Breast cancer (BC) is currently the most frequent and lethal cancer among women, and therefore, identification of novel biomarkers and potential anticancer agents for BC is crucial. Chelidonine is one of the main active ingredients of Chelidonium majus, which has been applied in Chinese medicine prescriptions to treat cancer. This paper aimed to evaluate the ability of chelidonine to trigger mitotic catastrophe in BC cells and to clarify its mechanism through the AKT/FOXO3/FOXM1 pathway. Bioinformatics analysis revealed that forkhead box O3 (FOXO3) was downregulated in different subtypes of BC. Factors such as age, stage, Scarff-Bloom-Richardson (SBR) grade, diverse BC subclasses, and triple-negative status were inversely correlated to FOXO3 levels in BC patients compared with healthy controls. Notably, patients exhibiting higher FOXO3 expression levels demonstrated better overall survival (OS) and relapse-free survival (RFS). Moreover, FOXM1 levels were negatively correlated with both OS and RFS in BC patients. These results revealed that FOXO3 might be considered a predictive biomarker for the prognosis of BC. By utilizing Gene Set Enrichment Analysis (GSEA), we delved into the main Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment pathways of FOXO3, and the results suggested that FOXO3 was mainly involved in cancer-related pathways and the cell cycle. Thereafter, MTT and flow cytometry (FCM) analysis indicated that chelidonine inhibited BC cell line proliferation and induced M phase arrest. It was found that chelidonine treatment induced MCF-7 cell apoptosis, significantly reduced the expression of survivin and promoted the expression of p53 and caspase-9. Further morphological observation illustrated depolymerization of the actin skeleton and shortening of actin filaments in BC cells, leading to the typical characteristics of mitotic catastrophe, such as abnormal mitosis and multinucleated cells. Western blot analysis demonstrated that chelidonine inhibited the expression of p-AKT to promote the expression of FOXO3 protein and weaken the expression levels of FOXM1 and polo-like kinase 1 (PLK1). Taken together, our present work proved that FOXO3 might be considered a potential therapeutic target for BC. Chelidonine emerges as a promising agent to treat BC by inducing M phase arrest of BC cells and hindering the AKT/FOXO3/FOXM1 axis, thereby inducing mitotic catastrophe in BC.

Unlike single-target Western medicines, traditional Chinese medicines (TCMs) exhibit diverse curative effects against multiple diseases through their “multicomponent” and “multitarget” manifestations. However, the material basis of the major therapeutic diseases and TCM underlying molecular mechanisms remain to be challenged. In the current study, we applied, for the first time, an integrated strategy that combines network pharmacology and experimental evaluation and explored and demonstrated the underlying possible mechanisms of a classic TCM formula, Huanglian Jiedu Decoction (HLJD), in the treatment of cerebral ischemia. First, the herb compound, protein compound, and GO-BP and KEGG pathways were constructed to predict the material basis of HLJD in the treatment of cerebral ischemia and explore the underlying molecular mechanisms. Network pharmacology analysis showed that HLJD treats cerebral ischemia mainly through its anti-inflammatory effect. We used molecular docking to verify that HLJD components have good binding activities to the arachidonic acid pathway enzymes, cyclooxylipase-2 (COX-2) and 5-lipoxygenase (5-LOX). Next, based on the prediction by the network pharmacology analysis, the rat model of middle cerebral artery occlusion (MCAO) was established to verify the efficacy of HLJD. The results showed that HLJD reduces the degree of brain injury in MCAO rats, probably by inhibiting the expression of the 5-LOX pathway and inflammatory response. In conclusion, our study demonstrates the effectiveness of integrating network pharmacology with an experimental study for material basis of the major therapeutic diseases and the underlying molecular mechanisms of TCM.

Objective To explore the mechanism by which Chaihu Shugan Powder inhibits LPS-induced inflammation in BV2 cells based on network pharmacology, molecular docking and in vitro experiments. Method The antidepressant mechanism of Chaihu Shugan Powder was predicted by network pharmacological analysis, and the key active ingredients were screened in combination with molecular docking technology. Further construct an LPS-induced inflammatory model of BV2 cells and provide intervention treatment with Chaihu Shugan Powder. The protein levels and mRNA expression levels of TNF-α, IL-1β, IL-6 and IL-10 in the cell supernatants and intracellular tissues of each group were detected by ELISA kits and qRT-PCR. The level of NO in the cell supernatant was detected by the Greiss method; The expressions of microglial polarization markers iNOS, CD86, CD206 and Arg-1, as well as key proteins of the TNF-α/NF-κB pathway in each group of cells were detected by Western blot. Result The results of network pharmacological analysis showed that a total of 118 intersection targets were obtained. Based on the degree value > 3 (median), 35 core targets were screened out, and the traceability analysis obtained 8 key components that played a role. The results of pathway enrichment indicated that the antidepressant effect of Bupleurum evacuation might be related to signaling pathways such as TNF, PI3K-Akt, Apoptosis and MAPK. The results of the CCK-8 experiment determined that the high, medium and low administration doses of Chaihu Shugan Powder were 125, 62.5 and 31.25μg/mL, respectively. The results of the Greiss method for detecting the NO level in the cell supernatant showed that different doses of Chaihu Shugan Powder could significantly reduce the release of NO induced by LPS ( P <0.001). ELISA and PCR results showed that the Chaihu Shugan Powder administration group could significantly adjust back the abnormal changes in the levels of various inflammatory factors ( P <0.001, P <0.01, P <0.05). The results of Western blot detection showed that compared with the Control group, the expressions of M1-type polarization markers iNOS and CD86 in microglia of the LPS group significantly increased ( P <0.001), while the expressions of M2-type polarization markers CD206 and Arg-1 significantly decreased ( P <0.001). The protein contents of TNF-α, TLR4, p-IKKα/β, p-IκBα, NF-κB p65 and c-fos proteins increased ( P <0.001); After treatment with Chaihu Shugan Powder, the abnormal expression of the above proteins could be significantly improved in all cases ( P <0.001， P <0.01， P <0.05). The findings from molecular docking indicated that all eight essential components demonstrated strong binding affinities with the proteins previously mentioned. Conclusion Chaihu Shugan Powder can exert an antidepressant effect by inhibiting the polarization of microglia to M1 type and regulating the TNF-α/NF-κB signaling pathway, thereby suppressing the expression of inflammatory factors.