Immunoregulatory mechanism studies of ginseng leaves on lung cancer based on network pharmacology and molecular docking

Panax ginseng is one of the oldest and most generally prescribed herbs in Eastern traditional medicine to treat diseases. Several studies had documented that ginseng leaves have anti-oxidative, anti-inflammatory, and anticancer properties similar to those of ginseng root. The aim of this research was to forecast of the molecular mechanism of ginseng leaves on lung cancer by molecular docking and network pharmacology so as to decipher ginseng leaves' entire mechanism. The compounds associated with ginseng leaves were searched by TCMSP. TCMSP and Swiss Target Prediction databases were used to sort out the potential targets of the main chemical components. Targets were collected from OMIM, PharmGKB, TTD, DrugBank and GeneCards which related to immunity and lung cancer. Ginseng leaves exert its lung cancer suppressive function by regulating the several signaling proteins, such as JUN, STAT3, AKT1, TNF, MAPK1, TP53. GO and KEGG analyses indicated that the immunoreaction against lung cancer by ginseng leaves might be related to response to lipopolysaccharide, response to oxidative stress, PI3K-Akt, MAPK and TNF pathway. Molecular docking analysis demonstrated that hydrogen bonding was interaction's core forms. The results of CCK8 test and qRT-PCR showed that ginseng leaves inhibit cell proliferation and regulates AKT1 and P53 expression in A549. The present study clarifies the mechanism of Ginseng leaves against lung cancer and provides evidence to support its clinical use.

According to the IARC, lung cancer was the malignant tumor with the highest mortality rate due to the poor prognosis 1 . Surgery is the first choice for lung cancer treatment, but most patients have already reached the middle and late-stage and have lost the best surgery chance when diagnosed. Therefore, radiotherapy and chemotherapy became the main clinical methods for treating lung cancers. However, conventional therapies have large toxic side effects, which interrupts most patients in completing an effective radiotherapy and chemotherapy cycle. Scholars have turned their attention to the development and utilization of traditional Chinese medicine, striving to develop safe and effective lung cancer adjuvant drugs.
Ginseng, dried roots of Panax ginseng C. A. Mey., is a precious Chinese herb with a long history of medicinal use. It was first published in "Shen Nong's Materia Medica" and listed as the top grade. Modern chemical and pharmacological studies have found that the extracts of ginseng leaves contained more ginsenosides than the roots and showed potent anticancer activity [2][3][4] . However, most of the researches are the anti-cancer effects of individual components of ginseng leaves. The polysaccharides in ginseng leaves inhibit tumor metastasis through activating macrophages and NK cells. Ginsenosides enhance the immune response of innate immune cells such as dendritic cells, phagocytes and NK cells by affecting the immune system and kill virus infects cells and tumor cells, regulate cytokines, complement and other immune molecules to produce immunoregulatory effects [5][6][7] . There is no report on the overall mechanism of multi-component and multi-target of ginseng leaves in the immunomodulation treatment of lung cancer. Traditional Chinese medicine has the characteristics of multiple components and multiple targets, with definite curative effect, relatively few adverse reactions, and low price 8 . It has attracted more and more attention from pharmaceutical workers. However, the features of www.nature.com/scientificreports/ Target Prediction database, two hundred and ten targets that were associated with the above active chemical compounds were discovered.
Screening mutual targets of drugs and diseases. The core active component targets of ginseng leaves were matched with the targets of lung cancer and immunity, resulting in the selection of 193 composite targets of ginseng leaves, lung cancer and immunity ( Fig. 2A) (Supplementary Table S1).
Screening and topological analysis of core targets. The PPI of the above 193 intersection targets was obtained on the STRING platform (Fig. 2B). The PPI map was taken when the data was import into Cytoscape 3.8.0. The PPI network was built with 166 nodes and 698 edges. The interaction is represented by each edge between proteins. Each Node represents the target protein molecules 15 . Then, according to the network topology's features, the median values for the DC, BC, CC, EC, LAC, NC were used to analyse potential drug targets 16 . Fifteen highly connected nodes (degree > 10, BC > 16.3058, CC > 0.5402, LAC > 5.4444, NC > 6.3355) were confirmed as significantly related targets with Network Analyzer in Table 2 and Fig. 3. The PPI network shows the  targets involved in lung cancer and immunity were HSP90AA1, JUN, STAT3 EGFR, MYC, VEGFA, CCND1,  TNF, MAPK1, AKT1, RELA, CDKN1A, TP53, IL2 and IL1B which were the major targets in lung cancer.

