Cucurbitacins mitigate vascular neointimal hyperplasia by suppressing cyclin A2 expression and inhibiting VSMC proliferation.

BACKGROUND: Restenosis frequently occurs after percutaneous angioplasty in patients with vascular occlusion and seriously threatens their health. Substantial evidence has revealed that preventing vascular smooth muscle cell proliferation using a drug-eluting stent is an effective approach to improve restenosis. Cucurbitacins have been demonstrated to exert an anti-proliferation effect in various tumors and a hypotensive effect. This study aims to investigate the role of cucurbitacins extracted from Cucumis melo L. (CuECs) and cucurbitacin B (CuB) on restenosis. METHODS: C57BL/6 mice were subjected to left carotid artery ligation and subcutaneously injected with CuECs or CuB for 4 weeks. Hematoxylin-Eosin, immunofluorescence and immunohistochemistry staining were used to evaluate the effect of CuECs and CuB on neointimal hyperplasia. Western blot, real-time PCR, flow cytometry analysis, EdU staining and cellular immunofluorescence assay were employed to measure the effects of CuECs and CuB on cell proliferation and the cell cycle in vitro. The potential interactions of CuECs with cyclin A2 were performed by molecular docking. RESULTS: The results demonstrated that both CuECs and CuB exhibited significant inhibitory effects on neointimal hyperplasia and proliferation of vascular smooth muscle cells. Furthermore, CuECs and CuB mediated cell cycle arrest at the S phase. Autodocking analysis demonstrated that CuB, CuD, CuE and CuI had high binding energy for cyclin A2. Our study also showed that CuECs and CuB dramatically inhibited FBS-induced cyclin A2 expression. Moreover, the expression of cyclin A2 in CuEC- and CuB-treated neointima was downregulated. CONCLUSIONS: CuECs, especially CuB, exert an anti-proliferation effect in VSMCs and may be potential drugs to prevent restenosis.

Formononetin ameliorates isoproterenol induced cardiac fibrosis through improving mitochondrial dysfunction.

Formononetin, an isoflavone compound, has been extensively researched due to its various biological activities, including a potent protective effect on the cardiovascular system. However, the impact of formononetin on cardiac fibrosis has not been investigated. In this study, C57BL/6 mice were used to establish cardiac fibrosis animal models by subcutaneous injecting of isoproterenol (ISO) and formononetin was orally administrated. The results showed that formononetin reversed ISO-induced heart stiffness revealed by early-to-atrial wave ratio (E/A ratio). Masson staining, western blot, immunohistochemistry and real-time PCR exhibited that the cardiac fibrosis and fibrosis-related proteins (collage III, fibronectin, TGF-ß1, α-SMA, and vimentin) and genes (Col1a1, Col3a1, Acta2 and Tgfb1) induced by ISO were significantly suppressed by formononetin. Furthermore, by combining metabolomics and network pharmacology, we found three important targets (ALDH2, HADH, and MAOB), which are associated with mitochondrial function, were involved in the beneficial effect of formononetin. Further validation revealed that these three genes were more abundance in cardiomyocyte than in cardiac fibroblast. The mRNA expression of ALDH2 and HADH were decreased, while MOAB was increased in cardiomyocyte upon ISO treatment and these phenomena were reversed by formononetin. In addition, we investigated mitochondrial membrane potential and ROS production in cardiomyocytes, the results showed that formononetin effectively improved mitochondrial dysfunction induced by ISO. In summary, we demonstrated that formononetin via regulating the expressions of ALDH2, HADH, and MAOB in cardiomyocyte to improve mitochondrial dysfunction and alleviate ß-adrenergic activation cardiac fibrosis.

Periplocymarin Alleviates Doxorubicin-Induced Heart Failure and Excessive Accumulation of Ceramides.

