The WD reflects a modern, but unhealthy human diet. The moderately high dietary cholesterol coupled with the ablation of the LDL receptor leads to hypercholesterolemia (dyslipidemia) and vascular inflammation, i.e., atherosclerosis. Moreover, the high saturated & trans-fat, plus the high sucrose contribute to the obese phenotype. Feeding male mice the WD results in a reduction in hepatic C18-22 ω3 and ω6 PUFA, a phenomenon seen in human NASH. Particularly relevant is the finding that male mice fed the WD results in a NASH phenotype that recapitulates many of the phenotypic features seen in human NASH patients, including obesity, dyslipidemia, hyperglycemia, hepatic damage, hepatosteatosis, and the induction of multiple markers of inflammation, oxidative stress and fibrosis.

西方饮食(WD)诱导的NASH小鼠模型构建

C57BL/6 male mice of 8 weeks were either fed a standard diet (STD) or a high-fat, cholesterol-rich diet (western diet; WD) containing 21% fat, 50% carbohydrate and 1.5% cholesterol for 22 weeks.

WD-induced NASH mouse model. (A) Increased bodyweight of WD group. (B) Increased fasting blood glucose of WD group.(C) Imparied glucose tolerance ability induced by western diet. (D) Area under curve of C.  (E-F）liver weight and ratio of liver weight and body weight. (G）Increased serum ALT levels of WD group. (H) Increased blood cholesterol of WD group. Data are means ±SEM; n=8-9 mice/group. *P <0.05, **P <0.01, ***P <0.001.

Analysis of H&E staining in WD-induced NASH mouse models. (A) Representative pictures of HE staining for liver steatosis . (B) The NAFLD activity score (NAS) was assessed by an external expert pathologist.

NASH小鼠模型用于评价治疗糖尿病的抗胰高血糖素受体（GCGR）抗体药物Crotedumab的药效

GCGR抗体Crotedumab减轻了WD诱导的NASH小鼠模型的代谢紊乱。(A)NASH模型建立及用药方案示意图。8周龄C57BL/6雄性小鼠喂食标准饲料(STD)或含21%脂肪、50%碳水化合物和1.5%胆固醇的高脂、富含胆固醇饲料（西方饮食；WD）22周。在第13周分析血糖水平和进行葡萄糖耐量试验。在第14周采集血液并分析ALT和血胆固醇，采血后给予Crotedumab(内部合成)治疗；(B)体重；(C)禁食血糖；(D-E)糖耐量实验及曲线下面积相对值；(F)肝脏重量；(G)肝重与体重的比值；(H-I)血清ALT、总胆固醇浓度；(J-K)肝脏组织切片HE染色及NAS评分。结果显示：应用药物后，禁食血糖和糖耐量试验血糖显著性降低；肝脏重量及ALT轻微降低；血清总胆固醇浓度降低；肝组织脂肪变性、气球样变有所缓解。以上结果说明应用胰高血糖素受体（GCGR）抗体能明显减轻西方饮食诱导的非酒精性脂肪性肝炎症状。数据为平均值±SEM，n = 10。

参考文献

1. Hernandez-Perez, E., Leon Garcia, P.E., Lopez-Diazguerrero, N.E., Rivera-Cabrera, F. & Del Angel Benitez, E. Liver steatosis and nonalcoholic steatohepatitis: from pathogenesis to therapy. Medwave 16, e6535 (2016).

2. Tsuchida, T., et al. A simple diet- and chemical-induced murine NASH model with rapid progression of steatohepatitis, fibrosis and liver cancer. J Hepatol 69, 385-395 (2018).

3. Farrell, G., et al. Mouse Models of Nonalcoholic Steatohepatitis: Toward Optimization of Their Relevance to Human Nonalcoholic Steatohepatitis. Hepatology 69, 2241-2257 (2019).