Enrichment analysis of GO and KEGG pathway. GO functional enrichment analysis of key targets
was performed relying on Bioconductor package in R software. The GO terms were screened according to the P (P < 0.05, Q < 0.05) value. There were 2363, 45, and 181 GO terms related to biological processes, cell components, and molecular functions, respectively (Fig. 4). As showed in Fig. 4, the top 10 biological processes were related to lipopolysaccharide, nutrient levels, oxygen levels, etc. For cellular components, the targets were  [17][18][19] . Comparing with the gene ontology and KEGG analysis outcomes, genes encoding proteins targeted by ginseng leaves should play a role in PI3K-Akt pathway, MAPK signaling pathway, TNF signaling pathway, Small cell lung cancer, Non-small cell lung cancer, apoptosis. These pathways involved cytokine receptor.
Active ingredients-shared key targets-signal pathway network diagram construction. The component-target-channel network diagram was formed to holistically make clear ginseng leaves' mechanism in Lung cancer immunity through Cytoscape 3.7.2. (Fig. 6). In the figure, the green Ellipse represented the ingredients in the drug, the yellow Diamond represented the targets of the ingredients, and the red V shape represented the pathways enriched in the targets. It demonstrated that each active compound could act upon multiple targets, each target can also respond to multiple active ingredients, and each target can have an effect on multiple pathways.     Table 3). The smaller the binding energy is, the better the ligand can bind to the protein. Mode 1 with the lowest binding energy was visualized using Pymol software (Fig. 7). The primary forms of interaction were hydrogen bonding and π-π stacking. This result indicated that their combination might play an important role in the treatment of lung cancer with ginseng leaves.   (Fig. 8A,B). Cytotoxic effect of ginseng leaves extract and F2 were confirmed against human Lung Adenocarcinoma Cells line A549.
Effect of ginseng leaves extract on the gene expression of AKT, STAT3 and P53 in A549. The mRNA expression levels of AKT1 and P53 were significantly different between the blank control group and ginseng leaves extract group (p < 0.05).Meanwhile, P53 was up-regulated expression, while AKT1 was down-regulated expression. Furthermore, there was no significant difference between groups in STAT3 mRNA expressions (Fig. 9).In conclusion, the results suggested that the above core targets could play important roles in ginseng leaves extract treatment of lung adenocarcinoma.

Discussion
Lung cancer is one of the most ordinary malignancies on earth, and its high morbidity and mortality seriously threaten the health of people all over the world 20,21 . TCM exerts its anticancer effects through apoptosis induction, proliferation inhibition, metastasis suppression, multidrug resistance reversal and immune function regulation. Moreover, TCM can ameliorate patients' quality of life 22,23 . A number of data show that ginseng leaves can inhibit tumor migration and invasion by regulating the crosstalk between tumor-related macrophages and non-small cell lung cancer, and improve the body's immune function [24][25][26][27] . Therefore, this paper researched the action mechanism from the point of immunomodulatory on lung cancer by the integrity of ginseng leaves. In this paper, the core active components in ginseng leaves were collected through data mining. The mechanism of inhibiting lung cancer through immune regulation was explored microscopically by network pharmacology. In the Venn diagram where the active ingredients of ginseng leaves intersect with diseases, there were 209 intersecting targets between drugs and lung cancer, and 193 intersecting targets between drugs and immunity. One hundred and ninety-three composite targets of ginseng leaves, lung cancer and immunity are the same as the immune intersection targets. The results showed that 92% of the treatment of ginseng leaves for lung cancer could be achieved by immune regulation.
