| BackgroundIt is of great significance for promoting the application of traditional Chinese medicine(TCM)in malignant tumors and the modernization of TCM by exploring the advantages of TCM in the treatment of malignant tumors and clarifying the anti-cancer mechanism of it.Maintenance therapy is an important treatment strategy for advanced non-small cell lung cancer(NSCLC),Maintenance chemotherapy can prolong progression-free survival(PFS),but its overall survival(OS)benefit is not obvious,its toxicity also increases significantly.The tutor's research group has been devoted to the research of TCM in the treatment of lung cancer for nearly 30 years,and has successfully developed the therapeutic scheme of Yiqi Chutan(Invigorating Qi and eliminating phlegm)and its corresponding formula.A lot of research has been done on the advantages of this scheme and its anti-cancer mechanism.The previous studies have showen that the patients with advanced NSCLC in the maintenance treatment period might could receive benefits from the scheme.In the research of anti-cancer mechanism,Yiqi Chutan Formula can inhibit the progression of lung cancer by inhibiting its metastasis,but there are still some unsolved problems:Firstly,what is the clinical efficacy of Yiqi Chutan Formula in the treatment of advanced non-small cell lung cancer?Second,what is the specific mechanism of Yiqi Chutan Formula on inhibiting lung cancer progression by preventing its metastasis?ObjectivesFirstly,we want to summarize the current evidence and understand the research status quo on TCM usage and its effect in the maintenance treatment period by literature review and meta-analysis.Secondly,we want to explore the effect of Yiqi Chutan Formula in the maintenance treatment of advanced NSCLC by a cohort study design and its mechanism of the formula on inhibiting the progression of NSCLC.In total,we want to provide the clinical and non—clinical evidence of Yiqi Chutan Formula on advanced NSCLC,in order to promote its clinical application in the maintenance treatment period.MethodsPart ?:The evidence of TCM maintenance therapy in advanced NSCLC.(1)The electronic databases were searched from their inception to August 2019,including CNKI,Wanfang database,Medline,PubMed,EMBASE et al.Randomized controlled trials(RCT)investigating the effectiveness of TCM maintenance versus chemotherapy maintenance on advanced NSCLC were collected.PFS,OS,quality of life,TCM syndrome score were meta-analyzed by Review Manager 5.3.Part ?:The effect of Yiqi Chutan Formula in the maintenance treatment of advanced NSCLC.(1)Patients with stage ?B-? or recurrent NSCLC who were hospitalized or outpatient treated in the First Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine from February 1,2012 to January 31,2019 were consecutively collected.The patients should also recived a response,that is,efficacy evaluation of the first-line chemotherapy is progression-free,including complete response(CR),partial response(PR)and stable disease(SD).At the maintenance period,they received TCM and/or chemotherapy and/or antiangionesis drugs.(2)According to the treatment scheme in maintenance period,the patients were divided into three groups:TCM maintenance group(TCM group),Western medicine maintenance group(WM group),Integrative nmaintenance group(patients received both TCM and western medicine,IT group).PFS curve and OS curve was carried out by Kaplan-meier method.Log-Rank test was used to evaluate the survival difference between them.The PFS and OS of patients maintained by chemotherapy alone or TCM alone,or TCM plus chemotherapy alone were also analyzed.The PFS and OS of adenocarcinoma subgroup were also analyzed by the above methods.(3)The hazard ratio(HR)and the relative 95%confidence intervals(CIs)of disease progression risk and mortality risk were calculated by Cox univariate and multivariate proportional risk regression model in different groups.Part ?:The effect of Yiqi Chutan Formula on peripheral blood bio-indicators in patients with advanced NSCLC.(1)Peripheral venous blood before and after treatment was collected from patients who participated in cohort study of the second part.Quantitative PCR was used to detect the expression levels of GRP78mRNA,GRP94 mRNA,P4HB mRNA,miR-21,miR-182 and miR-382 in peripheral blood.Pearson analysis was used to examine the correlation between the expression levels of different genes.The changes before and after treatment were analyzed by t-test.