Doxorubicin-driven cardiotoxicity could result in dilated cardiomyopathy and heart failure (HF). Previously, we showed that periplocymarin exerted a cardiotonic role by promoting calcium influx and attenuating myocardial fibrosis induced by isoproterenol (ISO) by improving the metabolism of cardiomyocytes. However, the impact of periplocymarin on doxorubicin (DOX)-triggered cardiomyopathy has not been investigated. In the current study, C57BL/6 mice were randomly divided into three groups, namely, the control, DOX, and DOX+periplocymarin groups. The cardiac function and apoptosis were measured. Our results revealed that periplocymarin administration greatly improved the DOX-induced cardiac dysfunction manifested by the ejection fraction (EF%), fractional shortening (FS%), left ventricular posterior wall thickness (LVPW), left ventricular anterior wall thickness (LVAW), left ventricular (LV) mass, and attenuated DOX-induced cardiomyocyte apoptosis assessed by hematoxylin and eosin (H&E) staining, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, and western blotting. Further study using H9c2 cells revealed that the pretreatment of periplocymarin suppressed DOX-induced apoptosis evidenced by annexin V staining. Moreover, liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis demonstrated that DOX lead to an accumulation in serum ceramide, and the pre-treatment of periplocymarin could reverse this phenomenon. Network pharmacology also demonstrated that ceramide metabolism was involved in the process. Consistently, real-time PCR showed that periplocymarin significantly abolished the induction of the genes involved in the de novo synthesis of ceramide, i.e., CerS2, CerS4, CerS5, and CerS6, and the induction was attributed to the treatment of DOX. Collectively, these results suggested that periplocymarin reduced cardiomyocyte apoptosis to protect hearts from DOX-induced cardiotoxicity and the de novo synthesis of ceramides was involved in this process.

Periplocymarin protects against myocardial fibrosis induced by ß-adrenergic activation in mice.

Periplocymarin is an effective component of Periplocae Cortex, which was wildly used as an ingredient in Traditional Chinese Medicine. Our group previously reported that periplocymarin exerted cardiotonic role via promoting calcium influx. However, its exact role in the pathogenesis of myocardial fibrosis has not been elucidated yet. The present study was aimed at determining the potential effect and underlying mechanism of periplocymarin in isoproterenol (ISO)-induced myocardial fibrosis. C57BL/6 mice were subcutaneously injected with ISO (5 mg/kg/day) or saline for 1 week. The early-to-atrial wave ratio (E/A ratio) measured by echocardiography revealed that ISO-induced heart stiffness was remarkably reversed by administration of periplocymarin (5 mg/kg/day). Masson trichrome staining exhibited that treatment of periplocymarin reduced the excessive deposition of extracellular matrix (ECM). Further investigations employing real-time PCR and western blot demonstrated that periplocymarin suppressed the expression of fibrosis related genes (Col1a1, Col3a1, Acta2 and Tgfb1) and proteins (Collagen I, Collagen III, α-SMA and TGF-ß1) induced by ISO. Metabolomics analysis demonstrated that periplocymarin ameliorated the disorders triggered by ISO and many of the differential metabolic substances were involved in amino acid, glucose and lipid metabolism. Further analysis using network pharmacology revealed that three key genes, namely NOS2, NOS3 and Ptgs2, may be the potential targets of periplocymarin and responsible for the disorders. Validation using heart tissues showed that the mRNA expression of NOS3 was decreased while Ptgs2 was increased upon ISO treatment, which were reversed by periplocymarin. Moreover, the expression of COX-2 (Ptgs2 encoded protein) was consistent with the aspect of Ptgs2 mRNA, while eNOS (NOS3 encoded protein) expression was unchanged. In vitro studies exhibited that periplocymarin exerts anti-fibrotic function via regulating at least eNOS and COX-2 in cardiomyocyte. Taken together, periplocymarin protects against myocardial fibrosis induced by ß-adrenergic activation, the potential mechanism was that periplocymarin targeted on, at least eNOS and COX-2, to improve the metabolic processes of cardiomyocyte and thus attenuated the myocardial fibrosis. Our study highlighted that periplocymarin is a potential therapeutic agent for the prevention of myocardial fibrosis.

Periplocymarin Plays an Efficacious Cardiotonic Role via Promoting Calcium Influx.

