Network pharmacology-based approach uncovers the pharmacodynamic components and mechanism of Fructus Tribuli for improving endothelial dysfunction in hypertension.

Fructus Tribuli (FT), a traditional Chinese medicinal herbal, has been used for the clinical treatment of cardiovascular diseases for many years and affects vascular endothelial dysfunction (ED) in patients with hypertension. This study aimed to demonstrate the pharmacodynamic basis and mechanisms of FT for the treatment of ED. The present study used ultra-high-performance liquid chromatography coupled with quadruple-time-of-flight mass spectrometry (UHPLC-Q-TOF/MS) to analyze and identify the chemical components of FT. The active components in blood were determined after the oral administration of FT by comparative analysis to blank plasma. Then, based on the active components in vivo, network pharmacology was performed to predict the potential targets of FT in treating ED. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were also performed, and component-target-pathway networks were constructed. Interactions between the major active components and main targets were verified by molecular docking. Moreover, spontaneously hypertensive rats (SHRs) were divided into the normal, model, valsartan, low-dose FT, medium-dose FT, and high-dose FT experimental groups. In pharmacodynamic verification studies, treatment effects on blood pressure, serum markers (nitric oxide [NO], endothelin-1 [ET-1,], and angiotensin Ⅱ [Ang Ⅱ)]) of ED, and endothelial morphology of the thoracic aorta were evaluated and compared between groups. Finally, the PI3K/AKT/eNOS pathway was analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot of the thoracic aorta of rats in each group to detect the mRNA expression of PI3K, AKT, and eNOS and the protein expression of PI3K, AKT, p-AKT, eNOS, and p-eNOS. A total of 51 chemical components were identified in FT, and 49 active components were identified in rat plasma. Thirteen major active components, 22 main targets, and the PI3K/AKT signaling pathway were screened by network pharmacology. The animal experiment results showed that FT reduced systolic blood pressure and ET-1 and Ang Ⅱ levels and increased NO levels in SHRs to varying degrees. The therapeutic effects were positively correlated with the oral dose of FT. Hematoxylin-eosin (HE) staining confirmed that FT could alleviate the pathological damage of the vascular endothelium. qRT-PCR and Western blot analysis confirmed that up-regulated expression of the PI3K/AKT/eNOS signaling pathway could improve ED. In this study, the material basis of FT was comprehensively identified, and the protective effect on ED was confirmed. FT had a treatment effect on ED through multi-component, multi-target, and multi-pathways. It also played a role by up-regulating the PI3K/AKT/eNOS signaling pathway.

Wang SY ，Sun XC ，Lv XY ，Li JN ，Han B ，Liu KL ，Wang S ，Sheng HG ，Zhang C ，Guo F ，Cui YD ... -  《-》

Network pharmacology-based prediction and validation of the active ingredients and potential mechanisms of the Huangxiong formula for treating ischemic stroke.

Huangxiong Formula (HXF) is composed of four herbs: Rheum palmatum L., Ligusticum striatum DC., Curcuma aromatica Salisb., and Acorus gramineus Aiton. HXF is clinically used for the treatment of ischemic stroke (IS). However, its molecular mechanism remains unclear. A network pharmacology-based strategy combined with experimental study in vivo and in vitro to were used to investigate the bioactive components, potential targets, and molecular mechanisms of HXF in the treatment of IS. The components of HXF were detected by ultra-performance liquid chromatography (UPLC). The potential active ingredients of HXF were acquired from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) and literature, and corresponding targets were discerned through the Swiss TargetPrediction database. IS-related targets were obtained from Genecards, Online Mendelian Inheritance in Man (OMIM), Therapeutic Target Database (TTD), and DisGeNET. The intersection of ingredient and disease targets was screened, and a herbal-compound-target network was constructed. A protein-protein interaction (PPI) network was created, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed. Based on these analyses, we established a compound-target-pathway (C-T-P) network. A cerebral ischemia-reperfusion (I/R) animal model was established, and the cerebral protective effect of HXF was assessed. The accuracy of the predicted targets was verified by real-time quantitative polymerase chain reaction (RT-qPCR). Hippocampal neuronal injury cell model induced by oxygen-glucose deprivation and reperfusion (OGD/R) was used to evaluate the protective effect of α-Asarone. Furthermore, molecular docking, drug affinity responsive target stability (DARTS) assay, and cellular thermal shift assay (CETSA) were performed to verify whether α-Asarone can bind to PI3K. A total of 44 active ingredients and 795 gene targets were identified through network pharmacology. Network analysis showed that naringenin, eupatin, kaempferol, and α-Asarone were possible drug candidates. SRC, AKT1, TP53, MAPK3, STAT3, HRAS, CTNNB1, EGFR, VEGFA, PIK3R1 could serve as potential drug targets. KEGG analysis implied that the PI3K/AKT signaling pathway might play an important role in treating IS by HXF. Moreover, HXF significantly reduced neurological impairment, cerebral infarct volume, brain index, and brain histopathological damage in I/R rats. The mRNA expression of the top 10 potential targets was verified in the brain tissue. The C-T-P network and UPLC analysis suggested that α-Asarone might be an important component of HXF and can inhibit oxidative stress and apoptosis in HT22 cells by activating the PI3K/AKT signaling pathway. Molecular docking, DARTS, and CETSA assay analysis confirmed that there were direct interactions between α-Asarone and PI3K. HXF had a therapeutic effect in IS with multi-component, multi-target, and multi-approach features. α-Asarone, identified as one of the major active components of HXF, could alleviate oxidative stress and apoptosis by targeting PI3K/AKT pathway.

