Retrospective Clinical Study Of Qingjin Desheng Tablets In The Treatment Of Non-small Cell Lung Cancer And Experimental Study On Regulating PI3K/AKT Signaling Pathway

Objective Qingjin Desheng tablets(drug composition: Ophiopogon Japonicus,Red Ginseng,Golden Cypress,Rhizoma Pinellinae Praeparata,Placenta Hominis,Gynostemma pentaphyllum,etc.)is created by Zhou Daihan,a Chinese medicine master,aiming at the pathogenesis of “deficiency,phlegm,blood stasis and toxin” of lung cancer.It has the effects of supplementing qi and removing phlegm,clearing the lung,detoxifying,reinforcing the healthy qi and inhibiting tumor.In order to provide more clinical and experimental evidence for the application of Qingjin Desheng tablets,we retrospectively study the effect of Qingjin Desheng tablets on Stage Ⅲ-Ⅳ Non-small Cell Lung cancer treatment through the clinical trial.In basic experiments,we studied the antitumor effect and possible mechanism of Qingjin Desheng tablets.Methods Part One 186 patients with stage Ⅲ-Ⅳ non-small cell lung cancer treated in the tumor center of the First Affiliated Hospital of Guangzhou University of traditional Chinese medicine from January 1,2017 to June 30,2020 were collected.According to whether the treatment period(chemotherapy,targeted drug therapy and immunotherapy)is combined with oral Qingjin Desheng tablets,they are divided into treatment group(98 cases)and control group(88 cases).The clinical data and overall survival(OS)of the patients were collected.Chi square test was used to compare the baseline of clinical data(including gender,age,smoking history,TNM stage,pathological type,degree of pathological differentiation,KPS score,gene mutation,TKI and immunotherapy,number of chemotherapy lines,and the number of treatment lines at the time of enrollment)between the treatment group and the control group.Kaplan-Meier method was used to analyze the OS of the two groups,and log-rank test was used to compare the survival curve.COX regression model was used for univariate and multivariate analysis between the two groups of clinical data and OS.Part Two(1)According to the standard method,we made the preparation of Qingjin Desheng tablets freeze-dried powder,and applied it to A549.MTT assay was used to detect the effect of QJDSP on the viability of A549,and the IC50 number of QJDSP was calculated for subsequent mechanism research.48 h IC50 was used as QJDSP medium dose group,1/2 times IC50 as low dose group,and 2 times IC50 as high dose group.Cell cloning assay was used to detect the effect of QJDSP on the cell proliferation.Wound healing and Transwell were used to detect the effect of QJDSP on the migration of A549.Flow cytometry was used to investigate the effect of QJDSP on cell cycle of A549.(2)To construct A549 subcutaneous tumor-bearing mice model and given QJDSP by gavage.The tumor growth rate,final tumor volume and weight of control group,positive drug group,QJDSP low-dose group,QJDSP medium-dose group and QJDSP high-dose group were monitored to explore the inhibitory effect of QJDSP on lung cancer growth.Part Three(1)We screened the potential signal pathways and target genes by high-throughput sequencing of tumor bearing mice in high-dose group and model group.By comparing the expression changes of tumor m RNA between the two groups,the m RNA with significant difference was selected.By comparing GO and KEGG databases,differential genes were analyzed in cell function enrichment and signal pathway enrichment.(2)Based on the sequencing results,A549 cells were transfected with small interfering RNA(si-PIK3R1).The effect of PI3 K on the plate cloning ability of A549 cells was studied on cell colonyforming experiment.Cell cycle experiment was used to detect the effect of PI3 K on A549 cell cycle.(3)Qingjin Desheng tablets and PI3 K specific inhibitor LY294002 were used to intervene A549 cells.The experimental groups were divided into control group,QJDSP group,LY294002 group(hereinafter referred to as "LY group")and LY294002+QJDSP group(hereinafter referred to as "LY+QJ group").The cell colony-forming experiment and cell cycle experiment were studied on A549 cells when QJDSP and LY294002 were used respectively and in combination.(3)Western blot and q PCR were used to further study the effects of si PI3 K,QJDSP and LY294002 on PI3K/AKT signaling pathway and the protein and m RNA levels of CDK2 and cyclin A2.(4)Western blot and q PCR further verified the effects of QJDSP on PI3K/AKT signaling pathway,CDK2 and cyclin A2 protein and m RNA levels of A549 subcutaneous transplanted tumors.Results Part One(1)There was no significant difference between the two groups at baseline.During the follow-up,3 patients in the control group were deleted,73 patients died and 12 patients survived.In the treatment group,4 patients were deleted,62 patients died and 32 patients survived.The median survival time of patients in the treatment group was 25 months and that of the control group was 21 months.The OS of patients in the control group was significantly lower than that in the treatment group( < 0.05).