Traditional Chinese medicine combined with chemotherapy in the treatment of advanced non-small cell lung cancer: Key drug screening and mechanism analysis

doi:10.21203/rs.3.rs-4648678/v1

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Research Article

Traditional Chinese medicine combined with chemotherapy in the treatment of advanced non-small cell lung cancer: Key drug screening and mechanism analysis

https://doi.org/10.21203/rs.3.rs-4648678/v1

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published 29 Jul, 2024

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In the course of clinical treatment for anti-tumor, the combination of traditional Chinese medicine (TCM) and other treatment schemes can reduce toxicity and increase efficiency. The purpose of this paper is to find out the key TCM and effective components for the treatment of non-small cell lung cancer (NSCLC) and analyze its therapeutic mechanism by analyzing the prescription of TCM combined with chemotherapy for NSCLC. Firstly, the prescriptions of TCM in the randomized controlled trials combined with chemotherapy for NSCLC were collected, and the core TCM was screened by frequency statistics, association rule analysis and cluster analysis. Then, the intersection targets of the potential effects of NSCLC and core Chinese medicine were collected, and PPI analysis and enrichment analysis were performed on the intersection targets to screen the core targets, components and pathways, and the core components were verified by molecular docking and cell experiments. In this study, 269 prescriptions were collected, among which the frequency of medication for Astragalus membranaceus (HQ, in Chinese), Wolfiporia cocos (FL, in Chinese), Atractylodes macrocephala (BZ, in Chinese) was over 100. Association rule analysis showed that they were highly correlated and clustered into the same category in cluster analysis. Their core components were Quercetin, Kaempferol and Isorhamnetin. The molecular docking results of the core components with the core targets AKT1 and EGFR obtained by PPI network analysis showed that they could bind stably. KEGG analysis screened 110 pathways including PI3K-Akt; the results of CCK-8 showed that Quercetin, Kaempferol and Isorhamnetin could effectively inhibit the proliferation of A549 cells, and Isorhamnetin had the best inhibitory effect. Isorhamnetin can inhibit the migration and invasion of A549 cells, induce apoptosis, G1 phase arrest, and decrease the expression of P-PI3K and P-AKT in A549 cells. In a word, the key TCM for the treatment of NSCLC include HQ, FL, BZ, etc. and its key components Quercetin, Kaempferol, Isorhamnetin have potential therapeutic effects on NSCLC according to the research results.

Non-small cell lung cancer

Traditional Chinese medicine

Medication rules

Network pharmacology

Molecular docking

Experimental validation

According to the report of the International Agency for Research on Cancer (IARC), the incidence (12.4 %) and mortality (18.7 %) of lung cancer in 2022 rank first (Bray et al. 2024), while the report of the National Cancer Center of China also shows that in 2022, China 's lung cancer will increase by 1.06 million and die by 730,000, which is also the cancer with the highest incidence and mortality (Zheng et al. 2022). Pathologically, lung cancer can be divided into small cell lung cancer and non-small cell lung cancer. NSCLC accounts for 80% - 85%, and 75% of patients are diagnosed at an advanced stage (St et al. 2020), which will make the patients miss the best treatment stage, so the 5-year survival rate of NSCLC patients is only 5.5% (Garon et al. 2019). At present, chemotherapy is one of the main treatment methods for NSCLC, but the survival rate of patients with advanced lung cancer receiving traditional chemotherapy is very low, and adverse reactions such as bone marrow suppression, liver and kidney function damage, and alopecia often occur, which seriously affects the quality of life of patients. Therefore, the methods of how to improve the therapeutic effect, patient survival rate and reduce adverse reactions are particularly important.

