| ObjectiveThe method of replenishing qi and removing the phlegm is the basic treatment method proposed by Professor Zhou Daihan to treat non-small cell lung cancer(NSCLC)patients with spleen deficiency and phlegm dampness.Qing Jin De Sheng Fang(QJDSF),as one of the representative prescriptions,has significant clinical effects.Its tablets are in-hospital preparations of the First Affiliated Hospital of Guangzhou University of Chinese Medicine.This study intends to objectively evaluate the clinical efficacy of Qing Jin De Sheng Pian(QJDSP)and study the molecular mechanism of its anti-tumor effect.Objectively evaluate the clinical efficacy of QJDSP combined with chemotherapy in the treatment of NSCLC patients through retrospective clinical trials.The anti-tumor effect of QJDSP is studied through cell experiment and animal experiment.Explore the molecular mechanism of QJDSP in the treatment of NSCLC through network pharmacology and transcriptome sequencing and carry out relevant verifications.Method1.A retrospective clinical trial of QJDSP combined with chemotherapy in the treatment of patients with advanced NSCLCThe NSCLC patients who had undergone chemotherapy at the Cancer Center of the First Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine in the past ten years were retrieved,and the patients were screened according to the admission criteria,and the patients were divided into treatment group and control group according to whether the patients took QJDSP during chemotherapy.The clinical information of the two groups of patients before and after treatment was collected to objectively evaluate the efficacy of QJDSP combined with chemotherapy.2.The effect of QJDSF solution on the function of NSCLC cells A549 and H1650 The effect of QJDSF solution on the proliferation of NSCLC cells A549 and H1650 and human normal lung epithelial cells beas-2b was detected by MTT experiment,and the half maximal inhibitory concentration(IC50)was calculated.The effect of QJDSF solution on the migration ability of NSCLC cells A549 and H1650 was detected by cell scratch test and Tranwell cell migration test.The effect of QJDSF solution on the invasion ability of NSCLC cells A549 and H1650 was detected by Transwell cell invasion experiment.The Annxin V/PI staining experiment was used to detect the effect of QJDSF solution on the apoptosis of NSCLC cells A549 and H1650.3.The effect of QJDSP on NSCLC cell A549 transplanted tumor in nude miceA nude mouse xenograft model of NSCLC cell A549 was established.After the model was established,it was randomly divided into control group(0 mg/kg QJDSP),low-dose group(0.555mg/kg QJDSP),medium-dose group(1.11mg/kg QJDSP),and high-dose group Group(2.22mg/kg QJDSP),positive drug group(2mg/kg cisplatin),the tumor size and weight of nude mice were measured every three days.After 21 days of intervention,blood was taken from the orbit and sacrificed by cervical vertebrae.The tumor and important organs were peeled off.The liver,kidney and tumor mass of each group of nude mice were made pathological sections,and the tissue morphology was observed by HE staining.The serum of each group of nude mice was used to detect indexes related to liver and kidney function.4.Explore the molecular mechanism of QJDSP in the treatment of NSCLC through network pharmacology and transcriptome sequencing and carry out relevant verificationsSearch the target of QJDSP through TCMSP database,and search the target of NSCLC through Dig See,Dis Ge NET,Drug Bank,Genecards,OMIM,pharm GKB,TTD database.After intersecting the targets of QJDSP and the therapeutic targets of NSCLC,we will further explore the treatment of NSCLC by QJDSP through the Kyoto Encyclopedia of Genes and Genomes(KEGG)and Gene Ontology(GO)enrichment analysis Molecular mechanism.Transcriptome sequencing was used to construct and sequence the m RNA library of nude mouse tumors in the control group and the high-dose group,and explore the molecular mechanism of QJDSP in the treatment of NSCLC through GO and KEGG enrichment analysis.Take the intersection of the two KEGG enrichment analysis results to further clarify the molecular mechanism of QJDSP's anti-tumor effect.The A549 and H1650 cells were divided into control group(0mg/ml QJDSF),high-dose group(0.08mg/ml QJDSF),high-dose + p53 inhibitor group(0.08mg/ml QJDSF+10?M/ml pifithrin-?),The apoptosis of each group was detected by Annexin V/PI staining experiment.The A549 and H1650 cells were divided into control group(0mg/ml QJDSF),low-dose group(0.02mg/ml QJDSF),high-dose group(0.08mg/ml QJDSF),high-dose + p53 inhibitor group(0.08mg/ ml QJDSF +10?M pifithrin-?),after24 hours of intervention,the proteins of A549 and H1650 cells in each group were extracted,and Western blotting(WB)experiments were used to detect the expression of related proteins in the p53 