司曲替尼对四氯化碳诱导的肝纤维化小鼠模型的影响及其作用机制

张欢; 吴翔宇; 赵倩雯; 芮法娟; 耿楠; 靳睿; 李婕

doi:10.12449/JCH260315

司曲替尼对四氯化碳诱导的肝纤维化小鼠模型的影响及其作用机制

DOI: 10.12449/JCH260315

张欢¹,
吴翔宇¹,
赵倩雯²,
芮法娟²,
耿楠²,
靳睿²,
李婕^{1, 2, , ,}

1.
南京中医药大学鼓楼临床医学院感染性疾病科，南京 210008
2.
南京大学医学院附属鼓楼医院感染性疾病科，南京 210008

基金项目:

北京肝胆相照公益基金会力肝专项研究项目 (iGandanF-1082024-LG004);

北京肝胆相照公益基金会力肝专项研究项目 (iGandanF-1082025-LG034);

北京肝胆相照公益基金会力肝专项研究项目 (iGandanF-1082025-LG019);

南京鼓楼医院临床研究专项资金项目 (2024-LCYJ-PY-52);

南京鼓楼医院临床研究专项资金项目 (2025-LCYJ-PY-02);

南京鼓楼医院国家自然科学基金青年培育项目 (2025-JCYJ-QP-016);

南京鼓楼医院国家自然科学基金青年培育项目 (2025-JCYJ-QP-017);

南京大学中医研究院课题项目和南京鼓楼医院医学发展医疗救助基金会资助项目 (ICM2024002);

南京大学中医研究院课题项目和南京鼓楼医院医学发展医疗救助基金会资助项目 (ICM2024032);

中国博士后科学基金 (2024M761417)

利益冲突声明：本文不存在任何利益冲突。

作者贡献声明：张欢负责设计论文框架，起草论文，实验操作以及研究过程的实施；赵倩雯负责数据整理，统计学分析；吴翔宇、芮法娟、耿楠和靳睿在论文修改与语言润色方面提供帮助；李婕负责拟定写作思路，指导撰写文章并最后定稿。

详细信息

通信作者:
李婕， lijier@nju.edu.cn （ORCID： 0000-0003-0973-8645）

计量
- 文章访问数: 18
- HTML全文浏览量: 6
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- 被引次数: 0
出版历程
- 收稿日期: 2025-09-15
- 录用日期: 2026-01-15
- 出版日期: 2026-03-25

Effect of sitravatinib on a mouse model of carbon tetrachloride-induced liver fibrosis and its mechanism

Huan ZHANG¹,
Xiangyu WU¹,
Qianwen ZHAO²,
Fajuan RUI²,
Nan GENG²,
Rui JIN²,
Jie LI^{1, 2
, ,
,}

1.
Department of Infectious Diseases，Nanjing Drum Tower Hospital Clinical College of Nanjing University of Chinese Medicine，Nanjing 210008，China
2.
Department of Infectious Diseases，Nanjing Drum Tower Hospital，Affiliated Hospital of Medical School，Nanjing University，Nanjing 210008，China

Research funding:

Beijing iGandan Foundation (iGandanF-1082024-LG004);

Beijing iGandan Foundation (iGandanF-1082025-LG034);

Beijing iGandan Foundation (iGandanF-1082025-LG019);

Nanjing Drum Tower Hospital Clinical Research Special Fund Project (2024-LCYJ-PY-52);

Nanjing Drum Tower Hospital Clinical Research Special Fund Project (2025-LCYJ-PY-02);

Nanjing Drum Tower Hospital Youth Cultivation Fund (2025-JCYJ-QP-016);

Nanjing Drum Tower Hospital Youth Cultivation Fund (2025-JCYJ-QP-017);

Project of Institute of Chinese Medicine, Nanjing University and Aid Project of Nanjing Drum Tower Hospital Health, Education & Research Foundation (ICM2024002);

Project of Institute of Chinese Medicine, Nanjing University and Aid Project of Nanjing Drum Tower Hospital Health, Education & Research Foundation (ICM2024032);

China Postdoctoral Science Foundation (2024M761417)

