Mei Si, Deng Zhe, Meng Fan-Ying, Guo Qian-Qian, Tao He-Yun, Zhang Lin, Xi Chang, Zhou Qing, Tian Xue-Fei
Faculty of Medicine, Hunan University of Chinese Medicine, Changsha, 410208, China.
College of Integrated Chinese and Western Medicine, Hunan University of Chinese Medicine, Changsha, 410208, China.
Chin J Integr Med. 2025 May 8. doi: 10.1007/s11655-025-4127-z.
To explore the underlying pharmacological mechanisms and its potential effects of Chinese medicine herbal formula Sini Powder (SNP) on hepatocellular carcinoma (HCC).
The active components of SNP and their in vivo distribution were identified using ultraperformance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry. Construction of component-target-disease networks, protein-protein interaction network, Gene Ontology function and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis, and molecular docking were employed to analyze the active components and anti-HCC mechanisms of SNP. Cell viability assay and wound healing assay were utilized to confirm the effect of SNP-containing serum (2.5%, 5.0%, 10%, 20%, and 40%), isoprenaline or propranolol (both 10, 100, and 1,000 µ mol/L) on proliferation and migration of HepG 2 or Huh7 cells. Meanwhile, the effect of isoprenaline or propranolol on the β 2 adrenergic receptor (ADRB2) mRNA expression on HepG2 cells were measured by real-time quantitative reverse transcription (RT-qPCR). Mice with subcutaneous tumors were either subjected to chronic restraint stress (CRS) followed by SNP administration (364 mg/mL) or directly treated with SNP (364 mg/mL). These two parallel experiments were performed to validate the effects of SNP on stress responses. Stress-related proteins and hormones were quantified using RT-qPCR, enzyme-linked immunosorbent assay, and immunohistochemistry. Metagenomic sequencing was performed to confirm the influence of SNP on the gut microbiota in the tumor-bearing CRS mice.
The distribution of the 12 active components of SNP was confirmed in various tissues and feces. Network pharmacology analysis confirmed the anti-HCC effects of the 5 active components. The potential anti-HCC mechanisms of SNP may involve the epidermal growth factor receptor (EGFR), proto-oncogene tyrosine-protein kinase Src (SRC) and signal transducer and activator of transcription 3 (STAT3) pathways. SNP-containing serum inhibited the proliferation of HepG2 and Huh7 cells at concentrations of 2.5% and 5.0%, respectively, after 24 h of treatment. Furthermore, SNP suppressed tumor progression in tumor-bearing mice exposed to CRS. SNP treatment also downregulated the expressions of stress-related proteins and pro-inflammatory cytokines, primarily by modulating the gut microbiota. Specifically, the abundance of Alistipes and Prevotella, which belong to the phylum Bacteroidetes, increased in the SNP-treated group, whereas Lachnospira, in the phylum Firmicutes, decreased.
SNP can combat HCC by alleviating stress responses through the regulation of gut microbiota.
探讨中药方剂四逆散（SNP）对肝细胞癌（HCC）的潜在药理机制及其潜在作用。
采用超高效液相色谱-四极杆飞行时间质谱联用技术鉴定SNP的活性成分及其体内分布。构建成分-靶点-疾病网络、蛋白质-蛋白质相互作用网络、基因本体功能和京都基因与基因组百科全书通路富集分析以及分子对接，以分析SNP的活性成分和抗肝癌机制。利用细胞活力测定和伤口愈合测定来确认含SNP血清（2.5%、5.0%、10%、20%和40%）、异丙肾上腺素或普萘洛尔（均为10、100和1000 μmol/L）对HepG 2或Huh7细胞增殖和迁移的影响。同时，通过实时定量逆转录（RT-qPCR）检测异丙肾上腺素或普萘洛尔对HepG2细胞β2肾上腺素能受体（ADRB2）mRNA表达的影响。对皮下肿瘤小鼠进行慢性束缚应激（CRS）后给予SNP（364 mg/mL）或直接用SNP（364 mg/mL）治疗。进行这两个平行实验以验证SNP对应激反应的影响。使用RT-qPCR、酶联免疫吸附测定和免疫组织化学对应激相关蛋白和激素进行定量。进行宏基因组测序以确认SNP对荷瘤CRS小鼠肠道微生物群的影响。
确认了SNP的12种活性成分在各种组织和粪便中的分布。网络药理学分析证实了5种活性成分的抗肝癌作用。SNP潜在的抗肝癌机制可能涉及表皮生长因子受体（EGFR）、原癌基因酪氨酸蛋白激酶Src（SRC）和信号转导及转录激活因子3（STAT3）通路。处理24小时后，含SNP血清分别在浓度为2.5%和5.0%时抑制HepG2和Huh7细胞的增殖。此外，SNP抑制了暴露于CRS的荷瘤小鼠的肿瘤进展。SNP治疗还下调了应激相关蛋白和促炎细胞因子的表达，主要是通过调节肠道微生物群。具体而言，在SNP治疗组中，属于拟杆菌门的Alistipes和普雷沃氏菌属的丰度增加，而属于厚壁菌门的毛螺菌属减少。
SNP可通过调节肠道微生物群减轻应激反应来对抗HCC。
