Momordicoside G Regulates Macrophage To Prevent Lung Injury And Lung Pre-carcinoma Lesions

The malignant progression of tumor is closely related to tissue damage.Malignant tumors often occur in chronic injury sites,and inflammation caused by chronic injury is a major risk factor for cancer development.Macrophages play an important role in the repair of tissue damage.They appear as different phenotypes in different environments.They are usually divided into classically activated M1-like and replaced activated M2-like.M1-like macrophages secrete IL-6,NO and ROS,and has the function of pro-inflammatory and tissue damage.M2-like macrophages mainly secrete IL-10 and TGF-β,and play a key role in anti-inflammatory and the repair of tissue damage.Momordicoside G is the main active ingredient extracted from Momordica charantia,has many pharmacological effects,including anti-inflammatory,anti-oxidant and anti-tumor.However,whether its tumor-preventing effect is related to the influence of macrophage phenotypes has not been reported.This study explores the contributions of macrophages to the effects of momordicoside G on lung injury and pre-carcinoma lesion.The experiment was divided into three parts: in vivo study,in vitro study and action mechanism.In vitro,LPS and IL-10 were used to polarize RAW264.7 into M1-like and M2-like macrophages,M1-like and M2-like macrophages were treated with different concentrations of Momordicoside G,macrophage phenotypes were detected by immunofluorescence;cell proliferation was examined by MTT assay;neutral red and bacterial phagocytosis assays were used to analyze the macrophages phagocytic capacity;cells were analyzed by laser holographic cell imaging and analysis system;Annexin V-FITC/PI staining was used to detect the effects of momordicoside G on cell apoptosis.In vivo studies,to evaluate the preventive effect of momordicoside G on lung injury and lung pre-carcinoma lesions,urethane-induced lung carcinogenic model was established.The pathological changes of lung were detected by HE staining.The effect of NLRP3 expression in lung tissue was examined with immunofluorescence.LPS-induced lung injury model was established to detect the effect of macrophage depletion by liposome-encapsulated clodronate(LEC)and adoptive transfusion M1-like and M2-like macrophage on lung injury.To evaluate the effect of momordicoside G on lung injury and its correlation with macrophages,the lung function was analyzed by maximal mid-expiratory flow using the animal respiratory metabolic measurement system;lung permeability was assessed by the evans blue dye extra-barrier technique and the wet/dry ratio of lungs;HE staining method was used to detect lung pathological changes in mice.In the mechanism study,to evaluate the effect of momordicoside G on different polarized phenotype macrophages function in vitro,acidic vesicular organelles in macrophages were stained by AO;cell autophagy was further estimated by immunofluorescence staining and western blot;ROS was determined by DCFH-DA;ELISA kits test IL-12,IL-10 and TGF-β levels;NO(nitric oxide)was determined by colorimetric assay kit.To verify the correlation between the preventive effect of lung injury and lung pre-carcinoma lesions of momordicoside G and macrophage phenotypes,in the urethane-induced lung cancer model,immunofluorescence was used to detect the macrophage phenotypes of lung alveolar cavity;the expression of IL-6,IL-12,IL-10 and TGF-β in alveolar lavage fluid were examined by ELISA.In the LPS-induced lung injury model,immunofluorescence and western blot were used to detect the effect of momordicoside G on the lung macrophage phenotypes.Finally,the underlying molecular mechanisms by which momordicoside G regulates macrophage polarization were predicted and explained using systems pharmacology approach.In vitro results showed that momordicoside G increased the phagocytic ability of macrophages and inhibited the proliferation of M1-like macrophages,altered cell morphologyand promoted apoptosis when administered at the dose that has no effect on cell viability in M2-like macrophages.In vivo results showed that in urethane-induced lung cancer model,urethane induced lung damage and pre-carcinoma lesions in a time-dependent manner,momordicoside G can significantly prevent lung injury and lung pre-carcinoma lesions,and these effects was macrophage phenotype-dependent.In the LPS-induced lung injury models,adoptive transfer of IL-10-induced M2-like macrophages improved lung injury,whereas LPS-induced M1-like macrophages aggravated lung injury.Momordicoside G reduces the damage promoting effect of M1-like macrophages,and improves lung function,lung permeability and lung injury in combination with M2-like macrophages.Mechanisms research showed that momordicoside G promoted autophagy in M1-like macrophages,decreased ROS,NO,IL-12 levels,and increased IL-10 and TGF-βlevels in vitro.In the urethane-induced lung cancer model,momordicoside G induced the expression of arginase in lung alveolar cavity macrophages,increased the levels of IL-10 and TGF-β and decreased the levels of IL-12 and IL-6 in alveolar lavage.Macrophage depletion by LEC following urethane injection significantly decreased lung tissue injury and carcinoma lesions.In LPS-induced lung injury,momordicoside G inhibits the expression of the M1-like marker iNOS and promotes the expression of the M2-like marker arginase.The network pharmacological results showed that momordicoside G decreased M1-like macrophage phenotype and maintained M2-like phenotype,these processes were related to regulating NF-κB、PPAR and other signaling pathways.In summary,M2-like macrophages play an important role in the repair of tissue damage.Timely healing of tissue damage can prevent lung pre-carcinoma lesions.Momordicoside G can selectively inhibit M1-like and promote its transformation into M2-like macrophages to promote lung injury repair and therefore prevent lung carcinogenesis.