| Background and objective:Primary liver cancer(PLC)is the sixth most common malignant cancer worldwide and the third leading cause of cancer death.The prognosis of liver cancer is poor,with a five-year survival rate of only 18%,posing a serious threat to people’s lives and health.Early stage liver cancer has a better prognosis with comprehensive treatment based on surgical resection,but many patients are already in advanced stage when they are found and lose the chance of surgical treatment,so they can only adopt interventional therapy,targeted therapy,chemotherapy,radiotherapy and other measures with poor treatment effect.In recent years,the emergence of immune checkpoint inhibitors(ICIs)has brought new options for cancer treatment and has shown great clinical efficacy in lung cancer,melanoma and colorectal cancer.In liver cancer,the treatment of ICIs has significantly improved the clinical efficacy compared with chemotherapy and targeted therapy,but the objective response rate(ORR)of ICIs monotherapy for liver cancer is less than 20%,which still cannot properly meet the needs of clinical treatment,and the combination of ICIs has become an important method to improve the efficacy.In the IMbrave150 trial,atelelizumab combined with bevacizumab resulted in an ORR of 30%for liver cancer treatment,which significantly improved the clinical efficacy.Therefore,the combination therapy strategy based on ICIs has become a hot research issue in the treatment of liver cancer,and several studies have shown that the combination of ICIs with tyrosine kinase inhibitors,chemotherapy,or dual ICIs has shown better clinical efficacy in the treatment of liver cancer.However,combination therapy inevitably increases side effects,and some patients have contraindications to combination therapy regimens,which limits the use of combination regimens.Meanwhile,although the combination regimen has achieved a current high ORR,a large proportion of patients with liver cancer are still ineffective.Therefore,the efficacy of ICIs in the treatment of liver cancer still needs further improvement.Huaier is a commonly used anticancer Chinese patent medicine in China,which is widely used in the treatment of liver cancer because of its effects on strengthening and consolidating the body’s resistance and promoting blood circulation to relieve blood stasis.Basic research has shown that huaier has the effects of inhibiting tumor growth,anti-tumor angiogenesis and enhancing immune function.At the same time,huaier also has a good safety profile.Therefore,the combination of huaier and ICIs was expected to achieve better clinical results in the treatment of liver cancer.Thus,this study aims to investigate the efficacy and potential mechanism of the combination strategy of huaier with ICIs in the treatment of liver cancer through basic experiments,and to provide an experimental basis for the clinical application of this combination strategy.The success of this study will help improve the efficacy of ICIs in the treatment of liver cancer,promote the application of ICIs,and provide more clinical options for the treatment of liver cancer.Method:Part 1:(1)MTT assay and clone formation assay were used to evaluate the effect of huaier on the proliferation of human liver cancer SK-Hep-1 cells.Then,animal experiments were used to evaluate the inhibitory effect of huaier on H22subcutaneous tumors.The experiments were divided into model group,huaier 1g/kg group,huaier 2g/kg group and huaier 4g/kg group.The model group was given saline by gavage,and the huaier group was given different doses of huaier by gavage every2 days.The tumor size was measured every 3 days,and the experiment was terminated after 3 weeks of intervention,and blood,tumor tissues and organ tissues were collected from the mouse.Animal safety evaluation was performed by liver and kidney function tests and HE staining of pathological sections of major organs.