| Objective:In vitro experiments were conducted to study the intervention effect and mechanism of Yishen Gukang Decoction on the proliferation and metastasis of lung cancer cells,and to provide experimental basis for further study of the anti-tumor effect of Yishen Gukang Decoction.Methods:Experiment 1 Effect of Yishu Gukang Decoction on the proliferation of lung cancer A549 cells:lung cancer A549 cells were taken and inoculated in a 96-well culture plate.Different concentrations of Yishu Gukang Decoction were added.After 24,48 and 72 hours of culture,CCK-8 method was used to detect the absorbance value,calculate the cell survival rate,and screen out the appropriate intervention concentration of Yishu Gukang Decoction in the low-dose and high-dose groups.Experiment 2 Effect of Yishu Gukang Decoction on the migration of lung cancer A549 cells:lung cancer A549 cells were taken and inoculated in a 6-well culture plate.After the cells were fully grown,3 scratches were made with the head of a spear.The intervention was divided into four groups:control group,Yishu Gukang Decoction low-dose group,Yishu Gukang Decoction high-dose group,and cyclophosphamide group.The width of the scratches was recorded under an inverted optical microscope at 0,24 and 48 hours,and the healing rate of the scratches was calculated.Experiment 3 Effect of Yishu Gukang Decoction on the Adhesion of Lung Cancer A549 Cells:Matrigel matrix glue was spread on the 96-well culture plate,and the cells of the 4 groups after intervention(control group,Yishu Gukang Decoction low-dose group,Yishu Gukang Decoction high-dose group,and cyclophosphamide group)were inoculated into the culture plate,respectively.After 1 hour,the unattached cells were absorbed,the absorbance value was detected by CCK-8 method,and the cell adhesion rate was calculated.Experiment 4 Effect of Yishu Gukang Decoction on invasion of lung cancer A549 cells:the upper compartment of Transwell compartment was coated with Matigel.The cells of the 4 groups after intervention(control group,Yishu Gukang Decoction low-dose group,Yishu Gukang Decoction high-dose group,and cyclophosphamide group)were inoculated into the upper chamber,and the medium was added into the lower chamber.After 24 hours,the upper compartment was removed,fixed,stained,photographed and cells in the lower compartment were counted.Experiment 5 Effect of Yishu Gukang Decoction on the morphology of lung cancer A549 cells:A549 cells were seeded on the cell slides in the 96-well culture plate,and divided into two groups(control group and Yishu Gukang Decoction group)for 24 hours.The treatment was fixed,dehydrated,dried,sprayed with gold,and observed by scanning electron microscope and photographed.Experiment 6 Effect of Yishu Gukang Decoction on lung cancer cells A549 cell transcriptome:the experimental points 2 group(control group and Yishu Gukang Decoction group)intervention for 24 hours,using the transcriptome sequencing technology,after Chinese medicine intervention differentially expressed genes,and through the GO(gene ontology)and KEGG(Kyoto encyclopedia)gene and genome analysis of differentially expressed genes function of enrichment.Cytoscape3.6.0 software was used to construct the differentially expressed gene protein interaction network to identify the key genes related to tumor metastasis.Results:Experiment 1:Starting from the intervention concentration of Yishu Gukang Decoction at 1 mg/mL,with the increase of drug concentration and action time,the inhibition of A549 cell proliferation gradually enhanced,showing a certain time and concentration dependent effect relationship.The optimal half inhibitory concentration was 4.18 mg/mL(24 h).In order to further observe the effect of Yishu Gukang Decoction on the migration,adhesion and invasion of lung cancer cells,Yishu Gukang Decoction with concentrations of 1 mg/mL and 4.18 mg/mL were used for intervention in subsequent experiments,which were the low-dose and high-dose groups of Yishu Gukang Decoction.Experiment 2:24 h,48 h compared with controls,Yishu Gukang Decoction low and high dose group and cyclophosphamide group cell migration rate were significantly decreased(P<0.01),and Yishu Gukang Decoction migration rate is lower than the high dose group Yishu Gukang Decoction low dose group(24 h:P<0.05,48 h,P<0.01),while the cyclophosphamide group cell migration rate is lower than the Yishu Gukang Decoction low and high dose group(P<0.01).Experiment 3:Compared with the control group,the adhesion ability of A549 cells to matrix was significantly inhibited in Yishu Gukang high-dose group and cyclophosphamide group,with inhibitory rates of 64.15%(P<0.01)and 36.56%(P<0.01),respectively.Compared with Yishu Gukang decoction low-dose group,the cell adhesion rate of Yishu Gukang decoction high-dose group was decreased(P<0.01),and compared with cyclophosphamide group,the cell adhesion rate of Yishu Gukang decoction high-dose group was decreased(P<0.01).Experiment 4:Compared with the control group,the number of invaded membrane cells in Yishu Gukang Decoction low-dose group,Yishu Gukang high-dose group and cyclophosphamide group were significantly reduced(P<0.01),and there was no statistical significance in the number of invaded cells between Yishu Gukang decoction low-dose group and Yishu Gukang high-dose group and cyclophosphamide group.Experiment 5:Scanning electron microscope results showed that the cells in the control group were normal and full,with wrinkled membrane and filopodia.In Yishu Gukang Decoction group,the wrinkle of cell surface membrane was significantly reduced,the filopodia was broken and the apoptotic bodies were formed.Experiment 6:Differences in genetic screening | log2FoldChange |>1.There are 440 differentially expressed genes,which cut gene has 136,raised 304 genes.One gene closely related to cancer,SOX2,was down-regulated in the Yishu Gukang Decoction group.A total of 307 results were obtained during GO functional enrichment,mainly involving biological processes(237 items),cellular components(56 items)and molecular functions(14 items).KEGG pathway analysis showed that the mechanism related to the inhibition of Yishu Gukang Decoction on lung cancer A549 cells mainly involved apoptosis,inflammation,immunity and other aspects.Through the construction of protein interaction network,six genes with the highest node degree,MMP9,MMP2 were found.Conclusion:1、Yishu Gukang Decoction may inhibit the growth and metastasis activity of lung cancer cells by reducing the proliferation,migration,invasion and adhesion of lung cancer A549 cells and promoting the apoptosis of tumor cells.2、The mechanism of Yishengukang Decoction in inhibiting the metastasis of lung cancer A549 cells may be related to the down-regulation of SOX2,MMP9 and CC12 gene expression. |
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