signaling pathway.WB experiment was used to detect the expression of related proteins on the p53 signal pathway in tumor tissues of nude mice in each group.Quantitative Real-time polymerase chain reaction(Q-PCR)experiment was used to detect the m RNA expression of p53 signaling pathway related genes in tumor tissues of each group.Results1.Retrospective clinical trials of QJDSP combined with chemotherapy in the treatment of advanced NSCLCThe NSCLC patients undergoing chemotherapy in the Cancer Center of the First Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine in the past ten years were searched and screened.A total of 221 patients were enrolled,including 116 in the treatment group and 105 in the control group.Statistical analysis found that the baselines of the two groups of patients before treatment were basically the same.After treatment,the Karnofsky performance status(KPS)of patients in the treatment group was significantly higher than that of the control group,and there was a statistical difference(P<0.05).After treatment,the objective remission rate and disease control rate of patients in the treatment group were higher than those in the control group,but there was no statistical difference(P>0.05).2.The effect of QJDSF solution on the function of NSCLC cells A549 and H1650MTT results showed that the inhibitory effect of QJDSF solution on A549,H1650,and beas-2b cells showed a positive correlation with time and concentration.The enhancement of the inhibitory effect was related to the increase of drug concentration and the extension of intervention time.The24 H,48H,72 H IC50 of NSCLC cell A549 were 0.124mg/ml,0.062mg/ml,0.041mg/ml,respectively.The 24 H,48H,72 H IC50 of NSCLC cell H1650 were 0.094mg/ml,0.077mg/ml,0.037 mg/ml,respectively.The 24 H,48H,72 H IC50 of human normal lung epithelial cells beas-2b were 4.571mg/ml,1.833mg/ml,1.602mg/ml,respectively.The results of cell scratch test,Transwell cell migration test and invasion test show that QJDSF solution can inhibit the migration and invasion of NSCLC cells A549 and H1650.The inhibitory effect and concentration show a positive correlation trend,and the differences between groups are statistically significant(P <0.05).Through Annexin V/PI staining experiments,it was observed that QJDSF solution can increase the proportion of early apoptotic cells in NSCLC cells A549 and H1650,and with the increase of the concentration,the proportion of early apoptotic cells increased,and the difference between groups was statistically significant(P< 0.05).3.The efficacy test of QJDSP on NSCLC cell A549 transplanted tumor in nude miceThe results of animal experiments showed that the volume of the dissected tumor in the middle and high dose groups was smaller than that in the control group,and the difference was statistically significant(P<0.05).The HE staining of the tumor body revealed that all tumor cells were stained with large nuclear stains with obvious heteromorphism.Compared with the model group,the number of tumor cells and nuclear divisions in the drug intervention group were relatively small,and the cell distribution was relatively loose.After treatment,the weight of nude mice in the middle and high-dose groups increased,and the weight of nude mice in each group of traditional Chinese medicine was statistically significant compared with the positive medicine group(P<0.05).The main organ indexes of nude mice with NSCLC cell A549 transplanted tumor were calculated.There was no significant difference in heart index,lung index,and kidney index among the groups(P>0.05).The liver index and spleen index of the nude mice in the positive drug group were significantly lower than those of the other 4 groups,and there were statistical differences(P<0.05).By testing the liver and kidney function of nude mice with NSCLC cell A549 transplanted tumors,there was no significant difference in Alanine transaminase(ALT)and Aspartate Transaminase(AST)of nude mice in each group(P>0.05).The mean value of UREA of QJDSP low,medium and high dose groups was lower than that of the control group,and the difference was statistically significant(P<0.05).Compared with the control group,the mean value of urea(Urea,UREA)in the positive drug group was not statistically significant(P>0.05).Compared with the control group,the average creatinine(Cr)of the QJDSP low,medium and high dose groups was not statistically different(P>0.05).The average Cr of the positive drug group was higher than that of the control group,and the difference was statistically significant(P<0.05).By HE staining the liver and kidney of nude mice with NSCLC cell A549 transplantation tumor,it was found that there was no significant difference in the morphology of liver and kidney in each group.4.Analyze the potential molecular mechanism of QJDSP in the treatment of NSCLC