(2)The potential anti-tumor immunomodulatory mechanism of huaier for the treatment of liver cancer was explored using network pharmacology.The Herb database and literature search were used to obtain the components of huaier,which were then entered into the Swiss Target Prediction database to obtain the potential targets of huaier.The keywords"hepatocellular carcinoma"and"liver cancer"were used to search the Gene Cards database,OMIM database,and NCBI gene database for liver cancer-related genes.Immune-related genes were obtained from Imm Port database and Innate DB database.The huaier targets,liver cancer-related genes and immune-related genes were entered into the online website venny 2.1.0 to construct a Venn diagram and obtain the common genes of all three.The obtained common genes were entered into the STRING database to construct protein-protein interaction(PPI)networks.The topological analysis of the PPI network was then performed using Cyto NCA plugin by setting"Betweenness","Degree","Closeness","Eigenvector", "LAC"and"Network"as filtering criteria to screen the core genes.The RNA-seq data of liver cancer and paraneoplastic samples were obtained from the TCGA database,and the data of normal controls were obtained from the GTEx database.The R package ggplot2 was used to map the differential expression of the core genes in tumor tissues and normal tissues.The obtained common genes were subjected to gene ontology(GO)enrichment analysis and Kyoto encyclopedia of genes and genomes(KEGG)pathway enrichment analysis.GO enrichment analysis was performed using the R language packages DOSE,cluster Profiler,enrichplot,colorspace,stringi,and ggplot2,and bubble plots and enrichment data results were calculated.KEGG enrichment analysis was performed using the R language DOSE,cluster Profiler,enrichplot,stringi,colorspace,ggplot2,and pathview packages,and bubble plots and enrichment data results were plotted.(3)Cellular experiments were used to evaluate the effect of huaier on IL-2.The study used human T-lymphocytic leukemia cells(Jurkat cells)that produced IL-2after induction by phorbol 12-myristate 13-acetate(PMA)and phytohaemagglutinin(PHA).It is widely used in immunological research.In this study,we first used the MTT method to screen for concentrations of huaier that had no inhibitory effect on Jurkat cells for subsequent experiments.According to the MTT results,huaier≤2mg/ml,it was not significantly cytotoxic to Jurkat cells,so huaier concentrations of 1mg/ml and 2 mg/ml were selected.The experiment was designed as blank group,model group,huaier 1 mg/ml group and huaier 2 mg/ml group,using a 12-well plate with 2×10~5cells per well.After 24 hours,50ng/ml PMA and 1μg/ml PHA were added to the model and huaier groups.huaier 1mg/ml group and huaier 2mg/ml group were added with corresponding doses of huaier solution.The same volume of complete medium was added to the blank group.After 24 hours of intervention,samples were collected,RNA was extracted,and IL-2 mRNA expression was measured.(4)Enzyme-linked immunosorbent assay(ELISA),quantitative real-time PCR(qPCR),and immunohistochemistry(IHC)were used to detect the expression of IL-2,IL-6,and TNF-αin the tumor tissues of the model group and the huaier group.(5)To explore the effect of huaier on immune cells,the number of CD4+T cells and CD8+T cells in spleen tissue of model group and huaier group was detected by flow cytometry,and the number of CD4+T cells and CD8+T cells in tumor tissue of model group and huaier group was detected by IHC.Meanwhile,because CD8+T cells are critical cells in anti-tumor immune regulation,and the experimental results showed that huaier can significantly up-regulate the expression of CD8+T cells.To further clarify the crucial role of CD8+T cells in the anti-tumor effect of huaier,anti-CD8αAb was used to antagonize CD8+T cells in mice,and the anti-tumor effect of huaier was observed.This part of the experiment was divided into model group,huaier group,anti-CD8αAb group and huaier+anti-CD8αAb group.The model group was given normal saline by gavage,and the huaier group was given 4g/kg huaier solution by gavage,0.2ml,every 2 days.Anti-CD8αAb group was given 50μg anti-CD8αAb by intraperitoneal injection every Monday and Friday.And huaier+anti-CD8αAb group was given huaier solution every 2 days and anti-CD8αAb was injected intraperitoneally twice a week(Monday and Friday).Tumor size was measured every 3 days.After 3 weeks of intervention,the experiment was terminated and the spleen tissue was collected to determine the number of CD8~+T cells by flow cytometry.