Apical sodium-dependent bile acid transporter inhibition with volixibat improves metabolic aspects and components of non-alcoholic steatohepatitis in Ldlr-/-.Leiden mice. Apical sodium-dependent bile acid transporter inhibition with volixibat improves metabolic aspects and components of non-alcoholic steatohepatitis in Ldlr-/-.Leiden mice

Abstract: Interruption of bile acid recirculation through inhibition of the apical sodium-dependent bile acid transporter (ASBT) is a promising strategy to alleviate hepatic cholesterol accumulation in non-alcoholic steatohepatitis, and improve the metabolic aspects of the disease. Putative disease-attenuating effects of the ASBT inhibitor volixibat (5, 15, and 30 mg/kg) were investigated in high-fat diet (HFD)-fed Ldlr-/-.Leiden mice over 24 weeks. Plasma and fecal bile acid levels, plasma insulin, lipids, and liver enzymes were monitored. Final analyses included liver histology, intrahepatic lipids, mesenteric white adipose tissue mass, and liver gene profiling. Consistent with its mechanism of action, volixibat significantly increased total bile acid excretion. At the highest dose, volixibat significantly attenuated the HFD-induced increase in hepatocyte hypertrophy, hepatic triglyceride and cholesteryl ester levels, and mesenteric white adipose tissue deposition, while total plasma bile acid levels remained constant. Non-alcoholic fatty liver disease activity score was significantly lower in volixibat-treated mice than in the HFD controls. Gene profiling showed that volixibat reversed the inhibitory effect of the HFD on metabolic master regulators, including peroxisome proliferator-activated receptor-γ coactivator-1β, insulin receptor, and sterol regulatory element-binding transcription factor 2. Volixibat may have beneficial effects on physiological and metabolic aspects of non-alcoholic steatohepatitis pathophysiology. Overall design: Experimental design: Mice (N = 75, between 16-18 weeks old) were matched by body weight and plasma cholesterol, triglyceride, and blood glucose levels were divided into five study groups, and maintained on either a chow diet (n = 10), a HFD (n = 20), or a HFD supplemented with volixibat 5, 15, or 30 mg/kg (n = 15 in each dose group) for 24 weeks (Fig 2). Food intake and body weight were monitored throughout the study to ensure accurate volixibat dosing. The schedule of assessments over the 24 weeks is shown in Fig 2, and included measurements of plasma cholesterol triglycerides, insulin, and liver enzymes, and determination of the lipoprotein profile. Bile acid species were measured in plasma and feces. Plasma samples (collected at weeks 0, 4, 8, 12, 16, 20, and 24) were obtained via a tail bleed after 5 hours of fasting (Fig 2). The mice were sacrificed at week 24 and the livers were isolated for histological evaluation of NASH parameters (as described above) [40] and lipid analysis. White adipose tissue depots were also collected and weighed. Gene expression analysis was carried out on liver tissue from eight mice in each group, selected as being representative of the group based on mean values of NASH parameters. All analyses were performed blindly.

摘要：通过抑制顶端钠依赖性胆汁酸转运蛋白（apical sodium-dependent bile acid transporter, ASBT）阻断胆汁酸再循环，是缓解非酒精性脂肪性肝炎（non-alcoholic steatohepatitis, NASH）肝内胆固醇蓄积、改善该病代谢表型的极具前景的策略。本研究针对ASBT抑制剂沃利昔班（volixibat，剂量分别为5、15、30 mg/kg）在高脂饮食（high-fat diet, HFD）喂养的Ldlr基因敲除Leiden小鼠（Ldlr-/-.Leiden mice）中的潜在疾病缓解效应开展了为期24周的探究。实验过程中监测了血浆与粪便胆汁酸水平、血浆胰岛素、脂质及肝酶水平。最终分析指标涵盖肝脏组织学检测、肝内脂质测定、肠系膜白色脂肪组织重量检测以及肝脏基因表达谱分析。

与沃利昔班的作用机制相符，其可显著提升总胆汁酸排泄量。在最高给药剂量下，沃利昔班可显著缓解高脂饮食诱导的肝细胞肥大、肝内甘油三酯及胆固醇酯水平升高，以及肠系膜白色脂肪组织沉积，同时血浆总胆汁酸水平保持稳定。沃利昔班给药组小鼠的非酒精性脂肪性肝病活动度评分（non-alcoholic fatty liver disease activity score, NAS）显著低于高脂饮食对照组。基因表达谱分析显示，沃利昔班可逆转高脂饮食对代谢核心调控因子的抑制作用，这些调控因子包括过氧化物酶体增殖物激活受体γ辅激活因子1β（peroxisome proliferator-activated receptor-γ coactivator-1β, PGC-1β）、胰岛素受体（insulin receptor, IR）以及固醇调节元件结合转录因子2（sterol regulatory element-binding transcription factor 2, SREBP-2）。沃利昔班或可对非酒精性脂肪性肝炎病理生理过程的生理与代谢异常发挥改善作用。

整体实验设计：
实验设计：选取75只16~18周龄的小鼠，按体重、血浆胆固醇、甘油三酯及血糖水平进行匹配分组，共分为5组，分别给予普通饲料（chow diet）喂养（n=10）、高脂饮食喂养（n=20），或高脂饮食联合5、15、30 mg/kg沃利昔班给药（每个剂量组n=15），持续干预24周（见图2）。整个实验过程中监测小鼠的进食量与体重，以确保沃利昔班给药剂量的准确性。24周实验期间的检测计划见图2，检测指标包括血浆胆固醇、甘油三酯、胰岛素及肝酶水平，以及脂蛋白谱分析。同时检测血浆与粪便中的胆汁酸组分。分别在第0、4、8、12、16、20及24周，对小鼠进行5小时空腹后尾静脉采血以获取血浆样本（见图2）。于第24周处死小鼠，分离肝脏以开展NASH相关参数的组织学评估（详见前文[40]）及脂质含量测定。同时收集白色脂肪组织并称重。针对每组中8只小鼠的肝脏组织开展基因表达分析，这些小鼠是根据NASH参数的平均值选取的组内代表性样本。所有分析均采用盲法进行。