Sodium tanshinone IIA sulfonate promotes endothelial integrity via regulating VE-cadherin dynamics and RhoA/ROCK-mediated cellular contractility and prevents atorvastatin-induced intracerebral hemorrhage in zebrafish.

Impaired vascular integrity leads to serious cerebral vascular diseases such as intracerebral hemorrhage (ICH). In addition, high-dose statin therapy is suggested to cause increased ICH risk due to unclear effects of general inhibition of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) on the vascular system. Here we evaluated the protective effects of sodium tanshinone IIA sulfonate (STS), which has high efficacy and safety in clinical studies of ischemic stroke, by using atorvastatin (Ator) induced ICH zebrafish embryos and human umbilical vein endothelial cells (HUVECs). By using double transgenic Tg(fli1a:EGFP)y1 & Tg(gata1a:dsRed)sd2 zebrafish, we demonstrated that STS effectively reduced the occurrence and area of hemorrhage induced by Ator in zebrafish and restored impairment in motor function. We further demonstrated that Ator-induced disruption in VE-cadherin (VEC)-containing cell-cell adherens junctions (AJs) in HUVECs by enhancing Src-induced VEC internalization and RhoA/ROCK-mediated cellular contraction. STS inhibited Ator-induced Src activation and subsequent VEC internalization and actin depolymerization near cell borders, reducing lesions between neighboring cells and increasing barrier functions. STS also inhibited the Ator-induced RhoA/ROCK-mediated cellular contraction by regulating downstream LIMK/cofilin and MYPT1/MLC phosphatase signaling. These results showed that STS significantly promoted the stability of cell junctions and vascular integrity. Moreover, we observed that regulations of both Src and RhoA/ROCK are required for the maintenance of vascular integrity, and Src inhibitor (PP2) or ROCK inhibitors (fasudil and H1152) alone could not reduce the occurrence Ator-induced ICH. Taken together, we investigated the underlying mechanisms of Ator-induced endothelial instability, and provided scientific evidences of STS as potential ICH therapeutics by promoting vascular integrity.

Amelioration of acute myocardial infarction by saponins from flower buds of Panax notoginseng via pro-angiogenesis and anti-apoptosis.

ETHNOPHARMACOLOGICAL RELEVANCE: The root of Panax notoginseng is traditionally used as an anti-hemorrhagic agent to promote blood circulation without causing "congealed" blood. Furthermore, the flower of P. notoginseng is a popular, traditional medicine taken daily for the preventing of hypertension and for reducing blood cholesterol profiles. Besides, the flower of P. notoginseng contains a higher level of saponins, particularly protopanaxadiol-type ginsenosides, as compared to the root. However, detailed pharmacological studies on this flower have rarely been conducted. MATERIAL AND METHODS: In this study, the saponins extracted from the flower of P. notoginseng (FS) were examined on the endothelial cell migration assay, chemically induced vascular insufficiency model in zebrafish larvae and myocardial infraction (MI) model in rats, for determination of their pro-angiogenic and therapeutic effects on MI treatment. RESULTS: Our results demonstrate that FS significantly promoted VEGF-induced migration of human umbilical vein endothelial cells (HUVECs) and partially restored defective intersegmental vessels (ISV) in a chemically induced vascular insufficiency model of zebrafish larvae. When compared to MI group, two weeks post-treatment of FS (25-50mg/kg/day) induced approximately 3-fold upregulation of VEGF mRNA expression and a concomitant increase in blood vessel density in the peri-infarct area of the heart. Moreover, TUNEL analysis indicates a reduction in the mean apoptotic nuclei per field in peri-infarct myocardium upon FS treatment. CONCLUSIONS: The pro-angiogenic effects of FS demonstrated in in vitro and in vivo experimental models suggest that the purified saponin preparation from flowers of P. notoginseng may potentially provide preventive and therapeutic agent for cardiovascular diseases.