BP analysis results showed that core active components were involved in reactive oxygen species metabolic process, response to lipopolysaccharide, response to oxidative stress, etc. Oxidative stress is characterized by the imbalance between reactive oxygen species (ROS) production and the capacity of the antioxidant system. Oxidative stress injury results in lipid peroxidation. The final product of lipid peroxidation can cause subsequent pathological consequences through the rearrangement of peroxyl radical, especially in the lungs 28 . Oxidative stress had been well documented to play a major role in cancer. Oxidative damage to DNA caused by ROS is thought to be one of the causes of cancer. Lipopolysaccharide (LPS) is a crucial component of the Gram-negative bacteria cell wall 29 . A change in the state or activity of the body caused by lipopolysaccharide stimulation. In vivo, endothelial cells, epithelial cells, monocytes and macrophages can be activated through the cell signal transduction system. Synthesizing and releasing various cell-stimulating factors, stimulating the body's active defense,  www.nature.com/scientificreports/ cause a series of inflammatory reactions, and exert the early immune response effect. Studies have confirmed that LPS can enhance the invasion and metastasis abilities of lung cancer and other malignant tumor cells 30,31 .
In the KEGG enrichment, Pathways associated with lung cancer and immunity and more enriched in 14 potential targets were PI3K-Akt pathway, MAPK signaling pathway, Non-small cell lung cancer.HSP90AA1, EGFR, MYC, VEGFA, CCND1, MAPK1, AKT1, RELA, CDKN1A, IL2 and TP53 targets are enriched in the PI3K-Akt pathway. This pathway is activated by various types of cellular stimuli or toxic insults to regulates fundamental cellular functions such as transcription, translation, proliferation, growth, and survival. PI3K/AKT pathway's deregulation is involved in lung tumorigenesis and it has been connected with high-grade tumors and advanced disease 32 . Deregulation of this pathway proceeds through a variety of mechanisms including upstream activation by tyrosine kinase receptors of PI3K, PIK3CA amplification as well as mutations in KRAS, PI3K or AKT 33 . The MAPK signaling pathway regulates the cell cycle, and the increase in its activity is positively correlated with the proliferative capacity of tumor cells, disease development and poor prognosis 34 . JUN, EGFR, MYC, VEGFA, TNF, MAPK1, AKT1, RELA, IL1B and TP53 are enriched in the MAPK signaling pathway. Studies have found that this effect may be related to the induction of Th1 cells to produce IFN-γ and promote cell proliferation 35,36 .
In the network topology analysis, the active ingredients of ginseng leaves have a greater possibility of acting on the immune regulation of lung cancer through HSP90AA1, JUN, STAT3 EGFR, MYC, VEGFA, CCND1, TNF, MAPK1, AKT1, RELA, CDKN1A, IL1B, IL2 and TP53. TP53 and JUN are important tumor suppressor genes and are new directions for gene targeted therapy of cancer 37,38 . TP53 strictly regulates the start of the cell cycle and can repair damaged DNA in time. When genes are mutated and signal pathways are abnormal, the cell cycle is out of control and certain genetic damages cannot be repaired and mutated, accelerating the progression of the tumor 39 . Studies have verified that the up-regulation of the HSP90AA1 gene reduced the body's immune surveillance and resistance to foreign substances, and involving DNA self-repair capabilities 40 . STAT3 is a transcription factor activated by various external stimuli and can induce cell growth, differentiation and survival 41 . It is mentioned that STAT3 activity is enhanced in up to 50% of all human tumors 42 . It is known that the activation of STAT3 in many malignant tumors is related to tumor proliferation, invasion, metastasis, and resistance to chemotherapy and radiotherapy 43,44 . In NSCLC, STAT3 is continuously activated in 22-65% of cases, and STAT3 activation is linked to poor prognosis, proliferation, and chemotherapy resistance [45][46][47][48] . EGFR as a transmembrane glycoprotein, which is one of the four members of the tyrosine kinase receptor ErbB family 49 is considered to be the driving gene of the tumor in the development of NSCLC 50,51 . VEGFA is a considerable target for anti-tumor therapy, and its expression is negatively associated with the differentiation and prognosis of lung cancer 52 . CCND1, the most important member of the cyclin family, is currently considered an oncogene and is overexpressed in human esophageal cancer, head and neck cancer, and breast cancer. The overexpression of CCND1 is considered an early incident in diverse carcinomas 53 .
