HEPATOXIC AND PROTECTIVE ACTIONS OF BILE SALTS

胆汁盐的肝毒性和保护作用

• 批准号: 3754345
• 负责人: DOUGLAS HEUMAN
• 金额: --
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3754345
• 关键词: cell membrane; chemical structure function; cholestane compound; cholesterol; cytotoxicity; flow cytometry; high performance liquid chromatography; laboratory rat; liver cells; membrane lipids; membrane permeability; scintillation spectrometry; tissue /cell culture; ursodeoxycholate; vesicle /vacuole

Bile salts are ionic sterol detergents which can disrupt membranes and damage cells. Hepatocellular injury by bile salts may be import in pathogenesis of cholestatic liver disease. Ursodeoxycholate (UDC), a hydrophilic bile salt which is a relatively poor detergent, improves liver function in patients with chronic cholestasis. We have shown that conjugates of UDC prevent cholestasis caused by taurochenodeoxycholate or taurodeoxycholate in vivo in bile fistula rats and block lysis of isolated rat hepatocytes, human erythrocytes, or cholesterol/phospholipid vesicles by these bile salts in vitro. We now propose to elucidate the mechanism of this potentially important protective interaction. Questions to be addressed include: i) how is the susceptibility of membranes to disruption by bile salts influenced by bile salt structure, composition of the aqueous phase, and composition of the membrane? ii) does UDC prevent membrane disruption by preventing partition of more toxic bile salts into membranes? iii) do UDC and other bile salts at non-disruptive concentrations alter physical properties of membranes (fluorescence anisotropy gradient)? iv) does UDC block the effects of more hydrophobic bile salts on exchange of cholesterol and phospholipid between membranes/ and v) how specific is the hepatoprotective action of UDC? Studies will employ a variety of in vivo (rat) and in vitro experimental systems. Membrane disruption will be assessed by leakage of permeability markers from vesicles or cytosolic enzymes from cells. Bile salt/membrane partition will be quantified using four complementary methods (rapid ultrafiltration, dialysis, density gradient ultracentrifugation, gel chromatography). Fluidity gradients of viable cells will be determined using a new flow cytometry method recently developed at this institution. We will quantify effects of UDC on bile salt-mediated exchange of cholesterol and phospholipid between membrane vesicles. Finally, we will investigate in primate hepatocyte culture the protective effects (if any) of UDC against a wide variety of cytotoxins. The ultimate purposes of this work ar to expand our basic understanding of bile salt:membrane interactions, to elucidate the mechanism of bile salt toxicity, and to establish a scientific rationale for use of UDC in treatment of human liver diseases.

胆盐是离子型甾醇去污剂，可破坏膜， 破坏细胞。 胆盐引起的肝细胞损伤可能是肝细胞损伤的重要因素。 胆汁淤积性肝病的发病机制。 熊去氧胆酸盐（UDC），a 亲水性胆汁盐是一种相对较差的去污剂， 慢性胆汁淤积症患者的肝功能。 我们已经证明 UDC的缀合物预防由牛磺鹅脱氧胆酸盐引起的胆汁淤积， 牛磺脱氧胆酸在胆瘘大鼠体内的作用和阻断溶解 分离的大鼠肝细胞、人红细胞或胆固醇/磷脂 囊泡由这些胆汁盐在体外。 我们现在建议阐明这一潜在重要的机制， 保护性互动 要解决的问题包括：i）如何 膜对胆汁盐破坏的敏感性受 胆汁盐结构、水相的组成和 膜？ii）UDC是否通过防止 将毒性更强的胆汁盐分配到细胞膜中？iii）做UDC和其他 非破坏性浓度的胆汁盐改变了 膜（荧光各向异性梯度）？iv）UDC是否阻止 更疏水的胆汁盐对胆固醇交换的影响， 膜之间的磷脂/和v） UDC保肝作用？研究将采用多种体内 (rat)和体外实验系统。 膜破裂将是 通过渗透性标记物从囊泡或胞质中的渗漏进行评估 细胞中的酶。 将定量胆盐/膜分配 使用四种互补的方法（快速超滤，透析， 密度梯度超离心，凝胶色谱法）。 流动性 将使用新的流式细胞术测定活细胞的梯度 这是该机构最近开发的一种方法。 我们将量化影响 UDC对胆盐介导的胆固醇和磷脂交换的影响 在膜囊泡之间。 最后，我们将在灵长类动物中进行研究 肝细胞培养的保护作用（如果有的话）UDC对广泛的 多种细胞毒素。 这项工作的最终目的是扩大 我们对胆盐的基本理解：膜相互作用，以阐明 胆盐毒性的机制，并建立科学的 使用UDC治疗人类肝脏疾病的基本原理。