Network Pharmacology-Based Approach Uncovers the Mechanism of GuanXinNing Tablet for Treating Thrombus by MAPKs Signal Pathway.

GuanXinNing tablet (GXNT), a traditional Chinese patent medicine, has been found to have remarkable antithrombotic effects and can effectively inhibit pro-thrombotic factors in previous studies. However, the mechanism of its antithrombotic effects remains little known. In this study, we first determined and identified the sources of each main compound in GXNT using liquid chromatography-mass spectrometry (LC-MS). Through the approach of network pharmacology, we predicted the action targets of the active components, mapped the target genes related to thrombus, and obtained potential antithrombotic targets for active ingredients. We then performed gene ontology (GO) enrichment analyses and KEGG signaling pathway analyses for the action targets, and constructed networks of active component-target and active component-target-pathway for GXNT. Additionally, we evaluated the pharmacodynamic effects of GXNT on thrombus using the rat thrombus model induced by FeCl3, observed the effects of antiplatelet aggregation via platelet assay, and further verified the results predicted by network pharmacology via Western blot. In total, 14 active ingredients were identified in GXNT, and 83 action targets were predicted, 17 of which are antithrombotic targets that potentially participate in processes including response to oxidative stress and positive regulation of blood vessel endothelial cell migration. KEGG pathway analyses revealed that the predicted action targets were involved in multiple signal pathways, such as MAPK, IL-17, and platelet activation. Pharmacodynamics study found that GXNT could significantly reduce the thrombus length and weight, lower platelet aggregation function, and decrease the levels of Fbg and PAI-1. In addition, GXNT could significantly increase 6-keto-PGF1α content and regulate the ratio of TXB2/6-keto-PGF1α, while not having dramatic effects on TXB2. GXNT was also observed to visibly inhibit maximum platelet aggregation. Herein, we further studied the thrombus-related MAPKs signaling pathway and found that GXNT could significantly reduce the phosphorylation levels of p38MAPK, ERK, and JNK proteins in platelet. This study revealed the pharmacodynamic material basis of GXNT and its potential multicomponent-multitarget-multipath pharmacological effects, confirmed the antithrombotic effects of GXNT, and showed that its mechanism may be related to inhibiting phosphorylation of p38, ERK, and JNK proteins in MAPKs signaling pathway, partially verifying the results from network pharmacology. The results from this study could provide a theoretical basis for the development and clinical application of GXNT.

Wang ML ，Yang QQ ，Ying XH ，Li YY ，Wu YS ，Shou QY ，Ma QX ，Zhu ZW ，Chen ML ... -  《Frontiers in Pharmacology》

Molecular targets and mechanisms of Guanxinning tablet in treating atherosclerosis: Network pharmacology and molecular docking analysis.

Guanxinning tablet (GXNT), a Chinese patent medicine, is composed of salvia miltiorrhiza bunge and ligusticum striatum DC, which may play the role of endothelial protection through many pathways. We aimed to explore the molecular mechanisms of GXNT against atherosclerosis (AS) through network pharmacology and molecular docking verification. The active ingredients and their potential targets of GXNT were obtained in traditional Chinese medicine systems pharmacology database and analysis platform and bioinformatics analysis tool for molecular mechanism of traditional Chinese medicine databases. DrugBank, TTD, DisGeNET, OMIM, and GeneCards databases were used to screen the targets of AS. The intersection targets gene ontology and Kyoto encyclopedia of genes and genomes enrichment analysis were performed in DAVID database. GXNT-AS protein-protein interaction network, ingredient-target network and herb-target-pathway network were constructed by Cytoscape. Finally, we used AutoDock for molecular docking. We screened 65 active ingredients of GXNT and 70 GXNT-AS intersection targets. The key targets of protein-protein interaction network were AKT1, JUN, STAT3, TNF, TP53, IL6, EGFR, MAPK14, RELA, and CASP3. The Kyoto encyclopedia of genes and genomes pathway enrichment analysis showed that pathways in cancer, lipid and atherosclerosis, and PI3K-Akt signaling pathway were the main pathways. The ingredient-target network showed that the key ingredients were luteolin, tanshinone IIA, myricanone, dihydrotanshinlactone, dan-shexinkum d, 2-isopropyl-8-methylphenanthrene-3,4-dione, miltionone I, deoxyneocryptotanshinone, Isotanshinone II and 4-methylenemiltirone. The results of molecular docking showed that tanshinone IIA, dihydrotanshinlactone, dan-shexinkum d, 2-isopropyl-8-methylphenanthrene-3,4-dione, miltionone I, deoxyneocryptotanshinone, Isotanshinone II and 4-methylenemiltirone all had good binding interactions with AKT1, EGFR and MAPK14. The results of network pharmacology and molecular docking showed that the multiple ingredients within GXNT may confer protective effects on the vascular endothelium against AS through multitarget and multichannel mechanisms. AKT1, EGFR and MAPK14 were the core potential targets of GXNT against AS.

Niu C ，Zhang P ，Zhang L ，Lin D ，Lai H ，Xiao D ，Liu Y ，Zhuang R ，Li M ，Ma L ，Ye J ，Pan Y ... -  《-》

Protective effects of GuanXinNing tablet (GXNT) on diabetic encephalopathy in zucker diabetic obesity (ZDF) rats.

