| In recent decades,new prevention and control measures are urgently needed to improve the situation that incidence and mortality of lung cancer have been increasing year by year,and lung cancer has become a leading killer of cancer that seriously threatens human life and health.Drug immunotherapy is a major breakthrough in the field of cancer treatment in this century.Its outstanding feature is that it can restore patients’immune ability to eliminate cancer cell and has the advantage of low toxicity and high efficiency in tumor treatment compared to traditional chemotherapy drugs that directly targets cancer cells.Current cancer immunotherapy strategies include activation of both innate and adaptive immune.The innate immune,as the first lines of defense,is mainly responsible for presenting antigens and also directly eliminates pathogens.Adaptive immune that can specifically recognize antigens and produce strong immune response is the main approach to eliminate pathogens,however,the immune response will not durable without the assistance of first-line immune.Macrophages,as the most abundant first-line immune cells in the tumor microenvironment,can be polarized into two phenotypes in different conditions:anti-tumor M1 macrophages and pro-tumor M2 macrophages.In tumor microenvironment,tumor-infiltrating macrophages(TIMs)are commonly described as M2 macrophages that stimulate angiogenesis,induce apoptosis of immune cells and transforms T cells into suppressive regulatory cells,thus contributing to tumor immune escape,tumor growth and metastasis.Studies have shown that targeting TIMs can not only reverse the tumor immunosuppressive microenvironment to independently exert the role of tumor immunotherapy,but also enhance the therapeutic effect in combination with other immunotherapies,however,the intensity and characteristics of immunotherapy remain unclear.Traditional Chinese medicine is an important source of medicine for immunomodulator.Zingiber,as food and medicine,has the functions of warming the stomach and dispelling cold,stopping vomiting,and regulating the spleen and stomach.6-Gingerol,the main bioactive component in ginger,has the pharmacological effects including anti-inflammatory,anti-oxidant,anti-lipidemic and anti-tumor.However,the effect of6-gingerol on macrophage polarization phenotypes and whether this effect is associated with its anti-tumor have not been reported.Our previous studies have shown that 6-gingerol has the lung cancer preventive actions.The aim of this study is to explore whether the lung carcinogenesis-preventing efficacy of6-gingerol is correlated to regulating macrophage phenotypes and its mechanism.This present study is divided into in vitro,in vivo and mechanism.In vitro,to explore the effect of 6-gingerol on macrophage phenotypes,the mouse peritoneal macrophages were isolated and stimulated by LPS and IL-4 to obtain M1-and M2-type,respectively.M1 and M2 macrophages were treated with different concentrations of 6-gingerol.MTT assay was used to to detect the proliferation of polarized macrophages;the effect of 6-gingerol on the macrophages morphology and volume was examined by laser holography.Neutral Red and bacterial phagocytosis assays were used to analyze the macrophages phagocytic capacity;the effect of macrophage phenotypes was detected by immunofluorescence.In vivo,urethane-induced lung carcinogenesis model was established to evaluate the preventive effect of 6-gingerol,the number and size of lung nodes were recorded during the experiment;the pathological changes of lung in mice were examined by HE staining;the effect of 6-gingerol on macrophage phenotypes in lung tissue were analyzed by immunofluorescence.M2 macrophages were depleted by liposome-encapsulated clodronate(LEC)to analyze the role of macrophages in lung carcinogenesis.In addition,we established an allograft model of Lewis lung cancer,the tumor weight and tumor volume were recorded and the effect of M1 and M2 macrophages on tumor growth was assessed;immunohistochemistry was carried to estimate intratumor macrophage and lymphocyte phenotypes.To further explore the mechanism by which 6-gingerol regulates macrophage phenotypes to prevent lung carcinogenesis,acidic vesicular organelles in peritoneal macrophages were stained by AO;the expression of autophagy related LC3-B and P62 protein was observed by