Study On The Mechanism Of Qilingchan Tablets Improving NSCLC Via Regulating Th17 Cell Differentiation And Interfering With IL-17/MAPK Signaling Pathway

Objective:To explore the potential pharmacodynamic mechanism of Qilingchan tablets(QLC)in improving the development and metastasis of non-small cell lung cancer(NSCLC),thereby providing more theoretical support for its clinical promotion and application.Methods:1.In order to better study the efficacy and mechanism of traditional Chinese medicine,the effective components of the three traditional Chinese medicines in Qilingchan were extracted in the early stage of this project,and after quality standard research and pilot scale-up experiments,a more controllable Qilingchan tablet was made;2.This project combined the method of systematic pharmacology with GEO analysis.First,search and screen the potential active ingredients of the three herbs in Qilingchan tablets through the network data platform,and conduct interactive analysis of the drug effect targets matched by each component and the NSCLC disease targets to obtain information on related biological processes,cell components,and signal pathways,then systematically explore the potential pharmacodynamic mechanism of Qilingchan tablets in the treatment of NSCLC;3.In in vitro experiments,A549 cells were used to observe the effects of Qilingchan tablets on the growth,proliferation,apoptosis,invasion,and migration of lung cancer.The CCK8 method was used to test the effects of Qilingchan tablets on the growth and proliferation of A549 cells;Annexin-V was used to detect the impact of Qilingchan tablets on A549 cell apoptosis;the IL-17/MAPK signaling pathway activators and inhibitors were used in the transwell and scratch experiments to explore whether the inhibitory effects of Qilingchan tablets on the invasion and migration of A549 cells were related to the IL-17/MAPK signaling pathway;the western blot and q RT-PCR methods were separately conducted to detect the protein and m RNA expression level of TRAF6,MAPK,pMAPK,MMP2,MMP9 under the intervention of Qilingchan tablets.4.In in vivo experiments,A549 cells were used to construct a subcutaneous tumor model with nude mice to observe the function of Qilingchan tablets on the growth and metastasis of lung cancer.Mice in the Qilingchan group were given different doses of Qilingchan tablets by gavage,and mice in the positive control group were given an intraperitoneal injection of DDP.In contrast,mice in the control group were given the same amount of normal saline gavage.Observing the general conditions of mice,measuring tumor volume,body weight,and tumor metastasis during the experiment;using flow cytometry to detect the distribution of Th17 cells in peripheral blood of mice;using immunofluorescence staining and immunohistochemistry to evaluate the pathological changes of mouse tumor tissue;western blot and q RT-PCR methods were used to separately detect the protein and m RNA expression level of TRAF6,MAPK,pMAPK,MMP2,MMP9 in tumor tissue.Results:1.The effective components of Qilingchan tablets meet the standards and are stable,which can pave the way for more in-depth basic and clinical research of Qilingchan;2.The results of systematic pharmacology and GEO analysis show that the three traditional Chinese medicine of Astragalus,Ganoderma,and Toadskin in Qilingchan contains 37 potentially active ingredients and 286 related drug targets.The potential pharmacodynamic mechanisms of Qilingchan tablets interfering with NSCLC mainly involve immune,inflammation processes,and signaling transduction pathways such as IL-17 and TNF signaling pathways are also involved;3.In vitro experiments showed that Qilingchan tablets could inhibit the proliferation of A549 cells and induce apoptosis.Among them,Qilingchan tablets of high dose exhibited superiority over the lower dose;meanwhile,IL-17 A could activate the MAPK signaling pathway and promote the invasion and migration of A549 cells,while Qilingchan tablets could inhibit A549 cell invasion and migration under the intervention of IL-17 A.At the same time,Qilingchan tablets can regulate IL-17/MAPK signaling pathway-related proteins at both protein and gene levels,such as TRAF6,pMAPK,MMP2,and MMP9,thus verifying that it can regulate the development of NSCLC diseases by regulating the IL-17/MAPK signaling pathway;4.In vivo experiments showed that the use of A549 cells could successfully construct a subcutaneous tumor model with nude mice,and the conditions of mice in the treatment groups were generally more stable than those in the control group;the weight loss of the mice in the treatment groups was less significant than that in the control group;The tumor volume and lung metastasis rate of mice in the treatment groups were significantly lower than that of the control group;combining the mouse peripheral blood flow cytometry,tumor tissue immunofluorescence and immunohistochemistry results,it could be concluded that Qilingchan tablets could regulate the differentiation of Th17 cells,reduce the expression of Th17 cells,and then reduce the tumor tissue IL-17 A expression.Meanwhile,Qilingchan tablets could interfere with the expression of TRAF6,pMAPK,MMP2,and MMP9 at both protein and gene levels,thereby affecting the development and metastasis of lung cancer via regulating the IL-17/MAPK signaling pathway.In addition,in comparison with a lower dose,the intervention effects of higher dose Qilingchan tablets were more significant.Conclusions:1.Qilingchan tablets could regulate the IL-17/MAPK signaling pathway and improve the proliferation,apoptosis,invasion,and metastasis of A549 cells;2.Qilingchan tablets could interfere with Th17 cell differentiation and regulate IL-17/MAPK signaling pathway to improve the development and metastasis of NSCLC disease.