Hydrogen Gas Inhibits The Progression Of Lung Cancer Through Regulating The CD47/CDC42 Pathway

Part One Proteomic analysis of hydrogen gas interference in lung cancer cellsPurpose: To investigate the effect of hydrogen gas intervention on the expression level of proteins in lung cancer A549 cells,in order to explore the underlying mechanism of hydrogen gas on lung cancer progression inhibition.Method: A549 cells were divided into two groups,the control group and the hydrogen gas(60%)group,and three replicate experiments were set up for each group.After 72 hours of hydrogen gas treatment,total proteins were extracted from the two groups and were subjected to non-labeled quantitative proteomics sequencing to determine the differentially expressed proteins among two groups.Then,these differentially expressed proteins were subjected to Gene Ontology,KEGG pathway and protein-protein interaction analysis.In addition,lung cancer A549 and H1975 cells were divided into control group and 60% hydrogen gas treatment group which were subjected to real-time fluorescent quantitative reverse transcription polymerase chain reaction(q RT-PCR)and western blotting assays to detect the relative expression levels of CD47 m RNA and protein.Results:1.Compared with the control group,hydrogen gas treatment induced 58 differently expressed proteins,including 35 upregulated proteins and 23 downregulated proteins;2.The functions of the 58 differentially expressed proteins are mainly enriched in ribonucleoprotein complex biogenesis”,“regulation of muscle system process”,“regulation of hypoxia-induced intrinsic apop-totic signaling pathway”,“ion channel binding”,“U6 sn RNA binding”,“calcium channel inhibitor activity”,“MICOS complex” and “myofibril”terms;3.Hydrogen gas treatment significantly reduced the expression of CD47 protein and m RNA,which may be a possible way for hydrogen to inhibit the progression of lung cancer.Summary: Hydrogen gas treatment can induce 58 differentially expressed proteins in lung cancer cells,including CD47,which was confirmed by western blotting and q RT-PCR assays.These results indicated that CD47 might be a possible a way through which hydrogen gas inhibits lung cancer progression.Part Two Hydrogen inhibits the growth,metastasis,invasion and immune escape of lung cancer cells by regulating CD47/CDC42 signalingPurpose: To investigate whether CD47/CDC42 is involved in hydrogen gas-induced suppression of lung cancer progression through in vitro experiments.Method: Lung cancer cells A549 and H1975 were used for this part experiments;transient transfection was used to upregulate and downregulate CD47 expression in cells;the effects of hydrogen/CD47 on the proliferation,metastasis,invasion,and apoptosis of lung cancer cells were measured by CCK-8,wound healing,Transwell chamber invasion test and flow cytometry assays;Western blotting was used to detect the expressions of apoptosis-related proteins,including Bcl-2,caspase3,cleaved caspase3,PARP,cleaved PARP,as well as CD47 and CDC42 proteins;The effects of hydrogen gas on the phagocytosis of phagocytes were evaluated by using the in vitro macrophage phagocytosis experiment.Results:1.Hydrogen gas intervention significantly inhibited the proliferation,metastasis and invasion of lung cancer cells,and induced apoptosis;2.CD47 knockout could cooperate with hydrogen gas treatment to inhibit lung cancer cell proliferation,metastasis and invasion,and induce apoptosis;3.CD47 overexpression could significantly impair the antitumor effect of hydrogen gas;4.Hydrogen gas intervention enhanced the phagocytosis of lung cancer cells by macrophages.Summary: Hydrogen gas intervention could inhibit the proliferation,metastasis and invasion and induce apoptosis of lung cancer cells via partly downregulating the expression of CD47,accompanied by enhanced phagocytosis of tumor cells by phagocytes.Part Three Hydrogen gas inhibits lung cancer growth and CD47 and CDC42 expression in vivoPurpose: To explore the effects of hydrogen gas treatment on the pathological changes of lung cancer tumors and the expression of CD47 and CDC42 using in vivo experiments.Method: Tumor-bearing mice were divided into control group,hydrogen group,and cis-platinum group;tumor tissues of each group were collected and stained with Hematoxylin & eosin(HE)staining and immunohistochemical staining(IHC)to evaluate the histopathology of tumor tissues and the expression of CD47 and CDC42 proteins,respectivelyResults:1.Consistent with cis-platinum treatment,hydrogen gas intervention could obviously alleviate tumor cell heterogeneity.2.Hydrogen gas intervention could significantly reduce the expression of CD47 and CDC42 in the tumor tissues from tumor-bearing miceSummary: This in vivo experiment further showed that hydrogen gas intervention could inhibit lung cancer progression by reducing the expression of CD47/CDC42.Hydrogen gas may be an effective treatment method for lung cancer patients with CD47 protein high expression.Conclusions:1.Hydrogen treatment significantly down-regulated the expression of CD47 at both protein and m RNA levels;2.Hydrogen gas intervention significantly inhibited the proliferation,metastasis and invasion of lung cancer cells,induced cell apoptosis,and enhanced the phagocytic effect of macrophages on lung cancer cells.3.CD47 knockout enhanced the effects of hydrogen gas on inhibiting cell proliferation,metastasis and invasion and inducing cell apoptosis in lung cancer;4.Overexpression of CD47 significantly weakened the anti-tumor effects of hydrogen gas;5.The effect of hydrogen gas intervention was consistent with that of cisplatin therapy,both of which could significantly alleviate tumor cell heterogeneity.