Mechanism Of Inhibiting NSCLC Proliferation By Regulating FXR1-IL-35 Axis Through Dendrobium Huoshanense Polysaccharide

Objective:Lung cancer is a serious risk to human health worldwide,with extremely high morbidity and mortality rates.Currently,clinically used chemotherapy has limited efficacy and strong toxic side effects,and some studies have shown that Chinese traditional precious Chinese herb Dendrobium huoshanense has shown certain advantages in lung cancer treatment.However,the detailed composition and potential molecular mechanism of its antitumor activity in lung cancer treatment are not well defined.Therefore,this study focuses on the inhibition of proliferation of NSCLC and the molecular mechanism by screening the active ingredients of Dendrobium huoshanense,provides experimental basis for the development of clinical therapeutic application and targeted precision cancer therapy of Dendrobium huoshanense.Methods:(1)Using the cancer HSP and Symmap databases,screening the active ingredients and targets of Dendrobium huoshanense,Cytoscape 3.9.1 software to create a network of interactions between the active compounds of Dendrobium huoshanense and their target genes;target-PPI network interactions were extracted from the STRING database as well as KEGG,GO and Hallmark enrichment analyses were performed.(2)IL-35 expression in NSCLC was retrieved by pan-cancer analysis and the HPA database;single cell transcriptome sequencing data of two LUAD groups were obtained from the GEO database to obtain IL-35 expression levels in the tumor microenvironment;analysis of expression-cell composition correlation elaborated that effect of IL-35 on the formation of immunosuppressive microenvironment;GSEA enrichment analysis of 576 LUAD cases from TCGA RNA-seq data from 576 LUAD patients from TCGA were subjected to GSEA enrichment analysis to screen out the pathways where IL-35 overlaps with the targets of the active ingredients of Dendrobium huoshanense,and to further obtain the core genes;differential expression analysis and survival analysis of core genes using GEPIA2.(3)Lewis Lung Cancer(LLC)tumor-bearing mouse model was constructed,and the general condition and organ coefficients of tumor-bearing mice were monitored after 21 days of continuous drug treatment;Flow cytometry was used to detect the frequency of iTr35 cells;the expression of p35 and EBI3 was detected by RT-PCR and Western blotting;ELISA was used to detect cytokine levels in serum;NK cell activity was detected by CCK8 assay.(4)Sequencing data from the immunotherapy cohort of tumor-bearing mice obtained from the TISMO database to analyze the efficacy of the active ingredient in combination with PD-1/PD-L1 monoclonal antibody for the treatment of NSCLC.Results:(1)Through network pharmacology analysis,obtained 8 active ingredients and 159 effective action targets of Dendrobium huoshanense including cDHPs.PPI network analysis showed that FXR1,DICER1,ACTR3,GART and TUBAIB were more tightly connected in their respective clusters.In KEGG enrichment analysis,drug targets were mainly enriched in pathogenic escherichia coli infection signaling pathway,gap junction signaling pathway,tight junction signaling pathway,apoptosis signaling pathway and colorectal cancer signaling pathway;Hallmark gene enrichment analysis showed that drug targets were enriched in mTORCl signaling pathway,apical junction signaling pathway,TNF-αsignaling pathway via NF-κB,interferon-γ signaling pathway,apoptosis signaling pathway,KRAS signaling pathway,etc;GO gene enrichment analysis showed that salmonella infection,cellular response to nitrogen compound,yersinia infection,gastrin signaling pathway,intracellular receptor signaling pathway,photodynamic therapy-induced NF-kB survival signal,Th17 cell differentiation,modulation of chemical synaptic transmission.(2)The results of pan-cancer analysis showed that IL-35 was significantly highly expressed in LUAD;immunohistochemical analysis of HPA database also showed that IL35 expression was elevated in lung cancer tissues compared with normal tissues;analysis of single cell sequencing data showed that IL-35 was mainly expressed at a high level in CD4+T cell population in tumor microenvironment;GSEA enrichment analysis showed that both NSCLC and SCLC pathways were activated in the IL-35 high expression group,The gene sets with relatively high enrichment scores were selected to screen the tight junction signaling pathway and NSCLC signaling pathway after intersecting with the target genes of Dendrobium huoshanense,and four genes overlapping in the common pathway,tight junction pathway and NSCLC pathway,FXR1,NF-κB2,ACTR3 and TUBA8,were screened;The results of differential expression analysis and survival analysis showed that FXR1 and ACTR3 were highly expressed in LUAD,while NF-κB2 and TUBA8 were not significantly differentially expressed in LUAD.Therefore,in this study,cDHPs were selected as the main active component of Dendrobium huoshanense to investigate the mechanism related to the anti-NSCLC of cDHP.(3)The levels of serum IFN-γ increased and IL-35 and IL-4 decreased,while p35 and EBI3 mRNA and protein levels decreased after cDHP administration(P<0.05).Relative reduction of iTr35 in the blood and spleen of mice after cDHPs treatment,while the activity of NK cells increased(P<0.05).(4)Analysis of sequencing data from the immunotherapy cohort of hormonal mice obtained from the TISMO database showed that the combination of cDHPs and PD-1/PD-L1 antibodies enhanced the efficacy of ICIs.Conclusion:We demonstrated through bioinformatics analysis and relevant animal experiments that cDHPs can effectively inhibit the proliferation of NSCLC,and the mechanism may be related to the regulation of the FXR1-IL-35 axis to reduce the expression of IL-35 and inhibit the differentiation of iTr35,thus enhancing the immune function of the body.Combination of cDHPs improves the therapeutic effect of PD-1/PD-L1 antibodies in patients with LUAD.