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胸腺素β4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/JCH260314
利益冲突声明：本文不存在任何利益冲突。
作者贡献声明：朱耘函负责设计论文框架，撰写文章；王思琪负责实验操作，研究过程的实施；景登娅负责数据收集，统计学分析，绘制图表；冯勤颖负责论文修改，拟定写作思路，指导撰写文章并最后定稿。
Effect of thymosin β4 on a mouse model of carbon tetrachloride-induced hepatic fibrosis and its mechanism
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摘要:
目的 本研究拟探讨胸腺素β4（Tβ4）通过调控血小板衍生生长因子（PDGF）表达及诱导肝星状细胞（HSC）凋亡，对四氯化碳（CCl4）诱导的肝纤维化小鼠模型的干预作用与潜在机制，为临床抗肝纤维化治疗提供新的实验依据。 方法 选取30只雄性C57小鼠，随机分为正常对照组、模型组，以及Tβ4低（3 mg/kg）、中（6 mg/kg）和高（12 mg/kg）剂量治疗组，每组6只。正常对照组自由摄食普通饲料，其余各组腹腔注射50% CCl4与橄榄油混合液建立肝纤维化模型，经超声及病理学验证造模成功后，对各治疗组小鼠连续皮下注射Tβ4干预4周。实验结束后取肝组织行苏木精-伊红及Masson染色观察组织病理变化，应用实时荧光定量聚合酶链反应检测血PDGF mRNA表达，原位末端转移酶标记（TUNEL）法检测HSC凋亡情况。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 与模型组比较，Tβ4中、高剂量治疗组小鼠肝纤维化程度均呈现不同程度的减轻。实时荧光定量聚合酶链反应结果显示，Tβ4可显著下调肝组织中PDGF mRNA表达，各剂量治疗组间差异有统计学意义（P<0.05）；Tβ4高剂量组与正常对照组PDGF mRNA表达差异无统计学意义（P<0.05）。TUNEL检测结果显示，Tβ4中、高剂量组HSC凋亡数量显著多于模型组。 结论 Tβ4可能通过抑制PDGF表达和促进HSC凋亡，从而改善CCl4诱导的小鼠肝纤维化，提示其在肝纤维化治疗中具有潜在应用价值。 -
关键词:
- 肝纤维化 /
- 胸腺素β4 /
- 小鼠， 近交C57BL
Abstract:Objective To investigate the therapeutic effect and potential mechanism of thymosin β4 （Tβ4） on carbon tetrachloride （CCl4）-induced hepatic fibrosis by regulating the expression of platelet-derived growth factor （PDGF） and inducing the apoptosis of hepatic stellate cell （HSC）， and to provide new experimental evidence for anti-hepatic fibrosis treatment in clinical practice. Methods A total of 30 male C57 mice were randomly divided into normal control group， model group， low-dose Tβ4 treatment group （3 mg/kg）， middle-dose Tβ4 treatment group （6 mg/kg）， and high-dose Tβ4 treatment group （12 mg/kg）， with 6 mice in each group. The mice in the normal control group were fed with a normal diet ad libitum， and those in the other groups were given intraperitoneal injection of 50% CCl4 mixed with olive oil to establish a model of hepatic fibrosis. After successful modeling confirmed by ultrasound and histopathology， the mice in each treatment group were given subcutaneous injection of Tβ4 for 4 consecutive weeks. Liver tissue was collected at the end of the experiment， and HE staining and Masson staining were used to observe histopathological changes； quantitative real-time PCR was used to measure the mRNA expression level of PDGF； TUNEL assay was used to assess the apoptosis of HSC. 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 model group， the middle- and high-dose Tβ4 treatment groups had varying degrees of alleviation of hepatic fibrosis. Quantitative real-time PCR showed that Tβ4 could significantly downregulate the mRNA expression level of PDGF in liver tissue， with a significant difference between the treatment groups （P>0.05）， and there was no significant difference in the mRNA expression level of PDGF between the high-dose Tβ4 treatment group and the normal control group （P>0.05）. TUNEL assay showed that the middle- and high-dose Tβ4 treatment groups had a significantly higher number of apoptotic HSCs than the model group. Conclusion Tβ4 may improve CCl4-induced hepatic fibrosis in mice by downregulating the expression of PDGF and promoting the apoptosis of HSC， suggesting that it has a potential application value in the treatment of hepatic fibrosis. -
Key words:
- Hepatic Fibrosis /
- Thymosin Beta 4 /
- Mice， Inbred C57BL
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注： a，正常对照组；b，模型组。
图 1 小鼠肝脏二维超声与弹性成像变化情况
Figure 1. Representative ultrasound and elastography images of mouse livers
注： a，正常对照组；b，模型组；c，Tβ4低剂量组；d，Tβ4中剂量组；e，Tβ4高剂量组。HE，苏木精-伊红。
图 2 各组小鼠肝组织HE染色结果（×40）
Figure 2. Representative HE stained liver sections from each group （×40）
注： a，正常对照组；b，模型组；c，Tβ4低剂量组；d，Tβ4中剂量组；e，Tβ4高剂量组。蓝色所示为胶原纤维。
图 3 各组小鼠肝组织Masson染色结果（×100）
Figure 3. Masson’s trichrome staining of liver tissues from each group （×100）
注：*P<0.05。PDGF，血小板衍生生长因子。
图 4 各组小鼠肝组织PDGF mRNA相对表达量
Figure 4. Relative mRNA expression of PDGF in liver tissues from each group
注： a，正常对照组；b，模型组；c，Tβ4低剂量组；d，Tβ4中剂量组；e，Tβ4高剂量组。红色荧光信号指示凋亡肝星状细胞。FITC，异硫氰酸荧光素。
图 5 各组小鼠肝组织荧光凋亡结果（FITC，×200）
Figure 5. Fluorescence assay for apoptosis detection in mouse liver tissues （FITC， ×200）
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