In this study, 28 active ingredients and 180 active ingredient targets were screened from the "Astragalus-Ligustrum" drug pair. By analyzing the "drug-active-component-target" network, 10 components, including quercetin, kaempferol, isorhamnetin, jaranol, methylnissolin, luteolin and eriodictyol, were identified as the main active components of "Astragalus-Ligustrum" drug pair in the treatment of NSCLC. The results suggest that the active ingredients were mainly flavonoids. Flavonoids are naturally occurring secondary metabolites, and more than 10,000 compounds have been reported [32], which are safe and easy to obtain and have a wide range of pharmacological activities. Numerous studies have shown that flavonoids have significant preventive and curative effects on many common cancers such as lung, breast, and colon cancers, and are considered as potential candidates for new anti-cancer therapies [33]. Quercetin and kaempferol are the common active ingredients of Astragalus and Ligustrum. Quercetin is a flavonol compound and an active ingredient in many herbal medicines with a wide range of anti-lung cancer mechanisms [34–35]. Kaempferol has been shown to have various biological activities such as antioxidant, anticancer, anti-inflammatory, atherosclerosis prevention, and protection of damaged cells [36–38]. In one study, kaempferol was found to target and destroy the nuclei of A549 lung cancer cells when combined with gold nanoclusters [39]. Isorhamnetin, hualanganin, astragalus rosmarinicus chroman-7-ol, (3R)-3-(2-hydroxy-3,4-dimethoxyphenyl) chroman-7-ol, 3,9-di-O-methylnisonic pea rosmarinicol, and 7-O-methyl-isomicroconvex sabinol are the active components from Astragalus. It has been shown that isorhamnetin can inhibit the proliferation and induce apoptosis of NSCLC cells, and its mechanism of action is related to the autophagic process[40]. Few studies have been reported on the rest of the active ingredients related to lung cancer. In addition, lignocaine and sageol are the active components from Ligustrum. Lignans significantly downregulated the expression levels of AIM2 mRNA and protein in NSCLC cells, thereby inhibiting the activation of AIM2 inflammatory vesicles, inducing G2/M phase arrest in cancer cells, and suppressing the EMT process to exert anti-tumor effects[41]. Sanguinarol exerted good antitumor effects on A549 human lung cancer cells by inducing mitochondria-mediated apoptosis, G2/M cell cycle arrest and inhibition of m-TOR/PI3K/Akt signaling pathway[42].
Among the top 10 active ingredients ranked by degree value, 8 active ingredients attributed to Astragalus and 4 active ingredients attributed to Ligustrum, indicating that "Astragalus-Ligustrum" drug pair plays a major role in the treatment of NSCLC. In the PPI network, TP53, SRC, STAT3, PIK3R1 and AKT1 were identified as the core targets in the network, which are the core targets of the "Astragalus-Ligustrum" drug pair in the treatment of NSCLC. TP53 is one of the first oncogenes identified, and mutations of TP53 are found in more than 50% of human tumors, mainly missense mutations[43, 44]. It has been reported that TP53-Mut is more common in squamous lung cancer than lung adenocarcinoma, with a mutation rate of 65%, and is highly correlated with smoking[45, 46]. TP53 mutations significantly increased immune checkpoint expression, activated effector T cells and increased interferon γ levels, and it is worth mentioning that the TP53/KRAS co-mutant subgroup showed increased PD-L1 expression and the highest PD-L1+/CD8A + ratio[47]. SRC is a member of the SRC tyrosine kinase family, and aberrant Src activation is associated with the development of several human tumors, including lung cancer, especially in adenocarcinoma, where SRC kinase activity shows high levels and the degree of kinase activity increases with tumor enlargement [48]. The association between SRC family kinase (SFK) member YES1 protein expression and The SFK inhibitor dasatinib acts synergistically with anti-PD-1 to inhibit tumor growth in experimental models of NSCLC[49]. STAT3, an important member of the signal transduction and transcriptional activator family, is a key signaling pathway molecule linking inflammation and tumor, and STAT3-targeted therapies are now expected to be a promising clinical strategy [50]. The AKT-STAT3 pathway is a downstream target of the EGFR signaling pathway, and Abdelhamed S et al found that inhibition of AKT and STAT3 activity could downregulate PD-L1 expression in gefitinib-resistant NSCLC, suggesting that STAT3 could be a potential therapeutic target for reversing acquired drug resistance in NSCLC[51]. PIK3R1, the p85 regulatory subunit of phosphatidylinositol 3 kinase, is considered as a tumor suppressor and is under-expressed in most tumors[52]. It is increasingly reported [53]that when PIK3R1 expression reduced in cancers such as lung and breast cancer, phosphorylation of PI3K and AKT enhanced, leading to increased classical AKT signaling in these tumorigenic phenotypes. AKT1 is one of the important isoforms of serine/threonine protein kinase, and studies suggest that its expression is upregulated as an oncogene in NSCLC, which is closely related to tumor progression and poor prognosis. It has been found [54]that the PI3K/AKT signaling pathway may affect the activity of immune cells in the tumor immune microenvironment and negatively regulate the expression of PD-L1 in NSCLC cells, but further studies are needed. In conclusion, TP53, SRC, STAT3, PIK3R1, AKT1 and other genes have been shown to be closely related to the development of NSCLC, and it is assumed that the "Astragalus-Ligustrum" drug pair mainly exerts regulatory effects through these targets to treat NSCLC. The molecular docking results showed that quercetin, kaempferol, isorhamnetin, and isorhamnetin were all used in the treatment of NSCLC. The molecular docking results showed that the core active ingredients such as quercetin, kaempferol, isorhamnetin, astragaloside, 7-O-methyl-isoconvex sarcosol, lignan, sage phenol and TP53, SRC, STAT3, PIK3R1, AKT1, PD-1 and PD-L1 had strong binding activities, which suggested that the results of this study were reliable.