As a traditional Chinese disease treatment plan, TCMs has the advantages of small side effects, non-invasiveness, low cost, and wide benefit population in tumor treatment. In recent years, many studies have shown that a variety of TCM components have anti-tumor activity (Chen et al. 2024; Yin et al. 2022; Yang et al. 2024), and the effect of reducing toxicity and increasing efficacy in the process of combined treatment with other treatment regimens is obvious. The multi-channel and multi-target treatment characteristics of TCMs can make up for the shortcomings of the current first-line treatment plans, such as single action pathway and large toxic and side effects. For example, Xiao et al. (Xiao et al. 2021) randomly divided 39 patients with stage III~IV NSCLC into TCM (Includes traditional Chinese medicine injection, traditional Chinese medicine decoction, capsule) combined with chemotherapy group and chemotherapy alone group. The results showed that the quality of life (QOL) score of the combined treatment group was higher, and the incidence of thrombocytopenia was significantly lower than that of the control group. There are also a number of studies that show that TCM has the effect of reducing toxicity and increasing efficiency (Wei et al. 2024; Zhang et al. 2022; Fei et al. 2023). Although modern pharmacological studies have shown that TCM can regulate TLR4/MyD88/NF-κB, P53, MAPK/TGF-β/Nrf2 and other signaling pathways (Zhao et al. 2024; Peng et al. 2020; Long et al. 2023), it can inhibit tumor proliferation, invasion and angiogenesis, so as to enhance drug sensitivity, reverse drug resistance, reduce adverse reactions and toxicity, and improve patient immunity (Zhang et al. 2024; Wang et al. 2020). However, due to the large number of components and targets of TCM, and the complex anti-tumor mechanism, and many components and mechanisms of action are not still clearly explained. Therefore, it is necessary to conduct in-depth research on it. In this study, the clinical randomized controlled literature of traditional Chinese medicine decoction combined with chemotherapy in the treatment of advanced NSCLC was excavated, and the prescriptions of traditional Chinese medicine were collected. The core TCM was screened out by frequency statistics, association rule analysis and cluster analysis. After that, the key targets and pathways of core TCM in the treatment of NSCLC were screened by network pharmacology analysis, and the key components were screened. In the end, they were verficated through molecular docking and cell experiment, to the results will provide reference for the treatment of NSCLC with TCM and the development of new drugs.

Materials

Quercetin, Kaempferol, CCK-8 kit were purchased from Aladdin (Shanghai, China); Isorhamnetin was purchased from Alfabiotech (Chengdu, China); Annexin V-FITC Apoptosis Detection Kit, Cell Cycle and Apoptosis Analysis Kit, Cell Plasma Membrane Staining Kit with DiO, Extracellular Matrix were purchased from Beyotime (Shanghai, China); PI, Recombinant Anti-GAPDH antibody, Prestained Protein Marker Ⅶ were purchased from Servicebio; Transwell chamber was purchased from NEST (Wuxi, China); Rabbit anti-PI3K, Rabbit anti-Phosphorylated PI3K, Goat Anti-Rabbit IgG were purchased from ZEN-BIOSCIENCE (Chengdu, China); Rabbit anti-AKT, Rabbit anti-Phosphorylated AKT were purchased from Proteintech (Wuhan, China); A549 cell line from ATCC (US).

Methods

Retrieval methodology

'Chinese traditional medicine ', ' non-small cell lung cancer ', ' NSCLC ', and ' chemical therapy ' were used as keywords to search CNKI, WanFang, VIP, Pubmed, Web of Science. The search date was from the establishment of the database to October 20, 2023.

Literature inclusion and exclusion criteria

Inclusion criteria: ①Type of study: RCT study; ②Subjects: patients with stage III~IV NSCLC diagnosed by histopathology and cytology; ③Intervention measures : the control group was only treated with conventional chemotherapy, and the experimental group was treated with traditional Chinese medicine decoction orally on the basis of the control group.

Exclusion criteria: ①review, cell or animal test literature; ②subjects with other diseases, TNM stage I~II; ③Literatures on other western medicine treatment methods other than chemotherapy or other traditional Chinese medicine treatment methods other than oral Chinese medicine decoction were used in the experiment; ④The composition of the drugs in the prescription is unknown.

Data extraction and collation

The included prescriptions were sorted into the Excel table. If the literature contained multiple prescriptions, the multiple prescriptions would be included in the statistics separately. Due to the dialectical medication, the addition and subtraction on the basic side only recorded the basic side, and finally removed the repeated prescription. For the same TCM, if there are different drug names, it is unified as the only drug name; for the same TCM due to the use of different parts, origin, processing methods and have different effects are recorded respectively.

Medication rule analysis

The collected prescriptions of TCMs were used for frequency statistics to find out high-frequency drugs; then the association rule analysis and cluster analysis were carried out through the ' arules ' and ' fmsb ' packages of R language. The association rules were screened with support > 0.1 and confidence > 0.6, and the core drugs of TCM for NSCLC were screened according to the results.

