| Objective: To observe the influence of Yiqi Huayu Jiedu Decoction on cell proliferation,apoptosis,cell cycle,cell migration,and the mTOR signaling pathways related proteins in HCT116 colorectal cancer cells;To observe the influence of Yiqi Huayu Jiedu Decoction and its different groups on mouse weight,tumors' inhibition rate,transfer rate,and the expression of mTOR signaling pathway related proteins in nude mouse model of colorectal cancer;And provide experimental basis for the clinical application of Yiqi Huayu Jiedu Decoction and transfer resistance of colorectal cancer.Methods(a)The cell experiments1.The cell proliferation and apoptosis :HCT116 cells were treated by different concentrations(10%,15%,20%)of Yiqi Huayu Jiedu Decoction serum,compared with the same concentration of blank rat serum and 10% fetal bovine serum(FBS),after 12 h,24h,48 h,72h,cell proliferation was detected by MTT method;After 24 h,48h,72 h,apoptosis rate was detected by Propidium iodide(PI)staining;After 48 h,the expression level of Bax,Bcl-x L and Caspase-3 protein in HCT116 cells were detected by Western blot.2.The cell cycle: HCT116 cells were treated by different concentrations(10%,15%,20%)of Yiqi Huayu Jiedu Decoction serum,after 48 h,cell cycle was detected by PI staining;The expression level of Cylin A2,Cyclin E1,Cyclin B1,and Cylin D1 in HCT116 cells were detected by Western blot.3.The cell migration:(1)Scratch experiment: After 6 orifice plate were full of cells,scratches along a straight line with 100?L point,HCT116 cells were treated by different concentrations(10%,15%,20%)drug-contained serum,after 24 h,48h,the distance scratches were measured.(2)Transwell experiment: HCT116 cells were treated by different concentrations(10%,15%,20%)drug-contained serum,after 24 h,cell migration ability was detected by transwell.4.Expression of mTOR signaling pathway related protein: HCT116 cells were treated by drug-contained serum,after 48 h,the expression level of AKT,p-AKT,mTOR,p-mTOR,S6K1,p-S6K1,4EBP1,p-4EBP1 protein were detected by Western blot.(b)The animal experimentsEstablish animal model of nude mouse colon cancer,and randomized into model group,Jianpi Yiqi group,Huayu Jiedu group,whole party group(Yiqi Huayu Jiedu group)and rapamycin group,each group of 10,the TCM group to fill the stomach,0.2 m L of each,once a day,five-day a week;Rapamycin 1 mg/Kg dose,intraperitoneal injection,2 times a week;After 4 weeks to be put to death animals,and observe the nude mice weight stable situation,tumors' inhibition rate,transfer rate;and the expression level of AKT,p-AKT,mTOR,p-mTOR,S6K1,p-S6K1,4EBP1,p-4EBP1 protein in intestinal transplantation tumor were detected by Western blot.Results(a)The cell experiments1.The cell proliferation and apoptosis:(1)cell proliferation after 12 h,15%,20% drug-contained serum on HC116 cells inhibition compared with the same concentration of blank rat serum groups had statistical significance(P < 0.05),after72 h,drug-contained serum on the strongest inhibitory effect of HCT116 cells,the cell proliferation inhibition rate of 10% group was 24.31%,and 15% group was 40.15%,and 20% group was 71.43%,compared with the same concentration of blank rat serum group differences were statistically significant(P < 0.01),suggesting Yiqi Huayu Jiedu Decoction drug-contained serum has good proliferation inhibition in HCT116 cells,and with time and concentration are positively correlated;(2)apoptosis:Compared with the corresponding concentration blank rat serum group,drug-contained serum treatment after 24 h,48h,72 h,late apoptosis rate of HCT116 cells increased,and the rate of apoptosis increased with the increase of concentration and time(P < 0.05),suggesting Yiqi Huayu Jiedu Decoction drug-contained serum can cause HCT116 cells apoptosis,and time,concentration were positively correlated.Compared with the blank of the rat serum group,the drug-contained serum raised the expression level of Bax,and decreased the expression level of Bcl-x L and Caspase 3(P < 0.05).2.The cell cycle: After 48 h treatment of Yiqi Huayu Jiedu Decoction drug-contained serum,10% and 15% drug-contained serum to block HCT116 cells in G1 phase and S phase,20% drug-contained serum to block HCT116 cells in G2.Compared with blank rat serum group,the expression of Cylin A2,Cyclin B1,Cyclin E1,Cylin D1 were in different degree in drug-contained serum group(P < 0.01).3.The cell migration:(1)Scratch experiment: After 24 h,48h treatment of different concentrations of drug-contained,drug-contained scratches were wider than the same concentration of blank rat serum groups(P < 0.05);After 48 h,the scratches distance of concentration of 10%,15% and 20% were 5.10 cm,5.56 m,6.03 cm,Same serum concentrations but different time,48 h of inhibition of migration ability were stronger than 24 h after treated by drug-contained serum,suggesting Yiqi Huayu Jiedu Decoction has certain inhibition of HCT116 cell migration,and concentration and time were positively correlated.