More Information

Corresponding author: LI Jie， lijier@nju.edu.cn （ORCID： 0000-0003-0973-8645）

摘要

摘要: 目的评估司曲替尼对四氯化碳（CCl₄）诱导的小鼠肝纤维化的治疗作用。方法将30只8周龄雄性C57BL/6J小鼠随机分为对照组、CCl₄模型组及司曲替尼低（5 mg/kg）、中（10 mg/kg）、高（20 mg/kg）剂量组。除对照组外，其余各组通过腹腔注射CCl₄诱导肝纤维化（持续4周）；自造模首日起，各司曲替尼剂量组每日灌胃相应浓度司曲替尼。检测各组小鼠血清总胆固醇（TC）、甘油三酯（TG）、丙氨酸氨基转移酶（ALT）水平；测定肝组织羟脯氨酸含量；通过苏木精-伊红、Masson及天狼星红染色观察肝组织病理改变；采用实时荧光定量聚合酶链反应和Western Blot分别检测肝组织中平滑肌肌动蛋白α与Ⅰ型胶原蛋白α1基因的mRNA及蛋白表达水平，以综合评估药物疗效。计量资料多组间比较采用单因素方差分析，进一步两两比较采用LSD‑t检验。结果与对照组相比，模型组TC、TG和ALT水平均显著升高（P值均<0.05）；与模型组相比，司曲替尼各剂量组TC、TG和ALT水平差异无统计学意义（P值均>0.05）。经司曲替尼干预后，肝组织羟脯氨酸水平下降，中、高剂量组与CCl₄模型组相比，差异均有统计学意义（P值均<0.05）。病理染色结果显示，司曲替尼治疗组胶原沉积减少，纤维间隔变薄、稀疏；司曲替尼高剂量组中，4只小鼠肝纤维化分期为S0～S1，2只为S2～S3，较CCl₄模型组肝纤维化程度有所缓解（S3～S4为主）。分子水平检测显示，司曲替尼可下调平滑肌肌动蛋白α与Ⅰ型胶原蛋白α1基因的mRNA及蛋白表达（P值均<0.05）。结论司曲替尼能有效缓解CCl₄诱导的小鼠肝纤维化，其作用机制可能与抑制肝星状细胞活化及胶原合成有关。
- 肝纤维化 /
- 司曲替尼 /
- 治疗学 /
- 小鼠，近交C57BL
Abstract: Objective To investigate the therapeutic effect of sitravatinib on carbon tetrachloride （CCl₄）-induced liver fibrosis in mice. Methods A total of 30 male C57BL/6J mice， aged 8 weeks， were randomly divided into control group， CCl₄ model group， and low- （5 mg/kg）， middle- （10 mg/kg）， and high-dose （20 mg/kg） sitravatinib groups. All mice except those in the control group were given intraperitoneal injection of CCl₄ for 4 consecutive weeks to induce liver fibrosis， and since the first day of modeling， the mice in the low-， middle-， and high-dose sitravatinib groups were given sitravatinib at the corresponding dose by gavage every day. The serum levels of total cholesterol （TC）， triglyceride （TG）， and alanine aminotransferase （ALT） were measured for the mice in each group； hepatic hydroxyproline content was measured； HE staining， Masson staining， and Sirius Red staining were used to observe liver histopathological changes； quantitative real-time PCR and Western blot were used to measure the mRNA and protein expression levels of α-smooth muscle actin （α-SMA） and collagen type I alpha 1 （Col1a1） in liver tissue. The therapeutic effect of sitravatinib was assessed based on the above results. A one-way analysis of variance was used for comparison of continuous data between multiple groups， and the least significant difference t-test was used for further comparison between two groups. Results Compared with the control group， the model group had significant increases in the levels of TC， TG， and ALT （all P<0.05）， and there were no significant differences in the levels of TC， TG， and ALT between the model group and the low-， middle-， and high-dose sitravatinib groups （all P>0.05）. Hepatic hydroxyproline content decreased after sitravatinib intervention， with a significant difference between the middle-/high-dose sitravatinib groups and the CCl₄ model group （both P<0.05）. Histopathological staining showed that the sitravatinib treatment groups had a reduction in collagen deposition， along with thinning and fragmentation of fibrous septa， and in the high-dose sitravatinib group， 4 mice had a fibrosis stage of S0—S1 and 2 mice had a fibrosis stage of S2—S3， suggesting a certain degree of alleviation of liver fibrosis degree compared with the CCl₄ model group （mainly S3—S4）. The measurement of related molecules showed that sitravatinib downregulated the mRNA and protein expression levels of α-SMA and Col1a1 （all P<0.05）. Conclusion Sitravatinib can effectively alleviate CCl₄-induced liver fibrosis in mice， possibly by inhibiting hepatic stellate cell activation and collagen synthesis.
- Hepatic Fibrosis /
- Sitravatinib /
- Therapeutics /
- Mice， Inbred C57BL

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注： HE，苏木精-伊红。

图 1 各组小鼠肝组织HE染色结果（×100）

Figure 1. HE staining of liver tissues in mice from each group of mice （×100）

下载: 全尺寸图片幻灯片

注： a，天狼星红染色（×100）；b，天狼星红染色相对表达量。

图 2 各组小鼠肝组织天狼星红染色结果

Figure 2. Sirius red staining of liver tissues from each group of mice

下载: 全尺寸图片幻灯片

注： a，Masson染色（×100）；b，Masson染色相对表达量。

图 3 各组小鼠肝组织Masson染色结果

Figure 3. Masson staining of liver tissues from each group of mice

下载: 全尺寸图片幻灯片

注： a，F4/80免疫组织化学染色（×200）；b，F4/80免疫组织化学染色相对表达量。CCl₄，四氯化碳。

图 4 各组小鼠肝组织F4/80免疫组织化学染色结果

Figure 4. Immunohistochemical staining of F4/80 in liver tissues of each group of mice

