Mechanism of Danshenyin in Inhibiting Platelet Activation in Rat Model of Hyperlipidemia Based on Proteomics / 中国实验方剂学杂志

ObjectiveTo decipher the mechanism of Danshenyin in regulating platelet activation in the rat model of hyperlipidemia by means of proteomics and molecular biology. MethodWistar rats were randomized into blank， model， and Danshenyin groups （n=10） according to the blood lipid level. The rats in the blank group were fed with a basic diet， and those in the model and Danshenyin groups with a high-fat diet. All the rats had free access to water and food. The treatment began at the 9th week. The rats in the Danshenyin group were administrated with Danshenyin by gavage at a crude drug dose of 3.6 g·kg-1. The rats in the model and blank groups were administrated with an equal volume of normal saline according to body weight. At the 12th week， the tissue samples were collected for the measurement of related indicators， and the blood lipid level was measured by an automatic biochemical analyzer. The whole blood viscosity and plasma viscosity were measured by an automatic hemorheometer. The platelet proteome was determined by liquid chromatography-mass spectrometry. Western blotting was employed to determine the protein levels of platelet membrane glycoprotein 4 （CD36）， focal adhesion kinase （FAK）， phosphatidylinositol 4-phosphate 5-kinase （PIP5K）， phosphorylated phosphatidylinositol 3-kinase （p-PI3K）， protein kinase B （Akt）， and phosphorylated protein kinase B （p-Akt）. ResultCompared with the model group， Danshenyin lowered the levels of total cholesterol （TC）， triglyceride （TG）， and low-density lipoprotein cholesterol （LDL-C） in the plasma （P<0.05）， elevated the level of high-density lipoprotein cholesterol （HDL-C） （P<0.05）， and reduced the platelet aggregation rate （P<0.05）. Compared with the blank group， the modeling up-regulated the expression of 44 proteins and down-regulated the expression of 12 proteins. Compared with the model group， Danshenyin up-regulated the expression of 21 proteins and down-regulated the expression of 22 proteins. Compared with the blank group， Danshenyin up-regulated the expression of 31 proteins and down-regulated the expression of 49 proteins. The gene ontology （GO） functional enrichment showed that the differentially expressed proteins were mainly involved in cholesterol transport and efflux， production of cytokines， dyslipidemia， and platelet activation. The Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway enrichment showed that the differentially expressed proteins were mainly involved in ECM-receptor interaction， peroxisome proliferators-activated receptors （PPAR）， focal adhesion， and PI3K/Akt signaling pathways. Danshenyin can significantly down-regulate the expression of CD36， FAK， PIP5K， PI3K， p-Akt （Ser473）， and p-Akt1/2/3 （Thr308）. ConclusionDanshenyin can restore the blood lipid level of hyperlipidemia rats and inhibit the platelet activation caused by abnormal lipid levels by down-regulating the CD36/PI3K/Akt signal cascade.

Constructing a competitive endogenous RNA network of EndMT-related atherosclerosis through weighted gene co-expression network analysis.

Atherosclerosis is a chronic inflammatory disease characterized by endothelial dysfunction and plaque formation. Under pro-inflammatory conditions, endothelial cells can undergo endothelial-to-mesenchymal transition (EndMT), contributing to atherosclerosis development. However, the specific regulatory mechanisms by which EndMT contributes to atherosclerosis remain unclear and require further investigation. Dan-Shen-Yin (DSY), a traditional Chinese herbal formula, is commonly used for cardiovascular diseases, but its molecular mechanisms remain elusive. Emerging evidence indicates that competing endogenous RNA (ceRNA) networks play critical roles in atherosclerosis pathogenesis. In this study, we constructed an EndMT-associated ceRNA network during atherosclerosis progression by integrating gene expression profiles from the Gene Expression Omnibus (GEO) database and weighted gene co-expression network analysis. Functional enrichment analysis revealed this EndMT-related ceRNA network is predominantly involved in inflammatory responses. ROC curve analysis showed the identified hub genes can effectively distinguish between normal vasculature and atherosclerotic lesions. Furthermore, Kaplan-Meier analysis demonstrated that high expression of IL1B significantly predicts ischemic events in atherosclerosis. Molecular docking revealed most DSY bioactive components can bind key EndMT-related lncRNAs, including AC003092.1, MIR181A1HG, MIR155HG, WEE2-AS1, and MIR137HG, suggesting DSY may mitigate EndMT in atherosclerosis by modulating the ceRNA network.