(6)As PI3K-Akt signaling pathway was most significantly enriched in KEGG results,and this pathway has an important immunomodulatory function.Therefore,this experiment was designed to investigate the effect of huaier intervention on the PI3K-Akt pathway.WB experiments were used to detect the expression of PI3K,p-PI3K,Akt,and p-Akt proteins in the tumor tissues of model group huaier group,and cell experiment was also conducted using PI3K inhibitor LY294002 to verify the critical role of this pathway in the immunomodulation played by huaier.The cell experiment was divided into blank group,huaier group,LY294002 10μmol/L group,and LY294002 20μmol/L group.A 12-well plate was used,and 2×10~5cells were seeded in each well.After 24 hours,huaier group,LY294002 10μmol/L group and20μmol/L group were added sequentially with PMA 50ng/ml,PHA 1μg/ml and huaier2mg/ml.The corresponding concentrations of LY294002 were added to the 10μmol/L and 20μmol/L LY294002 groups,respectively,and the same volume of complete medium was added to the blank group.After 24 hours,RNA was extracted and IL-2mRNA was measured.(7)The expression of PD-L1 in the tumor tissues of the model and huaier groups was detected by qPCR and IHC.Part 2:(1)MTT assay and colony formation assay were used to evaluate the effect of huaier on the proliferation of human umbilical vein endothelial cell(HUVEC).(2)The cell assay was used to investigate the effect of huaier on VEGFA.SK-Hep-1 cells were stimulated with 100μg/ml LPS to secrete VEGFA mRNA,and then treated with huaier to observe the effect of huaier on VEGFA mRNA expression.(3)ELISA,qPCR and IHC were used to detect the expression of VEGFA in the tumor tissues of the model group and the huaier group,and the expression of CD31protein in the tumor tissues of the model group and the huaier group was detected by IHC.Part 3:(1)Animal experiments were conducted to verify the effect of huaier combined with anti-PD-L1 Ab on the growth of H22 subcutaneous tumor.The experiment was divided into model group,huaier group,anti-PD-L1 Ab group,and huaier+anti-PD-L1 Ab group.Model group was given normal saline by gavage,and huaier group was given 4g/kg huaier solution by gavage,every 2 days.Anti-PD-L1 group was injected intraperitoneally with 100ug anti-PD-L1 Ab,every 3 days.The huaier+anti-PD-L1 group received huaier and anti-PD-L1 Ab simultaneously.Tumor size was measured every 3 days and tumor volume was calculated.After 3 weeks of drug treatment,the experiment was terminated and blood,tumor tissue and organ tissue samples were collected from the mice.The safety of animal experiments was evaluated by liver and kidney function tests and HE staining of pathological sections of major organs.(2)The expressions of IL-2,IL-6 and TNF-αin the tumor tissues of the four groups were detected by qPCR and IHC.(3)To explore the effect of huaier combined with anti-PD-L1 Ab treatment on immune cells,flow cytometry was used to detect the changes in the numbers of CD4~+T cells,CD8~+T cells and Treg cells in the spleen tissues of the four groups,and IHC was used to detect the staining results of CD4~+T cells and CD8~+T cells in the tumor tissues of the four groups.(4)The expression of PD-L1 in the tumor tissues of the four groups was detected by qPCR and IHC.(5)The expression of VEGFA in the tumor tissues of the four groups was detected by qPCR.(6)The tumor tissue samples of the model group and huaier+anti-PD-L1 Ab group were used for transcriptome sequencing,and the differentially expressed genes were screened according to the standard of|log2fc|≥1 and q<0.05.The differentially expressed genes were used for GO enrichment analysis and KEGG enrichment analysis.