of gene expressions of renal efflux transporter Mrp4 and the down-regulation of the renal reabsorption transporters including Asbt and organic solute transporter β (Ostβ), were the potential mechanism of the greatly elevated BAs urinary excretion. At the decompensatory stage, the liver lesion and the necrosis of hepatocytes aggravated apparently, and the mRNA expressions of most of the functional genes in liver down-regulated significantly. Meanwhile, in liver tissue, the concentrations of CDCAs that has higher hepatotoxicity and the composition of β-MCAs that is the Fxr antagonist increased, which might result in the EE-induced cholestasis developing from the compensatory stage to the decompensatory stage. Based on the results from molecular docking study, the binding mode and the docked value between EE-Fxr and estradiol-Fxr were similar.   In rats with T2DM, the concentrations and compositions of the individual BAs in serum exhibited significantly different characteristics compared to the control, and the most apparent BAs characteristics in the serum of T2DM rats was the significantly increased ratio of 12α-hydroxylate (OH) BAs to non-12α-OH BAs. Moreover, the mRNA expression ratio of hepatic Cyp8b1 increased profoundly, which could be the key factor contributing to the significantly increased ratio of 12α-OH BAs, and the secondary BAs level in T2DM rats decreased remarkably. In colon, pancreas and liver tissue of rats with T2DM, the levels of BAs that are the agonist of Fxr and Tgr5 decreased remarkably. Furthermore, the mRNA expressions of some functional proteins that belong to the Fxr and Tgr5-mediated signaling pathways down-regulated significantly, which might have negative effects on the glucose metabolic disorder in rats with T2DM and promote the development of T2DM.Conclusion    Under the circumstance of cholecystectomy, estrogen-induced intrahepatic cholestasis and T2DM, the concentrations and compositions of BAs in vivo exhibited significant metabolic disorder, which could be ascribed to the alterations of the signaling pathways that are consist of the relevant nuclear reporters, enzymes and transporters. The alterations of the BAs and they-mediated signaling pathways in the context of these three pathophysiological conditions were further associated with the occurrence and the development of the cholecystectomy relevant diseases, estrogen-induced intrahepatic cholestasis and T2DM. The findings from the current study provided new evidence for the illumination of the relationships between BAs metabolic disorder and the relevant diseases.
Pages152
URL查看原文
Language中文
Document Type学位论文
Identifierhttp://ir.lzu.edu.cn/handle/262010/343812
Collection药学院
Affiliation药学院
First Author AffilicationSchool of Pharmacy
Recommended Citation
GB/T 7714
张帆. 三种病生理条件下大/小鼠体内胆酸盐的变化及机制研究[D]. 兰州. 兰州大学,2019.
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