Study On Mechanism Of Pinellia And Ginger Synergistic Anti-proliferation And Pro-apoptosis Of Ovarian Cancer

OBJECTIVE:Pinellia and ginger are the common drug compatibility in Synopsis of the golden Chamber.And the main syndromes are different,the dosage,proportion,decocting method and taking method of pinellia and ginger are different.Pinellia and ginger are widely used in vomiting caused by various causes,such as vomiting after chemotherapy,pregnancy vomiting,and so on.At present,a large number of studies have shown that pinellia,ginger and their active ingredients have anti-tumor effects.Therefore,based on the previous work of Xiaobanxia Decoction on anti-chemotherapy vomiting,this study puts forward the following hypotheses:1.The compatibility of Pinellia and ginger also have a certain inhibitory effect on tumor growth;2.The compatibility of Pinellia and ginger has a certain synergistic effect on anti-tumor growth.Firstly,this study combed the literature of Pinellia and ginger,and deeply observed the antitumor mechanism of Pinellia and ginger by using SKOV3 cell line and experimental techniques such as network pharmacological screening,cell biology and molecular biology.METHOD:1.Literature research:Using pubmed,CNKI,Wan Fang,VIP and China biomedical database,with search terms such as"Pinellia","ginger","Pinellia","ginger","antitumor"terms,formulate search strategies according to different databases,and screen the collected literature,and summarize the antitumor properties of Pinellia,ginger and its active components,and which tumors can be inhibited.And verified by MTT method.2.Study on the synergistic effect of Pinellia and ginger:In order to explore what kind of compatibility proportion of pinellia and ginger has the most significant anti-tumor effect,First of all,the drug containing serum of pinellia,ginger,small pinellia soup（meridian,modern）,ginger Banxia decoction,pinellia dry ginger powder（water and slurry water）were prepared.CCK8 method was used to observe the ability of different compatibility to inhibit the proliferation of ovarian cancer cells,flow cytometry was used to observed its effect on inducing apoptosis,and WB techniques were used to observe the effect of pinellia and ginger on the expression of Bcl-2,Bax,and caspase-8 caspase-9,caspase-12,and caspase-3.The freeze-dried powder of Pinellia and ginger was prepared,and the IC50 values of Pinellia and ginger against SKOV3 cell proliferation was calculated by CCK8.according to the IC50value of Pinellia and ginger,Pinellia and ginger were mixed in different proportions to detect the inhibitory effect on SKOV3 cells under different compatibility conditions.Compusyn analysis software was used to calculate the combined index（CI）,inhibitory effect coefficient（Fa）,dose-reduction index（DRI）of Pinellia and ginger against SKOV3 cell proliferation,and to explore the dose and compatibility ratio of pinellia and ginger at different inhibitory effects.The flow cytometry and WB technology were used to observe the effect of pinellia and ginger on the induction of apoptosis under semi-inhibition conditions.The appropriate proportion was selected for the nude mouse model of ovarian cancer to observe its antitumor effect in vivo.3.Network pharmacology analysis and molecular docking:TCMSP database was used to obtain the main active components of Pinellia and ginger according to oral availability≥30%and drug likeness≥0.18,and the mol2 format of relevant active ingredient was obtained.Pharm Mapper database was used to obtain the relevant action target of pinellia and ginger.The Dis Ge NET and OMIM databases were used to collect related disease targets for ovarian cancer.The potential targets of pinellia and ginger acting on ovarian cancer were obtained through the intersection of the two,the common targets were uploaded to the STRING database for protein interaction analysis,and the"TCM-component-target-pathway"network was constructed using cytoscape software.The GO analysis and the KEGG analysis were performed using the KOBAS database.Select the interested signal pathways and potential targets,use autodock for molecular docking,select