Network pharmacology and experimental analysis to reveal the mechanism of Dan-Shen-Yin against endothelial to mesenchymal transition in atherosclerosis.

Atherosclerosis is a chronic inflammatory disease characterized by the formation of plaque and endothelial dysfunction. Under pro-inflammatory conditions, endothelial cells adopt a mesenchymal phenotype by a process called endothelial-to-mesenchymal transition (EndMT) which plays an important role in the pathogenesis of atherosclerosis. Dan-Shen-Yin (DSY) is a well-known traditional Chinese medicine used in the treatment of cardiovascular disease. However, the molecular mechanism whereby DSY mitigates atherosclerosis remains unknown. Therefore, we employed a network pharmacology-based strategy in this study to determine the therapeutic targets of DSY, and in vitro experiments to understand the molecular pharmacology mechanism. The targets of the active ingredients of DSY related to EndMT and atherosclerosis were obtained and used to construct a protein-protein interaction (PPI) network followed by network topology and functional enrichment analysis. Network pharmacology analysis revealed that the PI3K/AKT pathway was the principal signaling pathway of DSY against EndMT in atherosclerosis. Molecular docking simulations indicated strong binding capabilities of DSY's bioactive ingredients toward PI3K/AKT pathway molecules. Experimentally, DSY could efficiently modify expression of signature EndMT genes and decrease expression of PI3K/AKT pathway signals including integrin αV, integrin ß1, PI3K, and AKT1 in TGF-ß2-treated HUVECs. LASP1, which is upstream of the PI3K/AKT pathway, had strong binding affinity to the majority of DSY's bioactive ingredients, was induced by EndMT-promoting stimuli involving IL-1ß, TGF-ß2, and hypoxia, and was downregulated by DSY. Knock-down of LASP1 attenuated the expression of integrin αV, integrin ß1, PI3K, AKT1 and EndMT-related genes induced by TGF-ß2, and minimized the effect of DSY. Thus, our study showed that DSY potentially exerted anti-EndMT activity through the LASP1/PI3K/AKT pathway, providing a possible new therapeutic intervention for atherosclerosis.

Dan-Shen-Yin Granules Prevent Hypoxia-Induced Pulmonary Hypertension via STAT3/HIF-1α/VEGF and FAK/AKT Signaling Pathways.

Traditional Chinese medicine (TCM) plays an important role in the treatment of complex diseases, especially cardiovascular diseases. However, it is hard to identify their modes of action on account of their multiple components. The present study aims to evaluate the effects of Dan-Shen-Yin (DSY) granules on hypoxia-induced pulmonary hypertension (HPH), and then to decipher the molecular mechanisms of DSY. Systematic pharmacology was employed to identify the targets of DSY on HPH. Furthermore, core genes were identified by constructing a protein-protein interaction (PPI) network and analyzed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes (KEGG) analysis. Related genes and pathways were verified using a hypoxia-induced mouse model and hypoxia-treated pulmonary artery cells. Based on network pharmacology, 147 potential targets of DSY on HPH were found, constructing a PPI network, and 13 hub genes were predicted. The results showed that the effect of DSY may be closely associated with AKT serine/threonine kinase 1 (AKT1), signal transducer and activator of transcription 3 (STAT3), and HIF-1 signaling pathways, as well as biological processes such as cell proliferation. Consistent with network pharmacology analysis, experiments in vivo demonstrated that DSY could prevent the development of HPH in a hypoxia-induced mouse model and alleviate pulmonary vascular remodeling. In addition, inhibition of STAT3/HIF-1α/VEGF and FAK/AKT signaling pathways might serve as mechanisms. Taken together, the network pharmacology analysis suggested that DSY exhibited therapeutic effects through multiple targets in the treatment of HPH. The inferences were initially confirmed by subsequent in vivo and in vitro studies. This study provides a novel perspective for studying the relevance of TCM and disease processes and illustrates the advantage of this approach and the multitargeted anti-HPH effect of DSY.

