Study On The Mechanism Of Zeqi Decoction In Inhibiting Non-Small-Cell Lung Carcinoma

Objective:Zeqi decoction is a kind of Chinese herbal compound which has been proved to be effective in the treatment of lung cancer,but its molecular mechanism is not clear.In this study,non-small-cell lung carcinoma cell lines H1299,H1650 and A549 were used to investigate the effects of Zeqi decoction on proliferation,migration and iron death of non-small-cell lung carcinoma in vitro,to clarify the molecular mechanism of Zeqi decoction in inhibiting non-small-cell lung carcinoma,and to provide theoretical and experimental basis for Zeqi decoction in treating non-small-cell lung carcinoma.Methods:1.Mass spectrometryThe samples of Zeqi tang were separated by UPLC Exion LCTM AD and Agilent Technologies SB-C18 column,and then analyzed by AB SCIEX High-performance liquid chromatography trap mass spectrometer(AB SCIEX).2.To study the effect of Zeqi soup on non-small-cell lung carcinoma in vitroH1299,H1650 and A549 non-small-cell lung carcinoma cell lines were used as control and treatment groups.(1)MTT assay was used to detect the effect of Zeqi Tang on the proliferation of H1299,H1650 and A549 cells.(2)cell scratch test and Transwell method were used to detect the effect of Zeqi decoction on the migration of H1299,H1650 and A549 cells.(3)The cell cycle of H1299,H1650 and A549 cells was Flow cytometry.(4)Cell senescence kit was used to detect the effect of Zeqi decoction on the senescence of H1299,H1650 and A549 cells.(5)Reactive oxygen species kit and q-PCR was used to detect the effect of Zeqi decoction on iron death of H1299,H1650 and A549 cells.3.Study on the molecular mechanism of Zeqi decoction inhibiting non-small-cell lung carcinomaNetwork pharmacology,RNA sequencing,q-PCR and western blotting were used to explore the potential targets and mechanism of Zeqi decoction in inhibiting non-small-cell lung carcinoma.TCMSP and Herb databases were used to screen the active compounds from the Chinese medicinal herbs,Pinellia ternata and Iwami Province,and Herb,swisstargetprediction and SEA databases were used to screen the related targets.The Genecard and OMIM databases were used to screen out non-small-cell lung carcinoma disease targets,which were then interacted with the selected drug targets.The network of protein interactions and signaling pathways was constructed using the String and David Databases.Three non-small-cell lung carcinoma cell lines,H1299,H1650 and A549,were divided into control group and treatment group.The total RNA of the extracted samples was sent to Meiji Biological Company for RNA sequencing and the experimental results were analyzed.The gene expression levels of GPX4 and ACSL4 in H1299,H1650 and A549 cells were detected by q-PCR.The protein expression levels of CCND1、CDC45、MMP2、MMP9、PTGS2、GPX4 和 SLC7A11 in H1299 and A549 cells were detected by Western blot.Results1.Analysis of components in Zeqi decoction by mass spectrometryA total of 1067 metabolites were detected in Zeqi decoction sample(lyophilized powder solution),and 9 substances were classified as first class,flavonoids,phenolic acids,alkaloids,terpenoids,quinones,lignans and coumarins,tannins,steroids and others,the top 10 ingredients were gallic acid acid,Glucuronic acid acid,Melastin A-7-O-Glucuronic acid,Baicalin(wogonin-7-O-o-glycoside),acacetin-7-O-Glucuronic acid,3-indoleacrylic acid,4-O-gallic acid,3-amino-2-naphthylformic acid,Baicalin and n-phenylene isomethylamine,and material abundance ratio was 12.1 million,7.9 million,5.9 million,5.7 million,5.7million,4.9 million,3.2 million,3.3 million,2.9 million,2.4 million,1.7 million,and4.3 million.Its main components are from Ze qi,Ban xia,Shi jianchuan,Huang qin and other traditional Chinese medicines.2.Experimental results in vitro(1)MTT assay showed that Zeqi decoction significantly inhibited the proliferation of non-small-cell lung carcinoma cell lines H1299,H1650 and A549(p< 0.001),and with the increase of the concentration,the inhibitory effect was more strong,in a dose-dependent manner.(2)the results of cell scratch test showed that Zeqi decoction significantly inhibited the migration of H1650 and A549(P< 0.001);zeqi decoction significantly inhibited the longitudinal migration of H1299,H1650 and A549 by tanswell test(P <0.001).(3)The Flow cytometry results showed that Zeqi decoction inhibited H1299,A549 and H1650 in G1 phase and effectively inhibited the proliferation of tumor cells.(4)the results of cell senescence experiment showed that the number of H1299,A549 and H1650 cells in Zeqi group was significantly more than that in control group,which promoted the formation of cell senescence.(5)The results of reactive oxygen species(ROS)experiment showed that the ROS accumulation in H1299,H1650 and A549 cells of Zeqi group was higher than that in the control group.According to the results of cell senescence experiment,Zeqi decoction may induce the death of lung cancer cells by iron and inhibit the proliferation of lung cancer cells..3.Molecular mechanism of Zeqi decoction in inhibiting non-small-cell lung carcinomanetwork pharmacological analysis showed that there were 59 common targets between lung cancer and Zeqi tang.The protein interaction network diagram showed that EGFR,AKT1,ESR1,KDR,PIK3R1,MMP2 were the key proteins,p I3K-Akt signaling pathway and EGFR tyrosine kinase inhibitor resistance are the main signaling pathways.A total of 39479 genes and 204245 transcripts were identified by RNA sequencing,including 179058 known transcripts,and 189,137 and 384 genes were identified as H1299,H1650 and A549 respectively.KEGG analysis of replication and repair,cell growth and death,endocrine system and cancer overview;go analysis shows that Celluar process,cell part and binding are its main functions.qPCR results showed that GPX4 gene expression was down-regulated in non-small-cell lung carcinoma cell lines H1299,H1650 and A549 treated with Zeqi decoction.The expression level of ACSL4 gene was up-regulated in non-small-cell lung carcinoma cell lines H1299,H1650 and A549,with statistical significance.WB results showed that the expression levels of CCND1,CDC45,MMP2,MMP9,PTGS2,GPX4 and SLC7A11 in H1299 and A549 cells were significantly down-regulated after treatment with Zeqi decoction.Conclusion:1.Zeqi decoction is mainly composed of flavonoids,phenolic acids,alkaloids,terpenoids and other substances.The higher concentrations are those with anti-cancer effects,such as brevifolin carboxylic acid,gallic acid,baicalin and other substances.2.Zeqi decoction can effectively inhibit the development of non-small-cell lung carcinoma,inhibit the proliferation and migration of H1299,H1650 and A549non-small-cell lung carcinoma cell lines,arrest cell cycle,and promote the process of cell senescence and iron death,and showed a dose-dependent.3.Network pharmacological screening of target genes was similar to RNA sequencing differential genes.The protein interaction network between Zeqi decoction and non-small-cell lung carcinoma revealed that the key proteins were EGFR,AKT1,ESR1,KDR,PIK3R1,MMP2,etc.PI3k-Akt and EGFR tyrosine kinase inhibitor resistance signaling pathways may be involved in the non-small-cell lung carcinoma.qPCR results showed that Zeqi tang promoted the iron death of H1299,H1650 and A549 cells.WB results showed that Zeqi inhibited the migration of non-small-cell lung carcinoma cells,blocked cell cycle and promoted iron death.Zeqi decoction regulates the gene expression of H1299,H1650 and A549,mainly in cell growth and death,endocrine and cancer,DNA replication and repair.