Targets collection

The components of core drugs (OB > 30 %, DL > 0.18) were collected through TCMSP (https://tcmspw.com/tcms-p.php) database), and the components with antitumor activity confirmed by existing experiments but not included in TCMSP were supplemented by literature review. The potential targets of the collected components were collected through the SwissTargetPrediction (http:// www.swisstargetprediction.ch/) database (Probability > 0.15).

NSCLC targets were collected from OMIM (https://www.omim.org/), GeneCards (https://www.genecards.org/), DisGeNET (https://www.disgenet.org/) and TTD (https://db.idrblab.net/ttd/) databases with the keyword ' non-small cell lung cancer '. The collected potential drug targets and NSCLC targets were deduplicated and intersected to obtain potential targets for drug treatment of NSCLC.

PPI network construction

The intersection targets were input into the STRING (https://cn.string-db.org/) database, and the species was set to ' Homo sapiens ', with the minimum action threshold > 0.4. The obtained PPI network was imported into Cytoscape 3.9.1, and the core targets were screened according to degree, betweenness centrality and closeness centrality, then the results were visualized.

Enrichment analysis

GO and KEGG analysis were performed through the ' Bioconductor ' package of R language to screen out the key pathways of key Chinese medicine in the treatment of NSCLC and visualize them.

Molecular docking

The protein structures of AKTI (PDB ID: 4ekl) and EGFR (PDB ID: 6lub) were obtained from PDB (https://www.rcsb.org/) database, and the active pockets of target proteins were predicted by DeepSite (https://playmolecule.com/) platform) (Jiménez et al. 2017). The protein was subjected to dehydration and hydrogenation by Pymol 2.6.0a0, and then molecular docking was performed by AutoDock 1.5.7.

A549 cell culture

The cells were cultured in F12K medium containing 10% fetal bovine serum at 37 °C, 5% CO₂, and the medium was changed once a day. The cells were passaged when the degree of fusion reached 80%.

Determined by cell viability assay

The cell viability was detected by CCK-8 method. A549 cells were inoculated in 96-well plates at 5000 cells / well, with 5 replicate wells in each group. After the cells adhered to the wall, Quercetin, Kaempferol and Isorhamnetin were mixed with a concentration gradient of 0, 50, 100, 150, 200, 250 and 300 μmol/L for intervention, and the cell viability was detected at 24, 48 and 72 h, respectively. 100 μL CCK-8 working solution was added to each well for detection. After incubation for 30 min at 37 °C in dark, the OD value at 450 nm was detected by microplate reader, and the maximum and minimum values of each group were removed. Cell viability = (experimental OD-blank OD) / (control OD-blank OD) × 100%. Finally, the components with the best inhibitory effect were selected for subsequent experiments.

Cell morphology observation

A549 cells were inoculated on polylysine-treated slides and treated with 0 and 270 μmol / L for 48 h after cell attachment. Then, the cells were stained with Dio/PI staining solution (binding buffer: Dio: PI = 300: 1: 90), incubated for 20 min at 37 °C in the dark, the staining solution was removed, washed three times with PBS, and the cell morphology was observed under laser confocal microscopy.

Cell migration

The effect of isorhamnetin on the migration of A549 cells was verified by scratch test. A549 cells were inoculated in a 6-well plate to make the overnight fusion rate reach 100 %. The scratches were scratched with a 10 μL pipette tip, and then PBS was used to wash away the cell debris. The drug was prepared into 0, 90, 180, 270 μmol/L with F12 K medium containing 1% fetal bovine serum. After 48 h of treatment, the cell migration rate of each group was calculated. Cell migration rate = (0 h scratch area-48 h scratch area) / 0 h scratch area × 100 %.

Cell invasion

The effect of isorhamnetin on the invasion ability of A549 cells was verified by transwell assay. The matrigel was prepared according to the kit instructions and added to the upper chamber. After digestion, the cells were made into cell suspension with complete medium containing 0, 90, 180, 270 μmol/L drugs, respectively, inoculated in the upper chamber and cultured for 24 h at 37 °C. The cells and matrigel in the upper chamber were wiped off with a cotton swab, fixed with tissue fixative for 10 min, stained with 0.4% crystal violet, and photographed under an inverted microscope.

