Studies On The Anti-colon Cancer Effect And Mechanism Of Pomolic Acid And Glucopyranose Ester

Objective:In this study,cell experiments in vitro,network pharmacological,proteomics,western blot and animal experiments in vivo were performed to explore the anti-tumor effect and their mechanism of active monomer components separated from Achyrocline satureioides(Lam.)DC in colon cancer.Methods:①The MTT assay was performed to explore the anti-colon cancer effect of active monomer components separated from Achyrocline satureioides(Lam.)DC:After chemical extraction,separation and purification,6 monomer components with high content were obtained from Achyrocline satureioides(Lam.)DC.MTT assay was performed to observe the effect on proliferation of colon cancer cell HT-29 of the 6 monomer components,the active monomer would be obtained as the research object for anti-colon cancer later.②Cell experiments in vitro were performed to study the anti-tumor effect of PA and Pomolic acid-28-O-beta-D-glucopyranosyl ester(PAO)in colorectal cancer:MTT assay was to observe the proliferation of HT-29 cells treated by PA and PAO,and the cell morphology was observed under an inverted microscope.Honest 33342 staining,AO/EB staining and AnnexinV-FITC/PI flow cytometry were used to observe the apoptosis of cells treated with PA,and PI single staining flow cytometry was used to determine the cell cycle changes of distribution caused by PA;Scratch experiments were performed to observe the migration of HT-29 cells.③Network pharmacological analysis was used to get the targets of PA in colorectal cancer:Based on the big data platforms,the potential targets of PA and PAO were collected and the disease targets were screened in colon cancer,the predicted targets of above of both were intersected by Venn and predicted target protein interactions network were established by string;The biological function and pathways of the targets were obtained by enrichment analysis.Finally,the PA and PAO molecules were docked with the core target to validate the targets.④Cell proteomics was performed to obtain the possible targets of PA and PAO:After protein extraction,alkylation,enzymolysis,desalting,LC-MS sample loading and preliminary screening of differential proteins,differential proteins were obtained.Biological function analysis and pathway analysis were performed,and finally the differential proteins related to cancer,apoptosis and cycle were screened.⑤Western blot assay was performed to detect and verify the targets:Based on the results of the above cell experiments in vitro,network pharmacology and proteomics experiments,the proteins Bax,Bcl2,Caspase 3,Jak1,Stat3,p-stat3 and autophagy related proteins LC3Ⅱ,LC3Ⅰ,Beclin1 and p62 were detected by western blotting.⑥The animal experiments in vivo were performed for a verification:a colon cancer cell HT-29 cell suspension was injected subcutaneously into the right forelimb of a nude mouse as a tumor source,and the second-generation tumor source was used to inoculate the tumor mass to construct a heterotopic xenograft tumor model.On the third day of inoculation of the tumor,the animals were administered orally once a day.After 21 consecutive days of administration,the animals were sacrificed by cervical dislocation.And the tumor volume and tumor weight were measured,and the tumor tissue and liver tissue were stained with HE to observe the pathological changes.Results:①The components of pomolic acid,1-(2-methylbutanone)-3-isopentene-resorcinol,ursolic acid and quercetin-7-O-β-D-glucoside showed inhibitory effects on HT-29 cells at the experimental concentration.Among them,PA showed the strongest anti-colon cancer effect.②Both PA and PAO could inhibit the proliferation of HT-29 cells in a time-dose-dependent manner in vitro.After drug administration,the HT-29 cells were more sparse,and the cell body is more slender than the control group.The survival rate of cell in the PA group was significantly reduced;Both PA and PAO could significantly promote the apoptosis of HT-29 cells,and a large number of HT-29 cells were arrested in the G0/G1 phase,cell scratch experiments showed that PA and PAO could significantly reduce the rate of scratch healing.③Network pharmacological analysis showed that the core targets of PA and PAO were ERBB2,PPARG,PTGS2 and TNF;MAPK3,STAT3 and MTOR.Further molecular docking results showed that PA can stably bind to ERBB2,PTGS2,and TNF;PAO can stably bind to MAPK3 and STAT3.That showed that ERBB2,PPARG,PTGS2 and TNF;MAPK3 and STAT3 are the targets of these substances.④Proteomics studies showed that after PA and PAO drugs treating to HT-29 cells,11 differential proteins were obtained,including 6 up-regulated proteins and 5 down-regulated proteins in PA group;13 differential proteins were obtained,of which 1 was up-regulated and 12 were down-regulated in the PAO group.There were five proteins GSTP1,MCM7,mTOR,TAB2,XPO1 with the same expression between PA and PAO group.These targets were involved in signal pathways including mTOR,autophagy-related,and cell cycle.⑤Western blot experiments showed that PA could increase the Bax/Bcl2 ratio and up-regulate the relative expression of proteins Caspase 3,LC3Ⅱ/Ⅰ,Beclinl and p62;but there is no effect on the relative expression of protein Jak1.Although it down-regulates the expression of Stat3 protein,there is no statistical difference:PA could can decrease the relative expression of protein p-stat3.PAO could also increase the Bax/Bcl2 ratio,increase the relative expression of p62 protein;can up-regulate the expression of Jakl protein,there is a significant difference at 48h,can decrease the relative expression of Stat3 protein,the relative expression of p-stat3 protein was down-regulated(the western blot results here are basically consistent with the above-mentioned network pharmacology and proteomics results,which are mutually confirmed).Conclusion:PA is one of the strongest monomer components on anti-colon cancer effect separated from Achyrocline satureioides(Lam.)DC.The mechanism of PA in anti-colon cancer action may be related to increasing the relative expression of protein Bax/Bcl2 and Caspase 3,and cause HT-29 cells apoptosis;The relative expression levels of autophagy-related protein LC3Ⅱ/Ⅰ,Beclinl and p62 proteins were up-regulated,which promote the occurrence of autophagy and lead cell to death;The mechanism of PAO anti-colon cancer is related to increasing the relative expression levels of Bax/Bcl2 and down-regulating the relative expression of protein Stat3.