Dendrobium huoshanense polysaccharide inhibits NSCLC proliferation and immune evasion via FXR1-IL-35 axis signaling pathway

Abstract

Dendrobium huoshanense has received special attention for its advantages in the treatment of lung cancer, but the underlying molecular mechanisms are not yet well understood. First, we obtained 8 active ingredients and 159 effective action targets of Dendrobium huoshanense using network pharmacology, and searching target interactions through STRING, constructing the PPI network and KEGG, GO and Hallmark enrichment analysis. Then, we combined target's enrichment analysis and GSEA enrichment analysis of IL-35, indicating the mechanism of cDHPs for non-small cell lung cancer (NSCLC) may be related to tight junction and NSCLC pathway. Further, FXR1 and ACTR3 were identified as core therapeutic targets, and high expression of FXR1 or ACTR3 was significantly associated with poor prognosis of patients. The analysis of single-cell data also indicated that the percentage of CD4-CTLA4-Treg cells may be increased by the expression of IL-35, resulting in a suppressive immune microenvironment. Next, In vivo experiment, we detected iTr35 by flow cytometry, detected IL-35 level by RT-PCR, Western blotting and ELISA, and detected NK cell activity to explore the immunomodulatory effects and anti-tumor mechanism of cDHPs. After cDHPs administration, the conversion of CD4⁺ T cells to iTr35 is inhibited, p35 and EBI3 in both protein and mRNA levels, the levels of IL-35 and IL-4 in serum decreased. The levels of IFN-γ, while the activity of NK cells in mice increased, enhancing the anti-tumor immune effect of the organism. Finally, analysis of sequencing data from the immunotherapy cohort of tumor-bearing mice obtained from the TISMO database shows that the combination of cDHPs and PD-1/PD-L1 antibodies improves effector and thus PD-1/PD-L1 antibody efficacy. These findings suggest that cDHPs inhibit NSCLC proliferation and immune escape via the FXR1-IL-35 axis signaling pathway.

Keywords: IL-35; Immune evasion; Tumor microenvironment; iTr35.

Fig. 1

Active ingredients and targets of dendrobium huoshanense, PPI network analysis. A The network of the relationship between the active ingredients and the targets of Dendrobium huoshanense. B The PPI network of Dendrobium huoshanense’s targets

Fig. 2

Results of KEGG, HALLMARK and GO enrichment analysis. A KEGG enrichment analysis of therapeutic targets (the top 12 results). B Chord Diagram of KEGG enrichment analysis. C HALLMARK enrichment analysis of therapeutic target. D GO enrichment analysis of therapeutic target

Fig. 3

Multi-omics analysis reveals that IL-35 is highly expressed in LUAD and IL-35 regulates the development of NSCLC. A Pan-cancer analysis of IL-35. B Immunohistochemical analysis of IL-35 in lung tissue from normal subjects and patients with LUAD. C IL-35 expression in single-cell sequencing cohort. D Expression and cell composition correlation between IL-35 expression in CD4-CCR7-FOS T cell and CD4-CXCL13-Tfh and composition of CD4-CTLA4-Treg. E Analysis of immune infiltration abundance of IL-35 in LUAD and LUSC. F: The GSEA enrichment analysis of IL-35

Fig. 4

Dendrobium huoshanense inhibits proliferation, migration and invasion of NSCLC through the FXR1-IL-35-ACTR3 signaling pathway. A Volcano map analysis of differentially regulated genes in LUAD. B The mRNA Expression of ACTR3, FXR1, NF-κB, TUBA8 in LUAD. C The Gehan-Breslow–Wilcoxon test showed that the protein expression of ACTR3, FXR1 predicted poor survival in LUAD patients, and TUBA8, NF-κB2 did not correlate with patient survival. D Correlations between ACTR3, FXR1, NF-κB2, TUBA8 and IL-12A, EBI3 mRNA levels, ACTR3, FXR1 and IL-12A, EBI3 mRNA levels are positively correlated, one point represents one sample. E Correlations between ACTR3, TUBA8 and EMT signatures, FXR1, NF-κB and NSCLC signatures

Fig. 5

cDHPs inhibit the development of NSCLC in vivo by suppressing IL-35 expression. A Curve of mice body mass. B Tumor volume curve. C Measurement of IL-35, IL-4, IFN-γ concentrations after treatment with cDHPs by ELISA. D, E After cDHPs treatment, p35, EBI3 protein levels and mRNA levels were detected by Western blot and RT-PCR, respectively. F Organ index for Spleen, liver, kidney and lung. *p < 0.05; ∗∗p < 0.01

Fig. 6

cDHPs inhibit the conversion of iTr35 by inhibiting IL-35 to prevent tumor immune evasion. A, B Detection of CD4⁺Foxp3⁻ Tconv and iTr35 levels in blood after treatment with cDHPs by flow cytometry, respectively. C, D Detection of CD4⁺Foxp3⁻ Tconv and iTr35 levels in spleen and after treatment with cDHPs by flow cytometry, respectively. E: Effects of dendrobium huoshanense polysaccharides on NK cell viability. *p < 0.05; ∗∗p < 0.01

Fig. 7

cDHPs activate the innate immune system to kill tumor cells by enhancing NK cell activity. A IL12A expression level, tight junction score and NSCLC score of baseline, non-responders and responders in homologous mouse immunotherapy cohorts. B Expression and cell composition correlation between IL-35 expression in CD4-CTLA4-Treg and composition of CD4-CD8-IFTH1, CD8-HSP1A1, CD8-SLC4A10 and DC-LAMP3 cells