Apoptosis and cell cycle

The effects of isorhamnetin on apoptosis and cell cycle of A549 cells were detected by flow cytometry. Apoptosis: A549 cells were seeded in a 6-well plate and added to a complete medium containing 0, 90, 180, 270 μmol/L drugs after cell adherence. After culture of 48 h, cells were collected and stained with Annexin V-FITC and PI according to the kit instructions. After incubation of 20 min in the dark, the apoptosis was detected.

Cell cycle arrest: Grouping and treatment methods are the same as apoptosis.

Western blot

The cells were treated with 0, 90, 180 and 270 μmol/L drugs, respectively. After 48 h, the total protein was extracted and quantified by BCA kit. The sample was loaded at 20 μg/well, and the protein was separated by SDS-PAGE electrophoresis at 200 V constant pressure and 400 mA constant flow membrane. BSA was blocked for 2 h, the primary antibody was incubated overnight at 4 °C, the secondary antibody was incubated at room temperature for 1 h, and the ECL developer was developed. The results were analyzed by Image J 1.54 f software.

Literature retrieval results

A total of 7714 articles were retrieved, including 2585 articles on CNKI, 3689 articles on Wanfang, 1142 articles on VIP, 60 articles on pubmed, and 238 articles on web of science. After removing the duplicate literature, the remaining 5310 articles were collected, 298 prescriptions were collected, finally, 269 prescriptions were remained after deleting the repeated prescriptions.

Medication rule analysis

A total of 295 traditional Chinese medicines were used from 269 prescriptions, and among them, the five most frequently used were HQ, FL, BZ, Ophiopogon japonicus (MD, in Chinese), Glycyrrhiza uralensis (GC, in Chinese) (Fig. 2A); the three drug pairs of BZ-HQ, Hedyotis diffusa(BHSSC, in Chinese)-HQ and BZ-FL were ranked the highest in support, and they were also the most frequently used in the analysis of association rules (Table1). After clustering analysis of TCMs, it was found that Codonopsis pilosula(DS, in Chinese), HQ, BZ and FL were clustered into the same category, which indicated that these TCMs have similar properties and functions (Fig. 2B). Due to the highest frequency and correlation of HQ, BZ and FL based on the above analysis results, they were selected as the core drugs for subsequent analysis.

Table 1 Correlated herb pairs (top 10)

couplet drugs	support	confidence	degree
BZ-HQ	0.29	0.74	79
BHSSC-HQ	0.29	0.81	78
BZ-FL	0.29	0.72	77
FL-HQ	0.29	0.66	77
MD-HQ	0.28	0.68	74
DS-HQ	0.27	0.71	72
GC-HQ	0.26	0.66	71
DS-BZ	0.25	0.65	66
DS-FL	0.23	0.62	63
CP-FL	0.18	0.72	47

* CP (Tangerine Peel, in Chinese)

Network Pharmacology Analysis

Core targets and component screening

A total of 40 active ingredients of core drugs that met the screening conditions were collected from TCMSP, and 9 were supplemented through literature review (Xu et al. 2018; Chen et al. 2019; Zhang et al. 2019; Dou et al. 2021; Xu et al. 2021; Acharya et al. 2022; Mao et al. 2022; Zhang et al. 2022; Jiang et al. 2023). The collected components were imported into the SwissTargetPrediction database, then 156 potential targets that met the screening conditions were obtained. A total of 1309,1490,3926 and 168 NSCLC targets were collected from GeneCards, OMIM, DisGeNET and TTD databases, respectively. After removing duplicate targets, 5549 NSCLC targets were obtained. After intersecting the potential targets of active ingredients and NSCLC targets, 116 intersection targets were obtained, that is, the potential targets of core TCM in the treatment of NSCLC (Fig. 2C). According to the intersection targets obtained above, three key components were screened by sorting the degree values of the components corresponding to these targets: Quercetin, Kaempferol and Isorhamnetin.

PPI network construction

Table 2 Key targets of herbal medicine against NSCLC (top 10)

Target	Betweenness	Closeness	Degree
AKT1	1955.66	0.01	65.00
EGFR	1547.19	0.01	60.00
ESR1	1281.93	0.01	52.00
PTGS2	1144.71	0.01	46.00
SRC	792.72	0.01	46.00
MMP9	356.01	0.01	41.00
GSK3B	393.78	0.01	37.00
PARP1	498.49	0.01	38.00
MCL1	257.82	0.01	32.00
KDR	214.27	0.01	32.00

The 116 intersection targets were imported into the STRING database to obtain the PPI network. The obtained PPI network was analyzed by Cytoscape and the screening condition was greater than the mean value, that is, degree > 14.85, closeness centrality > 0.0042, betweenness centrality > 129.01. The key targets of TCMs components in the treatment of NSCLC were screened (Table 2). Because the degree, betweenness centrality and closeness centrality of EGFR and AKT1 were significantly higher than those of other targets, they were identified as the core targets of Astragalus, Atractylodes and Poria in the treatment of NSCLC (Fig. 2D). Then, they were molecularly docked with the three core components (Quercetin, Kaempferol and Isorhamnetin) screened above.