the binding mode with the lowest binding energy,and analyze its binding characteristics.4.experimental verification:SKOV3 cell was used to detect the effect of Pinellia and ginger on intracellular lipid peroxides and Fe²⁺were detected by immunofluorescence.The content of glutathione in cells was detected by enzyme-linked immunolabeling technology,the oxidative phosphorylation process of cell mitochondria and the function of complex I,Ⅱ,Ⅲwere detected by O2K technology,and the expression of PI3K/AKT/m TOR were detected by WB.RESULTS:1.Although there are few related studies,the anti-tumor research of Pinellia and ginger are also fully in line with the theory of traditional Chinese medicine.Moreover,the effects of active components of Pinellia ternata and ginger on tumor have also been widely studied.Among them,alkaloids,proteins,polysaccharides,cyclic dipeptides and volatile oil in Pinellia ternata showed antitumor activity;The anti-tumor activity of volatile oil and ginger oleoresin in ginger is relatively obvious.Literature research shows that Pinellia and ginger has inhibitory effects on colorectal cancer,liver cancer,ovarian cancer,cervical cancer,breast cancer and so on.MTT assay was used to detect SKOV3 cells,He La cells,A549 cells and Caco-2 cells.It was found that Pinellia and ginger had the most obvious inhibitory effect on SKOV3 cells.2.CCK8 method showed that the compatibility of Pinellia ternata and ginger had more obvious inhibitory effect on the proliferation of SKOV3 cells,and flow cytometry showed that the compatibility of Pinellia ternata and ginger had more obvious effect on inducing apoptosis（P<0.05）.WB detection showed that Pinellia ternata,ginger and compatibility groups had certain inhibitory effects on bcl-2,Caspase-3,8 and 9.（P<0.05）.CCK8 test showed that the IC50 values of Pinellia and ginger freez-dried powder inhibiting SKOV3proliferation were 125.3±15.4μg/ml（24h）and 95.15±9.63μg/ml（24h）,respectively.Compusyn analysis showed that Pinellia combined with different concentrations of ginger had obvious synergistic effect on promoting SKOV3 cell apoptosis,but ginger combined with different concentrations of Pinellia ternata did not show synergistic effect.The proportion of Pinellia ternata and ginger is different under different inhibition coefficient.When the inhibition coefficient is 0.5,the dosage of Pinellia ternata and ginger is 83.5ug/ml and 85.9ug/ml.Close to 1:1 ratio.3.Using TCMSP database,16 active components and 360 targets of Pinellia ternata and ginger were obtained according to the standard of OB>30%and DL>0.18.Using Dis Ge NET and OMIM database,2930 genes related to ovarian cancer were obtained.Pinellia ternata and ginger act on 99 genes related to ovarian cancer.KEGG analysis results show that"pathways in cancer"and"PI3K/Akt/m TOR"are the two most important signal pathways.The related targets in the"pathways in cancer"pathway are also closely related to PI3K/Akt/m TOR and oxidative stress.Molecular docking of related targets showed that many components could bind to related targets,and their binding sites were quite different,resulting in synergistic or antagonistic effects.4.Immunofluorescence showed that pinellia and ginger can significantly promote the production of ROS in SKOV3 cells,increase the content of intracellular iron ion,and reduce the ratio of GSH/GSSG（P<0.05）.The analysis of cell oxidative phosphorylation process showed that Pinellia and ginger had obvious inhibitory effect on complex I and II,which was also a high incidence site of free radical production.At the same time,the expression of NQO1 and GSTP1 decreased significantly.WB analysis of PI3K,Akt,m TOR showed that Pinellia and ginger could inhibit the phosphorylation of Akt and m TOR.（P<0.05）CONCLUSION:Ginger can synergistically promote the anti-proliferation and induced-apoptosis of Pinellia ternata to ovarian cancer SKOV3 cells,but there is no fixed compatibility ratio between them.When FA=0.5,the ratio of the two is 1∶1.The mechanism of its synergistic anti ovarian cancer may be related to the induction of ROS production,iron death and inhibition of PI3K signaling pathway.