Protection Mechanism of Addition and Subtraction Therapy of Danshenyin Combined with Wendantang for Myocardial Ischemia in Patients with Stable Angina / 中国实验方剂学杂志

Objective:To observe the efficacy of addition and subtraction therapy of Danshenyin combined with Wendantang in the treatment to stable angina pectoris （SAP） with stagnation of phlegm and blood stasis， and to explore its protection mechanism for myocardial ischemia. Method:One hundred and thirty-two patients were randomly divided into control group and observation group equally. Finished the 63 cases study both in control group and observation group after dropout， loss of follow-up and withdrawal. Patients in control group and observation group got antianginal drugs and the treatment of drug therapy to control the risk factors. All patients were treated with anti-angina drugs and risk factors control drugs. Patients in control group got Danlou Tablets by oral administration， 5 tablets/time， 3 times/day. Patients in observation group got dispensing decoction pieces of Danshenyin and Wendantang 1 dose/day. The treatment continued for 3 months in both groups. Scores of angina attack were graded every week. Before and after treatment， electrocardiogram treadmill exercise test was made to evaluate myocardial ischemia of coronary heart disease， and scores of phlegm stasis block syndrome and Seattle Angina questionnaire （SAQ） were graded for TCM symptoms and quality of life. Levels of hemorheology index， interleukin-6 （IL-6）， tumor necrosis factor-<italic>α</italic> （TNF-<italic>α</italic>）， intercellular adhesion molecule-1 （ICAM-1）， Cystatin C （CysC）， homocysteine （Hcy）， ischemic modified albumin （IMA） and macrophage migration inhibitory factor were detected. In addition， safety was evaluated. Result:After treatment， scores of times， duration， degree of angina pectoris， nitroglycerin dosage of angina pectoris and nitroglycerin dosage in the observation group were lower than those in the control group （<italic>P</italic><0.01）. Total exercise time， duration of ST depression for 1.0 mm， occurrence time of stable angina pectoris， metabolic equivalent and scores of Duke in the observation group were more than those in the control group （<italic>P</italic><0.01）. Score of stagnation of phlegm and blood stasis in the observation group was lower than that in the control group （<italic>P</italic><0.01）， while score of SAQ was higher than that in the control group （<italic>P</italic><0.01）. Levels of IL-6， TNF-<italic>α</italic>， ICAM-1， CysC， IMA， Hcy， MIF， whole blood viscosity （low cut， high cut）， whole blood reducing viscosity， plasma viscosity， platelet aggregation rate and fibrinogen （FIB） in the observation group were lower than those in the control group （<italic>P</italic><0.01）. Effect of angina pectoris in observation group was superior to that in control group （<italic>Z</italic>=2.091， <italic>P</italic><0.01）. No adverse reactions related to Danshenyin combined with Wendantang were found. Conclusion:Addition and subtraction therapy of Danshenyin combined with Wendantang based on the routine western medicine treatment can control the attack of angina pectoris， relieve the symptoms of phlegm and stasis block syndrome， and improve the quality of life for patients with SAP， showing superior clinical efficacy and safety. In addition， it can improve the hemorheology of patients， inhibit the inflammatory reaction， reduce the stenosis or obstruction of lumen in order to improve the degree of myocardial ischemia.

Protective Effect of Modified Danshenyin and Erchentang on Intimal Injury Caused by Carotid Atherosclerosis / 中国实验方剂学杂志