(2)At the same time,in COX regression analysis,we found that whether Qingjin Desheng tablets were combined during treatment,gender and immunotherapy were correlated with the survival time of patients.That is,during the treatment period,combined with oral administration of Qingjin Desheng tablets,compared with the patients who didn’t,the risk of death could be reduced by 35.3%.Immunotherapy can reduce the risk of death by 54.8%.Part Two(1)The cell test showed that QJDSP could inhibit the activity,proliferation,cloning capacity and migration of A549 in a time and dose-dependent manner.A549 cells were treated with different doses(0.1mg/ml-0.9mg/ml)of QJDSP for 24 h,48h and 72 h.In the same time,compared with the control group,the cell viability decreased with the increase of QJDSP concentration.At the same concentration,the viability of A549 cells decreased with the extension of QJDSP treatment time.QJDSP inhibited the growth of A549 cells in a time-dependent and dose-dependent manner.There were significant differences between QJDSP concentrations at the same time point,and there were significant differences between QJDSP concentrations at the same time point.The results of cell clone experiment showed that the inhibition of QJDSP on cell proliferation was enhanced with the increase of QJDSP concentration.The healing rate of control,QJDSP-1× and QJDSP-2× groups were observed 24 hours later.The healing rate of 0mg/ml group was significantly higher than that of the experimental group.Transwell experiment found that the number of A549 cells through the chamber was significantly reduced after QJDSP treatment.Flow cytometry showed that compared with the control group,QJDSP could significantly block A549 in S phase.(2)The animal experiment,QJDSP could effectively control the tumor growth rate,tumor size and weight of tumor-bearing nude mice.After 6 days of administration,compared with the model group,the growth rate of transplanted tumor volume in high-dose QJDSP group slowed down,and there was a significant difference between the two groups,indicating that high-dose QJDSP could inhibit the growth of transplanted tumor volume in nude mice,and the inhibitory effect increased with the increase of administration time.Compared with the model group,the tumor weight of highdose QJDSP group decreased,and QJDSP could inhibit the tumor weight.Part Three(1)355 differentially expressed genes were obtained by transcriptome sequencing of tumor tissue samples of tumor bearing nude mice,including 93 up-regulated genes and 262 down-regulated genes.Through comparative GO analysis,it is found that Biological Process is mainly enriched in DNA replication,G1/S transition of mitotic cell cycle,positive regulation of cell proliferation,protein phosphorylation and negative regulation of phosphorylation.Cell components are mainly enriched in cytoplasm,chromatin,nucleoplasm,immune synapse and cytoplasmic perinuclear region.Molecular functions are mainly concentrated in protein binding,transport activity,protein kinase activity,kinase activity and protease binding.KEGG pathway enrichment analysis showed that these differentially expressed genes were related to cell cycle,apoptosis,PI3K/AKT signaling pathway,FOXO signaling pathway,HIF1 signaling pathway,Ras signaling pathway,Rap1 signaling pathway,TNFα signal pathway and MAPK signal pathway,etc.(2)The results of cell cloning experiment showed that knocking down the expression of PI3 K and intervening A549 cells with QJDSP,LY294002 and LY294002 combined with QJDSP could inhibit the proliferation of A549 cells.Cell cycle experiment showed that compared with the control group,knockdown of PI3 K expression and intervention with QJDSP,LY294002 and LY294002 combined with QJDSP could significantly block A549 cells in S phase.(3)In order to further study the mechanism,we used Western blot and q PCR detection.It was found that knockdown of PI3 K expression,QJDSP,LY294002 and LY294002 combined with QJDSP intervention could inhibit the activity of PI3K/AKT pathway and the expression of CDK2 and cyclin A2.(4)Western blot and q PCR were used to detect A549 subcutaneous transplanted tumors in tumor bearing mice.It was found that QJDSP could also inhibit the activity of PI3K/AKT pathway and the expression of CDK2 and cyclin A2,which were confirmed the results of cell experiment each other.Conclusion 1.Oral administration of Qingjin Desheng tablets during treatment may be an independent prognostic factor for OS in patients with stage III-IV non-small cell lung cancer,which could help prolong the overall survival time of patients with lung cancer.2.QJDSP inhibited the activity,proliferation and migration of A549 in a time and dose-dependent manner,and induced S phase arrest in A549 cells.QJDSP could effectively inhibit the growth of lung cancer transplantation tumor in vivo.3.Qingjin Desheng tablets might inhibit the levels of CDK2 and cyclin A2 protein and m RNA by inhibiting the activity of PI3K/AKT pathway,induce S-phase arrest of A549 cells to inhibit cell proliferation and tumor.