Tumor necrosis factor TNF is called death ligand and can bind to extracellular death receptor to initiate the apoptosis program 54 . TNF-α interacts with TNFR1 and TNFR2 receptors and activates downstream intracellular signaling pathways. TNF-α induces both apoptosis and necrosis of cells through the TNFR1 55 . MAPK1 is chiefly involved in the regulation of cell proliferation, differentiation and apoptosis 56 . RelA transcription factors is a member of the NF-κB family of transcription factors and control gene expression in response to stimuli such as inflammation 57 . The abnormal expression of Myc is also associated with lung cancer, which was significantly overexpressed in more than 70% of NSCLC 58 . Early Myc activity is causally related to cancer mainly through its ability to drive tumor cell proliferation to participate in biosynthesis, cell metabolism; angiogenesis, invasion and metastasis 59 . CDKN1A plays a key role in the progression of the cell cycle and regulates both G1/S and G2/M checkpoints. CDKN1A, as an oncogene, promotes cancer cell proliferation by inhibiting apoptosis 60,61 . Interleukin-1β (IL-1β) is an inflammatory cytokine that belongs to the IL-1 family 62 . IL-1β is an effective driver of tumor progression. It is highly expressed in metastatic non-small cell lung cancer and drives tumor growth, invasion and metastasis 63 . Interleukin-2 (IL-2) is a cytokine signaling molecule necessary for the differentiation, growth and proliferation of T lymphocytes. It has been shown to improve the survival rate of patients with non-small cell lung cancer 64 . IL2 stimulates the proliferation and activation of immune cells with anti-tumor activity 65 . In summary, multiple targets and pathways are closely related to the occurrence, development, and prognosis of lung cancer, confirming the characteristics of multi-component, multi-target and multi-channel treatment of ginseng leaves.
In the component-target-pathway network, four core active components and 15 key potential targets are involved, all of which play an important part in anti-lung cancer through immune regulation. Molecular docking results indicate that the components had effective free energy against key targets. These findings verify the reliability of the active ingredients screened by network pharmacology and their interaction with lung cancer and immunity targets. Ginsenoside F2 and STAT3 have the best affinity. It has been noted that F2 induces apoptosis by causing an accumulation of ROS and activating the ASK-1/JNK signaling pathway 66 . F2 induces apoptosis of breast cancer by activating the intrinsic apoptotic pathway and mitochondrial dysfunction. However, the potential anti-cancer effect of ginsenoside F2 in lung cancer cells has not been appraised 67 . In this study, F2 inhibits the proliferation of A549 by the CCK8 experiment, but its mechanism of action is not yet understood. Further study will be made later on the basis of this finding.
Based on the research strategy of network pharmacology, this article integrates various database information and uses molecular docking for preliminary verification, and clarifies that the core components of ginseng leaves have multi-component and multi-target mechanism characteristics in inhibiting lung cancer through immunomodulation. However, Chinese medicine is not a single set of chemical components. Data mining and network pharmacology are based on the existing big data to make reasonable predictions about the internal mechanism of ginseng leaf inhibiting lung cancer through immune function. In the later stage, the above mentioned potential www.nature.com/scientificreports/ medicinal ingredients, target points, and mechanism of action need to be experimentally verified to provide a more scientific basis for the development and utilization of ginseng leaves and clinical research.

Methods
Screening of core active ingredients and targets. All the chemical constituents of Ginseng Folium were retrieved based on the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) 68 . According to the pharmacokinetics (ADME) parameters in the TCMSP database, oral availability (OB) ≥ 30% and drug-like activity (DL) ≥ 0.18 were set to screen the core active ingredients. Search for the active ingredients of ginseng leaves in the "Chinese Pharmacopoeia (2020)", and add the important active ingredients initially excluded by TCMSP as candidate active ingredients. TCMSP database and Swiss Target Prediction databases 69 were used to retrieve the gene targets for active ingredients. Obtained targets were then mapped to the UniProt database for normalization.
