| Background and objectivesBackground and purposeWith the deterioration of the environment and the arrival of an aging society,the number of lung cancer patients is on the rise year by year.The recurrence and metastasis of lung cancer are the main causes of death.Therefore,the molecular mechanism underlying lung cancer metastasis is the key to the prevention and treatment of lung cancer.Scientific research shows that:Tumor-associated macrophage(TAM)polarization type is closely related to the occurrence and development of tumors;macrophages are divided into M1 type and M2 type;have plasticity and heterogeneity;follow Different types of environment differentiate into different types,in which M2 macrophages can promote the proliferation and metastasis of lung cancer cells.Most studies show that the energy metabolism of tumor cells is dominated by glycolysis,and its reason is not only It is associated with the activation of protooncogenes(AKT,c-Myc,ErbB2,and RAS,etc.)and tumor suppressor gene mutations(such as PTEN and P53,etc.)and also with a variety of immune cells in the immune microenvironment of tumors.In the early stages of lung cancer,tumor cells mainly provide their own energy through glycolysis,and the infiltrating macrophages are mainly M1-type,and their main role is to inhibit tumor growth.With the development of tumors,in advanced tumor tissues,Some tumor cells provide a large amount of energy through their metabolism of fatty acids.At this time,M2 macrophages gradually occupy a dominant position,which facilitates the secretion of various cytokines and helps tumors The cysts resist adverse environmental conditions and assist tumor cells in invasion and migration;the process of tumor development is also the process of transition from M1 to M2macrophages.Pleural effusion is one of the complications of lung cancer,and is also the case of advanced lung cancer.An important feature is that pleural effusions are rich in cellular metabolites,proteins,nucleic acids,etc.Lactic acid,which is the final product of the glycolysis pathway,has a significantly higher pleural effusion content,and our laboratory studied pleural effusions earlier.The macrophage type M2phenotype derived from malignant pleural effusion was found to be significantly higher than the inflammatory phenotype of pleural effusion,so this study was based on previous studies to study the thoracic volume of patients with lung cancer.The effect of small molecules of lactic acid in the liquid on polarization of macrophage cells is intended to elucidate from the perspective of metabonomics how lactic acid drives the macrophages to polarize in the M2 direction under hypoxic conditions,thereby promoting the transfer of lung cancer molecules The mechanism provides new ideas for the treatment of lung cancer metastasis in the future.MethodsThe 132 cases of pleural effusions without any treatment were first diagnosed by the Department of Ultrasound in the First Affiliated Hospital of Zhengzhou University.The w hole course was placed in an ice box,immediately centrifuged at4°C and 3000 rpm,and the supernatant was stored in a refrigerator at-80°C.The lymphocytes in the extraction.Markers such as CD14,CD163,CD206,and 7AAD were used to detect the proportion of CD14~+CD163~+M2 macrophages in CD14~+macrophages of lymphocytes in pleural effusions by flow cytometry.According to the gold standard for clinical pathological diagnosis,50 cases of cancerous pleural effusion and 80 cases of inflammatory pleural effusion were diagnosed in 200 cases of pleural effusions without any other complications.The lactic acid expression was measured using a lactic acid kit.The relationship between the expression of differential metabolites and the ratio of M2 macrophages and the prognosis of patients was examined and verified using the TCGA database.CD14+cells were isolated from peripheral blood of healthy individuals.After 7days of culture,lactic acid(0,12.5 m M,25 mM,37.5 m M and 50 mM)was added at a gradient concentration in a hypoxic and normoxic environment.After 24 hours of incubation,flow cytometry and PCR were performed.Two methods were used to detect the proportion of M2 macrophages,and the metabolites and metabolic pathways were detected using non-targeted metabolomics.ResultsFlow cytometry results showed that the proportion of M2 macrophages in cancerous pleural effusions was significantly higher than that of inflammatory pleural effusions.Quantitative detection of 63 pleural effusion specimens using the lactic acid kit confirmed that there was a significant difference in the expression of lactic acid between cancerous pleural effusions and inflammatory pleural effusions.At the same time,the patient’s pleural effusion supernatants were analyzed using spss software.There is a certain correlation between the content of lactic acid and the ratio of CD14~+CD163~+/CD14~+,and there is also a certain correlation with the prognosis of clinical patients.The level of lactic acid was correlated with the proportion of M2 macrophages in the in vitro experimental cell lines.The level of lactic acid was correlated with the proportion of M2 macrophages in the in vitro experimental cell lines.Metabolomic results of the effect of lactic acid on macrophages show that lactic acid is reprogrammed by modulating macrophage metabolism,arachidonic ethanolamide,ciclopirox,isoleucine,leucine,palmitoyl serinol,adenine,trans-cinnamic acid,citraconic acid,2-propylvaleric acid,phenylalanine,EPA,myristic acid,triethylamine,MG(0:0/18:1(9Z)/0:0)When metabolites change,lactic acid can bind to macrophage surface G protein-coupled receptor GPR81,cause norepinephrine elevation,and activate PI3K-AKT and MAPK pathways to drive macrophage.The cells are polarized in the M2 direction and lead to poor patient outcomes.This result was corroborated by metabonomic detection of benign and malignant pleural effusions and TCGA database analysis.ConclusionLactic acid can bind macrophage surface G protein-coupled receptor GPR81,cause norepinephrine elevation,activate PI3K-AKT and MAPK/ERK pathways and drive macrophages to M2 direction polarization,And lead to a poor prognosis of the patient. |
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