Study On The Active Ingredients Of Bupleurum Bupleurum Inhibiting The Growth Of Gastric Cancer And Their Mechanisms

Background and PurposeGastric cancer(gastric cancer,GC)is a kind of malignant with highincidence and mortality.Despite significant progress in the fields of genome,molecular metabolism and treatments,the 5-year survival rate of gastric cancer patients is still very low.At present,the molecular mechanism that regulates the pathogenesis and development of GC is still unclear.Therefore,it is urgent to study the specific pathogenesis and drug targets of GC to improve the clinical treatment of GC.Traditional Chinese medicine has its unique advantages in preventing and treating gastric cancer,and it is also effective to improve immunity.To explore the effectiveness of Chinese medicine in treating cancer,many clinical and laboratory studies have been performed in recent decades.Bupleurumis a common and important Chinese herbal medicine with multiple properties,including immune regulation,protection of the liver and gallbladder,as well as antipyretic,anti-inflammatory,sedative,analgesic,antiviral and antitumor effects.A large number of literatures showed that Bupleurum combined with other drugs haspositive effect on the treatment of gastric cancer,and in vitro experiments have also proved that has certain inhibitory effect on gastric cancer cells.However,the active ingredients of Bupleurum effective to GC and their underlying mechanisms remain elusive.Network pharmacology is an emerging method that combines systems biology,bioinformatics,and pharmacology.A comprehensive method based on network pharmacology is employed to identify common target genes,construct the pharmacology of "origin-component-target-pathway",and systematically predict the active ingredients against gastric cancer,targets and signaling pathways.ObjectiveThis study mainly constructed a pharmacological network of"origin-component-target-pathway",and systematically predicted the active components of Bupleurum,targets and signaling pathways that may be effective to treat gastric cancer.Furthermore,in vivo and in vitro experiments were performed to verify the active ingredients of Bupleurum against gastric cancer and its underlying mechanism was investigated.Methods1.A comprehensive method based on network pharmacology was used to identify common target genes,and a network pharmacology method was used to construct a pharmacological network of Bupleurum "origin-component-target-pathway",and systematically predict the potential active ingredients against gastric cancer,targets and signaling pathways.2.CCK-8 was employed to measure cell viability and draw cell growth curve.Plate cloning experiment was used to verify the effect of isorhamnetin,an active ingredient of Bupleurum,on gastric cancer cell proliferation.Hoechest staining and flow cytometry to detect cell apoptosis.Western blot was used to detect the expression levels of EGFR/PI3K/AKT,p53/Bc1-2/Bax,MAPK signal pathway related proteins.3.Nude mouse acute toxicity test was used to determine isorhamnetin LD50.Nude mouse tumor formation experiment was used to verify the anti-tumor effect of isorhamnetin in vivo.TUNEL was used to detect tumor tissue cell apoptosis.Results1.There were 17 common ingredients available in the TCMSP database,including baicalin,isorhamnetin,kaempferol,flavonoids,saikosaponin Cand quercetin.There 17 ingredients that meet the requirements of OB≥30%and DL index≥0.18,were selected as candidate biologically active ingredients.The effective active ingredients of Bupleurum,their corresponding gene targets and the target genes for gastric cancer disease treatment were selected for Venn analysis.160 intersected target genes were obtained.KEGG pathway was analyzed to identify the main factors related to gastric cancer.A total of 31 top-ranked pathways were screened,the main pathways including EGFR,P53 signaling pathway,PI3K/Akt signaling pathway,and MAPK signaling pathway.2.Invitro assays showed that isorhamnetin hadsignificant inhibitory effects on GC cell proliferation(P<0.001).After treatment with 4,8 or 16 μmol/L isorhamnetin for 3,5 or 7 days,GC cell count was significantly reduced(P<0.05).Compared with the control group,the number of GC cell colonies in the 4,8,and 16μmol/L isorhamnetin groups was significantly reduced.Hoechst 33342 staining data showed that the apoptotic cells were reduced in volume and turned to be round.The apoptotic rate was significantly increased(P<0.05)upon isorhamnetin treatment.Compared with those in control group,the protein levels of EGFR,p-PI3K,p-AKT,Bcl-2,p-ERK in the isorhamnetin group were significantly down-regulated,while the protein levels of p53 and Bax were significantly up-regulated(P<0.05).3.Compared with the model group,the tumor mass and tumor volume in the isorhamnetin group were significantly reduced,and the cell apoptotic rate in tumor tissues was significantly increased(P<0.05).Conclusion1.The network pharmacology predicts that Bupleurum chinense inhibits gastric cancer,mainly by regulating PI3K/AKT,p53and ERK MAPK signaling pathways to inhibit the proliferation and survival of gastric cancer.2.Isorhamnetin can regulate the proliferation and apoptosis by PI3K/AKT,p53and ERK MAPK signaling pathways.3.Isorhamnetin inhibits tumor tissue proliferation and promotes cell apoptosis.