Disease-associated gene mining. Targets related to lung cancer and immunization were collected from the OMIM database, PharmGkb database, TTD database 70 , DrugBank database and GeneCards database 71 . The gene name's standardization and definition of the species as "human" were executed using the UniProt database. The Venn diagram of the active components, lung cancer targets and immune targets was drawn by using the bioinformatics platform Jvenn 72 .
Protein-protein interaction network construction and analysis. The potential targets were formed by STRING database to acquire the targets-PPI network. Selection parameters were set to "Homo sapiens" for species and the confidence level was set at 0.900 for the minimum required interaction score. The PPI network was then visualised by the Cytoscape software. The network analyser plugin in Cytoscape was used to calculate topological parameters in the network. Finally, the key immune regulatory target of ginseng leaves suppressing lung cancer was obtained. Network construction. The compound-target-pathway network was constructed using Cytoscape 3.7.2.

Gene ontology (GO) and the Kyoto encyclopedia of genes and genomes (KEGG) enrichment. Enrichment analysis of Potential
The topological properties were analysed using the Network Analyzer plug-in for Cytoscape to confirm the key components and targets 74 .
Molecular docking makes sure the interaction between targets and key compounds. The target protein structure was obtained from the RCSB PDB database. The 2D structure of ginseng leaves' active substance was sought by the PubChem Database 75 . ChemBio Draw 3D and Autodock Tool were applied to optimize the structure of key compounds and targets, including 3-dimensional chemical structures creation, energy minimization, and format transformation 76 . PyMol was used to process the protein, including getting rid of the ligands, rectifying protein structure, and doing away with water 74 . Docking was completed by R 4.0.2 software and Autodock Vina, and the molecules with the lowest binding energy in the docking conformation were selected to observe the binding effect by matching with the original ligands and intermolecular interactions.
Cell culture. A549 cells were obtained from the American Type Culture Collection and were plated in RPMI 1640 medium with 10% fetal calf serum and penicillin with streptomycin. Cells were all kept at 37 °C, with 5% CO 2 .
Cytotoxicity. Cytotoxicity was assayed using CCK8, taking ginseng leaves extract, ginsenoside F2 and cyclophosphamide as the different experimental groups and A549 cells as the blank control group. A549 cells were plated in 96-well plates in 100 μL of medium containing 10% heat-inactivated FBS. After 24 h, the cells were treated with cyclophosphamide, different concentrations of ginseng leaves extract, F2 and incubated for an additional 24 h. At the indicated time points,10 μL CCK8 reagent was added to the culture medium 1 h before analysis. Optical density (OD)450 values were measured using a microplate reader.
Gene expression using quantitative real-time PCR. In this trial, ginseng leaves extract was used as the different experimental groups, and A549 cells as the blank control group. After being placed into a six-well plate, the A549 cells were add to culture medium containing best concentrations of ginseng leaves extract and then cultured for 48 h. Next, Total RNA, was isolated utilizing Trizol reagent (Invitrogen), whose concentration and purity were detected using SPECTROstar Nano Microplate Reader (BMG Labtech). According to the manufacturer's protocol, the RNA was reverse-transcribed into cDNA using the extracted RNA sample as a template. The primer sequences were designed in the laboratory by Sangon Biotech (Sangon, China) based on the mRNA sequences obtained from the NCBI database and were purchased from Beijing Huada Gene and Biological Company, China (Table 4). Real-time PCR was then performed using an Applied Biosystems QuantStudio 5 Real-Time PCR system. Data reported as fold mRNA expression (2 −ΔΔCt ) and was normalized to β-actin.  TGA CCA TGA ACG AGT TTG AGTA GAG GAT CTT CAT GGC GTA GTAG   STAT3  TCG GCT AGA AAA CTG GAT AACG TGC AAC TCC TCC AGT TTC TTAA   P53  TTC CTG AAA ACA ACG TTC TGTC AAC CAT TGT TCA ATA TCG