| ObjectiveThrough data analysis of clinical trial literature on Chinese medicine for advanced non-small cell lung cancer(NSCLC),we investigated the evidence and treatment protocols as well as essential drugs to offer data-driven guidance for NSCLC clinical treatment.Using the core drug pairs as a starting point,we analyzed the characteristics of drug pairings and their clinical applications.We revealed the chemical basis underlying these pairings and verified the scientific rationality of traditional Chinese medicine pairing.Network pharmacology predicted the potential mechanism of action of the core drug pairs for treating NSCLC.The efficacy and key targets of the core drug pairs were then further validated through in vivo experiments in order to better guide clinical practice.Methods1.We retrieved and organized clinical trial literature from CNKI,Wanfang Data Knowledge Service Platform,China Science and Technology Journal Database,and China Biomedical Literature Database on TCM treatment of NSCLC in China from 1981 to 2020.Literature screening was performed based on strict criteria,and the collected data was normalized to create a "Database of Chinese Medicine Formulas for the Treatment of Middle and Late Stage NSCLC".Excel 2019,SPSS Modeler 18.0,SPSS Statistics 26.0,Gephi 0.9.2,and other software were employed for statistical processing and analysis of lung cancer evidence types,medication frequencies,properties,flavors,channels,association rules,systematic clustering,and complex networks.2.We collected core pairs of Chinese herbal drugs listed in Pharmacopoeia of People’s Republic of China,Compendium of National Standards for Chinese Herbal Drugs,and Chinese Herbal Drug Standards of People’s Republic of China,and summarized their effectiveness and clinical applications.Ultra-performance liquid chromatography(UPLC)was utilized to ascertain and examine alterations in the quantities of pertinent components in both single-decoction and co-decoction of the core pair.3.The TCMSP database was utilized to screen the active components and action targets of the core drug pairs,while the Genecards database,OMIM database,and Drugbank database were utilized to gather the pertinent targets of NSCLC.The Venny 2.1.0 online program was utilized to create a Wayne diagram for the purpose of identifying shared drug and disease targets.Said targets were then imported into the String database,ultimately leading to the construction of a PPI network diagram.We utilized Cytoscape 3.8.2 to analyze network topology parameters and identify key targets.The Metascape platform was utilized to conduct GO enrichment and KEGG pathway enrichment analyses on the identified key targets.Relevant 3D structures were obtained from the PubChem and RCSB protein structure databases.Strong binding activity between targets and compounds was achieved through molecular docking using PyMOL,AutoDockTools-1.5.6,and AutoDock Vina software.Molecular dynamics simulations were conducted using the Gromacs 2018.4 program for the complexes that exhibited successful outcomes in molecular docking.Finally,from a bioinformatics perspective,we conducted survival analysis and examined differential expression of the genes and proteins of the core targets using the Kaplan-Meier Plotter,GEPIA2,and HPA online databases,respectively.4.The Lewis lung cancer C57BL/6 mouse transplantation tumor model was created and randomly divided into various treatment groups:model group,cisplatin group,drug-pair low-dose group,drug-pair high-dose group,and drug-pair high-dose combined cisplatin group.Each group underwent 14 consecutive days of treatment.The body mass of the mice was recorded and used to calculate the tumor inhibition rate and immune organ index.The HE staining method was utilized to observe the histopathological and morphological changes of tumors in each group of mice.Immunohistochemistry was used to detect the expression of Ki-67 in the tumor tissues of mice in each group.The protein immunoblotting method was used to detect changes in PD-L1 and p-AKT protein expression levels in tumor tissues of the loaded mice.Results1.A total of 307 eligible articles were screened out,involving 483 formulas.The common syndrome of intermediate and advanced NSCLC was the deficiency of both Qi and Yin,with the common syndrome elements of Qi deficiency,Yin deficiency,phlegm,blood stasis,pathogenic heat(fire),toxin,and pathogenic dampness.The frequently used medicinals mainly had the functions of tonifying deficiency,clearing heat,resolving phlegm and relieving cough and dyspnea,promoting urination and draining dampness,and activating blood and resolving stasis.The high-frequency medicinals were Astragali Radix,Glycyrrhizae Radix et Rhizome,Ophiopogonis Radix,Fritillariae Thunbergii Bulbus,and Poria,which were mainly cold,bitter,sweet,and pungent,with tropism at lung,spleen,and stomach.The association rule analysis yielded 13 rules with strong association.Ten common factors were extracted from the factor analysis,and cluster analysis classified the medicinals into 5 groups.Complex network analysis suggested that the core formula was modified Liujunzi Tang and Yiqi Yangyin Jiedu prescription.2.By analyzing the organized Astragalus and Ligustrum compound preparation,we found that its efficacy primarily focuses on toning qi and nourishing yin,often combined with nourishing the liver and kidney,toning qi and blood,resolving blood stasis and detoxifying toxins,invigorating the blood and opening up collaterals,strengthening tendons and bones,dispelling wind and removing dampness,regulating menstruation,eliminating blemishes,and nourishing hair.These preparations were utilized in various diseases,including malignant tumors,liver conditions,gynecological issues,blood disorders,and deficiency labor,among others.The precision,repeatability,and stability tests of the UPLC content determination method were satisfactory,with accurate and dependable results.The study revealed that the combined administration of both medications significantly improved the solubility of trichothecene isoflavone glucoside and mangosteen glucoside in Astragalus,as well as terpineoside in Ligustrum.3.We screened 28 active ingredients and 570 targets of "Astragalus-Ligustrum",792 targets related to NSCLC,and 180 common targets by taking the intersection.Additionally,GO enrichment analysis revealed 221 biological processes,77 cellular components,and 95 molecular