Study On The Mechanism Of Inhibition Of Lung Cancer By Shuangshen-Granules Based On The Regulation Of Tumor Associated Macrophage Cell Polarization By Tumor Exosomes

BackgroundLung cancer ranks first in the world in morbidity and mortality.Although more and more lung cancer patients are diagnosed and treated at an early stage,new immunotherapy drugs are available and radiation therapy techniques are being improved,the total survival of advanced lung cancer has not been significantly extended,and the quality of life of patients has not been significantly improved.The five-year survival rate of patients with stage IA lung cancer is 83%,that of patients with stage IIIA is only 36%,and that of patients with advanced lung cancer is only 3.5%.The overall mid-life of lung cancer has not yet exceeded 1 year.How to reduce the recurrence and metastasis of early lung cancer patients after surgery,prolong the survival of patients with advanced lung cancer,or inhibit the progress of lung cancer are still medical problems that need to be solved urgently.With a large number of clinical studies confirming the effectiveness of traditional Chinese medicine in the prevention and treatment of lung cancer,as well as basic experimental researches on the mechanism of traditional Chinese medicine to prevent and treat lung cancer,the status of traditional Chinese medicine in the prevention and treatment of lung cancer has gradually increased.More and more studies have found that the mechanism of traditional Chinese medicine to prevent and treat lung cancer is closely related to the regulation of tumor immune micro-environment.Lung cancer belongs to the category of "Feiji" in Chinese medicine.Shuangshen-granules are made by pseudo-ginseng,American ginseng,Cordyceps sinensis.Pseudo-ginseng can promote blood circulation,remove blood stasis and replenish blood.American ginseng can strengthen the spleen,replenish Qi but not dryness,and can nourish Yin and generate fluid.Cordyceps sinensis can tonify lung and kidney.The three drugs have the effects of supplementing Qi,strengthening spleen and nourishing Yin,tonifying lung and kidney.Clinical studies have also proved that the benefiting Qi activating blood method is an important treatment for the prevention and treatment of lung cancer in traditional Chinese medicine,which is widely used in clinical practice and has achieved good results.However,due to the complexity of clinical evidence and the diversity of traditional Chinese medicine dialectical prescriptions,the theory of the prevention and treatment of lung cancer by the traditional Chinese medicine "benefiting Qi and Yin,promoting blood circulation " has not been well verified at the cell molecular level.Our pre-experimental studies found that Shuangshen-granule inhibited the growth of transplant tumors in mice with Lewis bearing mice,and performed full transcriptional group sequencing analysis on lung cancer mice with Shuangshen-granules intervention and mice without Shuangshen-granules intervention.Results showed differences in the expression of macrophage migration inhibitors(Macrophage migration inhibitory factor,MIF),macrophage marker protein Mannose receptor C type 1(MRC1)/CD206,KLF4,KLF2 and other Kruppel-like factors(KLF)families,Matrix metalloproteinases(MMPs)families,and non-coded RNAs such as microRNA-34a,microRNA-21,after Shuangshen-granules intervention.These differential genes are closely related to tumor-related macrophages(TAMs)ideotypes and functional regulation,and based on the important role of TAMs polarization in regulating tumor microenvironment to affect tumor growth,we started with TAMs to explore whether the role of Shuangshen-granules suppression of lung cancer was related to affecting TAMs ideotypes and functions.ObjectiveThis study will explore whether the mechanism of Shuangshen-granules inhibit tumor growth is related to the regulation of TAMs phenotype and function,and initially explores the mechanism of Shuangshen-granules affecting TAMs phenotype,through in vivo and in vitro experiments,with the help of modern molecular biology technology.Then provide an objective basis for the prevention and treatment of lung cancer by the method of Chinese medicine called "benefiting Qi and Yin,promoting blood circulation".Methods1.Build a model of Lewis bearing mice,intervene in the mice with high,medium and low doses of Shuangshen-granules,observe the volume change of the transplant tumors in each group of mice,the quality of the transplant tumor,and verify the inhibition effect of the Shuangshen-granules on Lewis lung cancer with the therapy of benefiting Qi for activating blood circulation.2.The effect of Shuangshen-granules on the ratio of M1 TAMs and M2 TAMs was observed by flow cytometry and immunofluorescence,respectively,to detect M1 and M2 tumor-related macrophages in mouse spleen,transplant tumor and cancer week tissue.3.Through Lewis and macrophages mixed injection,the tumor microenvironment model of M1 TAMs over-expression was constructed to observe the volume and mass of transplanted tumor,and the effect of M1 TAMs ratio and M2 TAMs ratio in mice was detected by flow cytometry,and the expression of Arginase-1(ARG1)and inducible Nitric Oxide Synthase(iNOS)in tumor tissue was detected by Western Blot method to explore the correlation between Shuangshen-granules inhibition of Lewis lung cancer and TAMs polarization.4.The correlation between transforming growth factor-?