(2)Using the data from KM Plotter database,the relationship between the expression of GRP78mRNA,GRP94 mRNA,P4HB mRNA,miR-21,miR-182 and miR-382 in NSCLC and the overall survival of the patients was analyzed by Cox multivariate proportional risk regression method.(3)Funrich and GeneMANIA software were used to analyze the biological functions,signaling pathways and protein protein interaction networks of target gene proteins of miR-21,miR-182 and miR-382.Part IV:The anti-progression mechanism of Yiqi Chutan Formula.(1)Mice with Lewis lung cancer was divided into five groups after models were successfully established by intravenous transplantation.The five groups include,model group,low-dose group of Yiqi Chutan Formula,high-dose group of Yiqi Chutan Formula,low-dose group of Yiqi Chutan Formula plus Taxus chinensis,high-dose group of Yiqi Chutan Formula plus Taxus chinensis,12-13 mice in each group.They were given gastric lavage daily after inoculation to explore the effects of Yiqi Chutan Formula on body weight,overall survival,tumour metastasis and the organs.Five of each group were executed on the 40th day.Lung metastases were taken and the expression of epithelial-mesenchymal transition markers,cancer-related fibroblast marker a-SMA in different groups were detected by immunohistochemistry.(2)TGF-beta was used to induce epithelial-mesenchymal transition of A549?H1299?H1650 lung cancer cell lines.Yiqi Chutan Formula extraction and 4-PBA,endoplasmic reticulum stress inhibitor were used to intervene.Western blot and quantitative PCR were used to detect the expression of epithelial-mesenchymal transition markers and endoplasmic reticulum stress markers.ResultsPart ?:The evidence of TCM maintenance therapy in advanced NSCLC..RCTs were included in the literature review.The results showed that there were no significant differences in PFS and OS between TCM maintenance therapy and maintenance chemotherapy.Compared with maintenance chemotherapy,TCM maintenance therapy could improve the score of TCM symptoms and improve the quality of life of patients with advanced NSCLC.The quality of all the included studies was relatively low.Part ?:The effect of Yiqi Chutan Formula in the maintenance treatment of advanced NSCLC.A total of 163 eligible patients with advanced NSCLC were collected,including 37 in the western medicine maintenance group,61 in the TCM maintenance group and 65 in the integrative maintenance group.The median follow-up time was 25.07 months.Of 163 patients,143 had disease progress,and 93 patients died.The median PFS(95%Cl)was 5.63(5.00,7.27)months,and the median OS(95%Cl)was 17.07(15.00,21.93)months.The median PFS of TCM group,WM group and IT group were 6.27 months,3.83 months and 6.70 months respectively(P=0.119).Their median OS were 21.83 months,13.40 months and 20.33 months respectively(P=0.041).TCM group versus IT group,PFS P=0.36,OS P=0.88;WM group versus TCM group,PFS P=0.28,OS P=0.03;WM group versus IT group,PFS P=0.027,OS P=0.024.The median PFS of patients received chemotherapy maintenance only and patients received TCM and chemotherapy maintenance was 3.57 months and 6.90 months(P=0.005),and their corresponding OS was 11.57 and 16.97 months(P=0.019).In the subgroup of lung adenocarcinoma,the PFS of TCM group,WM group and IT group were 5.63 months,4.90 months and 6.90 months respectively(P-0.410),and the corresponding OS was 21.83 months,16.03 months and 17.73 months respectively(P=0.065).The result of univariate Cox regression analysis showed that compared with WM group,the risk of disease progression in TCM group decreased(HR,95%Cl:0.75,0.48-1.17),and the risk of death also decreased by 45%(HR,95%Cl:0.55,0.32-0.94);Compared with WM group,the risk of disease progression and risk of death in IT group decreased by 37%(HR,95%Cl:0.63,0.41-0.99),and 43%(HR,95%Cl:0.57,0.34-0.97)respectively.The result of multivariate Cox regression showed that compared with WM group,the benefits of TCM group in reducing the risk of disease progression were higher(HR,95%Cl:0.53,0.31-0.91),IT group also can reduce the risk of disease progression(HR,95%Cl:0.46,0.27-0.78).Compared with WM group,the risk of death can be reduced by about 50%in the TCM group and IT group.Compared with TCM