Research On The Mechanism Of Qiyu Sanlong Recipe In Inhibiting Tumor Growth Of Mice Bearing Lewis Lung Carcinoma Based On Wnt/?-catenin Pathway

1 Objective To observe the anti-tumor effect of QYSL prescription on lewis lung cancer model mice,to explore the target and its mechanism of the prescription for non-small cell lung cancer,and to provide the adjuvant therapy of QYSL for clinical application based on Wnt / ?-catenin signaling pathway.Lung cancer provides experimental basis.2 Method 2.1 Modeling LLC lung cancer cell line was subcultured and transplanted into mice.The cells of logarithmic growth phase,trypsin digestion,centrifugation and centrifugation for 5 min were collected and diluted.The transplanted tumor model of C57 BL / 6 mice was established by injecting 0.2 m L of cell suspension into the left axillary of C57 BL / 6 mice by adjusting the cell concentration to 1×105 / m L syringe.2.2 Animal grouping and drug administration Mice bearing tumor were randomly divided into six groups: model group,high,middle and low dose group(4 times the daily dose of clinical adults),chemotherapy group,combined group(QYSL + cisplatin).,8 mice in each group The drug was administered on the second day of inoculation,as follows:(1)in the model group,0.2 m L/10 g saline was intragastrically perfused for 21 days,and 0.4 m L saline was injected intraperitoneally once a week;(2)in the chemotherapy group,0.2 m L/10 g of normal saline was intragastrically perfused for 21 days,and 0.4 ml of DDP solution was injected intraperitoneally once a week;(3)QH: 80.48g·kg-1·d-1 in the high dose group of Qiyu Sanlong recipe was given by stomach 21 days..Once a week,0.4 m L saline was injected intraperitoneally;(4)QM: 40.24g·kg-1·d-1 in the middle dose group of Qiyu Sanlong recipe was given by stomach 21 days.Once a week,0.4 m L saline was injected intraperitoneally;(5)QL: 20.12 g·kg-1·d-1 in the low dose group of Qiyu Sanlong recipe was given by stomach 21 days.Once a week,0.4 m L saline was injected intraperitoneally;(6)CQH: 80.48g·kg-1·d-1 Qiyu Sanlong recipe was given by stomach 21 days,and DDP solution was intraperitoneally injected once a week.2.3 Detection indicators The tumor volume and mass of lung cancer bearing mice were measured,the tumor inhibition rate was calculated,the ultrastructure of tumor cells was observed by Western blot and RT-q PCR was used to detect Wnt /?-catenin signal pathway Wnt series protein.Changes of ?-catenin and transcription level of downstream target genes.3 Results 3.1 Effect of QYSL on the Survival of mice bearing Lewis Lung Carcinoma The survival status of mice in the model group was good,the mental state,activity and diet of the mice in the three groups of high,middle and low QYSL were good,the survival state of the mice in the chemotherapy group was the worst and the weight loss was severe.The survival status of mice in the combined group was better than that in the QYSL group,but it was significantly better than that in the chemotherapy group.3.2 Effects of QYSL on the changes of tumor mass and tumor cell in lung cancer The tumor volume and tumor mass changes: chemotherapeutic agents could significantly reduce tumor volume and tumor mass(P < 0.01).QYSL had a certain inhibitory effect on tumor volume and mass,but the difference was not statistically significant(P > 0.05).The tumor inhibition rate of combination group and chemotherapy group was similar.The tumor cell changes: typical malignant characteristics was showed in the model group,apoptosis characteristics was showed in each QYSL group,especially in high dose group,apoptosis body appeared,chemotherapy group showed typical crescent apoptosis;In the combined group,chromatin was concentrated,edge set,organelle structure disappeared and necrosis.3.3 Effect of QYSL on the expression of CD44V6 and Survivin protein in tumor cells Compared with the model group,CD44V6,Survivin high dose group,chemotherapy group and combination group had statistical significance P < 0.05,compared with the low dose group,all the drug groups except the middle dose group had statistical significance(P < 0.05),and compared with the chemotherapy group,there was no significant difference between the other groups(P > 0.05).The results suggest that CD44V6 is mainly expressed in the membrane of tumor cells,and survivin is mainly expressed in the cytoplasm and nucleus of tumor cells.3.4 Effect of QYSL on Wnt of Wnt/?