Objective:To discuss the clinical efficacy of modified Danshenyin and Erchentang in treating carotid atherosclerosis （CAS）， and the effect on intimal injury. Method:Patients （151 cases） were divided into control group （75 cases） and observation group （76 cases）. Specifically， 69 cases in control finished the treatment （4 cases fell off in follow-up， and 2 cases were eliminated）， and 69 cases in observation group finished the treatment （3 cases fell off in follow-up， and 4 cases were eliminated）. Patients in both group got atorvastatin calcium tablets， 10 mg/time， 1 time/day， and aspirin enteric-coated tablets， 100 mg/time， 1 time/day. Patients in control group got Hedan tablets， 2 tablets/time， 3 times/day. Patients in observation group got modified Danshenyin and Erchentang， 1 dose/day. The treatment lasted for 4 months. Before and after treatment， color Doppler ultrasound of carotid artery was detected， and carotid intima-media thickness （IMT）， plaque number， plaque area， plaque thickness and hemodynamics were recorded. Levels of nitric oxide （NO）， endothelin-1 （ET-1）， von Willebrand factor （vWF）， soluble intercellular adhesion molecule-1 （sICAM-1）， vascular endothelial growth factor （VEGF）， matrix metalloproteinase-9 （MMP-9）， triglyceride （TG）， total cholesterol （TC）， high-density lipoprotein cholesterol （HDL-C）， low-density lipoprotein cholesterol （LDL-C）， whole-blood low-shear viscosity （LBV）， whole-blood high-shear viscosity （HBV）， plasma viscosity （PV）， platelet aggregation rate （PAR）， fibrinogen （FIB）， homocysteine （Hcy）， interleukin-6 （IL-6）， IL-10， tumor necrosis factor-<italic>α</italic> （TNF-<italic>α</italic>）， serum superoxide dismutase （SOD）， malondialdehyde （MDA）， oxidized low density lipoprotein （ox LDL） and circulating glutathione peroxidase （GSH-Px） were detected before and after treatment. And the safety was evaluated. Result:After treatment， IMT， number， area and thickness of plaque in observation group were less than those in control group （<italic>P</italic><0.01）. Peak systolic velocity and end diastolic velocity in observation group were higher than those in control group （<italic>P</italic><0.01）， while pulsatility index and resistance index were lower than those in control group （<italic>P</italic><0.01）. And levels of ET-1， vWF， sICAM-1， VEGF， MMP-9， TG， TC， LDL-C， LBV， HBV， PV， PAR， FIB， Hcy， IL-6， TNF-<italic>α</italic>， MDA and ox-LDL were lower than those in control group （<italic>P</italic><0.01）， whereas levels of NO， HDL-C， IL-10， SOD and GSH-Px were higher than those in control group （<italic>P</italic><0.01）. And there was no adverse reaction caused by traditional Chinese medicine. Conclusion:Modified Danshenyin and Erchentang can reduce plaque， improve hemodynamics and hemorheology， and regulate blood lipid metabolism and vascular endothelial factor， with anti-inflammatory and anti-oxidation damages. It can protect vascular intima， and inhibit the occurrence and development of CAS， with a safety in clinical use.

Effect of Method of Regulating Qi to Dissipate Blood Stasis and Phlegm on Cardiac Rehabilitation in Patients with Qi Deficiency and Blood Stasis Type Chronic Heart Failure / 中国实验方剂学杂志

Objective: To observe effect of method of regulating Qi to dissipate blood stasis and phlegm on degree of heart failure, ventricular remodeling, disease progression in patients with Qi deficiency and blood stasis type chronic heart failure (CHF).Method: One hundred and thirty-four patients with CHF were randomly divided into control group (62 cases) and observation group (62 cases) by random number table. The patients in control group got spironolactone tablets, 20 mg/time and qd. Benazepril, 20 mg/time and qd. Bisoprolol, 10 mg/time and qd. And digaoxin tablets if necessary. Based on the treatment in control group, patients in observation group additionally received Danshenyin and Xuefu Zhuyutang, 1 dose/day. The treatment course was 3 months in both groups. Before and after treatment, scores of Lee heart failure score were graded, cardiac function classification of the New York Heart Association (NYHA), 6 mins' walking test (6 MWT), scores of Qi deficiency and blood stasis and Minnesota living with heart failure questionnaire (MLHFQ) were evaluated. Echocardiography, left ventricular ejection fraction (LVEF), Left ventricular end-diastolic diameter (LVEDd), left ventricular end-stolic diameter (LVEDs), interventricular septum thickness at end-diastole (IVSd) and left ventricular myocardial mass index (LVMI) were recorded. Levels of matrix metalloprotein-9 (MMP-9), tissue inhibitor of matrix metalloprotease-1 (TIMP-1), transforming growth factor-β1 (TGF-β1), N-terminal pro-B-type na-triuretic peptide (NT-proBNP), galectin-3 and copeptin were detected.Result: Ridit analysis showed that after treatment, effect on cardiac function in observation group was better than that in control group (PPPPPβ1, NT-proBNP, galectin-3 and copeptin in observation group were lower than those in control group, while level of TIMP-1 was higher than that in control group (PConclusion: Based on the routine western medicine treatment, additional Danshenyin and Xuefu Zhuyutang can ameliorate symptoms of heart failure, relieve degree of heart failure, improve exercise tolerance and quality of life, inhibit ventricular remodeling, improve cardiac rehabilitation and delay progress of the disease for the CHF patients with Qi deficiency and blood stasis.