A network pharmacology approach and experimental validation to investigate the anticancer mechanism and potential active targets of ethanol extract of Wei-Tong-Xin against colorectal cancer through induction of apoptosis via PI3K/AKT signaling pathway.
Wei-Tong-Xin (WTX), derives from the Chinese herbal decoction (CHD) of Wan-Ying-Yuan in ancient China, has been shown to be effective therapeutic herbal decoction for treating gastrointestinal diseases. Present studies have demonstrated that WTX had potential to alleviate the symptoms of gastrointestinal inflammation, gastric ulcer and improve gastric motility.
The study primarily focused on exploring the therapeutic effect and possible pharmacological mechanism of WTX on colorectal cancer (CRC) based on network pharmacology, in vitro and in vivo experiments.
Firstly, colorectal cancer and WTX associated with targets were searched from GeneCards database and TCM Systems Pharmacology Database and Analysis Platform (TCMSP) respectively. The protein-protein interaction (PPI) network also was constructed to screening key targets. In addition, the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were applied to predict the underlying biological function and mechanism involving in the anti-colorectal cancer effect of WTX. Next, CCK-8, colony formation and transwell assays were performed to verify the influence of proliferation and metastasizing ability of HCT116 cells after treated with WTX. Cell cycle, apoptosis and reactive oxygen species (ROS) were analysis by flow cytometry. Hoechst 33258 staining was conducted to observe nuclear morphology changes. Protein expression of apoptosis and PI3K/AKT signaling as well as mRNA expression of ferroptosis and apoptosis were determined by Western Blotting and RT-qPCR. The effects of WTX and LY294002 combination on the PI3K/Akt/mTOR signaling pathway were measured by Western Blotting. Finally, the xenograft tumor mouse model was established by subcutaneous injection of CT26 cells to measure tumors volume and weight. Hematoxylin and eosin (HE) staining and immunohistochemical analysis were used to observe the pathological changes and the protein expression in tumor tissues.
There were 286 potential treatment targets from 130 bioactive compounds in WTX, 1349 CRC-related targets were identified. Eleven core targets (TP53, AKT1, STAT3, JUN, TNF, HSP90AA1, IL-6, MAPK3, CASP3, EGFR, MYC) were found by PPI network analysis constructed of 142 common targets. The results of KEGG enrichment displayed PI3K/AKT signaling pathway as core pathway. After the treatment of WTX, the inhibitory of viability, metastases and cell cycle arrest at G2/M phase were observed in HCT116 cells. Moreover, WTX induced an increase in the expression of apoptosis proteins (Bak, cytochrome c, cleaved caspase-9/caspase-9 and cleaved caspase-3/caspase-3) and the levels of ROS and MDA, a decrease in the expression of PI3K/AKT signaling related proteins (PI3K, p-PI3K, p-AKT/AKT and p-mTOR/mTOR) and the level of SOD. WTX treatment significantly reduced the tumor weight, increased cleaved caspase-3 positive area and decreased that of ki67 in xenograft mouse model.
Through a network pharmacology approach and in vitro experiments, we predicted and verified the effect of WTX on colorectal cancer cells mainly depended on the regulation of intrinsic apoptosis via PI3K/AKT signaling pathway, and further animal experiments proved that WTX has a good anti-colon cancer effect in vivo.
Lin F
，Zhang G
，Yang X
，Wang M
，Wang R
，Wan M
，Wang J
，Wu B
，Yan T
，Jia Y
... -
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Uncovering the pharmacological mechanism of Wei-Tong-Xin against gastric ulcer based on network pharmacology combined with in vivo experiment validation.
The prescription of Wei-Tong-Xin (WTX) is improved based on the prescription "Wanyingyuan", a famous decoction documented in the book of Huatuozhongzangjing in the Han dynasty. Many years of clinical verification have demonstrated that WTX can be used to treat gastrointestinal diseases, especially gastric ulcer (GU). However, the potential pharmacological mechanism is undefined.
This research was conducted to explore the pharmacological mechanisms under the consideration of the therapeutical effect of WTX against GU by combining the network pharmacology strategy and in-vivo verified experiments.
A prediction network describing the relationship between WTX and GU was established based on information collected from multiple databases. Then, the intersecting protein-protein interaction (PPI) network of the drug-disease overlapping gene targets was constructed, and several key targets related to both WTX and GU were obtained. Besides, the Gene Ontology (GO) biological enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed to investigate the key target genes and pathways of WTX against GU. Then, the candidate targets and signaling pathways of network pharmacology were validated in a rat model of GU induced by indomethacin following the results and available proof.
