| Background:Non-small cell lung cancer(NSCLC)is the mean diagnosis of lung cancer.Despite the NSCLC treatments are abundant,some patients still face variable clinical dilemmas such as targeted gene negativity,acquired drug-resistant mutations,and adverse reactions.which make the combination of multiple therapies a major strategy for NSCLC treatment in clinic.Local therapies such as minimally invasive ablation provide survival benefits for patients in both early and advanced NSCLC,with less invasive and repeatable implementation,making it a better choice for combination with other therapies as well.Especially,cryoablation is more suitable for the treatment of advanced lung cancer due to its clear ablation range and immune effect.Our previous study found that traditional Chinese medicine(TCM)combined with cryoablation has better clinical efficacy in the treatment of advanced NSCLC,however,its mechanism of action is still unclear and this therapy needs to be optimized.In TCM theory.phlegm,blood stasis,and poison are the basic pathogenesis of lung cancer and the activating blood circulation and detoxification is the common therapy of TCM in the treatment of NSCLC.Therefore,we selected representative formulas of activating blood circulation and detoxification to verify the efficacy of TCM combined with cryoablation in the NSCLC treatment,and used multi-omics technology to systematically explore its mechanism and screen potential targets,promoting the clinical application and optimization of this combined therapy in the treatment of NSCLC.Purposes:To investigate the efficacy and tumor metabolic mechanism of cryoablation and TCM(activating blood circulation and detoxification)alone or two therapies in combination in the treatment of NSCLC.To investigate metabolic alteration of tumor and blood after treatment of NSCLC with cryoablation and TCM(activating blood circulation and detoxification)in combination.To investigate the mechanism of cryoablation combined with TCM(activating blood circulation and detoxification)in the treatment of NSCLC by integrated analysis of metabolomics and proteomics.Methods:① Mass spectrometry of Chinese herbs was performed to analyze the chemical components of Xihuangwan(XHW)and Xingxiaowan(XXW),who are the representative formulas of activating blood circulation and detoxification.②The subcutaneous tumor model of C57BL/6J mice beared with green fluorescent protein-labeled Lewis lung carcinoma were given interventions of XHW/XXW and cryoablation alone or combinedly in different durations,respectively,during which we observed the body weight.subcutaneous tumor volume,fluorescence intensity and weight of tumor,and organ metastasis of mice,then comparing the tumor growth and metastasis of mice between groups.③To analyze the differential metabolite expression and major metabolic pathways in tumor tissues after the intervention of TCM and cryoablation alone or combinedly by non-targeted metabolomics;to compare the differences in metabolic changes of tumor tissues and peripheral blood after the intervention of TCM combined with cryoablation.To screen the potential targets and investigate the main mechanisms of TCM combined with cryoablation in tumor metabolism eventually.④ To analyze the differentially expressed proteins(DEPs)and its function of tumor tissues treated with TCM and cryoablation by the TMT quantitative proteomics,thus exploring the significant metabolic pathways and signal pathways.⑤ Through integration of metabolomics and proteomics,we analyzed the association of metabolites and proteins which differentially expressed in tumor tissues after intervention with TCM and cryoablation,obtained significant metabolic pathways that were co-enriched with differentially expressed metabolites and proteins,and performed specific pathway analysis of these enriched metabolic pathways.Results:① The mass spectrometry analysis of Chinese medicine identified 26 active ingredients of XHW and 19 active ingredients of XXW,with 16 same compounds between the two drugs.② The representative formulas of blood activation and detoxification method,XHW and XXW,could inhibit tumor volume and weight in the early stage of intervention,but no significant difference in the anti-tumor efficacy of the two drugs was observed.The tumor growth of mice in the cryoablation group and the TCM combined with cryoablation group were significantly inhibited in the advanced stage of intervention,which compared with those in the model and the TCM group.The inhibited rate of tumor in the TCM combined with cryoablation group was higher than that of cryoablation group.③ The metabolomics analysis showed that compared with the model group,the differentially expressed metabolites(DEMs)identified in the tumor tissues of the XHW and XXW groups were steroids and steroid derivatives as well as amino acids,peptides,and analogues.The metabolic pathway co-regulated by XHW and XXW was steroid hormone synthesis and Aldosterone-regulated sodium reabsorption.The XXW also regulated fatty acid biosynthesis and unsaturated fatty acid biosynthesis metabolic pathways.XHW also regulated Cortisol synthesis and secretion and Arachidonic acid metabolism.A total of 332 DEMs were identified in the tumor tissues of the cryoablation group compared to the model group,mainly enriched in 15 metabolic pathways.A total of 7 DEMs in the tumor tissues of TCM combined with cryoablation group were the same as those in the TCM group,and 28 DEMs were the same as those in the cryoablation group.DEMs identified in peripheral blood of the mice in the TCM combined with cryoablation group were mainly enriched in glycerophospholipids and fatty acid metabolism.A total of 24 DEMs were commonly identified in the tumor tissues at different durations of the TCM combined with cryoablation intervention.Eight metabolic targets were screened by ROC analysis in the treatment of TCM combined with cryoablation,and 23 metabolic pathways were enriched at both different time points.④ The proteomic analysis showed that 87 proteins were overexpressed and 25 proteins were down-regulated in the tumor tissues of the TCM combined with cryoablation group compared with the model group.These DEPs were significantly enriched to a total of 22 GO terms,74 structural domains,and four KEGG pathways.⑤ The integrated analysis of metabolomic and proteomic of tumor tissues after TCM combined with cryoablation displayed that mainly amino acids and hydroxyl acid analogues of DEMs were strongly associated with DEPs.These DEMs and DEPs were jointly enriched in a total of 31 metabolic pathways,among which 10 metabolic pathways were mainly enriched.Conclusions:① Both XHW and XXW,the representative formulas of blood activation and detoxification,could inhibit the tumor growth of Lewis lung carcinoma transplanted mice to some extent,and cryoablation alone or in combination with TCM more significantly inhibit the progression of lung cancer.② Metabolomic analysis indicates that TCM(activating blood circulation and detoxification)regulates steroids and amino acids metabolism in tumor tissues.Cryoablation mainly regulates nucleotide,glucose and amino acids metabolism in tumor tissues.③ Cryoablation and TCM(activating blood circulation and detoxification)commonly regulate steroids and nucleosides metabolism in tumor tissues,while cryoablation also regulates abnormal amino acids and glucose metabolism.And changes of glycerol phospholipid and fatty acid metabolism in peripheral blood might be related to the effects of TCM.④ The integration of multi-omics reveals that cryoablation combined with TCM(activating blood circulation and detoxification)regulates pyrimidine metabolism,glycerophospholipid metabolism,glutathione metabolism,and glycan biosynthesis,reducing nucleotide synthesis,energy supply of oxidation and activity of proliferation and metastasis of cancer cells,thus inhibiting the progression of lung cancer. |
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