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益脾养肝方对大鼠肝癌前病变肝干细胞恶性转化的影响和作用机制
DOI: 10.3969/j.issn.1001-5256.2022.04.023
伦理学声明：本研究方案于2019年3月13日经由陕西中医药大学伦理委员会审批，批号：SUCMDL20211115001，符合实验室动物管理与使用准则。
利益冲突声明：本研究不存在研究者、伦理委员会成员、受试者监护人以及与公开研究成果有关的利益冲突。
作者贡献声明：鞠迪负责课题设计，资料分析，撰写论文；李汨、韩曼负责收集数据，修改论文；房冰莹负责整理数据，统计分析；李京涛、闫曙光负责拟定写作思路，指导撰写文章并最后定稿。
Effect of Yipi Yanggan prescription on malignant transformation of liver stem cells in rats with liver precancerous lesion and its mechanism of action
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摘要:
目的 探讨益脾养肝方对二乙基亚硝胺(DEN)诱导的肝癌前病变中肝干细胞恶性转化的影响及可能的分子机制。 方法 将35只雄性SD大鼠随机分为正常对照组(空白组)、DEN模型组(模型组)、DEN+益脾养肝方组(益脾养肝方组)和DEN+护肝片组(护肝片组)，空白组5只，其余3组各10只。腹腔注射DEN诱导肝癌前病变模型，给药16周后处死。检测血清ALT、AST、Alb水平；取肝组织观察并记录其大小、外观等变化，计算肝重比(肝脏指数)；HE染色、天狼星红染色观察大鼠肝组织病理形态学改变；免疫组化检测OV6和谷胱甘肽S转移酶(GST-Pi)的表达；实时定量PCR检测EpCAM、CD133和CD90 mRNA的表达；Western Blot检测PI3K、Akt、mTOR蛋白及其磷酸化水平的表达。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 与模型组相比，益脾养肝方组和护肝片组肝脏病理形态显著改善，肝脏指数、ALT和AST水平降低、Alb水平升高(P值均＜0.05)；同时GST-Pi、OV6、p-PI3K、p-Akt、p-mTOR蛋白和EpCAM、CD133、CD90 mRNA表达均明显降低(P值均＜0.05)。与护肝片组相比，益脾养肝方组对肝脏的保护作用更显著，其肝脏指数、ALT和AST水平显著降低、Alb水平显著升高(P值均＜0.05)；且GST-Pi、OV6、p-PI3K、p-Akt、p-mTOR蛋白和EpCAM、CD133、CD90 mRNA表达水平均显著降低(P值均＜0.05)。 结论 益脾养肝方可通过抑制肝干细胞恶性转化改善DEN诱导的大鼠肝癌前病变，其作用机制可能与PI3K/Akt/mTOR信号通路有关。 Abstract:Objective To investigate the effect of Yipi Yanggan prescription on the malignant transformation of liver stem cells in liver precancerous lesion induced by diethylnitrosamine (DEN) and its possible molecular mechanism. Methods A total of 35 male Sprague-Dawley rats were randomly divided into normal control group (blank group), DEN model group (model group), DEN+Yipi Yanggan prescription group (Yipi Yanggan prescription group), and DEN+Hugan tablet group (Hugan tablet group), with 5 rats in the blank group and 10 rats in the other three groups. Intraperitoneal injection of DEN was performed to establish a model of liver precancerous lesion, the rats were sacrificed after 16 weeks of administration. The serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and albumin (Alb) were measured; liver tissue was collected to observe the changes in size and appearance and calculate liver weight ratio (liver index); HE staining and Sirius Red staining were used to observe the pathological and morphological changes of rat liver tissue; immunohistochemistry was used to measure the expression of OV6 and glutathione S-transferase-Pi (GST-Pi); RT-PCR was used to measure the mRNA expression of EpCAM, CD133, and CD90, and Western blot was used to measure the protein expression of PI3K, Akt, and mTOR and their phosphorylation level. A one-way analysis of variance was used for comparison 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 Yipi Yanggan prescription group and the Hugan tablet group had significant improvements in liver pathology and morphology, significant reductions in liver index and the levels of ALT and AST, and a significant increase in the level of Alb (all P < 0.05), as well as significant reductions in the protein expression levels of GST-Pi, OV6, p-PI3K, p-Akt, and p-mTOR and the mRNA expression levels of EpCAM, CD133, and CD90 (all P < 0.05). Compared with the Hugan tablet group, the Yipi Yanggan prescription group showed a more significant protective effect on the liver, with significant reductions in liver index and the levels of ALT and AST, and a significant increase in the level of Alb (all P < 0.05), as well as significant reductions in the protein expression levels of GST-Pi, OV6, p-PI3K, p-Akt, and p-mTOR and the mRNA expression levels of EpCAM, CD133, and CD90 (all P < 0.05). Conclusion Yipi Yanggan prescription can improve liver precancerous lesion induced by DEN in rats by inhibiting the malignant transformation of liver stem cells, and its mechanism of action may be associated with the PI3K/Akt/mTOR signaling pathway. -