There are 243 targets obtained from the 65 active ingredients in WTX, and 1362 disease targets related to GU were identified. Then, 6 key targets were determined with the PPI interaction network, which was structured from 126 overlapping gene targets. GO and KEGG analyses revealed that the phosphoinositide-3-kinase/protein kinase B (PI3K/AKT) signaling pathway might play a crucial role in the therapeutic mechanism of GU. In vivo verified experiments, WTX significantly reduced the ulcer area and improved the histopathological appearance of gastric tissues. Moreover, down-regulated the protein levels of IL6, TNF-α, and Caspase 3 in the gastric tissues while up-regulating the expression of p-PI3K, p-AKT, p-P53, and VEGFA compared to the model group.
WTX, an ancient traditional Chinese medicine (TCM) compound prescription, may affect the inflammatory response and apoptosis process by regulating PI3K/AKT signaling pathway and related gene targets. Therefore, it is an effective drug candidate for the modern treatment of GU.
Liu W
，Zhang X
，Ma T
，Wang J
，Lv X
，Wu B
，Yan T
，Jia Y
... -
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Mechanism of Bazhen decoction in the treatment of colorectal cancer based on network pharmacology, molecular docking, and experimental validation.
Bazhen Decoction (BZD) is a common adjuvant therapy drug for colorectal cancer (CRC), although its anti-tumor mechanism is unknown. This study aims to explore the core components, key targets, and potential mechanisms of BZD treatment for CRC.
The Traditional Chinese Medicine Systems Pharmacology (TCMSP) was employed to acquire the BZD's active ingredient and targets. Meanwhile, the Drugbank, Therapeutic Target Database (TTD), DisGeNET, and GeneCards databases were used to retrieve pertinent targets for CRC. The Venn plot was used to obtain intersection targets. Cytoscape software was used to construct an "herb-ingredient-target" network and identify core targets. GO and KEGG pathway enrichment analyses were conducted using R language software. Molecular docking of key ingredients and core targets of drugs was accomplished using PyMol and Autodock Vina software. Cell and animal research confirmed Bazhen Decoction efficacy and mechanism in treating colorectal cancer.
BZD comprises 173 effective active ingredients. Using four databases, 761 targets related to CRC were identified. The intersection of BZD and CRC yielded 98 targets, which were utilized to construct the "herb-ingredient-target" network. The four key effector components with the most targets were quercetin, kaempferol, licochalcone A, and naringenin. Protein-protein interaction (PPI) analysis revealed that the core targets of BZD in treating CRC were AKT1, MYC, CASP3, ESR1, EGFR, HIF-1A, VEGFR, JUN, INS, and STAT3. The findings from molecular docking suggest that the core ingredient exhibits favorable binding potential with the core target. Furthermore, the GO and KEGG enrichment analysis demonstrates that BZD can modulate multiple signaling pathways related to CRC, like the T cell receptor, PI3K-Akt, apoptosis, P53, and VEGF signaling pathway. In vitro, studies have shown that BZD dose-dependently inhibits colon cancer cell growth and invasion and promotes apoptosis. Animal experiments have shown that BZD treatment can reverse abnormal expression of PI3K, AKT, MYC, EGFR, HIF-1A, VEGFR, JUN, STAT3, CASP3, and TP53 genes. BZD also increases the ratio of CD4+ T cells to CD8+ T cells in the spleen and tumor tissues, boosting IFN-γ expression, essential for anti-tumor immunity. Furthermore, BZD has the potential to downregulate the PD-1 expression on T cell surfaces, indicating its ability to effectively restore T cell function by inhibiting immune checkpoints. The results of HE staining suggest that BZD exhibits favorable safety profiles.
BZD treats CRC through multiple components, targets, and metabolic pathways. BZD can reverse the abnormal expression of genes such as PI3K, AKT, MYC, EGFR, HIF-1A, VEGFR, JUN, STAT3, CASP3, and TP53, and suppresses the progression of colorectal cancer by regulating signaling pathways such as PI3K-AKT, P53, and VEGF. Furthermore, BZD can increase the number of T cells and promote T cell activation in tumor-bearing mice, enhancing the immune function against colorectal cancer. Among them, quercetin, kaempferol, licochalcone A, naringenin, and formaronetin are more highly predictive components related to the T cell activation in colorectal cancer mice. This study is of great significance for the development of novel anti-cancer drugs. It highlights the importance of network pharmacology-based approaches in studying complex traditional Chinese medicine formulations.
Lu S
，Sun X
，Zhou Z
，Tang H
，Xiao R
，Lv Q
，Wang B
，Qu J
，Yu J
，Sun F
，Deng Z
，Tian Y
，Li C
，Yang Z
，Yang P
，Rao B
... -
《Frontiers in Immunology》
ResearchGate
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钛学术
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