through network pharmacology and transcriptome sequencing244 therapeutic targets of QJDSP were found through the TCMSP website.Search the NSCLC in 7 databases,take the targets that appear in at least two databases,and get a total of 787 targets.After the intersection,89 potential therapeutic targets are obtained.These targets are used for protein interaction(protein protein).interaction,PPI)network analysis,GO enrichment analysis,KEGG enrichment analysis.Transcriptome sequencing resulted in 355 differentially expressed genes,of which 93 were up-regulated genes and 262 were down-regulated genes.These genes were analyzed by PPI network analysis,GO enrichment analysis,and KEGG enrichment analysis.Intersecting the KEGG enrichment analysis results of network pharmacology and transcriptomics KEGG enrichment analysis results,a total of 16 tumor-related signal pathways were obtained,namely apoptosis,cell cycle,p53 signaling pathway,PI3K-Akt signaling Pathway,Fox O signaling pathway,VEGF signaling pathway,TNF signaling pathway,NF-?B signaling pathway,HIF-1 signaling pathway,NOD-like receptor signaling pathway,T cell receptor signaling pathway,B cell receptor signaling pathway,Ras signaling pathway,Rap1 signaling pathway,Toll-like receptor signaling pathway,neurotrophin signaling pathway.Cell experiments found that it can significantly induce apoptosis in A549 and H1650 cells.Previous studies have shown that its anti-tumor effect may be related to p53 protein.Therefore,it is speculated that the anti-tumor effect of QJDSP may be related to the induction of NSCLC cell apoptosis by p53 signaling pathway.The Annexin V/PI staining experiment found that the early apoptosis rate of A549 and H1650 cells in the high-dose group was higher than that in the control group,and the difference was statistically significant(P<0.05).The early apoptosis rate of the high-dose group plus p53 inhibitor group was lower than that of the high-dose group,and the difference was statistically significant(P<0.05).WB experiments were performed to detect the protein expression of p53,bcl-2,bax,cytc,pro-caspase 3,cleaved-caspase 3 in the p53 apoptosis signal pathway,and it was found that the high-dose histone expression of A549 cells was compared with the control group,P53,cleaved-caspase 3,cytc,bax protein expression increased,bcl-2,pro-capase3 and other protein expression decreased,the difference was statistically significant(P<0.05).The high-dose histone expression of A549 cells compared with the high-dose plus p53 inhibitor group showed that the expression of cleaved-caspase 3,cytc,and bax protein decreased,and the expression of bcl-2,pro-capase3 protein increased,the difference was statistically significant(P<0.05).Compared with the control group,the high-dose histone expression of H1650 cells increased the expression of p53,cleaved-caspase 3,cytc,and bax,while the expression of bcl-2,pro-capase3 and other proteins decreased,the difference was statistically significant(P<0.05).The high-dose histone expression of H1650 cells was compared with the high-dose plus p53 inhibitor group.It was found that the expression of cleaved-caspase 3,cytc,and bax protein decreased,and the expression of bcl-2,pro-capase3 protein increased,the difference was statistically significant(P<0.05).The tumor protein content was detected by the WB experiment,and it was found that the tumor protein expression of the medium-dose group,high-dose group,and positive drug group compared with the control group,the expression of p53,cleaved-caspase 3,cytc,and bax increased,the expression of bcl-2,pro-capase3 protein decreased,the difference was statistically significant(P<0.05).The expression of m RNA in the tumor was detected by Q-PCR experiment,and it was found that the tumor m RNA expression of the medium-dose group,high-dose group,and positive drug group compared with the control group,the expression of p53,caspase 3,cytc,and bax increased,the expression of bcl-2 decreased,the difference was statistically significant(P<0.05).Conclusion1.The KPS score of patients in the QJDSP combined chemotherapy group was higher than that of the chemotherapy group.The objective response rate and disease control rate of the QJDSP combined chemotherapy group were not statistically different from the chemotherapy group.2.QJDSF solution can inhibit the proliferation,migration and invasion of NSCLC cells A549 and H1650,and has a weak inhibitory effect on the proliferation of human normal lung epithelial cells beas-2b,indicating that its inhibitory effect on cell proliferation is selective.3.QJDSF solution can induce apoptosis of NSCLC cells A549 and H1650 through p53 signaling pathway in vitro.4.QJDSP solution can inhibit NSCLC cell A549 transplanted tumor volume increase in nude mice through p53 signaling pathway in vivo. |
PDF Full Text Request