非酒精性脂肪性肝病(Nonalcoholic fatty liver disease, NAFLD)是一种脂肪在肝脏中过度累积的疾病。这种脂肪堆积不是由大量饮酒引起的。非酒精性脂肪性肝炎(Non-alcoholic steatohepatitis, NASH)是NAFLD的一种，除肝脏脂肪堆积外，还有肝炎、肝细胞损伤、纤维化及肝脏瘢痕形成的病理变化，进而可能导致肝硬化或肝癌。NASH进展的机制仍不清楚，目前还缺乏有效的治疗方法。

NASH的临床症状相当复杂，根据疾病过程包括肥胖、胰岛素抵抗、脂肪性肝炎、肝细胞气球样变和纤维化。临床前实验中经常使用的NASH模型有遗传动物模型、饮食诱导的动物模型及遗传与饮食诱导相结合构建的动物模型。然而，很难在短时间内模拟人类疾病的所有发病特征。

为了解NASH发生、发展的致病机制并开发创新疗法，我们开发了几种NASH不同发病阶段的小鼠模型。

• 西方饮食（WD）：饮食成分中高脂、高果糖和高胆固醇，该饮食诱导的NASH模型发展为肥胖、糖耐量受损和肝脏脂肪变性。
• 高脂肪蛋氨酸胆碱缺乏饮食（HFMCD）：饮食成分中含有60 kcal%脂肪，蛋氨酸、胆碱缺乏，该饮食诱导的NASH模型显示出肝损伤、肝脂肪变性和纤维化增加，伴随NAS评分增加。
• 四氯化碳(CCl4)注射：引起明显的肝脏炎症和纤维化。虽然很难获得完美的模型，但根据目标特点选择目前可用的合适模型是一个很好的选择。

饮食和化学诱导的NASH小鼠模型比较

高脂肪蛋氨酸胆碱缺乏饮食(HFMCD)诱导的NASH小鼠模型

HFMCD饮食是NASH的经典饮食模型。尽管饲料中含60 kcal%脂肪，但缺乏蛋氨酸和胆碱。蛋氨酸和胆碱都能促进脂肪以磷脂的形式由肝脏通过血液输送出去，提高脂肪酸在肝中的利用，防止脂肪在肝脏的异常堆积。

C57BL/6 male mice of 8 weeks were either fed a standard diet (STD) or a High-Fat Methionine-Choline-Deficient for 4 or 6 weeks.  

高脂肪蛋氨酸胆碱缺乏饮食（HFMCD）诱导的NASH模型。将野生型C57BL/6J小鼠随机分为4组，分别给予标准饲料（STD）和高脂肪蛋氨酸胆碱缺乏（HFMCD）饲料并喂养4周及6周后检测各项指标。(A)小鼠体重；(B)肝脏重量；(C)肝脏重量与体重的比值；(D-E)血清ALT、AST浓度；(F-G)肝组织切片HE染色及NAS评分；(H-I)肝组织切片天狼星红染色及肝纤维化程度统计。结果显示：HFMCD诱导组与STD组相比，肝脏重量明显增加；血清ALT、AST浓度显著升高；肝组织切片HE染色显示广泛的肝细胞脂肪变性、气球样变和小叶内炎症；肝组织天狼星红染色显示明显的肝纤维化。以上结果说明HFMCD诱导能成功建立NASH小鼠模型。数据为平均值±SEM，n = 5。

Analysis of H&E staining, index of liver function and NAFLD activity score the HFMCD-induced NASH mouse model. (A) Representative pictures of H&E staining showing reduced hepatic steatosis and inflammation after celastrol treatment. (B) ALT/AST change after MCD induction. Statistic data of NAFLD activity score (NAS). Values are expressed as mean ± SEM. **p<0.01, ***p<0.001.

参考文献

1. Hernandez-Perez, E., Leon Garcia, P.E., Lopez-Diazguerrero, N.E., Rivera-Cabrera, F. & Del Angel Benitez, E. Liver steatosis and nonalcoholic steatohepatitis: from pathogenesis to therapy. Medwave 16, e6535 (2016).

2. Tsuchida, T., et al. A simple diet- and chemical-induced murine NASH model with rapid progression of steatohepatitis, fibrosis and liver cancer. J Hepatol 69, 385-395 (2018).

3. Farrell, G., et al. Mouse Models of Nonalcoholic Steatohepatitis: Toward Optimization of Their Relevance to Human Nonalcoholic Steatohepatitis. Hepatology 69, 2241-2257 (2019).