Schisantherin A protects against 6-OHDA-induced dopaminergic neuron damage in zebrafish and cytotoxicity in SH-SY5Y cells through the ROS/NO and AKT/GSK3ß pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: The fruit of Schisandra chinensis (Turcz.) Baill, has been traditionally used in management of liver diseases and ageing associated neurodegeneration. The bioactive compound from this medicinal plant would be valuable for its potential use in prevention and treatment of Parkinson׳s disease. AIM OF THE STUDY: The overall objective of the present study was to understand the neuroprotective effect of schisantherin A, a dibenzocyclooctadiene lignan from the fruit of S. chinensis (Turcz.) Baill, and to elucidate its underlying mechanism of action. MATERIAL AND METHODS: This study investigated the protective effect of schisantherin A against selective dopaminergic neurotoxin 6-hydroxydopamine (6-OHDA)-induced neural damage in human neuroblastoma SH-SY5Y cells and zebrafish models. Oxidative stress and related signaling pathways underlying the neuroprotective effect were determined by multiple biochemical assays and Western blot. RESULTS: Pretreatment with schisantherin A offered neuroprotection against 6-OHDA-induced SH-SY5Y cytotoxicity. Moreover, schisantherin A could prevent 6-OHDA-stimulated dopaminergic neuron loss in zebrafish. Our mechanistic study showed that schisantherin A can regulate intracellular ROS accumulation, and inhibit NO overproduction by down-regulating the over-expression of iNOS in 6-OHDA treated SH-SY5Y cells. Schisantherin A also protects against 6-OHDA-mediated activation of MAPKs, PI3K/Akt and GSK3ß. CONCLUSION: These findings demonstrate that schisantherin A may have potential therapeutic value for neurodegenerative diseases associated with abnormal oxidative stress such as Parkinson׳s disease.

L-tetrahydropalamatine inhibits tumor necrosis factor-α-induced monocyte-endothelial cell adhesion through downregulation of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 involving suppression of nuclear factor-κ B signaling pathway.

OBJECTIVE: To investigate whether I-tetrahydropalmatine (I-THP), an alkaloid mainly present in Corydalis family, could ameliorate early vascular inflammatory responses in atherosclerotic processes. METHODS: Fluorescently labeled monocytes were co-incubated with human umbilical vein endothelial cells (HUVECs), which were pretreated with I-THP and then simulated with tumor necrosis factor (TNF)-α in absence of I-THP to determine if I-THP could reduce thecytokine-induced adhesion of monocytes to HUVECs. Then I-THP were further studied the underlying mechanisms through observing the transcriptional and translational level of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) and the nuclear translocation of nuclear factor (NF)-κ B in HUVECs. RESULTS: L-THP could block TNF-α-induced adhesion of monocytes to HUVECs and could significantly inhibited the expression of ICAM-1 and VCAM-1 on cell surface by 31% and 36% at 30 µ mol/L. L-THP pretreatment could also markedly reduce transcriptional and translational level of VCAM-1 as well as mildly reduce the total protein and mRNA expression levels of ICAM-1. Furthermore, I-THP attenuated TNF-α-stimulated NF-κ B nuclear translocation. CONCLUSION: These results provide evidences supporting that I-THP could be a promising compound in the prevention and treatment of the early vascular inflammatory reaction in atherosclerosis by inhibiting monocyte adhesion to vascular endothelial cell through downregulating ICAM-1 and VCAM-1 in vascular endothelial cell based on suppressing NF-κ B.

Recent advances in structure-based drug design and virtual screening of VEGFR tyrosine kinase inhibitors.

During the past decade, developments in computational processing and X-ray crystallography have allowed virtual screening become integrated into drug discovery campaigns. This review focuses on the recent advancements in the drug discovery of VEGFR2 tyrosine kinase inhibitors (VEGFR2 TKIs) by using in silico methodologies. An introduction for the methodology framework of pharmacophore modeling, molecular docking and structure-based design are provided. We discuss the recent studies on the structures of VEGFR2 protein kinase in different binding modes, and the insights on molecular interactions gained from knowledge of the co-crystal structures complex with structurally diverse VEGFR2 inhibitors. We provide some aspects of model construction and molecular docking techniques. Several representative examples of successful applications on VEGFR2 virtual screening for hit discovery, lead optimization and structure-based design are also presented.

In vivo angiogenesis screening and mechanism of action of novel tanshinone derivatives produced by one-pot combinatorial modification of natural tanshinone mixture from Salvia miltiorrhiza.

