Shuyu pills inhibit immune escape and enhance chemosensitization in hepatocellular carcinoma

Figure 1 Finger-print of Shuyu pills. The finger-print of Shuyu pills was determined by high resolution mass spectrometry. A: The 20 pharmaceutical ingredients were labeled according to the chromatographic retention time and their structures were analyzed by mass spectrometry; B: Chemical structure formulae of 20 compounds. Chemical structures and formulae of 20 compounds from PubChem (https://pubchem.ncbi.nlm.nih.gov/), numbered according to the compounds information in Table 1.

Figure 2 Shuyu pills inhibited the growth of hepatocellular carcinoma in vivo. The xenograft mouse model was established in BALB/c nude mice that were then randomly divided into four groups (n = 6): Control group (0.9% normal saline, daily), Shuyu pills (SYP) (200 mg/kg, daily), Cisplatin (DDP) (5 mg/kg, once a week), and SYP (200 mg/kg, daily) + DDP (5 mg/kg, once a week). The body weight of each mouse and the tumor volume were measured every 2 d, with the latter calculated as follows: Maximum tumor length × width (2 × 0.5). A: Representative images of the tumors at the end of treatment; B: Average tumor volumes, measured every 2 d; C: Tumor weights at the end of treatment; D: Average body weights of the mice, measured every 2 d. ^bP < 0.01 vs Control group; ^dP < 0.01 vs SYP group; ^fP < 0.01 vs SYP + DDP group. SYP: Shuyu pills; DDP: Cisplatin.

Figure 3 Shuyu pills inhibited the expression of hypoxia-inducible factor-1 alpha, programmed cell death 1, and programmed cell death 1 ligand 1. Nude mice injected subcutaneously with human hepatocellular carcinoma cells were treated with Shuyu pills (SYP), Cisplatin (DDP), or a combination of the two for 14 d, following which the tumor tissues were harvested as indicated. A–C: Western blot and quantitative reverse transcription polymerase chain reaction assays were used to respectively detect the protein and mRNA expression levels of hypoxia-inducible factor-1 alpha (HIF-1α), programmed cell death protein 1 (PD-1), and programmed cell death 1 ligand 1 (PD-L1) in the tumor tissue; D: Representative protein expression patterns of HIF-1α, PD-1, and PD-L1 as measured by western blot assay. Data are presented as the mean ± standard error of the mean, and comparisons between two groups were performed using the least significant difference test or Dunnett’s T3 method. ^aP < 0.05 and ^bP < 0.01 vs Control group; ^cP < 0.05 and ^dP < 0.01 vs SYP group; ^eP < 0.05 and ^fP < 0.01 vs SYP + DDP group. SYP: Shuyu pills; DDP: Cisplatin; HIF-1α: Hypoxia-inducible factor-1 alpha; PD-1: Programmed cell death 1; PD-L1: Programmed cell death 1 ligand 1.

Figure 4 Effects of Shuyu pills on the expression of CD4+ T cells and CD8+ T cells in subcutaneous hepatocellular carcinoma xenografts. Nude mice injected subcutaneously with human hepatocellular carcinoma cells were treated with Shuyu pills (SYP), Cisplatin (DDP), or SYP + DDP for 14 d. The tumor tissues were then collected and their expression of CD4+ T cells and CD8+ T cells was measured using the immunofluorescence assay. A: Immunofluorescence images of CD4+ T cells and CD8+ T cells; B: Quantitative analysis of the CD4+ T-cell and CD8+ T-cell immunofluorescence intensities; C and D: Protein expression levels of CD4+ T cells and CD8+ T cells as measured by western blot assay. Data are presented as the mean ± standard error of the mean, and comparisons between two groups were performed using the least significant difference test or Dunnett’s T3 method. ^bP < 0.01 vs Control group; ^cP < 0.05 and ^dP < 0.01 vs SYP group; ^fP < 0.01 vs SYP + DDP group. SYP: Shuyu pills; DDP: Cisplatin.