The Inhibitory Effects Of FuzhengQuxie Formula And Its Component(-)-Guaiol In Lung Cancer Epithelial-Mesenchymal Transition

Objective FuzhengQuxie Formula is an effective compound for clinical treatment of lung cancer.However,its effective components and mechanism of action are still unclear.The purpose of this study was to investigate the potential application value and possible molecular mechanism of FuzhengQuxie Formula and its effective component(-)-Guaiol in the treatment of lung cancer.Methods A subcutaneous xenograft model of Lewis lung cancer mice was constructed to clarify the role of FuzhengQuxie Formula in suppressing lung cancer,and to explore its regulation of tumor-associated macrophages(TAM)in the immune microenvironment Then,in-depth study on the effective component of(-)-Guaiol,an effective component of FuzhengQuxie Formula,which was selected from the previous work of our group.First,it is clear that the anti-lung cancer effect of(-)-Guaiol is related to the M2 phenotype of tumor-associated macrophages in vivo,but not M1 phenotype.Secondly,M2 macrophage was successfully constructed in vitro.The optimal effect concentration of(-)-Guaiol was screened by CCK-8 assay.The inhibitory effect of(-)-Guaiol on M2 macrophage was detected by flow cytometry.Then,the co-culture system of macrophage and lung cancer cells was established in vitro.The proliferation,scratch and invasion experiments were used to observe the inhibitory effect of(-)-Guaiol on the biological behavior of lung cancer under co-culture system.Subsequently,bioinformatics analysis was used to screen the possible molecular mechanisms of M2 macrophages to promote lung cancer.The critical signal molecules of(-)-Guaiol were screened by ELISA.Real-time quantitative PCR and Western blotting were used to detect the expression of lung cancer epithelial-mesenchymal transition(EMT)markers,key signaling molecules and downstream signaling pathway molecules.Finally,using key signaling molecule antagonists to block their signaling.Observing the changes in the downstream signaling pathway and the markers of EMT.Verifying the molecular mechanism of(-)-Guaiol by inhibiting the function of M2 macrophage to suppress EMT in lung cancer.Results A subcutaneous xenograft model of Lewis lung cancer mice was established,and low-dose and high-dose FuzhengQuxie Formula were administered intragastrically.At the end of the experiment,compared with the model control group,low-dose and high-dose FuzhengQuxie Formula could effectively inhibit the subcutaneous xenograft volume(P<0.001)and tumor weight of Lewis lung cancer mice(P<0.05,P<0.001).Repressing epithelial-mesenchymal transition molecular markers,promoting E-cadherin RNA expression(P<0.001),inhibiting N-cadherin(P<0.05),Vimentin(P<0.001),and snail(P<0.001)RNA expression.It also inhibited the expression of M2 macrophage-associated mRNA Arg-1(P<0.01),IL-10(P<0.01),IL-13(P<0.001)and CD206 protein Second,construct a macrophage clearance model.The results showed that macrophages participated in the growth of tumors and promoted the growth of transplanted tumors in Lewis lung cancer mice(P<0.05).(-)-Guaiol can effectively inhibit the volume of transplanted tumors in Lewis lung cancer mice(P<0.05)and tumor weight(P<0.05).The tumor and spleen were further separated into single cell suspensions,and macrophage cells were identified by flow cytometry.The results showed that(-)-Guaiol significantly inhibited the expression of surface membrane molecule CD206 of M2 macrophages in tumors and spleen(P<0.001),and showed no significant regulation on M1 macrophages(P>0.05).Therefore,the M2-type macrophage-induced activation model was established in vitro.The CCK-8 assay showed that the IC50 of(-)-Guaiol on M2 macrophages and LLC cells was 232.1 ?M and 174.6 ?M,respectively.The morphology of M2 macrophages after(-)-Guaiol treatment was similar to that of uninduced activated macrophages.The results of flow cytometry showed that 40 ?M and 60 ?M(-)-Guaiol had significant inhibitory effect on M2 macrophages(P<0.001).Establish a co-culture system of macrophages and lung cancer cells in vitro,CCK-8 experiments show that(-)-Guaiol can inhibit the proliferation of lung cancer cells in co-culture system.Wound healing test shows that(-)-Guaiol can inhibit the migration ability of lung cancer cells in co-culture system.Transwell experiments show that(-)-Guaiol can inhibit the invasion ability of lung cancer cells in co-culture system.RT-PCR results showed that(-)-Guaiol could inhibit the epithelial-mesenchymal transition of lung cancer in co-culture system,increase the expression of E-cadherin RNA(P<0.001),and decrease the RNA expression level of N-cadherin and Vimentin(P<0.001).The results of Western Blot experiments showed that(-)-Guaiol increased the protein expression level of E-cadherin and decreased the protein expression levels of N-cadherin and Vimentin.Finally,through the GEO database and TCGA database analysis,the molecular mechanism of M2 macrophage acting on lung cancer may be through the secretion of signaling molecules involved in the regulation of cytokine receptor signaling pathway.It was confirmed by RT-PCR and ELISA that(-)-Guaiol could inhibit the RNA expression of IL-10 in M2 macrophages(P<0.05)and secretion level(P<0.05).Western Blot results showed that(-)-Guaiol inhibited the expression of IL-10 and p-STAT3 protein in lung cancer cells under co-culture conditions.IL-10 antagonist can significantly inhibit the expression of p-STAT3 in the downstream signaling pathway,and the epithelial-mesenchymal transition of lung cancer cells is reversed.In vivo,ELISA results showed that(-)-Guaiol reduced IL-10 levels in peripheral blood of mice(P<0.001).RT-PCR results showed that(-)-Guaiol inhibited IL-10 RNA expression in transplanted tumors(P<0.001),Western Blot results showed that(-)-Guaiol inhibited the expression of IL-10 and p-STAT3 protein in transplanted tumors.Conclusion 1.FuzhengQuxie Formula inhibited the growth and EMT process of subcutaneous xenografts in Lewis lung cancer mice model,and inhibited the phenotype and function of M2 macrophages.2.(-)-Guaiol can effectively inhibit the phenotype and function of M2 macrophage,thereby preventing the growth of subcutaneous xenografts in Lewis lung cancer mice model,and resricting the proliferation,migration and invasion of lung cancer in vitro.3.IL-10 is a critical signaling molecule for M2 macrophage-mediated EMT.(-)-Guaiol can effectively reduce the secretion of IL-10 by M2 macrophages.4.(-)-Guaiol inhibits the EMT process of lung cancer by targeting M2 macrophages,and the IL-10/STAT3 pathway is involved in the regulation of signaling pathway.