| BackgroundLung cancer is the most common malignant tumor in the world,with the highest morbidity and mortality among all cancers.Non-small cell lung cancer(NSCLC)is the most common pathological type of lung cancer,and adenocarcinoma is the most common pathological type of NSCLC.With the proposal of the concept of tumor immune microenvironment(TIME),more and more studies have proved that TIME is closely related to the prognosis of NSCLC patients.How to improve TIME to enhance tumor immunity has become a new research hotspot of lung cancer.In recent years,Chinese medicine has been shown certain advantages in regulating and reshaping TIME.Qijiafuzheng formula(QJFZF)is a traditional Chinese medicine(TCM)compound used in clinical treatment of lung cancer for many years.QJFZF is formulated according to Professor Li Zhong’s ’Fuzhenggushe’ method.Previous studies have shown that QJFZF has inhibitory effect on tumor and can improve the clinical symptoms of NSCLC patients.However,its impact on the survival of lung cancer patients and the regulation of TIME are still not clear.Therefore,it is hoped that this study will preliminarily explore these problems by QJFZF.Objective(1)To analyze the characteristics of TIME in lung adenocarcinoma by bioinformatics analysis and network pharmacology,and to predict the targets and mechanisms of QJFZF on TIME in lung adenocarcinoma.(2)To observe the inhibitory effect of QJFZF on Lewis lung carcinoma mouse model and the regulation of immune function by animal experiments.(3)Using animal experiments to verify the predicted targets in the results of bioinformatics analysis and network pharmacology,and analyze the mechanism of QJFZF regulating TIME in lung adenocarcinoma.(4)Based on the real-world research(RWS)method,a retrospective cohort study was conducted to evaluate the effect of dialectical treatment of TCM based on QJFZF on the survival of NSCLC patients.Methods(1)The mRNA transcriptome data and clinical data of LUAD project were downloaded from TCGA database,and the stromal cell score and immune cell score of LUAD samples were obtained from ESTIMATE database.According to the medians of the two type of scores,the OS between high-and low-score groups were analyzed by Log-rank test.Cibersort algorithm was used to analyze the difference of immune cell content between different groups,and differential expressed genes(DEGs)were screened by limma package.(2)The TCMIO and TRRUST databases were used to obtain the herb targets of QJFZF.The herb targets were intersected and mapped with DEGs of lung adenocarcinoma TIME to obtain the common targets.The STRING database was used to construct the PPI network of the common targets,and CytoHubba plug-in was used to screen the key targets in the network.The correlation between the key targets and tumor purity and the degree of immune cell infiltration was analyzed by TIMER database.(3)The Lewis lung carcinoma subcutaneous transplanted mouse model was established and divided into the model control group,the TCM group,the cisplatin group and the TCM combined western medicine group,the normal control group was also set,with six mice in each group.Tumor-bearing mice were treated with normal saline,QJFZF,cisplatin,and QJFZF combined with cisplatin.During the drug intervention,the volume of subcutaneous transplanted tumor was measured.After the drug intervention was completed,the mice were sacrificed and the complete thymus and spleen were taken to calculate the organ index.The proportion of CD3,CD4,CD8 cells in peripheral blood was detected by flow cytometry.(4)HE staining was used to observe the morphology of tumor tissue,immunohistochemical staining was used to observe the infiltration of CD8 positive cells,and western blot was used to detect the expression levels of STAT1 and CXCL10 in tumor tissue.(5)A retrospective study was conducted with NSCLC patients as the research obj ects.The cases treated with integrated TCM and western medicine were from Dongzhimen Hospital,Beijing University of Chinese Medicine.The cases treated with western medicine were from The Surveillance Epidemiology and End Results(SEER)database.Propensity score matching(PSM)was used to match the two cohort samples.The clamp value was set to 0.001,and the matching ratio was set to 1:1.Log-rank test was used to compare the overall survival(OS)of 6 the matched cohort.Results(1)Mechanism prediction of QJFZF regulating TIME in lung adenocarcinoma:i.Analysis of TIME characteristics of lung adenocarcinoma:TCGA-LUAD tumor samples were divided into high-and low-score groups according to immune cell score and stromal cell score.The survival analysis between groups showed that the OS of the high immune cell score group was significantly better than that of the low immune cell score group(P<0.05),and there was no significant difference in OS between the stromal cell score groups.The results of immune cell content analysis showed that the contents of memory B cells,CD8+T cells and CD4+T cells in the samples of the high immune score group were significantly higher than those in the low immune score group(P<0.05).A total of 752 DEGs were screened out between high-and low-immune cell score groups,and biological enrichment analysis showed that DEGs mainly involved:a.MF:antigen bindings cytokine activity cytokine receptor activity,protein bindings and chemokine activity et al;b.BP:immune response-activating cell surface receptor signaling pathway,regulation of lymphocyte activation,and T cell activation et al;c.CC:T cell receptor complex,plasma membrane receptor complex,external side of plasma membrane et al.KEGG pathway enrichment analysis showed that DEGs were mainly related to hematopoietic