The potential targets of "Astragalus-Ligustrum" drug pair for the treatment of NSCLC mainly involved protein phosphorylation, enzyme-linked receptor protein signaling pathway, cellular response to growth factor stimulation, transmembrane receptor protein tyrosine kinase signaling pathway and other biological processes through GO bio-functional analysis. The enriched cellular fractions are mainly localized in the membrane raft and associated protein kinase complex fractions and are predominantly involved in biological functions such as kinase binding and activity. These biological processes are reflected in the targets and related pathways involved in drug resistance against NSCLC. Among them, protein kinases are a class of phosphotransferases that mainly contribute to protein phosphorylation, which is involved in most of the important life processes in cells. It has been demonstrated that flavonoids have inhibitory effects on mitogen-activated protein kinases, cytokinin-dependent protein kinases, PI3K and Akt/protein kinase B, thereby interfering with cell signaling, inducing apoptosis and inhibiting cancer cell proliferation, promoting oncogene expression and suppressing oncogene expression[55, 56]. KEGG enrichment analysis revealed that KEGG enrichment analysis showed that “Astragalus-Ligustrum” drug pair treatment of NSCLC involves several classical signaling pathways clearly associated with lung cancer, including PI3K-Akt, FoxO, PD-1/PD-L1, C-type lectin receptor, TNF, IL-17, T-cell receptor, Toll-like receptor, NOD-like receptor, NF-κB, etc. The PI3K-Akt pathway is currently one of the most important regulatory pathways in NSCLC and plays an important role in promoting NSCLC proliferation and metastasis, inducing epithelial-mesenchymal transition (EMT), and influencing acquired drug resistance in lung cancer [57–59]. There are many types of pathways in the entire KEGG database that are directly or indirectly related to PI3K-Akt. Upon activation of PI3K-Akt signaling, it is transmitted to downstream pathways, leading to the activation of a series of pathways, which include mTOR, VEGF, MAPK, FoxO, NF-κB, etc. PI3K-Akt can also be activated by multiple upstream pathways, which PI3K-Akt can also be activated by multiple upstream pathways, and these upstream signals can originate from PD-1/PD-L1, Toll-like receptors, B-cell receptors, JAK/STAT and other signaling pathways, so PI3K-Akt signaling pathway can be downstream of multiple signaling processes, or it is a very critical step of many signaling pathways. Another report showed that the PI3K-Akt pathway mediates immune escape of tumor cells by affecting the activity of immune cells in tumor microenvironment and the expression of PD-L1, which is involved in the formation of an immunosuppressive microenvironment [60]. The pathways mainly attributed to the immune system in the present findings are PD-1/PD-L1, NOD-like receptors, C-type lectin receptors, Toll-like receptors, T-cell receptors, and IL-17, and there are numerous studies showing that these pathways are closely related to the development of NSCLC [61]. Besides, TNF is a classical inflammatory, tumor and immune-related signaling pathway, and TNF-α may act as a tumor promoter gene, and in NSCLC TNF-α may regulate the cell cycle by activating the NF-κB signaling pathway, which in turn affects cell proliferation and apoptosis [62]. It is worth noting that these signaling pathways are mostly related to immune response and inflammation, suggesting that the treatment of NSCLC disease with "Astragalus-Ligustrum" drug pair may involve multiple pathways and targets, but focus on immune and inflammatory aspects. In combination with core target analysis, TP53, SRC, STAT3, PIK3R1 and AKT1 also play important roles in the PD-1/PD-L1 signaling pathway. Cancer immunotherapy is an increasingly successful strategy for the treatment of patients with advanced or conventional treatment-resistant cancers, and T cells are key mediators of tumor destruction, where they are critical for the specificity of tumor-expressed antigens. Based on this, further study will subsequently focus on the mechanisms associated with the PD-1/PD-L1, the negative regulatory signaling pathway for T cell activation.
In conclusion, 28 active ingredients and 180 active ingredient targets were screened from the "Astragalus-Ligustrum" drug pair. 10 components, including quercetin and kaempferol were identified as the main active components in the treatment of NSCLC. It affects protein phosphorylation, cellular response to growth factor stimulation and other biological processes, regulates PI3K-Akt, PD-1/PD-L1 and other lung cancer-related pathways, and then intervenes in the process of NSCLC. Further molecular kinetic results showed that the complex could bind to the target protein in the active pocket through van der Waals, hydrophobic and Pi bonds between the protein and small molecules. The binding activity between PD-L1 and the main active ingredient may be one of the important mechanisms of the anti-NSCLC action of "Astragalus-Ligustrum" drug pair. The predicted results are close to the modern pharmacological research results, which also support the feasibility of this study, and fit the holistic and systematic characteristics of Chinese medicine. Given that this is only an exploration at the level of molecular mechanism, basic experiments still need to be used subsequently to further validate its accuracy.