Shuyu pills inhibit immune escape and enhance chemosensitization in hepatocellular carcinoma.

Background: Hepatocellular carcinoma (HCC) is characterized by dysregulation of the immune microenvironment and the development of chemoresistance. Specifically, expression of the programmed cell death protein 1 (PD-1)/programmed cell death 1 ligand 1 (PD-L1) axis, an immune checkpoint, may lead to tumour immune escape, resulting in disease progression. The latest research shows that tumour immune escape may be caused by the upregulation of PD-L1 mediated by hypoxia-inducible factor-1 alpha (HIF-1α), and simultaneous inhibition of HIF-1α and PD-L1 has the potential to enhance the host's antitumour immunity. Moreover, inhibition of the PD-1/PD-L1 axis may mitigate tumour chemoresistance. Shuyu pills (SYPs) contain immunity-enhancing and antitumour components, making them a potential HCC treatment.

Aim: To investigate the efficacy of SYPs for HCC treatment simultaneous HIF-1α and PD-L1 inhibition and the mechanism involved.

Methods: A subcutaneous xenograft tumour model was first established in BALB/c nude mice by the subcutaneous injection of 1 × 10 SMMC-7721 cells. Male mice (male, 5 weeks old; = 24) were then randomly divided into the following four groups ( = 6): Control (0.9% normal saline), SYP (200 mg/kg), SYP + cisplatin (DDP) (200 mg/kg + 5 mg/kg DDP weekly intraperitoneal injection), and DDP (5 mg/kg cisplatin weekly intraperitoneal injection). The dose of saline or SYPs for the indicated mouse groups was 0.2 mL/d intragastric administration. The tumour volumes and body weights of the mice were measured every 2 d. The mice were euthanized by cervical dislocation after 14 d of continuous treatment, and the xenograft tissues were excised and weighed. Western blot assays were used to measure the protein expression of HIF-1α, PD-1, PD-L1, CD4+ T cells, and CD8+ T cells in HCC tumours from mice. Quantitative reverse transcription polymerase chain reaction was used for real-time quantitative detection of PD-1, PD-L1, and HIF-1α mRNA expression. An immunofluorescence assay was conducted to examine the expression of CD4+ T cells and CD8+ T cells.

Results: Compared to mice in the control group, those in the SYP and SYP + DDP groups exhibited reduced tumour volumes and tumour weights. Moreover, the protein and mRNA expression levels of the oncogene HIF-1α and that of the negative immunomodulatory factors PD-1 and PD-L1 were decreased in both the SYP and SYP + DDP groups, with the decrease effects being more prominent in the SYP + DDP group than in the SYP group (HIF-1α protein: Control SYP, = 0.0129; control SYP + DDP, = 0.0004; control DDP, = 0.0152, SYP + DDP DDP, = 0.0448; HIF-1α mRNA: control SYP, = 0.0009; control SYP + DDP, < 0.0001; control DDP, = 0.0003, SYP SYP + DDP, = 0.0192. PD-1 protein: Control SYP, = 0.0099; control SYP + DDP, < 0.0001, SPY SYP + DDP, = 0.0009; SYP + DDP DDP, < 0.0001; PD-1 mRNA: control SYP, = 0.0002; control SYP + DDP, < 0.0001; control DDP, = 0.0003, SPY SYP + DDP, = 0.0003; SYP + DDP DDP, = 0.0002. PD-L1 protein: control SYP, < 0.0001; control SYP + DDP, < 0.0001; control DDP, < 0.0001, SPY SYP + DDP, = 0.0040; SYP + DDP DDP, = 0.0010; PD-L1 mRNA: Control SYP, < 0.0001; control SYP + DDP, < 0.0001; control DDP, < 0.0001, SPY SYP + DDP, < 0.0001; SYP + DDP DDP, = 0.0014). Additionally, the quantitative and protein expression levels of CD4+ T cells and CD8+ T cells were simultaneously upregulated in the SYP + DDP group, whereas only the expression of CD4+ T cells was upregulated in the SYP group. (CD4+ T cell quantitative: Control SYP + DDP, < 0.0001, SYP SYP + DDP, = 0.0005; SYP + DDP DDP, = 0.0002. CD4+ T cell protein: Control SYP, = 0.0033; Control SYP + DDP, < 0.0001; Control DDP, = 0.0021, SYP SYP + DDP, = 0.0004; SYP + DDP DDP, = 0.0006. Quantitative CD8+ T cells: Control SYP + DDP, = 0.0013; SYP SYP + DDP, = 0.0347; SYP + DDP DDP, = 0.0043. CD8+ T cell protein: Control SYP + DDP, < 0.0001; SYP SYP + DDP, < 0.0001; SYP + DDP DDP, < 0.0001). Finally, expression of HIF-1α was positively correlated with that of PD-1/PD-L1 and negatively correlated with the expression of CD4+ T cells and CD8+ T cells.

Conclusion: SYPs inhibit immune escape and enhance chemosensitization in HCC simultaneous inhibition of HIF-1α and PD-L1, thus inhibiting the growth of subcutaneous xenograft HCC tumours.