functions associated with the identified pathways.KEGG enrichment analysis identified 221 relevant signaling pathways,mainly involving the expression of PI3K-Akt,PD-L1,and signaling pathways such as PD-1 checkpoints,T-cell receptors,and Toll-like receptors in cancer.The findings from molecular docking analysis revealed that all active ingredients exhibited significant binding activity with TP53,SRC,STAT3,PIK3R1,AKT1,PD-1,and PD-L1.Notably,huarachein displayed weak binding affinity towards PD-1 receptor protein.Based on the molecular dynamics simulation analysis results,it has been confirmed that kaempferol exhibits higher structural stability and binding affinity towards PD-L1.The Kaplan-Meier Plotter analysis indicated that lung cancer patients with low expression levels of TP53,SRC,AKT1,PD-1,and PD-L1 exhibited better overall survival rates compared to those with high expression levels.However,patients with high expression levels of PIK3R1 displayed better overall survival rates than those with low expression levels.These differences were statistically significant(P<0.05).The GEPIA2 analysis revealed a significantly higher expression level of TP53 in lung squamous carcinoma specimens compared to normal specimens.Additionally,there was a significant difference in the expression of PIK3R1 and PD-L1 in lung glandular and squamous carcinoma specimens(P<0.05).The HPA database validation indicated lower expression levels of PIK3R1 in lung cancer tissue as compared to normal lung tissue.However,elevated expression levels were found for TP53.SRC,STAT3 AKT1,PD-1 and PD-L1.4.Changes in body mass of mice:During the pre-intervention period,there was no significant difference in the body weight of mice in each group.From days 1 to 13,the body weight of mice in the model group,low-dose drug-pairing group,and high-dose drug-pairing group increased gradually.The body weight of mice in the drug-pairing high-dose group increased from the 7th day and was higher than that of the other four groups.On the other hand,the body weights of mice in the cisplatin group and the combination group decreased significantly,starting from day 10,and were accompanied by reduced activity and diet.Tumor Weight and Tumor Suppression Rate:The tumor weights of mice in the cisplatin group,the high-dose group,and the combined treatment group were significantly lower than those of the other four groups,as compared to the tumor weight of mice in the model group.Furthermore,the tumor weight of mice in the combined group was lower than that of mice in the cisplatin group,and these differences were statistically significant(P<0.01).The combination treatment group exhibited the highest tumor suppression rate at 73.01%,followed by the cisplatin group at 59.59%.The drug-pair high and low dose groups showed rates of 42.30%and 3.86%,respectively.The results indicated that the immune organ index in the cisplatin group tended to decrease compared with the model group,but the difference was not statistically significant(P>0.05).However,the thymus and spleen indices of mice in the high-dose drug pair group were significantly higher than those in the cisplatin and model groups(P<0.01).The HE staining results indicated that the tumor cells in the combined drug-treated group showed significant necrotic areas in a sheet-like pattern,with sparse arrangement,structural disorder,and small cytoplasmic vacuoles.The results of the Ki-67 immunohistochemistry experiments showed that the expression of Ki-67 in the cisplatin group,the high-dose drug pair group,and the combination group was significantly different from that in the model group(P<0.01).The Western Blot assay results demonstrated a significant reduction in PD-L1 expression within the tumor tissues of the remaining groups,as compared to the model group(P<0.05).Additionally,the drug-pair-low dose group exhibited a statistically significant reduction in p-AKT expression when compared to the model group.In the model group,there was no statistically significant difference in the expression level of p-AKT protein(P>0.05).However,the cisplatin group,drug-pair high-dose group,and combination group had a significant decrease in expression of p-AKT protein compared to the model group,with statistically significant differences(P<0.05).Conclusion1.The site of disease in advanced NSCLC primarily involved the lung and spleen,with deficiency of qi and yin being the fundamental deficiencies and mixed solid elements including phlegm,blood stasis,heat(fire),toxicity,and dampness.Benefitting qi and nourishing yin.strengthening the spleen and resolving phlegm,clearing heat and removing toxins,and activating blood circulation and removing blood stasis were the fundamental therapeutic principles in TCM for treating middle and late stage NSCLC.The central pair was "Astragalus-Ligustrum" according to the pattern formula created by the association rule analysis.2."Astragalus-Ligustrum" was commonly paired medicines in recent traditional Chinese medicine formulas.Astragalus served as the primary medicine while Ligustrum served as the auxiliary medicine.Their combination was found to be beneficial in balancing qi and yin,resulting in effective therapeutic outcomes in clinical application.The content of active ingredients of Astragalus and Ligustrum underwent changes post-decoction.It was probable that the increase in trichostatin glucoside and aristolochicin content in Astragalus comprised the material basis for the efficacy.3."Astragalus-Ligustrum" contained active components that were relevant in treating NSCLC via the targeting of PIK3R1,AKT1,PD-1,PD-L1,and regulating biological processes like protein phosphorylation,as well as signaling pathways like PI3K-Akt,PD-1/PD-L1.This indicated that drug combinations were identified by multiple compounds,targets,and pathways of action.This provided a scientific foundation and research direction for further clarification of their molecular mechanisms.4."Astragalus-Ligustrum" improved the mental status and prevented weight loss in Lewis lung cancer mice while also safeguarding their immune organs to a certain degree.In combination with cisplatin,"Astragalus-Ligustrum" notably inhibited tumor tissues in mice.By down-regulating the expression of PD-L1 and p-AKT,"Astragalus-Ligustrum" exerted anti-tumor effects,thereby validating the results of the network pharmacology screening of targets. |
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