(7)Western blotting was used to detect the protein expressions of PI3K,p-PI3K,Akt and p-Akt in the tumor tissues of the four groups to evaluate the effect of huaier combined with anti-PD-L1 Ab treatment on the PI3K-Akt signaling pathway.Results:Part 1:(1)Huaier can inhibit the proliferation and clone formation of SK-Hep-1 cell and inhibit the growth of H22 subcutaneous tumors,among which the tumor suppressive effect of huaier 4g/kg was the most significant.The safety evaluation showed that huaier was not significantly toxic to the liver and kidney functions and major organs of mice,and the safety was favorable.(2)Using a network pharmacology approach,the 159 common genes of huaier-associated targets,liver cancer-associated genes,and immune-associated genes were obtained.By performing network topology analysis on the common genes,25 of them were screened as core genes,including IL-2,IL-6,TNF,VEGFA,PIK3CA,Akt1,JAK2,STAT3,MAPK1,MAPK3,MAPK14,MAP2K1,EGFR,FGF2,JUN,SRC,PTGS2,PPARG MMP9,GSK3B,HIF1A,AR,ESR1,TP53,HSP90AA1.Then,the expression data of the above genes in liver cancer and normal control samples were obtained from the TCGA database,and the analysis revealed significant differences in the expression of most of the core genes in liver cancer tissues and normal tissues.GO enrichment analysis of the common genes revealed a total of 471biological processes,81 cellular components,and 176 molecular functions.175pathways were obtained by KEGG enrichment analysis,including PI3K-Akt signaling pathway,MAPK signaling pathway,PD-L1 expression and PD-1 checkpoint pathway in cancer,etc.(3)Huaier could promote the secretion of IL-2 mRNA by Jurkat cells.At the same time,the results of animal experiments showed that huaier promoted the expression of IL-2,TNF-αand IL-6 in tumor tissues.(4)The flow cytometry results showed that huaier could upregulate the number of CD8~+T cells in spleen tissue,and the IHC results of tumor tissue showed that huaier could increase the number of CD4~+T cells and CD8~+T cells.In the animal experiment of huaier combined with anti-CD8αAb in the intervention of subcutaneous cancer,the anti-tumor effect of huaier was inhibited after treatment with anti-CD8αAb,and the tumor volume of anti-CD8αAb group and huaier+anti-CD8αAb group had no significant change compared with the model group.Flow cytometry analysis of spleen tissue showed that CD8~+T cells in the anti-CD8αAb group and huaier+anti-CD8αAb group were significantly lower than those in the model group(P<0.01 and P<0.01,respectively).(5)Huaier upregulated the expression of p-PI3K and p-Akt proteins in tumor tissues,and had no significant effect on the expression of PI3K and Akt proteins.In the cellular assay,the secretion of IL-2 mRNA by Jurkat cells promoted by huaier was significantly inhibited after the use of PI3K inhibitor,and the expression of IL-2mRNA was significantly downregulated in the LY294002 20μmol/L group compared with the huaier group(P<0.01).(6)Huaier up-regulated the expression of PD-L1 in tumor tissues.Part 2:(1)Huaier inhibited HUVEC cell proliferation and clone formation.(2)Huaier downregulated the expression of VEGFA mRNA in SK-Hep-1 cell induced by LPS.(3)Huaier downregulated the expression of VEGFA mRNA and VEGFA protein in tumor tissues and decreased the microvascular density of tumor tissues.Part 3:(1)The animal experiment results of huaier combined with anti-PD-L1 Ab treatment showed that huaier group and anti-PD-L1 Ab group had significant tumor suppressive effect,and the difference was statistically significant compared with model group(P<0.05 and P<0.001),and the tumor suppressive effect of huaier+anti-PD-L1 Ab group was further enhanced,and the difference was significant compared with model group(P<0.0001).The safety evaluation showed that the safety of the four groups was excellent,and no significant pathological changes were observed in liver and kidney functions and organ tissues.