Enrichment analysis

A total of 110 KEGG signaling pathways were enriched, including PI3K-Akt signaling pathway, EGFR tyrosine kinase inhibitor resistance, FoxO signaling pathway and other signaling pathways closely related to malignant tumors (Fig. 2E). 1982 GO entries was enriched by GO functional enrichment, involving 1703 biological processes, 90 cellular components, and 189 molecular functions (Fig. 2F). According to the results of KEGG enrichment, both the significance and the number of enriched intersection targets are higher in the PI3K-Akt signaling pathway, and existing studies have also shown that it is closely related to tumors (He et al. 2021; Khezri et al. 2022; et al. 2022). Therefore, we will futher verify it by Western blot experiments. In addition, we plotted the component-target-PI3K/Akt signaling pathway according to the corresponding relationship between PI3K-Akt pathway, targets and components (Fig. 2G).

Molecular docking

Table 3 Docking binding energy

Components	AKT1 kcal/mol	EGFR kcal/mol
Quercetin	-6.53	-6.64
Isorhamnetin	-6.58	-6.79
Kaempferol	-6.25	-6.50

The results of molecular docking showed that the binding energies of the two core targets and the three key components were all less than -1.2 kcal/mol, which indicated that they could be spontaneously and stably combined, and that Quercetin, Kaempferol and Isorhamnetin could act on AKT1 and EGFR targets to play an anti-NSCLC role. Among them, the binding energy of EGFR and Isorhamnetin was the lowest (-6.79 kcal/mol) (Fig. 2H, Table 3). From the diagram, it could be found that the main forces between isorhamnetin and EGFR are Carbon Hydrogen Bond, Pi-Sigma, Pi-Sulfur and Pi-Alkyl. Isorhamnetin can form Pi-Alkyl bond with Lysa745, Alaa743 and Vala726 of EGFR, Pi-Sigma bond with Meta790, Leua718 and Leua844, Pi-Sulfur bond with Meta766, and Carbon Hydrogen Bond with meta793, Glna791, Glua792, Lysa745 and Leua718.

Cell experiment

Determined by cell viability assay

In this experiment, the effects of Quercetin, Kaempferol and Isorhamnetin on the viability of A549 cells at 6 different concentrations and 3 time points were investigated. The results showed that the viability of A549 cells and the three components showed the dependence between dose and time. The difference between each treatment group and the blank group was statistically significant (P < 0.05), and the inhibitory effect of Isorhamnetin was the strongest. Therefore, the subsequent experiments focused on the effect of Isorhamnetin on A549 cells (Fig. 3A-D). The uncontrolled proliferation of cells is a major feature of tumors. When the tumor proliferates to a certain extent, it will transfer from the original lesion to other parts by means of tumor blood vessels. Therefore, the inhibition of tumor cell proliferation can effectively control tumor progression. It can be seen from the result that the viability of A549 cells was significantly inhibited after treatment with 100 μmol/L Isorhamnetin for 48 h.

Cell morphology observation

From the inverted microscope and confocal microscope, it could be found that the cell morphology of the blank group was full and irregular paving stone, while the cell morphology of the treatment group became elongated and slender. In the laser confocal microscope image, it could also be observed that the PI dye could not penetrate the membrane due to the integrity of the plasma membrane, , while the Dio dye could penetrate the membrane due to its lipophilicity in the normal group, so the cells in the normal group only have green fluorescence in the cytoplasmic region, while there is no red fluorescence in the nuclear region; however, for the treatment group, due to the influence of isorhamnetin, the cells began to apoptosis and the cell membrane was damaged. Therefore, most cells not only had green fluorescence in the cytoplasmic region, but also had red fluorescence in the nuclear region (Fig. 3E-H). The above results suggest that isorhamnetin will shrink the cytoplasm of A549 cells and recover the pseudopods, thus their morphology will change. At the same time, it will also promote the apoptosis of the cells and lead to the damage of the cell membrane.

