Study On The Mechanism Of Xiaoyan Decoction Combined With Apatinib Mesylate On Gastric Cancer

Objective:To investigate the effects of Xiaoyan Decoction and Xiaoyan Decoction combined with Apatinib Mesylate on human gastric cancer MGC-803 cells and MFC gastric cancer bearing mice and its mechanism.Method:1.Network pharmacological study on Xiaoyan Decoction in the treatment of gastric cancer:The active components and targets of Xiaoyan Decoction were screened by TCMSP,TCMID and BATMAN-TCM databases;Using Drug Bank,Gene Cards and OMIM databases to mine gastric cancer disease targets;The intersection gene of Xiaoyan Decoction and gastric cancer was obtained by combined screening;Using Cytoscape 3 7.2 draw the network diagram of drug compound intersection target with software;Using String data analysis platform to build PPI network,and using MCODE plug-in to analyze PPI network cluster module;Using Metascape database for KEGG enrichment analysis and GO enrichment analysis;Using Kaplan Meier plotter database to explore the impact of key gene targets on OS in patients with gastric cancer and draw the survival curve.2.Study on the biological behavior of Xiaoyan Decoction on human gastric cancer MGC-803 cells:The effects of different concentrations of Xiaoyan Decoction on the proliferation of human gastric cancer MGC-803cells were detected by CCK-8,and the effects of different concentrations of Xiaoyan Decoction on the apoptosis and cycle of human gastric cancer MGC-803 cells were detected by flow cytometry.3.Antitumor effect and mechanism of Xiaoyan Decoction combined with Apatinib on human gastric cancer MGC-803 cells:CCK-8 was used to detect the effects of Blank group,Xiaoyan Decoction group,Apatinib group and Combined group on the proliferation of human gastric cancer MGC-803 cells;The expression levels of p-PI3K,p-AKT,Cleaved Caspase-9,Bax,Bcl-2,VEGFA and VEGFR-2 in MGC-803 cells after intervention were detected by Western blot.4.Antitumor effect and mechanism of Xiaoyan Decoction combined with Apatinib on MFC gastric cancer bearing mice:25 BALB/c mice were randomly divided into5 groups(n=5).Blank control group,Tumor bearing model group,Xiaoyan Decoction group,Apatinib group and Combined group were set up.Except for Blank control group,the other 20mice constructed MFC gastric cancer mouse transplantation model,when the tumor volume grows to 40-50mm~3,it will be administered in groups,and the administration period is 14 days.The living conditions of mice in each group were observed,the tumor volume and mass of mice were measured and noted down,as well as the protein expression levels of PI3-K/Akt signal pathway related indexes(p-PI3K,p-AKT,Cleaved Caspase-9,Bax,Bcl-2)and angiogenesis related indexes(MMP-9,MMP-2,VEGFA,VEGFR-2)in tumor tissues in different groups were detected by Western blot.Result:1.Network pharmacological analysis results:41 active components,266 target genes and 1163 gastric cancer target genes of Xiaoyan Decoction were screened,and 116intersection genes were finally obtained.After excluding the active components without intersection genes,35 compounds were obtained.TP53,JUN,AKT1,MAPK3,RELA,MAPK1,ESR1,MAPK14 and IL6 are the key targets of Xiaoyan Decoction in the treatment of gastric cancer.The results of go enrichment analysis showed that it was related to the signal pathway of apoptosis,response to oxidative stress,response to chemical stress and response to inorganic substances.The results of KEGG pathway enrichment analysis showed that it was mainly related to PI3K-Akt signal pathway,IL-17 signal pathway,TNF signal pathway and platinum resistance in cancer.The results of survival analysis showed that the overall survival of patients with high expression of TP53,AKT1,MAPK3,RELA and ESR1 was significantly lower than that of low expression group,while the overall survival of patients with high expression of JUN,MAPK1 and MAPK14 was significantly higher than that of low expression group.2.Effect of Xiaoyan Decoction on the biological behavior of human gastric cancer MGC-803 cells:(1)The results of CCK-8 showed that Xiaoyan Decoction significantly inhibited the proliferation of MGC-803 cells in a dose-dependent manner(P<0.05),and its IC50 was 63.56mg/ml.