Diabetic encephalopathy (DE) is a complication of diabetes that leads to cognitive and behavioral decline. Utilizing safe and effective complementary and alternative medications for its management is a wise choice. Previous studies have shown that GuanXinNing Tablet (GXNT), an oral preparation primarily derived from two Chinese herbs, Salvia miltiorrhiza Bge. and Ligusticum chuanxiong Hort., exerts a beneficial neuroprotective effect. In this study, we explored the protective effects of GXNT on DE in male Zucker diabetic fatty (ZDF) rats induced by a high-fat diet, aiming to ascertain its significance and potential mechanisms. ZDF rats were induced to develop type 2 diabetes (T2DM) with DE by a high-fat diet and treated with GXNT for 8 weeks until they were 20 weeks old. Throughout the experiment, the animals' vital parameters, such as body weight, were continuously monitored. Cognitive function was evaluated using the Y maze test. Biochemical kits were employed to analyze blood glucose, lipids, and vascular endothelial-related factors. Cerebrovascular lesions were assessed using magnetic resonance angiography (MRA) imaging. Brain lesions were evaluated using hematoxylin and eosin (H&E) staining and ultrastructure observation. IgG and albumin (ALB) leakage were detected using immunofluorescence. GXNT demonstrated an enhancement in the overall well-being of the animals. It notably improved cognitive and behavioral abilities, as demonstrated by extended retention time in the novel heterogeneous arm during the Y-maze test. GXNT effectively regulated glucose and lipid metabolism, reducing fasting and postprandial blood glucose, glycated hemoglobin (HbA1c), and total cholesterol (TC) levels. Additionally, it exhibited a protective effect on the vascular endothelium by reducing the serum TXB2/PGI2 ratio while elevating NO and PGI2 levels. Moreover, GXNT ameliorated stenosis and occlusion in cerebral vessel branches, increased the number of microvessels and neurons around the hippocampus, and improved microvascular occlusion in the cerebral cortex, along with addressing perivascular cell abnormalities. Immunofluorescence staining showed a decrease in the fluorescence intensity of IgG and ALB in the cerebral cortex. GXNT demonstrated a highly satisfactory protective effect on DE in ZDF rats. Its mechanism of action could be based on the regulation of glucolipid metabolism and its protective effect on the vascular endothelium.

Li Y ，Chen J ，Tu H ，Ma Q ，Wang M ，Chen J ，Chen M ... -  《BMC Complementary Medicine and Therapies》

Guanxinning Tablet Alleviates Post-Ischemic Stroke Injury Via Regulating Complement and Coagulation Cascades Pathway and Inflammatory Network Mobilization.

Wang Y ，Li Y ，Zhou Y ，Gao Y ，Zhao L ... -  《Drug Design Development and Therapy》

Vasodilatory Effect of Guanxinning Tablet on Rabbit Thoracic Aorta is Modulated by Both Endothelium-Dependent and -Independent Mechanism.

Vasodilatory therapy plays an important role in the treatment of cardiovascular diseases, especially hypertension and coronary heart disease. Previous research found that Guanxinning tablet (GXNT), a traditional Chinese compound preparation composed of Salvia miltiorrhiza (Danshen) and Ligusticum chuanxiong (Chuanxiong), increase blood flow in the arteries, but whether vasodilation plays a role in this effect remains unclear. Here, we found that GXNT significantly alleviated the vasoconstriction of isolated rabbit thoracic aorta induced by phenylephrine (PE), norepinephrine (NE), and KCl in a dose-dependent manner with or without endothelial cells (ECs). Changes in calcium ion levels in vascular smooth muscle cells (VSMCs) showed that both intracellular calcium release and extracellular calcium influx through receptor-dependent calcium channel (ROC) declined with GXNT treatment. Experiments to examine potassium channels suggested that endothelium-denuded vessels were also regulated by calcium-activated potassium channels (Kca) and ATP-related potassium channels (KATP) but not voltage-gated potassium channels (kv) and inward rectifying potassium channels (KIR). For endothelium-intact vessels, the nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) contents in vascular tissue obviously increased after GXNT treatment, and pretreatment with the NO synthase inhibitor Nw-nitro-L-arginine methyl ester (L-NAME) or guanylyl cyclase inhibitor methylthionine chloride (MB) significantly inhibited vasodilation. An assessment of NO-related pathway protein expression revealed that GXNT enhanced the expression of phosphorylated endothelial NO synthase (eNOS) in a dose-dependent manner but had no effect on total eNOS, p-Akt, Akt, or PI3K levels in human umbilical vein ECs (HUVECs). In addition to PI3K/AKT signaling, Ca2+/calmodulin (CaM)-Ca2+/CaM-dependent protein kinase II (CaMKII) signaling is a major signal transduction pathway involved in eNOS activation in ECs. Further results showed that free calcium ion levels were decreased in HUVECs with GXNT treatment, accompanied by an increase in p-CaMKII expression, implying an increase in the Ca2+/CaM-Ca2+/CaMKII cascade. Taken together, these findings suggest that the GXNT may have exerted their vasodilative effect by activating the endothelial CaMKII/eNOS signaling pathway in endothelium-intact rings and calcium-related ion channels in endothelium-denuded vessels.

Ling Y ，Shi J ，Ma Q ，Yang Q ，Rong Y ，He J ，Chen M ... -  《Frontiers in Pharmacology》