Cell migration

The results of scratch test showed that the cell migration rates were 39.30%, 32.40%, 21.02% and 15.17% after 48 h treatment with Isorhamnetin at concentrations of 0, 90, 180 and 270 μmol / L, respectively. Although there was no significant difference between the 90 μmol/L group and the control group, the inhibition of cell migration ability became stronger with the increase of drug concentration. When 180 and 270 μmol/L were compared with the control group, the difference was statistically significant (P < 0.05), indicating that the higher the concentration of Isorhamnetin, the more significant the inhibition of A549 cell migration ability (Fig. 4A-H, M). NSCLC is easy to metastasize, which is closely related to poor prognosis. It can be seen from this experiment that although the low concentration group has no obvious inhibitory effect on the migration of A549 cells, with the increase of administration dose, the viability and migration ability of A549 cells are gradually inhibited. Its migration ability is concentration-dependent, indicating that isorhamnetin can effectively inhibit the migration of A549 cells.

Cell invasion

The results of this experiment showed that with the increase of dosage, the fewer cells penetrated the membrane. The number of A549 cells in the 0, 90, 180 and 270 μmol/L groups was (451 ± 23.52), (210 ± 17.52), (119.67 ± 4.93) and (71.67± 13.65), respectively. The difference between the administration group and the control group was statistically significant (P < 0.05) (Fig. 4I-L, N). Therefore, Isorhamnetin can effectively inhibit the invasion of A549 cells, and the higher the concentration, the stronger the inhibitory effect on its invasion ability.

Apoptosis and cell cycle

The effect of Isorhamnetin on the apoptosis of A549 cells was detected by FITC and PI staining. The results showed that the apoptosis rates of 0,90,180 and 270 μmol/L groups were 7.29%, 15.04%, 23.87% and 30.74 %, respectively. The early apoptosis rate of 90 μmol/L treatment group was 11.94%, while the late apoptosis rate was 3.10%. The early apoptosis rate of 180 μmol/L treatment group was 18.67%, and the late apoptosis rate was 5.20%. The early apoptosis rate of 270 μmol/L treatment group was 20.51%, and the late apoptosis rate was 10.23%. It can be seen that with the increase of drug concentration, the number of apoptotic cells also increased, and the number of late apoptotic cells also increased synchronously. It shows that with the increase of Isorhamnetin concentration, its effect on A549 cells gradually changes from proliferation inhibition to direct killing, and the higher the concentration, the more obvious the effect (Fig. 5A-D). This is also consistent with the results of cell morphology observation, indicating that isorhamnetin can induce apoptosis of A549 cells.

PI staining was used to detect the effect of isorhamnetin on the cell cycle of A549 cells. The results showed that the proportion of G1 phase cells in the blank group was 63.17%, while the proportion of G1 phase cells in the 90, 180 and 270 μmol/L groups was 77.73%, 83.49% and 90.92%, respectively. It can be seen that with the increase of drug concentration, the G1 phase arrest is more obvious (Fig. 5E-H). This result further indicated thatA549 cells were arrested in G1 phase, and the process of DNA synthesis and cell division became slower with the increase of drug concentration. It is suggested that isorhamnetin can inhibit DNA replication and promote cell apoptosis, thereby reduce the rate of cell proliferation and controlling tumor diseases.

Western blot

The results of Western blot showed that there was no significant difference in the expression of AKT and PI3K between the blank group and each treatment group (P > 0.05), while the expression of P-AKT and P-PI3K decreased with the increase of drug concentration. Compared with the blank group, there was no statistical significance in the 90 μmol/L group,but there was statistical significance in the 180 and 270 μmol/L groups (P < 0.05) (Fig. 5I-K). From the previous theoretical analysis, it can be seen that the PI3K-Akt signaling pathway has a regulatory effect on a series of biological processes such as proliferation, apoptosis and migration of tumor cells, and the two targets of PI3K and AKT are the core targets of the pathway. It can be seen from the results that the expression of P-PI3K and P-Akt in each treatment group decreased. Although there was no significant difference in the expression between the 90 μmol/L group and the blank group, but the expression decreased with the increase of drug concentration, showing a dose-dependent manner. It was further proved that isorhamnetin can regulate the PI3K-Akt signaling pathway by inhibiting AKT1 and PI3K phosphorylation, thereby inhibit cell growth and proliferation and promoting apoptosis.