group,IT treatment can not reduce the risk of death.Part ?:The effect of Yiqi Chutan Formula on peripheral blood bio-indicators in patients with advanced NSCLC.In TCM maintenance group,the expression of GRP78 mRNA,GRP94 mRNA and P4HB mRNA decreased after treatment,the expression of miR-182 and miRNA-382 were mainly up-regulated,while the expression of miR-21 was poorly consistent.The consistency of GRP94mRNA,P4HBmRNA,miR-21,miR-182 and miR-382 in integrative maintenance group was poor except GRP78mRNA,which is upregulated after treatment.Compared the expression changes of miR-21 between the TCM maintenance group and the integrative maintenance group,the increase level of miR-21 in the TCM maintenance group was lower than that in the integrative maintenance group.The expression levels of P4HBmRNA and GRP78mRNA were negatively correlated with the survival period of lung adenocarcinoma,and with the survival of lung squamous cell carcinoma.There was no significant correlation between GRP94mRNA expression and survival of lung squamous cell carcinoma and adenocarcinoma.The expression of miR-21 was negatively correlated with the survival time of lung adenocarcinoma,but not with the survival time of lung squamous cell carcinoma.There was no significant correlation between the expression of miR-182 and the survival time of lung adenocarcinoma,but positive correlation between the expression of miR-382 and the survival time of lung adenocarcinoma and squamous cell carcinoma.The function of miR-21 target genes is mainly signal transduction and cell-to-cell communication.The signal pathways involved include:integrin family pathway,vascular endothelial growth factor/vascular growth factor receptor pathway,TRAIL pathway and Glypican pathway.miR-21 targeted genes which are closely related to metastasis include RECK,TNFPSF11B,TGFB1,PDCD4,SPRY2 and FOFX2.Part ?:The anti-progression mechanism of Yiqi Chutan Formula.The weight gain of mice in the low-dose group of Yiqi Chutan Formula was greater than that in the model group and that in the low-dose group of Yiqi Chutan Formula plus Taxus chinensis.The median survival time of model group was 51.5 days,and the median survival time of low-dose group of Yiqi Chutan Formula was 56 days.Compared with model group,there was an increased trend,but with no statistical significance.The survival time of low-dose group of Yiqi Chutan Formula was significantly longer than that of low-dose group of Yiqi Chutan Formula plus Taxus chinensis.The low-dose group of Yiqi Chutan Frormula n had the most biggest effect on inhibiting lung metastasis,but without statistical significance.Yiqi Chutan Formula had no significant effect on heart,liver and kidney,but it might have an effect on spleen.Compared with the model group,the expression of Vimentin in metastasis was decreased in the low-dose group of Yiqi Chutan Formula,which indicated that Yiqi Chutan Formula could inhibit the epithelial-mesenchymal transformation of tumor cells.Compared with the model group,the expression of a-SMA and TGF-beta in the metastatic lesions was decreased in the low-dose group of Yiqi Chutan Formula,which indicated that Yiqi Chutan Formula could inhibit the activation of cancer associated fibroblast cells(CAFs).Yiqi Chutan Formula can inhibit the migration and invasion of A549 cells,it also could inhibit the epithelial-mesenchymal transformation of A549,H1650 and H1299 cell lines.TGF-beta induced the expression of endoplasmic reticulum stress markers GRP94,XBP-1s and ATF4.Yiqi Chutan Formula could inhibit the expression of GRP94,XBP-1s and ATF4 induced by TGF-beta.The inhibitor of endoplasmic reticulum stress,4-PBA,could inhibit epithelial-mesenchymal transition induced by TGF-beta.ConclusionWhether used alone or in combination with western medicine maintenance therapy,Yiqi Chutan Formula may reduce the risk of disease progression and death in advanced NSCLC,prolong its PFS and PS.Compared with western medicine maintenance,its advantages are mainly reflected in the long-term benefits.Yiqi Chutan Formula could inhibit the metastasis of lung cancer.Its mechanism may be related to its inhibition on activation of CAFs,regulation on endoplasmic reticulum stress response,and inhition on epithelial-mesenchymal transition pathway,which deserves further study. |
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