维生素A缺乏联合CCl4诱导制备稳定的小鼠慢性肝纤维化模型

doi:10.12122/j.issn.1673-4254.2025.07.20

摘要/Abstract

摘要：

目的采用维生素A（VA）缺乏饲料饲喂联合低浓度CCl₄诱导，制备一种稳定的慢性肝纤维化动物模型。方法 126只Balb/c小鼠随机分3组，分别给予VA正常饲料（VAN组）、500 IU/kg VA（VAD组1）及200 IU/kg VA（VAD组2）饲料饲养，4周后每组选取6只小鼠检测血清视黄醇水平；将剩余108只小鼠平均分为未造模组与造模组，每组再分为VAN组、VAD组1、VAD组2，18只/组。造模组腹腔注射5% CCl₄ 10 mL/kg，2次/周，共8周。检测血清视黄醇、ALT/AST、肝脏指数；进行肝组织HE、Masson染色、纤维化程度评分和氧化应激水平检测。结果饲养4周后，VAD组的血清视黄醇水平均低于VAN组，VAD组2更低（P<0.05）。CCl₄造模8周，肝脏指数及ALT/AST增高，肝脏表面有颗粒状突起，肝索结构紊乱、假小叶形成，可见大量蓝染纤维组织和纤维间隔；VAD组较VAN组更显著，但VAD组2肝脏坏死严重，假小叶纤维间隔处大量炎性细胞浸润，2只小鼠死亡。CCl₄停止给药，VAN造模组肝脏指数及ALT/AST基本恢复，肝索结构恢复正常，纤维化自发逆转；VAD造模组血清ALT/AST、肝脏大体及病理无明显变化，VAD组1仍可见肝索结构紊乱和假小叶，VAD组2腹腔黏连严重，仍可见假小叶及大量炎性细胞浸润，纤维化分期评分为3；CCl₄造模组（VAD组1）氧化损伤指标8-OHdG高于CCl₄造模组（VAN组）。结论以含有500 U/kg VA的饲料饲喂小鼠4周，10 mL/kg CCl₄干预8周，可获得中重度的肝纤维化模型，成功率100%，停药8周无自发逆转，可建立一种稳定的肝纤维化动物模型。

关键词: 维生素A缺乏, 四氯化碳, 慢性肝纤维化, 动物模型

Abstract:

Objective To prepare a stable mouse model of chronic liver fibrosis induced by dietary vitamin A (VA) deficiency combined with CCl₄ injections. Methods A total of 126 Balb/c mice were randomized into 3 groups for feeding with a normal VA diet or a VA-deficient diet containing 500 or 200 IU/kg VA. After 4 weeks of feeding, half of the mice in each group were given intraperitoneal injections of 5% CCl₄(10 mL/kg, twice a week) for 8 weeks. Serum retinol, ALT/AST and liver index of the mice were examined, liver tissue pathologies were observed with HE and Masson staining, and liver fibrosis score and oxidative stress level were evaluated. Results Four weeks of VA-deficient feeding, especially at 200 IU/kg, significantly lowered serum retinol level of the mice. CCl₄ injections for 8 weeks obviously increased liver index and ALT/AST and caused obvious liver fibrosis in all the mice, but liver pathologies were more severe in the 2 VA-deficient groups; severe liver necrosis with inflammatory cell infiltration was observed in 200 IU/kg VA group, where 2 mice died. After discontinuation of CCl₄, the mice with normal dietary VA showed gradual recovery of the liver index, ALT/AST, liver cord structure and liver fibrosis; the mice with VA deficiency, however, showed no significant improvements in these parameters, and the mice with 200 IU/kg VA still had serious abdominal adhesion, false lobules and massive inflammatory cell infiltration with a fibrosis stage score of 3. The oxidative damage index 8-OHdG was significantly higher in 500 IU/kg VA group than in normal VA group after CCl₄ modeling. Conclusion Feeding with diet containing 500 IU/kg VA for 4 weeks and 10 mL/kg CCl₄ injections for 8 weeks can result in stable moderate to severe liver fibrosis in mice without spontaneous reversal at 8 weeks of drug withdrawal.

Key words: vitamin A deficiency, CCl₄, chronic liver fibrosis, animal models

阳亭亭, 赵丽. 维生素A缺乏联合CCl₄诱导制备稳定的小鼠慢性肝纤维化模型[J]. 南方医科大学学报, 2025, 45(7): 1527-1534.

Tingting YANG, Li ZHAO. A stable mouse model of chronic liver fibrosis induced by vitamin A deficiency and intraperitoneal CCl₄ injection[J]. Journal of Southern Medical University, 2025, 45(7): 1527-1534.

