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-1αand 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 via 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 1×107 SMMC-7721 cells.Male mice(male,5 weeks old;n=24)were then randomly divided into the following four groups(n=6):Control(0.9%normal saline),SYP(200 mg/kg),SYP+cisplatin(DDP)(200 mg/kg+5 mg/kg DDP weekly via intraperitoneal injection),and DDP(5 mg/kg cisplatin weekly via intraperitoneal injection).The dose of saline or SYPs for the indicated mouse groups was 0.2 mL/d via 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,PD1,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 HIF1α 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 vs SYP,P=0.0129;control vs SYP+DDP,P=0.0004;control vs DDP,P=0.0152,SYP+DDP vs DDP,P=0.0448;HIF-1αmRNA:control vs SYP,P=0.0009;control vs SYP+DDP,P<0.0001;control vs DDP,P=0.0003,SYP vs SYP+DDP,P=0.0192.PD-1 protein:Control vs SYP,P=0.0099;control vs SYP+DDP,P<0.0001,SPY vs SYP+DDP,P=0.0009;SYP+DDP vs DDP,P<0.0001;PD-1 mRNA:control vs SYP,P=0.0002;control vs SYP+DDP,P<0.0001;control vs DDP,P=0.0003,SPY vs SYP+DDP,P=0.0003;SYP+DDP vs DDP,P=0.0002.PD-L1 protein:control vs SYP,P<0.0001;control vs SYP+DDP,P<0.0001;control vs DDP,P<0.0001,SPY vs SYP+DDP,P=0.0040;SYP+DDP vs DDP,P=0.0010;PD-L1 mRNA:Control vs SYP,P<0.0001;control vs SYP+DDP,P<0.0001;control vs DDP,P<0.0001,SPY vs SYP+DDP,P<0.0001;SYP+DDP vs DDP,P=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 vs SYP+DDP,P<0.0001,SYP vs SYP+DDP,P=0.0005;SYP+DDP vs DDP,P=0.0002.CD4+T cell protein:Control vs SYP,P=0.0033;Control vs SYP+DDP,P<0.0001;Control vs DDP,P=0.0021,SYP vs SYP+DDP,P=0.0004;SYP+DDP vs DDP,P=0.0006.Quantitative CD8+T cells:Control vs SYP+DDP,P=0.0013;SYP vs SYP+DDP,P=0.0347;SYP+DDP vs DDP,P=0.0043.CD8+T cell protein:Control vs SYP+DDP,P<0.0001;SYP vs SYP+DDP,P<0.0001;SYP+DDP vs DDP,P<0.0001).Finally,expression of HIF-1αwas 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 via simultaneous inhibition of HIF-1α and PD-L1,thus inhibiting the growth of subcutaneous xenograft HCC tumours.