Key words:
- Liver Neoplasms /
- Yipi Yanggan Recipe /
- Pathologic Processes /
- Precancerous Conditions
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图 1 肝组织HE染色和天狼星红染色(×100)
Figure 1. Images of liver sections stained by Hematoxylin-Eosin (HE) and Sirius Red staining(×100)
图 2 GST-Pi及OV6在DEN诱导肝癌前病变组织中的表达
注：GST-Pi免疫组化染色(×200)，OV6免疫组化染色(×400)。
Figure 2. Immunohistochemistry staining of GST-Pi and OV6 in DEN-induced liver precancerous lesion
图 3 PI3K/Akt/mTOR在DEN诱导肝癌前病变组织中的表达
Figure 3. Expression of PI3K/Akt/mTOR signaling pathway- related protein in DEN-induced liver precancerous lesion
表 1 引物序列表
Table 1. Primer sequences used in RT-PCR
基因 上游 下游 CD90 ATCCAGCATGAGTTCAGCCT ATCCTTGGTGGTGAAGTTGG CD133 AACCACAACGGAAGTCAGCT TGACATCCCCAGTTTGGTTT EpCAM CGCAGCTCAGAAAGACTGTG CTCGGGATCATACAGACCGT β-actin CAACCTTCTTGCAGCTCCTC TTCTGACCCATACCCACCAT 
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表 2 各组大鼠肝组织中胶原沉积半定量分析
Table 2. Semi-quantitative analysis of collagen deposition in liver tissues of rats in various groups
组别 动物数(只) 胶原沉积光密度值(107) 空白组 5 1.49±0.45 模型组 9 12.54±5.361) 护肝片组 9 4.59±1.042) 益脾养肝方组 10 2.32±0.512)3) F值 20.29 P值 ＜0.05 注：与空白组相比，1)P＜0.05；与模型组相比，2)P＜0.05；与护肝片组相比，3)P＜0.05。
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表 3 各组大鼠血清ALT、AST、Alb和肝脏指数比较
Table 3. Comparison of ALT, AST, Alb and liver index in various groups
组别 动物数(只) ALT(U/L) AST(U/L) Alb(g/L) 肝脏指数(%) 空白组 5 26.67±4.60 82.48±14.54 36.68±2.43 2.78±0.18 模型组 9 46.67±6.521) 228.89±16.881) 20.46±2.791) 5.79±0.621) 护肝片组 9 38.15±1.362) 196.91±17.972) 24.98±1.312) 4.65±0.392) 益脾养肝方组 10 29.97±3.832)3) 157.17±21.522)3) 27.81±1.472)3) 3.44±0.722)3) F值 16.28 49.15 38.90 22.32 P值 ＜0.05 ＜0.05 ＜0.05 ＜0.05 注：与空白组相比，1)P＜0.01；与模型组相比，2)P＜0.05；与护肝片组相比，3)P＜0.05。
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表 4 各组大鼠肝组织中OV6及GST-Pi含量半定量分析
Table 4. Semi-quantitative analysis of OV6 and GST-Pi in liver tissues of rats in various groups
组别 动物数(只) OV6光密度值(×107) GST-Pi光密度值(×108) 空白组 5 0.57±0.19 1.27±0.78 模型组 9 10.71±3.811) 6.71±1.321) 护肝片组 9 5.17±1.412) 4.11±1.272) 益脾养肝方组 10 2.21±0.922)3) 1.79±0.882)3) F值 38.21 33.81 P值 ＜0.05 ＜0.05 注：与空白组相比，1)P＜0.05；与模型组相比，2)P＜0.05；与护肝片组相比，3)P＜0.05。
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表 5 各组大鼠肝组织中EpCAM、CD133和CD90 mRNA相对表达量比较
Table 5. Comparison of mRNA expression levels of EpCAM, CD133 and CD90 in various groups
组别 动物数(只) EpCAM CD133 CD90 空白组 5 1.24±0.32 1.18±0.15 1.07±0.26 模型组 9 18.59±2.731) 20.66±3.841) 21.00±3.511) 护肝片组 9 10.37±2.362) 9.17±1.902) 12.04±1.612) 益脾养肝方组 10 6.13±1.602)3) 5.26±1.302)3) 9.11±2.142)3) F值 129.40 110.20 70.61 P值 ＜0.05 ＜0.05 ＜0.05 注：与空白组相比，1)P＜0.05；与模型组相比，2)P＜0.05；与护肝片组相比，3)P＜0.05。
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表 6 各组大鼠肝组织中PI3K/Akt/mTOR信号通路相关蛋白半定量分析
Table 6. Semi-quantitative analysis of PI3K/Akt/mTOR signaling pathway-related protein in liver tissues of rats in various groups
组别 动物数(只) p-mTOR mTOR p-PI3K PI3K p-Akt Akt 空白组 5 1 1 1 1 1 1 模型组 9 4.9±0.411) 1.08±0.16 10.05±0.981) 1.06±0.22 5.72±1.461) 1.31±0.07 护肝片组 10 2.64±0.282) 1.07±0.19 5.74±0.712) 1.12±0.32 2.54±0.212) 1.34±0.16 益脾养肝方组 9 2.08±0.142)3) 0.89±0.23 3.40±0.752)3) 0.93±0.32 1.86±0.192)3) 1.02±0.31 F值 40.35 0.27 58.61 0.22 15.38 1.19 P值 ＜0.05 ＞0.05 ＜0.05 ＞0.05 ＜0.05 ＞0.05 注：与空白组相比，1)P＜0.05；与模型组相比，2)P＜0.05；与护肝片组相比，3)P＜0.05。
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