肝细胞癌（HCC）是全球死亡率较高的常见消化系统癌症之一。木香芳颗粒剂（MJF）的主要成分为生物碱、黄酮类和多糖类。 MJF已经在临床治疗乙型肝炎、肝硬化和肝细胞癌方面使用了30多年。以往的研究很少关注MJF在治疗肝细胞癌的肿瘤免疫学机制。本研究旨在探讨MJF在肝细胞癌治疗中对肿瘤免疫学的作用机制。使用分子网络相关的高性能液相色谱-电子喷雾离子化-飞行时间-质谱法鉴定MJF的可吸收成分，并利用网络药理学和通路富集分析筛选出潜在的抗HCC靶点。将40只雄性小鼠在口服应用MJF7天后随机分为空白组、模型组和MJF组（1.8、5.4和10.8 g / kg / d）。计算平均体重增长量、脾脏和胸腺指数。用苏木精和伊红染色肿瘤组织，并用酶联免疫吸附试验测量干扰素γ（IFN-γ）、肿瘤坏死因子α（TNF-α）、白细胞介素-2、天冬氨酸氨基转移酶、丙氨酰转移酶、甲胎蛋白（AFP）、Fas和FasL等指标。利用实时荧光定量PCR评估相关Bax和Bcl2 mRNA表达，利用Western印迹评估转化生长因子β1（TGF-β1）和Decapentaplegic同源蛋白4（SMAD4）等蛋白的表达。将HepG2细胞系用10mg/mL、20mg/mL、30mg/mL、40mg/mL的MJF处理，另外3个组用TGF-β1抑制剂（LY364947）和不同剂量的MJF处理。利用实时荧光定量PCR评估相关的TNF-α、IFN-γ、Bax和Bcl2 mRNA表达，利用Western印迹评估TGF-β1、SMAD2、p-SMAD2、SMAD4和SMAD7等蛋白的表达。MJF可以提高H22肿瘤负荷小鼠的体重增长和肿瘤抑制率，保护免疫器官和肝功能，降低HCC指标AFP，影响免疫和细胞凋亡，通过增加TGF-β1、SMAD2、p-SMAD2和SMAD4的相对表达和降低SMAD7来上调TGF-β1 / SMAD信号通路，减少免疫因子TNF-α和IFN-γ，减少凋亡因子Fas、FasL和Bcl2 / Bax，并抑制HepG2细胞中LY364947的效应。MJF通过激活TGF-β1 / SMAD信号通路，影响免疫和凋亡细胞因子来抑制HCC，这可能是MJF调节免疫逃避和细胞凋亡。 ©作者(s) 2023。 百世登出版集团股份有限公司。版权所有。

Hepatocellular carcinoma (HCC) is one of the most common digestive system cancers with high mortality rates worldwide. The main ingredients in Mu Ji Fang Granules (MJF) are alkaloids, flavonoids, and polysaccharides. MJF has been used in the clinical treatment of hepatitis, cirrhosis and HCC for more than 30 years. Few previous studies have focused on the mechanism of MJF on tumor immu-nology in the treatment of HCC.To explore the mechanism of action of MJF on tumor immunology in the treatment of HCC.The absorbable ingredients of MJF were identified using Molecule Network related to High Performance Liquid Chromatography-Electron Spray Ionization-Time of Flight- Mass Spectrometry, and hub potential anti-HCC targets were screened using network pharmacology and pathway enrichment analysis. Forty male mice were randomly divided into the Blank, Model, and MJF groups (1.8, 5.4, and 10.8 g/kg/d) following 7 d of oral administration. Average body weight gain, spleen and thymus indices were calculated, tumor tissues were stained with hematoxylin and eosin, and Interferon gamma (IFN-γ), Tumor necrosis factor α (TNF-α), Interleukin-2, aspartate aminotransferase, alanine aminotransferase, alpha-fetoprotein (AFP), Fas, and FasL were measured by Enzyme-linked Immunosorbent Assay. Relevant mRNA expression of Bax and Bcl2 was evaluated by Real Time Quantitative PCR (RT-qPCR) and protein expression of Transforming growth factor β1 (TGF-β1) and Mothers against decapentaplegic homolog (SMAD) 4 was assessed by Western blotting. The HepG2 cell line was treated with 10 mg/mL, 20 mg/mL, 30 mg/mL, 40 mg/mL of MJF, and another 3 groups were treated with TGF-β1 inhibitor (LY364947) and different doses of MJF. Relevant mRNA expression of TNF-α, IFN-γ, Bax and Bcl2 was evaluated by RT-qPCR and protein expression of TGF-β1, SMAD2, p-SMAD2, SMAD4, and SMAD7 was assessed by Western blotting.It was shown that MJF improved body weight gain and tumor inhibition rate in H22 tumor-bearing mice, protected immune organs and liver function, reduced the HCC indicator AFP, affected immunity and apoptosis, and up-regulated the TGF-β1/SMAD signaling pathway, by increasing the relative expression of TGF-β1, SMAD2, p-SMAD2 and SMAD4 and decreasing SMAD7, reducing immune factors TNF-α and IFN-γ, decreasing apoptosis cytokines Fas, FasL and Bcl2/Bax, and inhibiting the effect of LY364947 in HepG2 cells.MJF inhibits HCC by activating the TGF-β1/SMAD signaling pathway, and affecting immune and apoptotic cytokines, which may be due to MJF adjusting immune escape and apoptosis.©The Author(s) 2023. Published by Baishideng Publishing Group Inc. All rights reserved.