(TGF-?),vascular endothelial growth factor(VEGF),MMP-9 and MIF in tumor tissue was discussed by flow cytometry and Western Blot method to detect the proportion of Regulatory T cells(Tregs)and memory T-cells in mice.5.To construct the stable transfected cell line of MIF+/+Lewis and the stable transfected cell line of shMIF Lewis,and observe the effect of Shuangshen-granules on MIF expression in Lewis cells6.Extraction of Lewis extracellular exosomes,qPCR method to detect microRNA-34a expression in exosomes,observe the effect of Shuangshen-granules on MIF downstream target gene microRNA-34a in Lewis cell-sourced exosomes,and explore the correlation between Shuangshen-granules affecting MIF expression and exosome microRNA-34a expression.7.Build a model of Lewis lung cancer cell origin exosome and macrophage co-culture,intervene in Lewis cells,MIF+/+Lewis cells,shMIF Lewis cells through Shuangshen-granules,and observe the effect of Lewis exosome on macrophage polarization after Shuangshen-granules intervention.8.To detect the ratio of M1 to M2 in macrophages that are interfered with the exosomes of tumor cell origin,as well as the expression of KLF4 genes in macrophages,and to explore the effects of Lewis lung cancer exosome on macrophage polarization and the correlation with KLF4 gene expression after intervention by Shuangshen-granules.Result1.After the intervention of Shuangshen-granules,the volume and weight of transplanted tumor decreased in the mice with Lewis lung cancer.With the increase of dosage of Shuangshen-granules,the effect of inhibiting tumor weight and volume was better.2.High,medium and low doses of Shuangshen-granules were able to reduce the proportion of M2 TAMs in mouse spleen and increase the ratio of M1 TAMs,but the effect of Shuangshen-granules in suppressing M2 TAMs ratio did not show a correlation with the dose gradient of Shuangshen-granules.The ratio of M1 TAMs in the spleen of mice was associated with the dose gradient of Shuangshen-granules,and the proportion of M1 TAMs increased with the dose of Shuangshen-granules.The expression of CD86 in transplanted tumor tissue was positively related to the dose gradient of Shuangshen-granules,and the expression of CD206 was negatively related to the dose gradient of Shuangshen-granules.3.The proportion of spleen M1 TAMs in the mouse model of Lewis lung cancer cells mixed with macrophages increased significantly,and the volume of mouse transplant tumor decreased,suggesting the success of molding.After the intervention of Shuangshen-granules on this mouse model,the proportion of M1 TAMs increased compared to the model group,and the volume of mouse transplant tumor decreased compared to the model group,suggesting that the effect of Shuangshen-granules in suppressing Lewis lung cancer may be related to the increase of M1 TAMs ratio.4.Shuangshen-granules could reduce the expression of ARG1,TGF-?,MMP-9,VEGF and MIF proteins in mouse transplant tumor tissues,and increase the expression of iNOS protein.The M1 TAMs over-expression model also performed the same role.However,the inhibition of VEGF,MIF,ARG1,TGF-? and MMP-9 expression by Shuangshen-granules and M1 TAMs over-expression model showed no synergy,and showed synergy in increasing the expression of iNOS.5.Shuangshen-granules could increase the proportion of CD8+ central memory T cells,which is proportional to M1 TAMs,and reduce the proportion of CD8+effect memory T cells,which is inversely proportional to M1 TAMs ratio.Shuangshen-granules could increase the proportion of CD4+CD25+T cells and reduce the proportion of CD4+CD25+Foxp3+Treg cells,but there was no obvious correlation with the ratio of M1 TAMs.6.The stable transfected cell technology of lentivirus successfully constructed MIF+/+Lewis and shMIF lewis stable transfected lung cancer cell strains,and verified MIF expression at gene and protein levels respectively.7.microRNA-34a expression decreased in MIF+/+Lewis exosome.The drug-containing serum of Shuangshen-granules could inhibit the expression of MIF+/+Lewis cell MIF proteins and genes in in-body,and increase the expression of microRNA-34a in MIF+/+Lewis cell-sourced exosomes.8.A co-culture model of MIF+/+Lewis,shMIF Lewis,Lewis cell-sourced exosomes and macrophages were successfully constructed.The proportion of M2 macrophages could be increased and the proportion of M1 macrophages could be increased in-body,and the exosomes of MIF+/+Lewis after the intervention of the drug-containing serum of Shuangshen-granules could inhibit this effect and increase the proportion of M1 macrophages.9.The exosomes of MIF+/+Lewis could increase the expression of KLF4 gene in macrophages.After the intervention of the drug-containing serum,MIF+/+Lewis exosome could reduce the expression of KLF4 gene.Conclusion1,Shuangshen-granules can inhibit lung cancer in mice,and its mechanism may be related to regulating the polarization of TAMs to M1 type.MIF in lung cancer cells may be one of the targets of Shuangshen-granules.2.Shuangshen-granules can regulate the polarization of TAMs through exosomes derived from lung cancer cells,and its mechanism may be related to MIF-miR-34a-KLF4 pathway.3.The molecular biological mechanism of "benefiting Qi and Yin,promoting blood circulation" therapy in the prevention and treatment of lung cancer may be related to the regulation of tumor cell exosomes,the regulation of TAMs polarization and function,and the remodeling of tumor immune microenvironment.