下载: 全尺寸图片幻灯片

注： a，ACTA2 mRNA相对表达量；b，COL1A1 mRNA相对表达量。ACTA2，平滑肌肌动蛋白α基因；COL1A1，Ⅰ型胶原蛋白α1基因。CCl₄，四氯化碳。

图 5 各组小鼠肝组织ACTA2、COL1A1 mRNA相对表达水平比较

Figure 5. Comparison of relative mRNA expression levels of ACTA2 and COL1A1 in liver tissues of each group of mice

下载: 全尺寸图片幻灯片

图 6 各组小鼠肝组织羟脯氨酸含量比较

Figure 6. Comparison of hydroxyproline detection in liver tissues of each group of mice

下载: 全尺寸图片幻灯片

注： a，Western Blot结果；b，α-SMA蛋白相对表达量；c，Col1a1蛋白相对表达量。α-SMA，平滑肌肌动蛋白α；Col1a1，Ⅰ型胶原蛋白α1。

图 7 各组小鼠肝组织α-SMA、Col1a1蛋白表达量

Figure 7. The protein expression levels of α-SMA and Col1a1 in the liver tissues of each group of mice

下载: 全尺寸图片幻灯片

表 1 PCR引物序列

Table 1. PCR primer sequences

基因	序列
18S RNA	上游引物：5'-CATTCGAACGTCTGCCCTATC-3'
18S RNA	下游引物：5'-CCTGCTGCCTTCCTTGGA-3'
ACTA2	上游引物：5'-CCCAGACATCAGGGAGTAATGG-3'
ACTA2	下游引物：5'-TCTATCGGATACTTCAGCGTCA-3'
COL1A1	上游引物：5'-CGATGGATTCCCGTTCGAGT-3'
COL1A1	下游引物：5'-AAGGGTGCTGTAGGTGAAGC-3'
注：PCR，聚合酶链反应；ACTA2，平滑肌肌动蛋白α基因；COL1A1，Ⅰ型胶原蛋白α1基因。

下载: 导出CSV

表 2 小鼠分组、造模及给药情况

Table 2. Grouping， modeling and drug administration of mice

分组	动物数（只）	给药方案
对照组	6	普通饲料喂养基础上予以玉米油腹腔注射，同时予以等量生理盐水灌胃，连续4周
CCl₄模型组	6	普通饲料喂养基础上予以CCl₄溶液腹腔注射，等量生理盐水灌胃，1次/1.5 d，连续4周
司曲替尼低剂量组	6	普通饲料喂养基础上予以CCl₄溶液腹腔注射，司曲替尼5 mg/kg灌胃，1次/1.5 d，连续4周
司曲替尼中剂量组	6	普通饲料喂养基础上予以CCl₄溶液腹腔注射，司曲替尼10 mg/kg灌胃，1次/1.5 d，连续4周
司曲替尼高剂量组	6	普通饲料喂养基础上予以CCl₄溶液腹腔注射，司曲替尼20 mg/kg灌胃，1次/1.5 d，连续4周
注：CCl₄，四氯化碳。腹腔注射体积分数为10%的CCl₄玉米油溶液（CCl₄与玉米油的比例为1∶9），注射剂量为5 mL/kg，1次/1.5 d，连续4周。

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表 3 各组小鼠血清TG、TC和ALT水平的比较

Table 3. Comparison of serum TG、TC and ALT levels of each group of mice

组别	动物数（只）	TG（mg/dL）	TC（mg/dL）	ALT（U/L）
对照组	6	21.68±7.69	162.10±26.76	63.89±12.50
CCl₄模型组	6	100.56±25.90^1）	243.84±44.73^1）	254.60±39.89^1）
司曲替尼低剂量组	6	99.50±16.00^1）	245.06±32.95^1）	245.74±74.11^1）
司曲替尼中剂量组	6	87.80±22.07^1）	228.33±43.47^1）	233.62±25.28^1）
司曲替尼高剂量组	6	84.23±23.71^1）	222.46±22.97^1）	224.37±33.62^1）
F值		15.76	5.58	4.83
P值		<0.001	0.002	0.005
注：与对照组比较，1）P<0.05。TG，甘油三酯；TC，总胆固醇；ALT，丙氨酸氨基转移酶；CCl₄，四氯化碳。

下载: 导出CSV

表 4 各组小鼠肝纤维化分期

Table 4. Liver fibrosis grades of mice in each group

组别	动物数（只）	肝纤维化分期（只）
组别	动物数（只）	S0	S1	S2	S3	S4
对照组	6	6	0	0	0	0
CCl₄模型组	6	0	0	0	3	3
司曲替尼低剂量组	6	0	1	2	2	1
司曲替尼中剂量组	6	0	2	2	1	1
司曲替尼高剂量组	6	1	3	1	1	0
注：CCl₄，四氯化碳。

下载: 导出CSV

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