Periplocymarin, which belongs to cardiac glycosides, is an effective component extracted from Periplocae Cortex. However, its cardiovascular effects remain unidentified. In the present study, injection of periplocymarin (5 mg/kg) through external jugular vein immediately increased the mean arterial pressure (MAP) in anesthetized C57BL/6 mice. Ex vivo experiments using mouse mesenteric artery rings were conducted to validate the role of periplocymarin on blood vessels. However, periplocymarin failed to induce vasoconstriction directly, and had no effects on vasoconstriction induced by phenylephrine (Phe) and angiotensin II (Ang II). In addition, vasodilatation induced by acetylcholine (Ach) was insusceptible to periplocymarin. Echocardiography was used to evaluate the effects of periplocymarin on cardiac function. The results showed that the injection of periplocymarin significantly increase the ejection fraction (EF) in mice without changing the heart rate. In vitro studies using isolated neonatal rat ventricular myocytes (NRVMs) revealed that periplocymarin transiently increased the intracellular Ca2+ concentration observed by confocal microscope. But in Ca2+-free buffer, this phenomenon vanished. Besides, inhibition of sodium potassium-activated adenosine triphosphatase (Na+-K+-ATPase) by digoxin significantly suppressed the increase of MAP and EF in mice, and the influx of Ca2+ in cardiomyocytes, mediated by periplocymarin. Collectively, these findings demonstrated that periplocymarin increased the contractility of myocardium by promoting the Ca2+ influx of cardiomyocytes via targeting on Na+-K+-ATPase, which indirectly led to the instantaneous rise of blood pressure.

The inhibition effect of uncarialin A on voltage-dependent L-type calcium channel subunit alpha-1C: Inhibition potential and molecular stimulation.

Cardiovascular diseases, such as hypertension and cardiac failure, have become the most major and global cause for threatening human health in recent years. Uncaria rhynchophylla as a traditional Chinese medicine is widely used to treat hypertension for a long history, whereas its medicinal effective components and potential action mechanism are uncertain. Therefore, twenty-four alkaloids (1-24) isolated from U. rhynchophylla were assayed for their relaxant effects against phenylephrine (Phe)-induced contraction of rat mesenteric arteries. Among them, we surprisingly found that uncarialin A (21) exhibited most potent relaxation effect against Phe-induced contraction (IC50 = 0.18 µM) in the manner of independent on endothelium-derived vasorelaxing factors and endothelium. All the experiments including measurement of Ca2+ in vascular smooth muscle cells (VSMCs) by fluorescence microscopy, whole-cell path clamp, molecular docking, and molecular dynamics, demonstrated that uncarialin A (21) could significantly inhibit L-type calcium channel subunit alpha-1C (Cav1.2) via the hydrogen bond interaction with amino acid residue Met1186, allowing the inhibition of Ca2+ inward current. Our results suggested that uncarialin A (21) could be served as a potential L-type Cav1.2 blocker in the effective treatment of cardiovascular diseases.

Simultaneous Quantitative Analysis of Multiple Biotransformation Products of Xian-Ling-Gu-Bao, a Traditional Chinese Medicine Prescription, with Rat Intestinal Microflora by Ultra-Performance Liquid Chromatography Tandem Triple Quadrupole Mass Spectrometry.

Xian-Ling-Gu-Bao (XLGB), a famous traditional Chinese medicine prescription consisted of six herbal medicines, was used for prevention and treatment of osteoporosis in China. As an oral formulation, the multiple components contained in XLGB were inevitably biotransformed by the intestinal microflora before absorption via the gastrointestinal tract. However, the dynamic profiles of biotransformation products of XLGB remain unknown. In this paper, a rapid and sensitive ultra-performance liquid chromatography tandem triple quadrupole mass spectrometry method was developed for the simultaneous quantitative analysis of multiple biotransformation products of XLGB with rat intestinal microflora. For 10 selected quantitative compounds, all calibration curves revealed good linearity (r2 > 0.99) within the sampling ranges considered. The whole intra- and inter-day precisions (as relative standard deviation) of all analytes were <13.5%, and the accuracies (as relative error) were in the range from -11.3 to 11.2%. The lower limits of quantification were 20, 10, 5, 20, 2, 2, 2, 5, 2 and 2 ng/mL for sweroside, timosaponin BII, epimedin C, asperosaponin VI, psoralen, isobavachin, icariside II, timosaponin AIII, isobavachalcone and icaritin, respectively. The matrix effects, extraction recoveries and stabilities were all satisfactory. Meanwhile, dynamic profiles of 21 additional biotransformation products were also monitored by their area-time curves. The analytical method was successfully applied to describe dynamic profiles of 31 biotransformation products of XLGB and the recipes with removal of a definite composed herbal medicine (Anemarrhenae Rhizoma or Rehmanniae Radix).

