| The incidence and mortality rate of lung cancer are increasing year by year,seriously endangering human life and health,and the incidence of lung cancer ranks second and fatality rate ranks first in the world.Currently,the conventional treatment for most lung cancer patients is surgery,chemotherapy,radiation therapy,or a combination of these.However,these methods have great limitations and seriously affect the quality of life of patients.In recent years,with the country’s vigorous development of the traditional Chinese medicine industry,more and more people have realized the importance of traditional Chinese medicine in the treatment of lung cancer.The advantage of traditional Chinese medicine in preventing the occurrence and metastasis of tumors lies in improving the overall environment of tumor patients,that is,the tumor-host microenvironment,preventing the potential of cancer cells from occurring,invading and metastasizing,and restoring their equilibrium through long-term use of traditional Chinese medicine.Psoralen is bitter,pungent and warm in nature,it belongs to the kidney and spleen meridians.Psoralea has the pharmacological effect of promoting bone marrow hematopoiesis,enhancing immunity and endocrine by regulating the nervous and blood system,among them,bavachalcone is its effective component and has antibacterial and minimal toxic side effects.Objective:This project aims to explore the effects of bavachalcone on lung cancer cells in vivo and in vivo,to clarify whether bavachalcone can inhibit the proliferation of lung cancer cells,how to inhibit the metastasis of lung cancer cells,whether it can inhibit the occurrence and metastasis of tumors by regulating the typing of macrophages in the tumor microenvironment,and how macrophages and CD4~+T cells are connected in tumor treatment,and other scientific issues,so as to provide new ideas and new methods for the treatment of lung cancer with traditional Chinese medicine.Methods:In this study,the killing effect of bavachalcone on lung cancer cells(NCI-H446 and A549)and human normal pulmonary bronchial epithelial cells(BEAS-2B)was explored through in vitro cell proliferation experiments,compared with clinical first-line chemotherapy drugs(cisplatin);an ultra-low adhesion plate 3D culture model was established to explore the inhibitory effect of bavachalcone on lung cancer cells;a tumor bearing model of subcutaneous lung cancer in nude mice was established,and the inhibitory effect of bavachalcone on two lung cancer cells(NCI-H446 and A549)and the side effects after cisplatin treatment were studied;the effects of bavachalcone on lung cancer cell metastasis were explored by migration and invasion experiments,and the effects of bavachalcone on metastasis-related proteins were detected by Western blot,immunohistochemistry and the construction of 3D culture models of lung cancer cells and normal epithelial cells.Bavachalcone stimulated macrophages(M(?))in vitro,detected their typing,sorted CD4~+T cells by magnetic bead sorting,and explored the connection between macrophages and CD4~+T cells through co-culture.Transcriptomics methods were used to detect and screen out genes with important biological significance and statistically significant differences in macrophages in tumorigenesis and development,which provided important strategies for the diagnosis of lung cancer and the treatment of traditional Chinese medicine.Results:Through CCK-8 experimental detection,it was found that bavachalcone(20μmol/L)had a strong killing effect on lung cancer cells(H446 and A549),and with the increase of drug dose and prolongation of time,the inhibitory effect gradually increased,and bavachalcone had a weak killing effect on normal lung epithelial cells.The 3D ultra-low adhesion plate culture method better simulates small cell lung cancer and lung adenocarcinoma tissues in vitro,and the inhibition rate of bavachalcone reaches 40%at 72 hours,and the chemotherapy drug(cisplatin)is below 20%.After three weeks of intratumor injection and administration,we can find that after the treatment of lung cancer with bavachalcone,the tumor mass decreases,and with the increase of drug concentration,the mass gradually decreases,and the effect on the body weight of nude mice is small.After three weeks of treatment,the tumor tissue was removed and immunohistochemical testing was performed,and we found that the positive rate of Ki67 was reduced,and bavachalcone could inhibit the proliferation of lung cancer cells;the fluorescence value of E-Cadherin increased,the fluorescence value of N-Cadherin,SNAIL and Vimentin decreased,and the bavachalcone inhibited the metastasis of lung cancer cells.Through scratch test and invasion test,bavachalcone has a strong killing effect on lung cancer cells(H446 and A549),thus inhibiting cell migration and invasion rate,and the inhibition effect gradually increases with the increase of drug dose and time.Western blot experiment and the construction of lung cancer cells and lung normal epithelial cells 3D culture model experimental detection showed that bavachalcone can reduce the content of MMP protein,SNAIL protein,Vimentin protein and N-Cadherin protein of lung cancer cells,increase the content of E-Cadherin protein,hinder the mesenchymal transformation of epithelial cells,and then inhibit the metastasis of lung cancer cells.C57 mice were injected with Lewis cells through the tail vein,transplanted the metastatic model,and after treatment by gavage administration(bavachalcone),the spleen and lung tissues of mice were taken to observe the effect after treatment,and immunofluorescence detection was performed,the positive rate of CD4 in the spleen of mice increased,the positive rate of CD86 in mouse lung tissues increased,and the positive rate of CD206 decreased;detecting the subpopulation of lymphocytes in mouse spleen tissue,we found an increased proportion of CD4~+T cells,and detecting mouse lung tissue macrophage typing,we found that the proportion of mouse M1 macrophages was elevated,inhibiting lung cancer cell proliferation and metastasis.Through HE staining of the heart,liver and kidney of mice,we did not find pathological damage,indicating that bavachalcone has few side effects on the body.By stimulating mouse bone marrow mononuclear macrophages(M(?))in vitro,we can find that bavachalcone can induce giant cell differentiation to M1 type(66.2%);the cytokine concentration of TNF-αsecreted by macrophages increases,and the concentration of IL-10 cytokine decreases.After magnetic bead sorting of CD4~+T cells,the proportion of CD4~+T can reach(91%),follow-up experiments can be carried out,CD4~+T cells stimulated by bavachalcone can promote the secretion of IFN-γ,after sorting and co culturing with macrophages,the proportion of M1 type macrophages can reach 73.5%after adding bavachalcone stimulation.The mechanism by which bavachalcone induce macrophage differentiation to M1 type may be related to changes in cell cycle,immune response,and TNF signaling pathway by transcriptomics.Conclusion:Bavachalcone can inhibit the proliferation and metastasis of lung cancer cells in vitro and in vivo,and has fewer side effects than the first-line chemotherapy cisplatin.Bavachalcone can inhibit the proliferation and metastasis of lung cancer cells through the typing of macrophages in the tumor microenvironment,can effectively increase the proportion of CD4~+T cell subsets,and the mechanism of bavachalcone inducing macrophage differentiation to M1 type may be related to the change of cell cycle,immune response and TNF signaling pathway,to delay the progression of lung cancer and provide new ideas for the prevention and treatment of lung cancer with traditional Chinese medicine. |
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