STAM-NASH模型构建

实验动物：C57BL/6小鼠，0-24h，雄性

造模试剂： High Fat Diet、 Streptozocin

STAM (NASH-HCC) 模型小鼠构建与特征分析。(A) 新生雄性小鼠于第2天注射STZ, 4周龄开始喂食HFD饲料。(B-D) 诱导后体重和血糖变化。(E) 诱导后第6、8、12周HE染色代表性图片。(F) 血液生化结果显示STAM模型特征。(G) NAFLD活动评分。

一、C57BL/6老龄鼠的NASH模型

模型诱导

模型验证

GAN饮食诱导的老龄鼠NASH模型表现出明显的代谢紊乱

GAN饮食诱导的老龄鼠NASH模型表现出代谢紊乱。(A-B)GAN饮食诱导组的体重增加。(C)GAN饮食诱导组的葡萄糖耐受能力受损。(D)C图曲线下面积。(E)GAN饮食诱导组的血浆胰岛素含量增加。N=6-10 mice per group. Data are expressed as mean ± SEM. **: p<0.01.

GAN饮食诱导12周后，老龄鼠表现出比年轻小鼠更严重的NASH表型

GAN饮食诱导的老龄鼠NASH模型。(A)诱导12周后H&E染色的代表性图片。(B)NAS（NAFLD acticity score）评分。

Data are expressed as mean ± SEM.N=6-10 mice per group. **: p<0.01.

GAN饮食诱导21周后，老龄鼠表现出比年轻小鼠更严重的纤维化

GAN饮食诱导的老龄鼠纤维化。(A)诱导21周后天狼星红染色的代表性图片。(B)天狼星红染色的纤维化评分。

Data are expressed as mean ± SEM. N=6-10 mice per group. **: p<0.01.

GAN饮食诱导21周后，老龄雄鼠肝脏中的免疫细胞浸润增加

GAN饮食诱导21周后，老龄雄鼠肝脏中的免疫细胞浸润增加。(A)流式细胞仪评估肝脏中单核细胞（CD11b^intF4 / 80^low）和kupffer细胞（CD11b⁺F4 / 80^hi）浸润比例。(B)肝脏中不同类型免疫细胞分析。

Data are expressed as mean ± SEM. N=6-10 mice per group. *P <0.05, **P <0.01, ***P <0.001.

模型诱导

模型验证

GAN饮食诱导的C57BL/6小鼠NASH模型。(A)治疗下的体重变化。(B)治疗后的葡萄糖耐受能力。(C)B图曲线下的面积。(D)诱导20周后H&E染色的代表性图片。(E)NAS（NAFLD acticity score）评分。

Data are expressed as mean ± SEM. N=9 mice per group. *p<0.05, **p<0.01,***p<0.001.

模型构建

模型验证

GAN饮食诱导的B-ob/0b小鼠NASH模型。(A)治疗下的体重变化。(B)治疗后的葡萄糖耐受能力。(C)B图曲线下的面积。

Data are expressed as mean ± SEM. N=9 mice per group. *p<0.05, **p<0.01,***p<0.001.

GAN饮食诱导B-ob/ob小鼠的NASH及纤维化。(A)诱导16周后H&E染色的代表性图片。(B)NAS（NAFLD acticity score）评分。(C)诱导16周后天狼星红染色的代表性图片。(D)天狼星红染色的纤维化评分。

Data are expressed as mean ± SEM. N=9 mice per group.

参考文献

1. Hansen, H.H., et al.,  Human translatability of the GAN diet-induced obese mouse model of non-alcoholic steatohepatitis. BMC Gastroenterol. 2020 Jul 6;20(1):210.

2 .Hansen, H.H., et al., Mouse models of nonalcoholic steatohepatitis in preclinical drug development. Drug Discov Today, 2017. 22(11): p. 1707-1718.

3. Ibrahim, S.H., et al., Animal Models of Nonalcoholic Steatohepatitis: Eat, Delete, and Inflame. Dig Dis Sci, 2016. 61(5): p. 1325-36.