(2)The expressions of IL-2 mRNA,IL-6 mRNA and TNF-αmRNA in the huaier group and anti-PD-L1 Ab group were not significantly different from those in the model group.However,the levels of IL-2 mRNA,IL-6 mRNA and TNF-αmRNA in the huaier+anti-PD-L1 Ab group were higher than those in the huaier group(P<0.05,P<0.01,P<0.05),and the levels of IL-2 mRNA,IL-6 mRNA and Tnf-αmRNA in the huaier+anti-PD-L1 Ab group were higher than those in the model group(P<0.05).The expression of IL-2 mRNA,IL-6 mRNA and TNF-αmRNA were also significantly upregulated(P<0.01,P<0.01,P<0.01).IHC results showed that the huaier group up-regulated the expression of IL-2,IL-6,and TNF-αproteins compared with the model group,and the huaier+anti-PD-L1 Ab group further increased the expression of IL-2,IL-6,and TNF-αproteins compared with the huaier and anti-PD-L1 Ab groups.(3)The flow cytometry results of spleen showed that the number of CD8~+T cells in spleen was up-regulated in the huaier group,and the increase of CD8~+T cells was further promoted in the huaier+anti-PD-L1 Ab group compared with the huaier group.Meanwhile,the number of Treg cells in the huaier group was lower than that in the model group,and the number of Treg cells in the huaier+anti-PD-L1 Ab group was further lower than that in the huaier group,but there was no statistical difference between the huaier+anti-PD-L1 Ab group and the anti-PD-L1 Ab group.IHC of tumor tissue suggested that the huaier group could promote the increase of CD8~+T cells and CD4~+T cells in tumor tissue,and the huaier+anti-PD-L1 Ab group further promoted the increase of CD8~+T cells and CD4~+T cells compared with the huaier group.(4)The qPCR results showed that PD-L1 mRNA was increased in the huaier+anti-PD-L1 Ab group compared with the model group.The IHC results showed that PD-L1 protein was increased in huaier group and huaier+anti-PD-L1 Ab group compared with model group.(5)The qPCR results showed that VEGFA mRNA expression was highest in model group,and VEGFA mRNA was downregulated in huaier,anti-PD-L1 Ab and huaier+anti-PD-L1 Ab groups compared with model group.(6)The RNA quality of the sequenced samples was qualified and met the detection requirements,and the quality control results of the data obtained by sequencing were acceptable.The correlation coefficients among the samples were larger,and the correlation among the samples was excellent.The sequencing data were analyzed,and a total of 443 up-regulated genes and 62 down-regulated genes were obtained.The results of GO enrichment analysis of differential genes were mainly shown in immune system process,inflammatory response,immune response,cell differentiation,cellular response to lipopolysaccharide,etc.The KEGG enrichment results were mainly shown in PI3K-Akt signaling pathway,MAPK signaling pathway,apoptosis,cytokine-cytokine receptor interaction,pathways in cancer,natural killer cell-mediated cytotoxicity,etc.(7)The p-PI3K and p-Akt proteins were increased in the huaier+anti-PD-L1 Ab group compared with the model group,and the PI3K and Akt protein expression did not differ between the groups.Conclusion:(1)Huaier inhibited the proliferation of SK-Hep-1 cells and suppressed the growth of H22 subcutaneous tumor without significant toxicity.(2)Huaier up-regulates the expression of IL-2,TNF-αand IL-6,promotes the infiltration of CD4~+T cells and CD8~+T cells.And CD8~+T cells play an important role in anti-tumor immune regulation.(3)Huaier activates PI3K-Akt signaling pathway,while huaier upregulates PD-L1 expression in tumor tissues.(4)Huaier inhibits the proliferation of HUVEC cells,downregulates VEGFA expression,inhibits tumor microvessels,and exerts anti-tumor angiogenic effects.(5)Huaier combined with anti-PD-L1 Ab therapy has significant tumor suppressive effect with excellent safety.(6)Huaier combined with anti-PD-L1 Ab treatment can further promote the secretion of anti-tumor cytokines,promote immune cell infiltration,upregulate PD-L1expression in tumor tissues and inhibit VEGFA expression.Among them,PI3K-Akt signaling pathway played an influential regulatory role in the treatment of liver cancer with huaier combined with anti-PD-L1 Ab.(7)Huaier has significant immunomodulatory effects on immune cells and cytokines to enhance anti-tumor immunomodulation,and when combined with anti-PD-L1 Ab,it can enhance anti-liver cancer efficacy. |
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