The Mechanism Of Action Of Three Monomers On Lung Cancer Cells Based On Network Pharmacology

According to the statistics of the composition of Chinese medicinal materials in the previous period,kaempferol,p-coumaric acid and protocatechuic acid were obtained by screening.The mechanism of action of kaempferol,p-coumaric acid and protocatechuic acid on lung cancer was analyzed by network pharmacology,and experiments were designed according to the results of predictive analysis to explore the signaling pathways of the three drugs on H446 small cell lung cancer cells and H1299 non-small cell lung cancer cells and mechanism of action.To provide theoretical basis for the experimental research of kaempferol,p-coumaric acid and protocatechuic acid in the prevention and treatment of lung cancer.According to the signal pathway analysis of network pharmacology,there are 54 intersection targets of kaempferol and lung cancer,30 intersection targets of p-coumaric acid and lung cancer,and 76 intersection targets of protocatechuic acid and lung cancer.The primary target of kaempferol and p-coumaric acid on lung cancer cells is ESR1,while the primary target of protocatechuic acid on lung cancer cells is RARB.The results of Clu GO signaling pathway analysis showed that the three monomers have important effects on the estrogen signaling pathway and the development of non-small cell lung cancer.The signaling pathway results of the Kyoto Encyclopedia of Genes and Genomes KEGG showed that 13 of the 16 core targets were located in the cancer signaling pathway.The targets involved are JUN,ESR1,RXRB,RXRG,RXRA,RARA.ESR1 and ESR1 complex can indirectly or directly up-regulate the expression of Cyclin D1 protein in the cell cycle,while RARB can inhibit the activation of estrogen signaling pathway.Molecular docking results showed that when kaempferol was docked with RARB,two hydrogen bonds were formed at GLU-165,and when protocatechuic acid was connected to ESR1 and RARB,two hydrogen bonds were formed at GLU-71 and GLU-180,respectively.Experiments were designed according to the results of network pharmacology analysis.First,the CCK-8 experiment was performed to detect the toxic effects of the three monomers on H446 and H1299 cells,and the half-inhibitory concentration was calculated,and the half-inhibitory concentration was used as the dosage for subsequent experiments.The effect of three monomers on cell migration was studied by cell scratch test,the effect of three monomers on cell growth was judged by HE staining,and the effect of the three monomers on apoptosis of lung cancer cells was observed by Hochest/PI experiment.According to the cell growth state,the effect of monomers on the expression of cell proliferation-related proteins was detected by immunofluorescence.Finally,Western Blot experiments were performed to detect the effect of monomers on the expression of related proteins in the estrogen signaling pathway of H446 cells.According to the signal pathway analysis of network pharmacology,the primary target of kaempferol and p-coumaric acid on lung cancer cells is ESR1,while the primary target of protocatechuic acid on lung cancer cells is RARB.According to the signal pathway results of KEGG,the Kyoto Encyclopedia of Genes and Genomes,ESR1 and ESR1 complex can indirectly or directly upregulate the expression of Cyclin D1 protein in the cell cycle,while RARB can inhibit the activation of estrogen signaling pathway.Molecular docking results showed that when kaempferol was docked with RARB,two hydrogen bonds were formed at GLU-165,and when protocatechuic acid was connected to ESR1 and RARB,two hydrogen bonds were formed at GLU-71 and GLU-180,respectively.According to the results of HE staining,the coumaric acid group and DMSO had little effect on the cells,which were thin and plump and grew in good condition.The cells in the kaempferol group were similar to those in the protocatechuic acid group,with cell death,most cells were fibrous,and some cells were shrinking.The scratch experiment showed that the reduction ratio of the scratch area of Kaempferol,p-coumaric acid and protocatechuic acid was significantly lower than that of the CK control group at the IC50 concentration.There is a decrease in the ability of cells to migrate.The apoptosis and immunofluorescence experiments showed that the three monomers all promoted the apoptosis of H446 cells,while p-coumaric acid and protocatechuic acid inhibited the proliferation of cells.Western Blot results showed that after kaempferol acted on H446 cells,the expression of SP1 was up-regulated,and the expression of ESR1 was down-regulated.The overall regulation made no significant change in the expression of downstream CCND1 protein,while the expression of Bcl-2was down-regulated.The protein expression was slightly different,which may be caused by the expression of proliferation-related proteins regulated by other pathways.Protocatechuic acid relatively up-regulated the expression of SP1 and down-regulated the expression of ESR1.The overall regulation made the expression of downstream CND1 up-regulated and the expression of Bcl-2 down-regulated.The results echo the results of immunofluorescence and apoptosis.Kaempferol down-regulated the expression of SP1 and up-regulated the expression of CCND1,while kaempferol and ESR1 were down-regulated at low and high concentrations,but up-regulated at moderate concentrations.P-coumaric acid down-regulated ESR1,CCND1 and Bcl-The expression of 2,that is,p-coumaric acid inhibited the proliferation of H1299 cells and promoted their apoptosis.Protocatechuic acid up-regulated the expression of SP1 and down-regulated the expression of Bcl-2.The results show that kaempferol,p-coumaric acid and protocatechuic acid affect cell proliferation and apoptosis through estrogen signaling pathway;ESR1,SP1 and coenzyme form a conjugate,which together regulate the target of downstream CCND1 gene.In vitro studies have shown that p-coumaric acid and protocatechuic acid inhibit cell proliferation;kaempferol,p-coumaric acid and protocatechuic acid promote apoptosis of lung cancer cells.The expression of ESR1 and SP1 proteins was inconsistent with the expression trend of CCND1 and Bcl-2.Further combined with the signal pathway analysis results of network pharmacology,some of the core targets of the three monomers are also located in the calcium signaling pathway,the MAPKA signaling pathway and the PIK/AKT signaling pathway,which may regulate the proliferation and progression of lung cancer cells.apoptosis.