-catenin Pathway initiation protein Compared with the model group,each medication group had statistical significance(P < 0.01),and compared with the chemotherapy group,QYSL,The protein level of Wnt1,Wnt2 and Wnt5 a in high dose group was significantly lower than that in combined group(P < 0.01,P < 0.01).QYSL had better inhibitory effect on tumor growth at high dose,and had a certain synergistic effect with cisplatin.3.5 Effect of QYSL on Wnt/?-catenin Pathway Nuclear transcription protein in tumor tissues Compared with the model group,the GSK3 ?-p-GSK3 ?,?-catenin group had statistical significance(P < 0.01),compared with the low-dose group,the high dose group of QYSL had statistical significance(P < 0.01),and compared with the chemotherapy group,the high dose group of QYSL had statistical significance(P < 0.01),and compared with the chemotherapy group,the high dose group of QYSL had statistical significance(P < 0.01).The combined group had statistical significance(P < 0.01).The combination of QYSL and chemotherapeutic drugs had synergistic effect.3.6 Effect of QYSL on the expression of Dvl-1 and SFRP-2 protein in tumor tissues Compared with the model group,Dvl-1 treatment group were statistically significant(P < 0.01);compared with the low dose group,middle dose group,chemotherapy group,combination group was statistically significant(P < 0.01);compared with the chemotherapy group,more than drug group had no statistical significance(P > 0.05).Compared with model group,SFRP-2 medium dose group had statistical significance(P < 0.05),high dose group had statistical significance(P < 0.01),compared with low dose group,each drug group had statistical significance(P < 0.01),and compared with chemotherapy group,medium dose group,middle dose group.3.7 Effect of QYSL on target gene of Wnt /?-catenin pathway C-myc m RNAs: compared with the model group and the low dose group,the high dose group and the chemotherapy group had statistical significance(P < 0.01).Cyclin D1 m RNAs: compared with the model group,each drug group had statistical significance(P < 0.05 P < 0.01),and compared with the low dose group,the middle dose group and the chemotherapy group had statistical significance(P < 0.01).Axin2 m RNAs: compared with the model group,each drug group had statistical significance(P < 0.01),compared with the low dose group and the chemotherapy group,QYSL had statistical significance P < 0.01.QYSL had synergistic effect with chemotherapy drugs.4 Conclusion 4.1 QYSL can improve the survival status of mice bearing Lewis Lung Carcinoma,inhibit lung tumor mild,high dose of tumor growth inhibition effect is better,there is a dose-effect relationship;4.2 The therapeutic effect of QYSL alone was milder compared with DDP,however the combination of QYSL decoction and chemotherapy exhibited an increased therapeutic effect compared with the treatments administered alone,and has a certain inhibitory effect on lung tumor.4.3 The anti-tumor mechanism of QYSL may be that the prescription can reduce the expression of Wnt1 ?Wnt2?Wnt5a protein,affect the binding of Wnt protein to cellular membrane receptor,regulate the expression of GSK3??p-GSK3???-catenin protein in Lewis Lung Carcinoma tissue.and decrease the level of ?-catenin-TCF complex in the nucleus of Lewis Lung Carcinoma cells.Thus,the transcriptional level of the downstream target gene of Wnt / ?-catenin,c-mycine D1,axin2 was affected,and finally,the growth,invasion and metastasis of Lewis Lung Carcinoma were affected.4.4 QYSL through the expression of beta-catenin pathway proteins regulate Wnt /?-catenin become an effective method for lung cancer adjuvant therapy,and get the ideal pressure in clinical use.