Zhao S ，Zhang P ，Yan Y ，Xu W ，Li J ，Wang L ，Wang N ，Huang Y ... -  《-》

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 ... -  《-》

Influence of electroacupuncture on ghrelin and the phosphoinositide 3-kinase/protein kinase B/endothelial nitric oxide synthase signaling pathway in spontaneously hypertensive rats.

To investigate the influence of electroacupuncture (EA) on ghrelin and the phosphoinositide 3-kinase/protein kinase B/endothelial nitric oxide synthase (PI3K/Akt/eNOS) signaling pathway in spontaneously hypertensive rats (SHRs). Eight Wistar-Kyoto rats were used as the healthy blood pressure (BP) control (normal group), and 32 SHRs were randomized into model group, EA group, EA plus ghrelin group (EA + G group), and EA plus PF04628935 group (a potent ghrelin receptor blocker; EA + P group) using a random number table. Rats in the normal group and model group did not receive treatment, but were immobilized for 20 min per day, 5 times a week, for 4 continuous weeks. SHRs in the EA group, EA + G group and EA + P group were immobilized and given EA treatment in 20 min sessions, 5 times per week, for 4 weeks. Additionally, 1 h before EA, SHRs in the EA + G group and EA + P group were intraperitoneally injected with ghrelin or PF04628935, respectively, for 4 weeks. The tail-cuff method was used to measure BP. After the 4-week intervention, the rats were sacrificed by cervical dislocation, and pathological morphology of the abdominal aorta was observed using hematoxylin-eosin (HE) staining. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of ghrelin, nitric oxide (NO), endothelin-1 (ET-1) and thromboxane A2 (TXA2) in the serum. Isolated thoracic aortic ring experiment was performed to evaluate vasorelaxation. Western blot was used to measure the expression of PI3K, Akt, phosphorylated Akt (p-Akt) and eNOS proteins in the abdominal aorta. Further, quantitative real-time polymerase chain reaction (qRT-PCR) was conducted to measure the relative levels of mRNA expression for PI3K, Akt and eNOS in the abdominal aorta. EA significantly reduced the systolic BP (SBP) and diastolic BP (DBP) (P < 0.05). HE staining showed that EA improved the morphology of the vascular endothelium to some extent. Results of ELISA indicated that higher concentrations of ghrelin and NO, and lower concentrations of ET-1 and TXA2 were presented in the EA group (P < 0.05). The isolated thoracic aortic ring experiment demonstrated that the vasodilation capacity of the thoracic aorta increased in the EA group. Results of Western blot and qRT-PCR showed that EA increased the abundance of PI3K, p-Akt/Akt and eNOS proteins, as well as expression levels of PI3K, Akt and eNOS mRNAs (P < 0.05). In the EA + G group, SBP and DBP decreased (P < 0.05), ghrelin concentrations increased (P < 0.05), and the concentrations of ET-1 and TXA2 decreased (P < 0.05), relative to the EA group. In addition, the levels of PI3K and eNOS proteins, the p-Akt/Akt ratio, and the expression of PI3K, Akt and eNOS mRNAs increased significantly in the EA + G group (P < 0.05), while PF04628935 reversed these effects. EA effectively reduced BP and protected the vascular endothelium, and these effects may be linked to promoting the release of ghrelin and activation of the PI3K/Akt/eNOS signaling pathway.

Zhang Y ，Zhong DL ，Zheng YL ，Li YX ，Huang YJ ，Jiang YJ ，Jin RJ ，Li J ... -  《Journal of Integrative Medicine-JIM》

Network pharmacology and experimental verification to explore the anti-superficial thrombophlebitis mechanism of Mailuo shutong pill.

Li S ，Xiao H ，Liu M ，Wang Q ，Sun C ，Yao J ，Cao N ，Zhang H ，Zhang G ，Xiao X ... -  《-》