| Objective:To observe the effect of N-butanol Fraction of Wenxia Formula Extract(NWXF)on the growth,migration and invasion of non-small cell lung cancer cells in vivo and in vitro,and how it regulates the expression of MMP2,MMP9 and its related transcription factor Spl,so as to explore the anti-tumor and anti metastasis mechanism of NWXF,so as to provide theoretical support and experimental basis for the study of the mechanism of traditional Chinese medicine intervention on lung cancer metastasis.Method:1.The components of NWXF were detected by UPLC-Q-Orbitrap MS,and the detected compounds were analyzed qualitatively.2.MTT assays were used to observe the inhibitory effect of NWXF on the proliferation of three kinds of lung cancer cells(human lung adenocarcinoma cell line A549,human lung adenocarcinoma cell line H1299,human lung large cell lung cancer cell line H460)and a normal human bronchial epithelial cell(HBE).The migration and invasion ability of A549 and H460 cells were detected by scratch test,migration and invasion test(Transwell).The expression of MMP2,MMP9 and related transcription factor Sp1 in A549 and H460 cells were detected by Western blot.The binding activity of Spl with MMP2 and MMP9 promoters were detected by ChIP assay.3.Thirty BALB/c nude mice were subcutaneously inoculated with 0.2mL of A549 tumor cell suspension in the right anterior axilla to make A549 xenograft tumor model,and were randomly divided into five groups:NWXF high-dose group,NWXF medium dose group,NWXF low-dose group,positive control group(cisplatin group)and model group(solvent control group).Observe the general condition of the animals,measure and record the weight and tumor volume of the animals every three days,a total of 35 days of intervention.Immunohistochemistry and Western blot was used to detect the expression of MMP2,MMP9 and related transcription factor Sp1.Aim to evaluate the effect of NWXF on the growth and invasion of transplanted tumor.Result:1.UPLC-Q-Orbitrap MS analysis showed that there were 71 compounds(with mzCloud best match score greater than 85)in the NWXF,including esters,phenols,ketones,alkaloids,etc.The chemical constituents of Angelica sinensis are ferulic acid,chlorogenic acid and angelic acid.Emodin,aloe emodin and catechin are the chemical constituents of rhubarb.Aconitine is the main component of Aconitum carmichaeli.Ginsenoside Rb1 Ginsenoside Rg3 and quercetin are the chemical constituents of ginseng.This proved that NWXF ensured the good retention of the effective components in the decoction process.2.NWXF had inhibitory effect on the proliferation of A549,H460,H1299 and HBE cells,and the cytotoxicity to HBE was lower than that of other lung cancer cell lines.In vitro migration and invasion assays,the number of cells migrating and invading to the bottom of the chamber decreased in the low,medium and high dose groups of NWXF,and the inhibition of migration and invasion ability gradually increased with the increase of concentration,and the difference was statistically significant compared with the blank control group(P<0.05).Western blot results showed that NWXF could down regulate the expression of matrix metalloproteinases MMP2 and MMP9 in A549 and H460,and reduce the expression of transcription factor Sp1(P<0.05).ChIP results showed that NWXF could reduce the binding activity of Spl and MMP2 promoter in A549 and H460(P<0.05),but had no significant effect on the binding activity of Spl and MMP9 promoter in A549 and H460(P>0.05).3.In vivo experiments there were no significant differences in animal weight and spleen weight between the model group and each dose group(P>0.05),but the tumor weight was significantly lower than that of the model group(P<0.05).Compared with the model group,the expressions of MMP2,MMP9 and Sp1 in each dose group were decreased(P<0.05).Conclusion:The anti-tumor mechanism of NWXF may be related to its regulation of SP1 mediated MMP2 expression. |