Cucurbitacins extracted from Cucumis melo L. (CuEC) exert a hypotensive effect via regulating vascular tone.

As an effective medicine for jaundice in traditional Chinese medicine, Cucumis melo L. has been widely used in China. However, its effect on vascular function is still unclear. In this study, we extracted the compounds of Cucumis melo L., and the major ingredients were identified as cucurbitacins (CuEC, cucurbitacins extracted from Cucumis melo L.), especially cucurbitacin B. We replicated the toxicity in mice by intraperitoneal injection of a high dose of CuEC (2 mg/kg) and demonstrated that the cause of death was CuEC-induced impairment of the endothelial barrier and, thus, increased vascular permeability via decreasing VE-cadherin conjunction. The administration of low doses of CuEC (1 mg/kg) led to a decline in systolic blood pressure (SBP) without causing toxicity in mice. More importantly, CuEC dramatically suppressed angiotensin II (Ang II)-induced SBP increase. Further studies demonstrated that CuEC facilitated acetylcholine-mediated vasodilation in mesenteric arteries of mice. In vitro studies showed that CuEC induced vasodilation in a dose-dependent manner in mesenteric arteries of both mice and rats. Pretreatment with CuEC inhibited phenylephrine-mediated vasoconstriction. In summary, a moderate dose of CuEC reduced SBP by improving blood vessel tension. Therefore, our study provides new experimental evidence for developing new antihypertensive drugs.

Systematic screening and characterization of Qi-Li-Qiang-Xin capsule-related xenobiotics in rats by ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry.

Qi-Li-Qiang-Xin capsule (QLQX), a well-known traditional Chinese medicine prescription (TCMP), is consisted of eleven commonly used herbal medicines, has been widely used for the treatment of chronic heart failure (CHF). However, the absorbed components and related metabolites after oral administration of QLQX are still remaining unknown. In the present work, a reliable and effective method using ultra performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC/Q-TOF-MS) was established to identify QLQX-related xenobiotics in rats. Based on a representative structure based homologous xenobiotics identification (RSBHXI) strategy, a total of eleven compounds (salvianolic acid B, formononetin, benzoylmesaconine, alisol A, sinapine thiocyanate, naringin, tanshinone IIA, ginsenoside Rg1, ginsenoside Rb1, astragaloside IV and periplocin), bearing different chemical core structures, were selected and investigated for their metabolism in vivo. And then, comprehensive metabolic profiles of the holistic multi-ingredients in QLQX were achieved. As a result, a total of 121 QLQX-related xenobiotics (47 prototypes and 74 metabolites) were identified or tentatively characterized, among them eight prototypes (mesaconine, hypaconine, songorine, fuziline, neoline, talatizamine formononetin, neocryptotanshinone) and two metabolites (calycosin-gluA, formononetin-guA) were relatively the main existing xenobiotics exposed in blood. All absorbed prototype constituents were mainly from six composed herbal medicines (Aconiti lateralis radix, Astragali radix, Ginseng radix, Alismatis rhizoma, Salvia miltiorrhiza radix, Periploca cortex). The main metabolic reactions were methylation, hydrogenation, hydroxylation, oxidization, sulfation and glucuronidation. This is the first study on in vivo metabolism of QLQX. These results enabled us to focus on several high exposure ingredients in the discovery of effective substances of QLQX, however further pharmacokinetic study on these QLQX-related xenobiotics are needed to be carried out.