图/表 7

表1 CCl4造模8周后对小鼠血清视黄醇、ALT/AST水平及肝纤维化程度的影响

Tab.1 Effects of CCl4 treatment for 8 weeks on serum levels of retinol， ALT/AST and liver fibrosis in mice (Mean±SD)

Index	Unmodeling group			CCl₄model group
Index	VAN group (n=6)	VAD group 1 (n=6)	VAD group 2 (n=6)	VAN group (n=6)	VAD group 1 (n=6)	VAD group 2 (n=4)
Serum retinol (μmol/L)	1.313±0.09	0.76±0.111^b	0.592±0.042^bc	1.278±0.073	0.695±0.095^b	0.514±0.034^abc
Liver index (%)	4.25±0.658	4.07±0.175	4.41±0.969	6.05±0.802^a	6.37±0.195^a	6.19±0.337^a
ALT (U/L)	42.03±8.576	45.22±9.212	53.17±6.61^bc	70.73±8.663^a	75.73±9.886^ab	86.38±10.34^abc
AST (U/L)	76.13±7.81	113.33±11.72^b	111.01±23.73^bc	136.20±13.12^a	158.72±24.34^ab	182.74±19.49^abc
Degree of fibrosis	0	0	0	2	3	3

图1 CCl4造模8周后各组肝脏组织大体标本、HE染色及Masson染色

Fig.1 Gross observation of mouse livers and HE staining (Original magnification: ×400) and Masson staining (×100) of the liver tissues in each group after 8 weeks of CCl4 modeling. The arrows from "Unmodeled group" to "CCl4 model group" respectively point to: The cytoplasm is lightly stained and presents a vacuolar phenomenon,The phenomenon of inflammatory cell infiltration and the formation of blue stained fibrous tissue.

表2 CCl4停药4周各组血清视黄醇、ALT/AST水平及肝纤维化程度

Tab.2 Effects of CCl4 on serum levels of retinol, ALT/AST and liver fibrosis in mice 4 weeks after CCl4 discontinuation (Mean±SD)

Index	Unmodeling group			CCl₄ model group
Index	VAN group (n=6)	VAD group 1 (n=6)	VAD group 2 (n=6)	VAN group (n=6)	VAD group 1 (n=6)	VAD group 2 (n=4)
Serum retinol (μmol/L)	1.325±0.073	0.733±0.122^b	0.587±0.053^bc	1.29±0.073	0.707±0.083^b	0.534±0.068^abc
Liver index (%)	4.10±0.748	3.98±0.226	4.28±0.859	6.32±1.228^a	6.08±0.585^a	5.85±0.947^a
ALT (U/L)	44.32±4.36	47.88±11.10	49.33±7.32	57.7±12.95^a	70.92±12.73^ab	90.33±20.40^abc
AST (U/L)	78.27±411.24	110.43±12.34^b	129.95±17.85^bc	101.43±17.53^a	147.92±22.29^ab	173.15±31.25^abc
Degree of fibrosis	0	0	0	2	3	3

图2 CCl4停药4周后各组肝脏组织大体标本、HE染色及Masson染色

Fig.2 Gross observation of mouse livers and HE staining (×400) and Masson staining (×100) of the liver tissues in each group 4 weeks after CCl4 discontinuation. The arrows from "Unmodeled group" to "CCl4 model group" respectively point to: The cytoplasm is lightly stained and presents a vacuolar phenomenon,The phenomenon of inflammatory cell infiltration and formation of pseudolobules.

表3 CCl4停药8周各组血清视黄醇、ALT/AST水平及肝纤维化程度

Tab.3 Serum levels of retinol, ALT/AST and liver fibrosis in mice 8 weeks after CCl4 discontinuation (Mean±SD)

Index	Unmodeling group			CCl₄ model group
Index	VAN group (n=6)	VAD group 1 (n=6)	VAD group 2 (n=6)	VAN group (n=6)	VAD group 1 (n=6)	VAD group2 (n=3)
Serum retinol (μmol/L)	1.37±0.08	0.78±0.13^b	0.60±0.10^bc	1.31±0.09	0.73±0.09^bc	0.55±0.10^abc
Liver index (%)	4.06±0.472	4.20±0.0.85	4.34±0.78	5.68±1.01^a	6.02±0.445^a	5.87±1.63^a
ALT (U/L)	45.1±8.95	48.67±7.86	49.52±10.58	49.33±0.09	72.78±0.09^ab	86.57±13.91^abc
AST (U/L)	76.40±12.19	114.85±9.84^b	123.0±25.21^bc	84.47±12.32	141.45±16.06^ab	197.4±67.92^abc
Degree of fibrosis	0	0	0	1	3	3

图3 CCl4停药8周后各组肝脏组织大体标本、HE染色及Masson染色

Fig.3 Gross observation of mouse livers and HE staining (×400) and Masson staining (×100) of the liver tissues in each group 8 weeks after CCl4 discontinuation. The arrows from "Unmodeled group" to "CCl4 model group" respectively point to: The cytoplasm is lightly stained and presents a vacuolar phenomenon,Disorder of hepatic cord structure and formation of pseudolobules.

图4 优选模型组肝脏氧化损伤的评估

Fig.4 Validation of oxidative damage of the liver in optimal model group.

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维生素A缺乏联合CCl₄诱导制备稳定的小鼠慢性肝纤维化模型

A stable mouse model of chronic liver fibrosis induced by vitamin A deficiency and intraperitoneal CCl₄ injection

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