(2)The results of Annexin V-FITC/PI double staining showed that the apoptosis rate of MGC-803cells increased after Xiaoyan Decoction of 0,20,40 and 60mg/ml acted on MGC-803 cells for24h(P<0.05).(3)The results of cell cycle detection by flow cytometry showed that with the increase of Xiaoyan Decoction concentration,the percentage of G0/G1 phase cells in MGC-803 cells gradually decreased,the percentage of S phase cells gradually increased(P<0.05),and the percentage of G2/M phase cells changed little(P>0.05).3.Effect of Xiaoyan Decoction combined with Apatinib on gastric cancer MGC-803 cells:(1)The results of CCK-8 showed that Xiaoyan Decoction combined with Apatinib significantly inhibited the proliferation of MGC-803 cells.Compared with Xiaoyan Decoction single drug group and Apatinib single drug group,the inhibition effect of combined drug group was more significant(P<0.0001).(2)Western blot showed that compared with the Control group,Xiaoyan Decoction group,Apatinib group and Combination group could effectively down regulate the expression of p-PI3K,p-AKT,Bcl-2,VEGFA and VEGFR-2 protein(P<0.05),and up regulate the expression of Cleaved Caspase-9 and Bax protein(P<0.05).Compared with the Xiaoyan Decoction group,the Combined group inhibited the expression of p-PI3K,p-AKT,Bcl-2 and VEGFA protein more significantly(P<0.05),and promoted the expression of Cleaved Caspase-9 protein more significantly(P<0.05);Compared with Apatinib group,the Combined group inhibited the expression of p-PI3K,p-AKT and Bcl-2 protein more significantly(P<0.05),and promoted the expression of Cleaved Caspase-9 and Bax protein more significantly(P<0.05).4.Effects of Xiaoyan Decoction combined with Apatinib on MFC gastric cancer bearing mice:(1)Living conditions of mice:Compared with the Tumor bearing model group,the mental status and quality of life of mice in Xiaoyan Decoction group,Apatinib group and Combined group were better.(2)Tumor volume of mice:Compared with the Tumor bearing model group(350.800±124.252)mm~3,the tumor volume of Xiaoyan Decoction group,Apatinib group and Combined group decreased significantly(P<0.05),which were(157.063±44.417)mm~3,(117.813±25.133)mm~3 and(66.175±16.867)mm~3 respectively.Compared with Xiaoyan Decoction group and Apatinib group,the Combined group had more obvious inhibitory effect on transplanted tumor(P<0.05).(3)Tumor weight of mice:Compared with the Tumor bearing model group(0.342±0.111)g,the tumor weight of Xiaoyan Decoction group,Apatinib group and Combined group decreased significantly(P<0.05),which were(0.117±0.037)g,(0.101±0.023)g and(0.042±0.016)g respectively.Compared with Xiaoyan Decoction group and Apatinib group,the Combined group had more obvious inhibitory effect on tumor,but the difference was not statistically significant(P>0.05).(4)Western blot detection:Compared with the Tumor bearing model group,Xiaoyan Decoction group,Apatinib group and Combined group can inhibit the expression of p-PI3K,p-AKT,Bcl-2,MMP-9,MMP-2,VEGFA and VEGFR-2 proteins(P<0.05),and promote the expression of Cleaved Caspase-9 and Bax proteins(P<0.05).Compared with Xiaoyan Decoction group and Apatinib group,the Combined group inhibited the expression of p-PI3K,p-AKT,Bcl-2,MMP-9,MMP-2 and VEGFR-2protein more significantly(P<0.05),and promoted the expression of Bax protein more significantly(P<0.05).Conclusion:1.Xiaoyan Decoction can play an anti-gastric cancer role by regulating PI3K-Akt signal pathway,IL-17 signal pathway and TNF signal pathway.2.Xiaoyan Decoction can effectively inhibit the proliferation,induce the apoptosis and S-phase block of human gastric cancer MGC-803 cells.3.Xiaoyan Decoction combined with Apatinib can effectively inhibit the growth and proliferation of human gastric cancer MGC-803 cells by regulating the protein expression of VEGF/PI3K/Akt signaling pathway.4.Xiaoyan Decoction combined with Apatinib can induce apoptosis and inhibit angiogenesis by regulating VEGF/PI3K/Akt signal pathway,so as to effectively inhibit the growth of transplanted tumor in gastric cancer mice and improve the quality of life of gastric cancer mice.