Zhang Yi-Bing, Bao Yong-Rui, Wang Shuai, Li Tian-Jiao, Tai He, Leng Jia-Peng, Yang Xin-Xin, Wang Bo-Cai, Meng Xian-Sheng
College of Pharmacy, Liaoning University of Traditional Chinese Medicine, Dalian 116600, Liaoning Province, China.
Department of Clinical Trail Institution Office, Dalian Municipal Central Hospital, Dalian 116033, Liaoning Province, China.
World J Gastrointest Oncol. 2023 Mar 15;15(3):504-522. doi: 10.4251/wjgo.v15.i3.504.
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 and 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, and 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 /, 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.
肝细胞癌（HCC）是全球死亡率极高的常见消化系统癌症之一。木鸡方颗粒（MJF）的主要成分是生物碱、黄酮类化合物和多糖。MJF已用于肝炎、肝硬化和HCC的临床治疗30多年。以往很少有研究关注MJF治疗HCC的肿瘤免疫学机制。
探讨MJF治疗HCC的肿瘤免疫学作用机制。
采用高效液相色谱-电喷雾电离-飞行时间-质谱联用的分子网络技术鉴定MJF的可吸收成分，并通过网络药理学和通路富集分析筛选潜在的抗HCC关键靶点。40只雄性小鼠在口服给药7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）。计算平均体重增加量、脾脏和胸腺指数，用苏木精和伊红对肿瘤组织进行染色，采用酶联免疫吸附测定法检测干扰素γ（IFN-γ）、肿瘤坏死因子α（TNF-α）、白细胞介素-2、天冬氨酸转氨酶、丙氨酸转氨酶、甲胎蛋白（AFP）、Fas和FasL。通过实时定量PCR（RT-qPCR）评估相关mRNA表达，采用蛋白质印迹法检测转化生长因子β1（TGF-β1）和母亲对脱磷酸化蛋白同源物（SMAD）4的蛋白表达。用10 mg/mL、20 mg/mL、30 mg/mL、40 mg/mL的MJF处理HepG2细胞系，另外3组用TGF-β1抑制剂（LY364947）和不同剂量的MJF处理。通过RT-qPCR评估TNF-α、IFN-γ等相关mRNA表达，采用蛋白质印迹法检测TGF-β1、SMAD2、磷酸化SMAD2、SMAD4和SMAD7的蛋白表达。
结果显示，MJF可改善H22荷瘤小鼠的体重增加和肿瘤抑制率，保护免疫器官和肝功能，降低HCC指标AFP，影响免疫和凋亡，上调TGF-β1/SMAD信号通路，其机制为增加TGF-β1、SMAD2、磷酸化SMAD2和SMAD4的相对表达，降低SMAD7，减少免疫因子TNF-α和IFN-γ，降低凋亡细胞因子Fas、FasL和/，并抑制LY364947对HepG2细胞的作用。
MJF通过激活TGF-β1/SMAD信号通路并影响免疫和凋亡细胞因子来抑制HCC，这可能是由于MJF调节了免疫逃逸和凋亡。
