Jiawei Xiaoyao San exerts anti-liver cancer effects via exosomal miRNA pathway

doi:10.12307/2025.061

Abstract

Abstract: BACKGROUND: Previous studies by our research group discovered that Jiawei Xiaoyao San has a significant anti-liver cancer effect, but the specific mechanism of action was unclear.
OBJECTIVE: To investigate the regulatory effects of the traditional Chinese medicine formula Jiawei Xiaoyao San on the levels of miRNAs in plasma exosomes of rats with diethylnitrosamine chronically induced primary liver cancer, based on high-throughput sequencing combined with bioinformatics.
METHODS: SD rats were randomly divided into a blank control group, a liver cancer model group, and a Jiawei Xiaoyao San treatment group. Liver cancer models were induced by continuous administration of diethylnitrosamine for 12 weeks. Starting from the 17th week, rats in the Jiawei Xiaoyao San treatment group were administered Jiawei Xiaoyao San once daily until the end of the 20th week, while rats in the blank control and liver cancer model groups were given an equivalent volume of saline. Anti-hepatocellular carcinoma effects were validated by assessing the morphological structure of rat liver tissues, along with the expression of the hepatocellular carcinoma markers, Glypican-3 protein and serum alpha-fetoprotein. Plasma exosomes from each group of rats were isolated using ultracentrifugation. High-throughput sequencing technology was used to screen for differentially expressed miRNAs in rat plasma exosomes. Bioinformatics was used to predict the potential biomarkers through which Jiawei Xiaoyao San exerts its anti-liver cancer effects via liver cancer-derived exosomal miRNAs, followed by functional analysis.
RESULTS AND CONCLUSION: (1) Jiawei Xiaoyao San significantly improved the morphological structure of liver tissues in a rat model of liver cancer. Compared with the liver cancer model group, the expression of liver cancer markers Glypican-3 protein and serum alpha-fetoprotein was significantly reduced in the Jiawei Xiaoyao San treatment group. (2) Bioinformatics analysis showed that in the Jiawei Xiaoyao San group, upregulated miR-223-3p in the liver cancer model group had target binding sites with genes E2F1 and NCOA1, which were closely related to liver cancer survival and prognosis. Therefore, Jiawei Xiaoyao San has a therapeutic effect on liver cancer, possibly by targeting negative regulation of NCOA1/E2F1 through liver cancer plasma-derived exosomal miR-223-3p, thereby playing anti-liver cancer effect.

Key words: liver cancer, Jiawei Xiaoyao San, exosomes, miRNA, bioinformatics, miR-223-3p

CLC Number:

Liu Xiaoming, Cheng Jinlai, Li Rushuang, Li Niuniu, Qin Qiuyun, Xia Meng, Yao Chun. Jiawei Xiaoyao San exerts anti-liver cancer effects via exosomal miRNA pathway[J]. Chinese Journal of Tissue Engineering Research, 2025, 29(19): 4052-4062.

Figures/Tables 4

2.1 实验动物数量分析参与此次实验的SD大鼠共有31只，在20周，1只肝癌模型组大鼠因肝腹水死亡，23只大鼠造模成功，肝癌模型组11只、加味逍遥散组12只与空白对照组7只进入结果分析。 2.2 加味逍遥散对肝癌模型大鼠的抗肝癌作用苏木精-伊红染色显示，空白对照组大鼠的肝细胞结构完整，胞质内颗粒清晰可见，且肝小叶中央静脉周围的肝细胞呈放射状排列；肝癌模型组肝细胞成团块状结节，伴有炎性细胞浸润；加味逍遥散治疗后肝小叶损伤且炎性细胞浸润等情况改善，见图1A。免疫组化结果显示，与空白对照组相比，肝癌模型组呈现出大量黄棕色，Glypican-3的累积吸光度值显著上升，提示Glypican-3的表达明显升高，加味逍遥散组Glypican-3的累积吸光度值有所降低，见图1B，C。大鼠体质量监测结果显示，空白对照组大鼠体质量持续稳定增长，模型组、加味逍遥散组大鼠总体体质量增长较缓，在造模期第4周由于毒性反应以及失血等影响，导致体质量有所下降。但模型组和加味逍遥散组间体质量没有显著统计学差异，见图1D。血清甲胎蛋白检测结果显示，空白对照组大鼠血清甲胎蛋白水平最低，肝癌模型组大鼠血清甲胎蛋白水平显著升高，加味逍遥散组治疗后甲胎蛋白水平有所降低，见图1E。 2.3 血浆外泌体表征透射电子显微镜观察显示，分离出的物质为杯状或圆形囊泡，见图2A。纳米流式报告分析显示，外泌体颗粒大小的中心值均在60-70 nm，其中空白对照组为1.18×1011 particles/mL，加味逍遥散组为9.99×109 particles/mL，肝癌模型组为8.87×1010 particles/mL，见图2B。Western blot检测显示肝癌模型组CD63的表达水平增加，加味逍遥散组CD63的表达水平被抑制，见图2C。以上3种检测方法结果提示从空白对照组、肝癌模型组以及加味逍遥散组提取到的样本被鉴定为外泌体。"

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