immunofluorescence;DCFH-DA,Mito-Tracker Green and Lyso-Tracker Red were used to evaluate the intracellular ROS,mitochondrial and lysosomal function,respectively in vivo.In the urethane-induced lung carcinogenesis model,the expression of autophagy proteins LC3-B and P62 in lung tissue was detected by Western blot.The immunofluorescence was carried to test scavenger receptors MSR1,CD36 and endoplasmic reticulum stress GRP78.Cytokine,including arginase 1,reactive oxygen species(ROS),L-arginine,nitric oxide(NO),colony-stimulating factor(CSF-1)and alveolar cavity,including interferon-gamma(IFN-γ),interleukin 12(IL-12),interleukin 10(IL-10),transforming growth factor-β1(TGF-β1)were quantified with ELISA kits.The immunofluorescence was used to detect the role of LEC,arginase inhibitor(nor-NOHA),colony stimulating factor 1 receptor(CSF-1R)inhibitor(Pexidartinib)on macrophage phenotypes.In the Lewis lung cancer allograft model,immunohistochemistry and western blot were applied to determine the intratumor macrophage and lymphocyte phenotypes to confirm the M2 macrophage-resetting efficacy of6-gingerol.Finally,the underlying molecular mechanisms by which 6-gingerol regulates macrophage polarization were predicted and explained using systems pharmacology approach.In vitro study showed that 6-gingerol that had no effect on cell proliferation within the concentration range did not change M1 polarization phenotype,but diminished the volume and decreased the phagocytic ability in M2 macrophages in a dose-dependent manner,and thus reset arginase~+M2macrophages toward iNOS~+M1 macrophages.In vivo result indicated that lung carcinogenesis was positively correlated with macrophage infiltration in lung interstitial in the urethane-induced lung carcinogenesis,and these infiltrating macrophages were mainly arginase~+M2 phenotype.6-Gingerol increased the percentage of iNOS~+M1macrophages,reduced the percentage of arginase~+M2 macrophages,significantly decreased the number and size of lung cancer nodes,improved the pathological abnormalities of lung tissue.The selective deletion of M2 macrophages by LEC has a significant preventive effect on urethane-induced lung carcinogenesis,whereas pexidartinib,which completely depleted macrophages,promoted lung carcinogenesis.In the Lewis lung cancer allograft model,6-gingerol aborted tumor promoting efficacy of adoptive transfer of M2 macrophages,synergistically prevented tumor proliferation with adoptive transfer of M1 macrophages,increased intratumor the percentage of CD8~+lymphocytes and decreased the percentage of Foxp3~+lymphocytes.Mechaniam study revealed that 6-gingerol promoted autophagy,enhanced mitochondrial and lysosomal functions in M2 macrophages,resulted in a reduction in the levels of arginase 1,ROS and an increase in the levels of L-arginine,NO,however,the levels of CSF-1 remained unchanged in vitro.LEC or nor-NOHA selectively suppressed M2 macrophages and further increased the efficacy of6-gingerol-promoted iNOS~+M1 macrophage polarization.However,pexidartinib,which depleted both M1and M2 macrophages,aborted the efficacy of 6-gingerol-promoted iNOS~+M1 macrophage polarization.In the urethane-induced lung carcinogenesis model,6-gingerol induced expression of MSR1,CD36 and GRP78 in lung tissue,increased the levels of IFN-γand IL-12 and decreased the levels of IL-10 and TGF-β1 in the alveolar cavity.The combination of 6-gingerol and LEC enhanced the lung carcinogenesis preventive efficacy.The lung cancer preventive effect of 6-gingerol was weakened when combined with pexidartinib.In the Lewis lung cancer allograft model,6-gingerol combined with LEC increased intratumor the percentage of iNOS~+macrophages and CD8~+T cells and decreased the percentage of Foxp3~+T cells,which exerted synergistic anti-tumor effects.Network pharmacological analysis indicated that 6-gingerol regulates the polarization of macrophages to M1 phenotype through PI3K-Akt,Jak-STAT,TNF signaling pathways.In summary,M2 macrophages play an integral role in lung carcinogenesis.6-Gingerol can prevent lung carcinogenesis by reprogramming M2 macrophages into M1 macrophages and exerte synergistic effect in combination with M2 macrophages depletion agents and M1 macrophages adoptive immunotherapy,and its mechanism involves many pathways besides mainly inhibiting arginase. |
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