Objectives:

2

To explore the underlying pharmacological mechanisms and its potential effects of Chinese medicine herbal formula Sini Powder (SNP) on hepatocellular carcinoma (HCC).

Methods:

2

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 μmol/L) on proliferation and migration of HepG 2 or Huh7 cells. Meanwhile

the effect of isoprenaline or propranolol on the β2 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.

Results:

2

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 r

eceptor (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.

Conclusion:

2

SNP can combat HCC by alleviating stress responses through the regulation of gut microbiota.