Lung cancer is the malignant tumor with the highest mortality rate. As one of the main first-line treatment options, chemotherapy has the disadvantages of poor prognosis and toxic and side effects. Therefore, it is of great significance to explore ways to improve the efficacy and reduce the toxicity of chemotherapy for the treatment of NSCLC. As a traditional disease treatment plan in China, TCM has the characteristics of multi-component, multi-target and multi-channel synergistic treatment of diseases, and has many clinical applications in the treatment of malignant tumors.

In this study, firstly, three core TCMs for the treatment of NSCLC were screened out by frequency statistics, association rules and cluster analysis. The frequency of these three Chinese medicines in the collected 269 clinical prescriptions was greater than 100, and the HQ reached 175 times, indicating their importance in the treatment of NSCLC. The results of association rule analysis also showed that the frequency of any two simultaneous occurrence of these TCMs was greater than 70, and the frequency of simultaneous occurrence of three TCMs was 52 times. Through cluster analysis, it was found that they were clustered into the same category, indicating that these TCMs have a strong correlation. Among them, HQ has the effects of tonifying qi, consolidating the exterior, diuresis and detoxification. It mainly contains Astragalus polysaccharides, saponins and flavonoids. Zhou et al. ' s research showed that various extracts of HQ had inhibitory effects on breast cancer cells, and can induce apoptosis and regulate PI3K/AKT/mTOR signaling pathway (Zhou et al. 2018). BZ has the functions of invigorating spleen and replenishing qi, drying dampness and promoting diuresis. The components of BZ include volatile oils, polysaccharides, lactones, vitamins, amino acids, etc. Chen et al. ' s research shows that various components extracted from BZ can induce apoptosis of colorectal cancer cells (Chen et al. 2023). Poria cocos has the effect of promoting water and dampness, invigorating spleen and calming heart. It contains polysaccharides, triterpenoids, sterols and other components.

In this study, the anti-NSCLC mechanism of these three TCMs was further analyzed by network pharmacology. Through the PPI network, it can be found that various components of these three TCMs play an anti-NSCLC role through numerous targets, and the core targets EGFR and AKT1 were also screened (Miriam et al. 2024; Chen et al. 2023; Gao et al. 2023; Miller et al. 2024). It is reported that the mutation rate of EGFR in Asian NSCLC patients is as high as 50% (Attili et al. 2020). The abnormal activation of PI3K-Akt signaling pathway is one of the most common mechanisms of EGFR-TKIs resistance (Wang et al. 2016). However, there is still a lack of effective treatment for EGFR-TKIs resistance mediated by abnormal activation of PI3K-Akt signaling pathway (Tan, 2020). AKT1 is an important target in the PI3K-Akt signaling pathway. It is regulated by PI3K and PTEN. Its abnormal activation will promote tumor cell invasion, migration, proliferation and anti-apoptosis. After activation, PI3K accumulates on the cell membrane and phosphorylates 3,4-diphosphate phosphatidylinositol (PIP2) to 3,4,5-triphosphate phosphatidylinositol (PIP3) (Tiwari et al. 2017). PIP3 binds to AKT and activates Thr308 and Ser473 sites. The activated AKT then activates the mTOR signaling pathway, regulates protein synthesis and cell growth, promotes cell proliferation, and inhibits apoptosis. PTEN is the first known tumor suppressor gene with phosphatase activity. It antagonizes PI3K and inhibits AKT activation by promoting dephosphorylation of PIP3 at D3 to generate PIP2(Li et al. 2016). The results of KEGG enrichment analysis showed that EGFR tyrosine kinase inhibitor resistance and PI3K-Akt signaling pathway, which are closely related to EGFR and AKT1, were significantly enriched. It also proved that there was a strong correlation between the three traditional Chinese medicines screened in the previous study and the enriched targets and pathways. They may form a network of mutual synergy and interaction through these targets and pathways, thus playing a therapeutic role in NSCLC. The results of GO enrichment analysis also prove this point.

