Explore The Mechanism Of Laoke Formula In The Treatment Of Non-small Cell Lung Cancer Based On Network Pharmacology

Objective:Lao Ke(LK)formula has achieved great success in clinical practice in the treatment of non-small cell lung cancer(NSCLC).However,little is known about its action mechanism.In our study,we retrospectively studied clinical data to compare the therapeutic effects between LK formula group and non TCM group,predicted active ingredients and investigated underlying action mechanism of the formula by applying network pharmacology methods.Experimental methods were applied to verify the effect of the formula on lung cancer cells.Methods:Patients diagnosed with NSCLC in Tianjin Medical University Cancer Institute and Hospital were retrospectively studied.Clinical characteristics,treatment plan and prognostic factors were collected from impatient and outpatient medical records.Kaplan-Meier method was used for survival rate.Multivariate Cox regression analysis were used to evaluate the prognostic factors.The chemical ingredients of the herbs and candidate NSCLC-related targets were collected from databases and literature.Network pharmacology was applied to construct “NSCLC-target-ingredient-LK formula” multilevel network to predict active ingredients and confirm potential action mechanism.Trypan blue cell count,CCK8 assay,wounding scratching assay and transwell migration assay were used to investigate the effects of aqueous extract of LK formula on lung cancer cells.The western blot method was used to verify the action target of aqueous extract of the formula and to explore the action mechanism.Results:1.The univariate analysis revealed that palliative resection treatment(p=0.013)and LK formula treatment(p<0.001)were protective factors.While high blood Cyfra21-1(p=0.043)and SCC(p=0.027)was linked with poor survival.Cox regression analysis showed that LK formula treatment(HR Exp[β]= 0.430,95% CI,0.281-0.658,p<0.001)were independent protective factors.Serum high SCC(HR Exp[β]=1.995,95% CI,1.170-3.400,p=0.011)was an independent predictor for poor survival.2.Survival analysis showed that the median survival time for all 155 patients was 21months(17-25 months),the median survival time was 28 months(21-35 months)vs15 months(12-18 months)in LK formula group and non TCM group.The 1-,2-,3-and 5-year overall survival rates was 90.9%,60.0%,38.2%,21.8% vs 60.0%,37.0%,15.0%,2.0% in two groups respectively.The two groups were statistically significant(p<0.001).3.Databases and literature search indicated that there were 328 ingredients in nine drugs of LK formula.There were 1728 action targets of the herbs and 2192 NSCLC-related targets.After projection the main action targets were EGFR,AKT1,NFκB1,TP53 and probably main active ingredients were kaempferol,luteolin,apigenin,quercetin,beta-sitosterol.4.Trypan blue cell count indicated that aqueous extract of LK formula showed a significant suppression effect on lung carcinoma cell proliferation after 48 hours in doses of 200ug/ml(p=0.0087),400ug/ml(p<0.0001)and after 72 hours in doses of100ug/ml(p=0.0326),200ug/ml(p=0.001),400ug/ml(p=0.001).CCK-8 assay indicated that aqueous extract of LK formula showed a significant suppression effect on lung carcinoma cell proliferation after 48 hours in doses of 300ug/ml(p<0.0001),600ug/ml(p<0.0001)and after 72 hours in doses of 150ug/ml(p=0.0288),300ug/ml(p=0.0039),600ug/ml(p=0.0024).The wound healing assay indicated that aqueous extract could also significantly suppress cell migration after 12 hours in dose of400ug/ml(p=0.0004)and after 24 hours in doses of 100ug/ml(p=0.0106),200ug/ml(p=0.0027),400ug/ml(p<0.0001).Transwell assay indicated that aqueous extract could significantly suppress cell migration after 16 hours in dose of 150ug/ml(p<0.0001),300ug/ml(p<0.001).And anti-migration time is shorter than that of antiproliferation.Western blot results showed that the aqueous extracts of LK formula could downregulate the expression of EGFR,AKT1,NFκB1 and upregulate the expression of P53 and p-P53.Conclusion:LK formula has obvious therapeutic effect on patients with NSCLC in clinical practice.Its main action targets are EGFR,AKT1,NFκB1 TP53 and probably main active ingredients are kaempferol,luteolin,apigenin,quercetin,beta-sitosterol.The aqueous extract of LK formula has a significant anti-proliferation and anti-migration effect on lung cancer cells.