lineage,cytokine-cytokine receptor interaction,cell adhesion molecules and chemokine signaling pathways et al.In addition,the PPI network of DEGs was constructed by STRING,and 20 most important molecules were screened out:CCR7,CTLA4,CD86,CD80 and FOXP3 et al.ii.Prediction of QJFZF regulating TIME mechanism of lung adenocarcinoma:64 tumor immune targets and 515 downstream regulatory target genes of QJFZF were obtained through TCMIO and TRRUST databases.After mapping these targets with DEGs of lung adenocarcinoma TIME,39 common targets were obtained.The method of network topology analysis screened out five key targets in the PPI network of common targets:CCL3,IL10,CXCL10,FOXP3 and CD86.According to the correlation coefficient,MCC value and fold change of each target,CXCL10 may be the core target of QJFZF in regulating TIME of lung adenocarcinoma.CXCL10 was regulated by transcription factor STAT1,and there was a positive correlation between them(r=0.76,P<0.001).GSEA enrichment analysis showed that JAK-STAT pathway was up-regulated when CXCL10 was highly expressed.(2)The effect of QJFZF on tumor growth and immune function in Lewis lung carcinoma mouse model:i.Effect of QJFZF on Lewis lung carcinoma mouse model:The tumor volume of TCM combined western medicine group was the smallest,which was significantly smaller than that of TCM group and model control group(P<0.05).The tumor volume of cisplatin group and TCM group were smaller than that of model control group(P<0.05).ii.The effect of QJFZF on immune function of Lewis lung carcinoma mouse model:The thymus index of TCM group and normal control group was significantly higher than that of cisplatin group(P<0.05).In terms of spleen index,the spleen index of TCM group and TCM combined western medicine group was significantly higher than that of cisplatin group and normal control group(P<0.05),and the spleen index of cisplatin group was significantly lower than that of model control group(P<0.05).Flow cytometry showed that there was no significant difference in the levels of CD3+and CD8+lymphocytes in peripheral blood among these groups.The levels of CD4+lymphocytes and CD4/CD8 in the TCM group were significantly higher than those in the model control group,cisplatin group and TCM combined western medicine group(P<0.05).The levels of IFN-γ and CXCL10 in peripheral blood of mice were detected by Procarta Plex multi factor technique.The results showed that the levels of IFN-γand CXCL10 in TCM combined western medicine group were significantly higher than those in model group(P<0.05).(3)QJFZF promotes CD8+T cell infiltration in TIME of lung adenocarcinoma through STAT1/CXCL10 axis:The results of immunohistochemical staining showed that the expression of CD8 in tumor tissues of TCM combined western medicine group,cisplatin group and TCM group was significantly higher than that of model control group(P<0.05).The average proportion of positive cells in TCM combined western medicine group was also significantly higher than that in TCM group and cisplatin group(P<0.05).Western blot results showed that the expression level of STAT1 in TCM combined western medicine group was significantly higher than that in model control group,cisplatin group and TCM group(P<0.05),and the expression level of STAT1 protein in TCM group and cisplatin group was also significantly higher than that in 8 model group(P<0.05).The expression level of CXCL10 protein in TCM combined western medicine group was significantly higher than that in model group and TCM group(P<0.05),and the expression level of CXCL10 protein in TCM and cisplatin group was significantly higher than that in model group(P<0.05).(4)Effect of QJFZF on survival of NSCLC patients:i.Integrated TCM and western medicine cohort:A total of 520 NSCLC patients were included in the integrated TCM and western medicine cohort.Survival analysis showed that the 1-year,3-year and 5-year survival rates were 73.1%,56.9%and 50.3%,respectively.Survival analysis between different clinical features showed that patients with younger age(<65 years),female,adenocarcinoma,NO and M0 had longer OS.ii.Western medicine treatment cohort:5022 NSCLC patients were included in the SEER database.The 1-year,3-year and 5-year survival rates were 43.5%,18.3%and 10.7%,respectively.Survival analysis between groups with different clinical features showed that patients with younger age,adenocarcinoma and squamous cell carcinoma,N0,M0 and early AJCC stage had longer OS.iii.Survival comparison of the two cohorts:A total of 122 samples were obtained from the two cohorts after PSM matching,and there was no significant difference in clinical characteristics between the two groups.The survival analysis results showed that the OS of patients in the integrated TCM and western medicine cohort better than that in the western medicine treatment cohort(P<0.05).Conclusion(1)OS of patients with lung adenocarcinoma with high immune cell infiltration was better than that of patients with low infiltration.QJFZF may regulate TIME of lung adenocarcinoma through multiple targets and pathways.(2)QJFZF can effectively inhibit the tumor growth of Lewis lung cancer mice,and can enhance the peripheral immune function of mice by increasing the thymus index,spleen index,the number of peripheral blood CD4+lymphocyte subsets,CD4/CD8 ratio and IFN-y.(3)QJFZF may promote the infiltration of CD8+T lymphocytes in TIME of lung adenocarcinoma by regulating STAT1/CXCL10 signaling axis,thus exerting anti-tumor effect.(4)On the basis of conventional western medicine treatment,the combination of QJFZF may be beneficial to the survival of NSCLC patients. |
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