BACKGROUND: Natural products present in low quantity in herb medicines constitute an important source of chemical diversity. However, the isolation of sufficient amounts of these low abundant constituents for structural modification has been a challenge for several decades and subsequently halts research on the utilization of this important source of chemical entities for drug discovery and development. And, pro-angiogenic therapies are being explored as options to treat cardio-cerebral vascular diseases and wound healing recently. The present study investigates the pro-angiogenic potential of tanshinone derivatives produced by one-pot synthesis using zebrafish model. METHODOLOGY/PRINCIPAL FINDINGS: In order to address the difficulty of chemical modification of low abundant constituents in herb medicines, a novel one-pot combinatorial modification was used to diversify a partially purified tanshinone mixture from Salvia miltiorrhiza. This led to the isolation of ten new imidazole-tanshinones (Compounds 1-10) and one oxazole-tanshinone (Compound 11), the structures of which were characterized by spectroscopic methods in combination with single-crystal X-ray crystallographic analysis. The angiogenesis activities of the new tanshinone derivatives were determined in an experimental model of chemical-induced blood vessels damage in zebrafish. Of all the tested new derivatives, compound 10 exhibited the most potent vascular protective and restorative activity with an EC50 value of 0.026 µM. Moreover, the mechanism underlying the pro-angiogenesis effect of 10 probably involved the VEGF/FGF-Src-MAPK and PI3K-P38 signalling pathways by gene expression analysis and a blocking assay with pathways-specific kinase inhibitors. CONCLUSIONS/SIGNIFICANCE: Taken together, our study demonstrated the more distinctive pro-angiogenic properties of 10 than other tanshinones and revealed 10 has potential for development as a pro-angiogenic agent for diseases associated with insufficient angiogenesis. Our results highlighted the great potential of adopting a newly modified one-pot approach to enhance the chemical diversity and biological activities of constituents from natural products regardless of their abundances.

Bisdemethoxycurcumin Increases Sirt1 to Antagonize t-BHP-Induced Premature Senescence in WI38 Fibroblast Cells.

Curcuminoids are well known for their capabilities to combat risk factors that are associated with ageing and cellular senescence. Recent reports have demonstrated that curcuminoids can extend the lifespan of model organisms. However, the underlying mechanisms by which these polyphenic compounds exert these beneficial effects remain unknown. In this study, t-BHP-induced premature senescence model in human fibroblasts was chosen to explore the protective effects of a curcuminoid, bisdemethoxycurcumin (BDMC), on cellular senescence. The results demonstrated that BDMC attenuated oxidative stress-induced senescence-like features which include the induction of an enlarged cellular appearance, higher frequency of senescence-associated ß -galactosidase staining activity, appearance of senescence-associated heterochromatic foci in nuclei, decrease in proliferation capability, and alteration in related molecules such as p16 and retinoblastoma protein. Notably, we found that BDMC treatment activated Sirt1/AMPK signaling pathway. Moreover, downregulating Sirt1 by the pharmacological inhibitor nicotianamine or small interfering RNA blocked BDMC-mediated protection against t-BHP-mediated decrease in proliferation. These results suggested that BDMC prevented t-BHP-induced cellular senescence, and BDMC-induced Sirt1 may be a mechanism mediating its beneficial effects.

Danshensu protects against 6-hydroxydopamine-induced damage of PC12 cells in vitro and dopaminergic neurons in zebrafish.

The overproduction of reactive oxygen species (ROS) has been implicated in the development of neurodegenerative diseases such as Parkinson's disease (PD) and Alzheimer's disease (AD). Previous studies have indicated that danshensu (beta-3,4-dihydroxyphenyl-lactic acid), a main hydrophilic component of the Chinese materia medica Radix Salviae Miltiorrhizae (Danshen, Pharmacopoeia of PR China), has ROS scavenging and antioxidant activities, however its mechanism of action was not clear. In this study, we investigated whether the protective effects of danshensu against neurotoxin 6-hydroxydopamine (6-OHDA)-induced oxidative stress involved the Nrf2/HO-1 pathways. Pretreatment with danshensu in PC12 cells significantly attenuated 6-OHDA-induced cytotoxicity and the production of ROS. Danshensu activated the nuclear translocation of Nrf2 to increase heme oxygenase-1 (HO-1), conferring protection against ROS. Danshensu induced the phosphorylation of Akt, and its cytoprotective effect was abolished by PI3K, Akt and HO-1 inhibitors. These results confirmed the crucial role of PI3K/Akt and HO-1 signaling pathways as the underlying mechanistic action of danshensu. Taken together, the results suggest that danshensu enhances HO-1 expression to suppress 6-OHDA-induced oxidative damage via PI3K/Akt/Nrf2 signaling pathways. Moreover, 6-OHDA-induced dopaminergic neuronal loss in zebrafish could be reduced by danshensu, further supporting the neuroprotective potential of danshensu.

