Study On The Mechanism Of Fuzheng Kangai Decoction Containing Serum On The Proliferation And Apoptosis Of Ovarian Cancer HO-8910 Cells

Purpose: To study and predict the potential targets,signal pathways and possible mechanisms of FZKAD in the treatment of ovarian cancer,explore the effects of FZKAD on the proliferation inhibition and apoptosis of ovarian cancer HO-8910 cells,and verify the FZKAD-mediated ovarian cancer HO-8910 cell apoptosis and expression of death-related proteins reveals.However,FZKAD treatment of ovarian cancer may be achieved by regulating the apoptosis of cancer cells.Material and method: The research is divided into three parts,the network pharmacology study of FZKAD in the treatment of ovarian cancer;the experimental study of FZKAD-containing serum on the proliferation and apoptosis of ovarian cancer HO-8910 cells;the experiment of the mechanism of FZKAD-mediated ovarian cancer HO-8910 cell apoptosis.The study details as follows: Experiment One 1.Build the database Enter "Red Ginseng","Licorice","Atractylodes","Zhe Fritillary","Pan Xia","Chenpi","Fructus Aurantii","Oldenlandia diffusa","Branch lotus","Tuckahoe","Bamboo Ru","Ginger",sifted out the active ingredients.Enter into the Drug Bank and Pub Chem database to obtain the target of traditional Chinese medicine.Enter "Ovarian Cancer" and "Ovarian Carcinoma" in Gene Cards and OMIM databases to obtain target genes for ovarian cancer disease.2.Data network analysis: Merge the self-built Chinese medicine and disease target library,take the intersection to get the candidate target,and use the STRING database to construct the PPI network diagram.Download the tsv format of the PPI results,count the targets,and take the intersection with the PPI network results again to get the drug-disease core target.Using the "Cluster Profiler" package of the R software,the GO and KEGG pathway enrichment analysis were performed respectively,and the traditional Chinese medicine-component-target-pathway network was successfully constructed.Experiment Two 1.Medicinal serum preparation and cell culture Twenty SPF SD rats were randomly selected from 10 labeled drug-containing group(FZKAD concentrated drug),and the other 10 labeled with control group(normal saline),and gavage simultaneously once a day for 10 days.At the end of the administration,4 hours were counted.After anesthesia,blood was taken and serum was prepared for use.Ovarian cancer HO-8910 cells are cultured in an incubator(saturated humidity,37°C,5% CO2),with a cell coverage of 95%,and passage 1:2 for later use.2.Study on Proliferation and Apoptosis of HO-8910 Cells Take 10% different concentrations of drug-containing serum,include 4.6g/ml,2.3g.ml,1.53g/ml,0.92g/ml,0.51g/ml,set up experimental groups 1-5.And take 10% blank serum as blank control group,intervention HO-8910 cells.At different time points of 12 h,24h,and 48 h,the cell proliferation was detected by the CCK8 method.Flow cytometry was used to detect the rate of apoptosis.3.The expression of FAS and FASL was detected by immunocytochemistry.Western-blot method was used to detect the expression of P53 protein.Results: Experiment One 1 Search the TCMSP,BATMAN-TCM,Drug Bank,and Pub Chem databases for 12 FZKAD Chinese herbs.Zhuru did not get the relevant ingredients and target proteins.The remaining 11 flavors excluded invalid molecules,removed duplicates,and obtained 171 valid molecules.Matched 441 target proteins.In the Gene Cards and OMIM databases,duplicate entries were removed and genes with Gifts values of 50 or higher were selected to obtain 286 disease genes.We have integrated drug discovery target proteins and disease target genes to obtain 93 important genes.2 Predictive analytics of R software found P53,AKT1,CASP3,STAT3,EGFR,EGF,CCND1,CASP8 and FAS,which are the core targets of FZKAD in the treatment of ovarian cancer.Core pathways include PI3K-AKT,MAPK,Fox O,HIF-1,AGE-RAGE signaling pathways and so on.Experiment Two 1 The result of HO-8910 cell proliferation Proliferation of HO-8910 cells in groups 1-5 changed with time and concentration of drug-containing serum.For 48 hours in the 4.6 g / ml intervention group,the cells received the strongest growth inhibition,with an inhibition rate of 35.5%.This was statistically significant compared to each group(P <0.05).2 The result of HO-8910 cell apoptosis The data show that the apoptosis rates in experimental groups 1-5 changed compared to the control group.There is no statistical significance between Group 1 and 2,Group 3 and 4(P> 0.05).However,there are Academic significance statistics in other groups(P <0.05)..The maximum apoptosis rate of HO-8910 cells was 36.5±3.65% at a concentration of 4.6 g / ml.3 Results of FAS and FASL expression in cells Immunohistochemical detection of FAS expression is predominantly in the cell membrane,with small amounts of free FAS in the cytoplasm.FASL can be expressed in the envelope and cytoplasm.The brown-yellow particles were more pronounced in experimental groups 1-3 and the distribution in groups 4-5 was reduced.4 The relationship between the concentration of drug-containing serum and the expression of FAS/FASL FAS and FASL were expressed in drug-containing serogroups 1-5 compared to the control group.Expression of FAS and FASL was most pronounced in Experimental Group 1 and the difference was statistically significant(P <0.05).The distributions of FAS proteins,brown-yellow particles in groups 2 and 3 were not very different,and the comparison was not statistically significant(P> 0.05).Intracellular FAS expression decreased with decreasing serum levels in the other groups,which was statistically significant(P <0.05).Expression of FASL proteins in groups 2,3,and 4 was reduced in the cytoplasm and envelope,resulting in lighter color.There were no statistically significant comparisons between the groups(P> 0.05).Very few light brown particles were seen in Group 5,which was not much different from the blank group and was not statistically significant in comparison(P> 0.05).5 Results of P53 expression in cells Western blots detect P53 protein.Compared to ?-actin in the reference group,the control group lacks a clear band and P53 expression is inadequate.In experimental groups 1 to 5,a black-gray band was observed and P53 protein was expressed.As the concentration of drug-containing serum decreased,the expression of P53 in HO-8910 cells decreased.6 The relationship between the concentration of drug-containing serum and the expression of P53.The concentration of drug-containing serum in experimental groups 1-5 decreased,and the expression of P53 in HO-8910 cells decreased.Differences between groups were statistically significant(P <0.05).That is,P53 expression in HO-8910 cells is upregulated when serum levels containing the FZKAD drug are high.Conclusion: 1.The core target genes for FZKAD in the treatment of ovarian cancer are P53,AKT1,CASP3,STAT3,EGFR,EGF,CCND1,CASP8 and FAS.And the core signaling pathways are PI3K-AKT,MAPK,Fox O,HIF-1 and AGE-RAGE.The potential mechanism of action includes growth,proliferation,inflammation,apoptosis,angiogenesis,etc.2.FZKAD inhibits the proliferation of HO-8910 cells,promotes apoptosis,and has a positive correlation with the concentration and intervention time of drug-containing serum.3.FZKAD can up-regulate the expression of FAS and increase the binding with FASL when cell protein is expressed,to affect the death receptor signaling pathway and make HO-8910 cells achieve apoptosis.4.FZKAD positively regulates the expression of P53,directly inhibits the proliferation of HO-8910 cells and induces their apoptosis.5.Among the core targets obtained by network pharmacology,P53 and FAS can induce the apoptosis of HO-8910 cells.And the results of network pharmacology analysis have reference value.