(2)Transwell experiment: After 24 h treatment of different concentrations of drug-contained,cell migration inhibition rate increased,and the migration inhibition rate increased with the increasing of concentration,after deal with 24 h,migration inhibition rate were 21.9%,23.3%,50.9%;15%,20% concentration group compared with the corresponding concentration blank rat serum group difference was statistically significant(P < 0.05).Yiqi Huayu Jiedu Decoction drug-contained serum can inhibit HCT116 cell migration,and concentration were positively correlated.4.Expression of mTOR signaling pathway related protein: The expression of AKT,mTOR,S6K1,4 EBP1 between groups had no statistical difference(P > 0.05),and the expression level of p-AKT,p-mTOR,p-S6K1,p-4EBP1 were in different degree,compared with FBS group and blank rat serum differences had statistical significance(P < 0.05),suggesting Yiqi Huayu Jiedu Decoction drug-contained serum can regulate phosphorylation level of mTOR signaling pathway related protein.(b)the animal experiments1.The weight : After treatment,weight reduced in different degree,the model group,Huayu Jiedu group,Jianpi Yiqi group had statistically difference compared with before treatment(P < 0.05);The comparison of difference before and after,Jianpi Yiqi group and the whole party group weight loss were less than the model group(P <0.05),Huayu Jiedu group weight loss was greater than the model group(P < 0.05),the Jianpi Yiqi and the whole party group can stable weight,while the Huayu Jiedu group have certain influence of losing weight of nude mice.2.Tumors' inhibition rate : For tumors' inhibition rate,the Jianpi Yiqi group compared with model group had no statistical difference(P > 0.05);Huayu Jiedu group and the whole party to tumors had a better inhibition,tumor inhibition rate were27.2%,34.2%,the difference is statistically significant compared with model group(P< 0.05),there was no statistically significant difference compared with rapamycin group(P > 0.05),shows that Huayu Jiedu and the whole party group have good effect of inhibitory,while the Jianpi Yiqi group is little.3.Transfer rate: The of groups in different degree,the model group and Jianpi Yiqi group overall transfer rate was 90%,the Huayu Jiedu group and the whole party group transfer rate is 70% and 60% respectively,all were lower than model group,the difference was statistically significant compared with model group(P < 0.05),the Huayu Jiedu group and the whole party group can inhibit the colon cancer metastasis of the nude mice.4.Expression of mTOR signaling pathway related protein: After different components TCM treatment on nude mice,the expression of AKT,mTOR,S6K1,4EBP1 protein between groups have no statistical difference(P > 0.05),but the expression of p-AKT,p-mTOR,p-S6K1,p-4EBP1 were in different degree,Huayu Jiedu group and the whole group performed significantly,compared with model group and Jianpi Yiqi group difference were statistically significant(P < 0.05),Huayu Jiedu group and the whole group regulating the phosphorylation level of mTOR signaling pathway related protein.Conclusions1.Yiqi Huayu Jiedu Decoction can inhibit cell proliferation,block the cell cycle,promote apoptosis,inhibit cell migration,may be by inhibiting mTOR related proteins AKT,mTOR,S6K1 and 4EBP1 phosphorylation,preventing pathways activated,that inhibiting cell proliferation by cutting the expression of Cylin A2,Cyclin B1,Cyclin E1,Cylin D1,inducing the HCT116 cells apoptosis by raising apoptosis protein Bax,cutting expression of Bcl-x L,Caspase-3,and inhibit migration.2.Yiqi Huayu Jiedu Decoction can stable weight of nude mice,inhibit colorectal cancer and transfer,its anti-tumor and resistance to transfer effects may be mediated by inhibiting AKT?mTOR?S6K1 and 4EBP1 phosphorylation,preventing mTOR signaling pathway activation.3.The advantage of Jianpi Yiqi group is stable in weight,The advantages of Huayu Jiedu group are inhibiting tumor growth and transfer;Huayu Jiedu party may be the main ingredients of anti-tumor growth and metastasis in Yiqi Huayu Jiedu Decoction,The main role of the Jianpi Yiqi group maybe reduce the drawbacks of Huayu Jiedu party in the injury,so,full compatibility effect optimal. |
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