The experimental results of CCK-8 showed that the three core components of quercetin, kaempferol and isorhamnetin screened in this study all had anti-NSCLC activity in a concentration-dependent and time-dependent manner, and isorhamnetin had the best inhibitory effect. Combined with scratch test and transwell test, it can be seen that isorhamnetin not only inhibits the proliferation, but also inhibits migrate and invade ability of A549 cells. It can be seen from the flow cytometry results that isorhamnetin inhibits the proliferation of A549 cells in two aspects: on the one hand, it promotes apoptosis, and on the other hand, it mediates cell cycle arrest. The rapid proliferation and metastasis of tumor cells are closely related to the disease progression of patients. If the proliferation and metastasis of tumor cells are inhibited, the speed and risk of deterioration of patients can be reduced. In order to verify whether isorhamnetin acts on A549 cells through the PI3K-Akt signaling pathway, this study performed Western blot experiments on PI3K and AKT, two key targets of the PI3K-Akt signaling pathway. PI3K has three expression types, among which PI3KI is most associated with the occurrence and development of cancer (Fruman et al. 2017). It can phosphorylate PIP2 to PIP3, and then transfer Akt to the plasma membrane to activate it. Akt contains three homologous genes: Akt1, Akt2 and Akt3.After activation, it will further activate its downstream target proteins to mediate the growth, invasion, metastasis and anti-apoptosis of tumor cells (Revathidevi et al. 2019). From the experimental results, it can be seen that the P-AKT/AKT and P-PI3K/PI3K in the treatment group were significantly lower than those in the control group, indicating that the AKT phosphorylation process was inhibited by isorhamnetin, indicating that the PI3K-Akt signaling pathway is one of the mechanisms by which isorhamnetin inhibits A549 cells.

From the results of KEGG and GO enrichment analysis, it can be seen that the inhibitory mechanism of the core traditional Chinese medicine screened in this study on NSCLC is not only related to the PI3K-Akt signaling pathway, but also the active components of these three traditional Chinese medicines against NSCLC are not only quercetin, kaempferol and isorhamnetin. In the existing reports, they also have many components with anti-tumor activity (Yang et al. 2024; Zhang et al. 2023; Qiao et al. 2023; Xie et al. 2024). Therefore, further research on these anti-tumor active drugs is needed to screen out more anti-tumor active components for new drug development and clinical application.

In this study, the medication rules of 269 prescriptions for the treatment of NSCLC were analyzed. The results showed that the frequency of HQ, BZ and FL was higher than 100, and the association rule analysis also showed that they were highly correlated and clustered into the same category in cluster analysis, indicating that they had potential synergistic effects in the treatment of NSCLC and might be the core Chinese medicine for the treatment of NSCLC. In this study, 116 potential targets of these three core TCMs for NSCLC were collected. Through PPI network construction and mapping relationship between components and targets, we found out three key components of these three TCMs, quercetin, kaempferol and isorhamnetin, as well as the core targets AKT1 and EGFR for the treatment of NSCLC. Molecular docking verified that they could be stably combined; at the same time, through enrichment analysis, it was found that the mechanism of these three Chinese medicines in the treatment of NSCLC may be related to the PI3K-Akt pathway. Subsequent cell experiments further proved that Quercetin, Kaempferol and Isorhamnetin all had the effect of inhibiting A549 cells, and Isorhamnetin had the best effect. Isorhamnetin also inhibited the proliferation, migration and invasion of A549 cells, induced apoptosis and cell cycle arrest, and inhibited the phosphorylation of AKT1 and PI3K.

Author contributions Qian Tang & Xuefang Zheng：Conceptualization, Funding acquisition, Project administration, Writing-review & editing.

Yu Tang：Methodology, Investigation, Writing-original draft, Preparation, Writing-review & editing.

Zhengping Xian：Investigation, Preparation, Technical supports, Writing-review & editing.

Fengjiao Wu：Technical supports.

Hongyu Cao：Software, Writing-review & editing.

Lihao Wang：Writing-review & editing.

Yanan Du：Writing-review & editing.

The authors declare that all data were generated in-house and that no paper mill was used.

Funding This work was financially supported by the National Natural Science Foundation of China (NO. 21571025，No.21601024，No.21601025), the subject construction project-the interdisciplinary project of Dalian University (DLUXK-2023-YB-007).

Data availability The data that support the findings of this study are available from the corresponding author upon reasonable request.

Competing interests The authors declare no competing interests.

Ethical approval Not applicable.

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Traditional Chinese medicine combined with chemotherapy in the treatment of advanced non-small cell lung cancer: Key drug screening and mechanism analysis

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