Enzyme kinetic and molecular docking studies for the inhibitions of miltirone on major human cytochrome P450 isozymes.

Previous studies have shown that major tanshinones isolated from Danshen (Salvia miltiorrhiza) inhibited human and rat CYP450 enzymes-mediated metabolism of model probe substrates, with potential in causing herb-drug interactions. Miltirone, another abietane type-diterpene quinone isolated from Danshen, has been reported for its anti-oxidative, anxiolytic and anti-cancer effects. The aim of this study was to study the effect of miltirone on the metabolism of model probe substrates of CYP1A2, 2C9, 2D6 and 3A4 in pooled human liver microsomes. Miltirone showed moderate inhibition on CYP1A2 (IC(50)=1.73 µM) and CYP2C9 (IC(50)=8.61 µM), and weak inhibition on CYP2D6 (IC(50)=30.20 µM) and CYP3A4 (IC(50)=33.88 µM). Enzyme kinetic studies showed that miltirone competitively inhibited CYP2C9 (K(i)=1.48 µM), and displayed mixed type inhibitions on CYP1A2, CYP2D6 and CYP3A4 with K(i) values of 3.17 µM, 24.25 µM and 35.09 µM, respectively. Molecular docking study further confirmed the ligand-binding conformations of miltirone in the active sites of these human CYP450 isoforms, and provided some information on structure-activity relationships for the CYPs inhibition by tanshinones. Taken together, CYPs inhibitions of miltirone were weaker than dihydrotanshinone, but stronger than cryptotanshinone, tanshinone I and tanshinone IIA.

[Use of zebrafish models for the research of traditional Chinese medicine].

Traditional Chinese medicine (TCM) exhibits a broad range of effects on biological activity that is probably due to interactions of complex chemical constituents with multiple targets in the body. Understanding the active chemical constituents in TCM is very important in providing rationales for the clinical usage of TCM. The zebrafish (Danio rerio) has recently become a popular model in the field of drug screening, specifically emerging as an important vertebrate model for in vivo high-content drug screening of multiple efficacy parameters and whole-organism toxicity. The authors also discussed the advantages of the zebrafish model for evaluating drug metabolism. Zebrafish usage in TCM screening should be a viable approach that helps identify active chemical markers, biological pathways and mechanistic actions of TCM.

Danshensu is the major marker for the antioxidant and vasorelaxation effects of Danshen (Salvia miltiorrhiza) water-extracts produced by different heat water-extractions.

Some of the major components of Danshen (Salvia miltiorrhiza), a widely used Chinese herbal medicine rich in phenolic acids, are thermosensitive and may degrade to other phenolic acids during extractions with heating. The chemical profiles of Danshen water-extract may vary with different heat water extraction at different temperatures, affecting the composition and bioactivity of the extracts. In this study, six water-extracts of Danshen obtained from heat reflux water extraction and microwave-assisted extraction with water (MAE-W) at different temperatures were tested for their composition and pharmacological effects. Among these extracts, the third-round MAE-W (100°C) extract had the highest phenolic acids and tanshinones contents, with the strongest antioxidant activity in 2,2-diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl (DPPH) assay and ferric reducing/antioxidant potential (FRAP) assay. This extract also showed the strongest inhibitory effects on 2,2'-azobis-2-amidinopropane (AAPH)-induced hemolysis in human red blood cells, hydrogen peroxide-induced apoptosis in rat heart H9c2 cells and the highest relaxation effects on rat basilar artery. The antioxidant effects of Danshen water-extracts linearly correlated to their relaxation effects (r=0.895-0.977). Through multiple linear regression analysis, danshensu was found to be the most significant marker in the antioxidant and vasodilation effects of Danshen water-extract, while tanshinone IIA as the marker on hydrogen peroxide-induced apoptosis in rat heart H9c2 cells. Danshensu is, therefore, a useful marker for the quality control of Danshen water-extracts in antioxidant and vasodilation, while tanshinone IIA for anti-apoptotic potential of different extracts.

Anti-angiogenic effect of furanodiene on HUVECs in vitro and on zebrafish in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: Furanodiene is an active ingredient of the traditional Chinese medicine, Rhizoma Curcumae, commonly used for the treatment of cancer in China. AIM OF THE STUDY: To investigate the anti-cancer property of Rhizoma Curcumae, this study describes the anti-angiogenic activities of furanodiene in human umbilical vein endothelial cells (HUVECs) in vitro and in zebrafish in vivo. MATERIALS AND METHODS: HUVECs were treated with different doses of furanodiene in the presence or absence of vascular endothelial growth factor (VEGF). The anti-proliferative effect of furanodiene was measured using the XTT assay. The anti-migration and anti-invasion activities of this compound were investigated with a wound-healing migration model and a three-dimensional cell invasion model, respectively. The effects of furanodiene on HUVEC differentiation were assessed by in vitro tube formation in Matrigel™. The expression of related proteins was detected by Western blot. Morphological observations of zebrafish were evaluated in transgenic Tg (fli1: EGFP) zebrafish embryos. RESULTS: Our results showed that furanodiene exposure could significantly inhibit the proliferation of HUVECs in a dose-dependent manner and inhibit VEGF-induced proliferation at a low dose. Relative to the VEGF-induced control, the number of invading and migrating cells was significantly reduced in the furanodiene-treated groups. Furanodiene also dramatically suppressed tube formation and p-Akt (Ser473), p-Erk 1/2 (Thr202/Tyr204), ICAM-1, p-p85 (Ser428) as well as p85 protein expression. Furthermore, exposure to furanodiene inhibited angiogenesis in the zebrafish model. CONCLUSIONS: This study demonstrated that furanodiene exposure exhibits a potential anti-angiogenic effect through suppression of endothelial cell growth, invasion, migration and tube formation via regulation of the PI3K pathway. This potential anti-angiogenic effect of furanodiene may play an important role in the anti-tumor activity of the traditional Chinese medicine, Rhizoma Curcumae.

Protective, antioxidative and antiapoptotic effects of 2-methoxy-6-acetyl-7-methyljuglone from Polygonum cuspidatum in PC12 cells.

Much correlative evidence indicates that the oxidative modification of protein by reactive oxygen species (ROS) is involved in normal aging as well as the pathogenesis of neurodegenerative diseases such as Alzheimer's disease. In this study, we explored the antioxidative and neuroprotective effects of a naphthoquinone, 2-methoxy-6-acetyl-7-methyljuglone (MAM), purified from the dried rhizome of POLYGONUM CUSPIDATUM (Chinese name Hu-Zhang). Pretreatments with MAM (24 h) were investigated for their protective effects against apoptosis induced by the oxidizing agent TERT-butyl hydroperoxide ( T-BHP) in PC12 cells. The results indicated that MAM pretreatments could effectively protect PC12 cells against cytotoxicity induced by T-BHP in a dose-dependent manner. Cell viability was determined by both MTT and LDH assays. Increasing concentrations of MAM enhanced cell viability significantly and completely prevented cell death induced by T-BHP at 2.5 µM. The corresponding extracellular lactate dehydrogenase (LDH) levels were also attenuated significantly by various concentrations of MAM. In addition, it was found that the antioxidative effect of MAM was stronger than those of resveratrol and lipoic acid. The antiapoptotic property of MAM was further investigated with Hoechst 33342 nuclear staining and TUNEL assay. Pretreatments of MAM were able to prevent the T-BHP-induced nucleus fragmentation and accumulation of apoptotic bodies (commonly accepted as markers of apoptosis) inside the cells in a dose-dependent manner. T-BHP induced the phosphorylation of ERK 1/2, JNK and p38 MAPK, which were all impeded by pretreatments with MAM, indicating that MAM may act as a potent antioxidant which significantly interferes with the MAPK apoptotic cascades, probably rescuing cells by inhibiting the death pathways.