H2O2参与水杨酸诱导丹参培养细胞中丹酚酸B合成的信号转导

过氧化氢(Hydrogen peroxide,H2O2)为活性氧(Reactive oxygen species,ROS)的一种,存在于许多生物体系中并介导植物中多种生理和生化过程。为了探讨H2O2作为信号分子在水杨酸(Salicylic acid,SA)诱导丹参培养细胞合成丹酚酸B(Salvianolic acid B,Sal B)过程中的作用,分别考察了SA和H2O2、过氧化氢酶(Catalase,CAT)、二甲基硫脲(2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide,DMTU)及咪唑(Imidazole,IMD)对苯丙氨酸解氨酶(Phenylalanine ammonia-lyase,PAL)和酪氨酸氨基转移酶(Tyrosine aminotransferase,TAT)的活性及Sal B含量的影响。结果表明,SA处理可有效地诱导丹参培养细胞中H2O2产生、PAL和TAT活性升高以及Sal B合成积累量的增加;外源施加10～30 mmol/L H2O2也可以有效促进PAL、TAT活性升高和Sal B合成积累量的增加;用H2O2的清除剂CAT处理发现,CAT对SA或外源H2O2诱导的Sal B合成积累具有消除作用,说明H2O2可能作为SA诱导Sal B积累过程中的上游信号分子起作用;用H2O2淬灭剂DMTU处理,可以有效抑制SA对Sal B合成的促进作用;用质膜烟酰胺腺嘌呤二核苷酸(Nicotinamide vadenine dinucleotide phosphate,NADPH)氧化酶(H2O2来源的主要酶)抑制剂IMD处理,可以抑制Sal B的合成,但这种抑制效果可以部分被外源施加的SA削弱,说明通过HADPH氧化酶产生的H2O2受阻时,SA诱导的Sal B合成积累也会受到抑制。表明H2O2是介导SA诱导丹参培养细胞中Sal B合成积累的信号分子。     

Ca2+对水杨酸诱发的丹参幼苗丹酚酸B生物合成的影响

为研究Ca2+在水杨酸诱导丹参幼苗丹酚酸B生物合成过程中的作用,分别用激光共聚焦显微镜和高效液相色谱仪检测胞外Ca2+通道抑制剂Vp和LaCl3,胞内Ca2+通道抑制剂LiCl以及胞内钙调素拮抗剂TFP处理前、后水杨酸诱导丹参叶片保卫细胞内Ca2+荧光强度和丹酚酸B含量的变化。结果表明,水杨酸 (SA) 处理后6 min即可诱发丹参幼苗叶片保卫细胞内Ca2+迸发,持续时间为2~3 min,丹参幼苗丹酚酸B生物合成量亦显著增加,且丹酚酸B合成量的增加发生在Ca2+迸发之后。胞外Ca2+通道抑制剂,胞内Ca2+通道抑制剂以及胞内钙调素拮抗剂均可抑制水杨酸诱导的Ca2+迸发和丹酚酸B的生物合成。结果表明水杨酸诱发的Ca2+对丹参幼苗丹酚酸B生物合成具有重要的调控作用。     

Effects of salvianolic acid B on proliferation,neurite outgrowth and differentiation of neural stem cells derived from the cerebral cortex of embryonic mice

Salvianolic acid B is isolated from Salvia miltiorrhiza,the root of which is widely used as a traditional Chinese medicine to treat stroke.However,little is known about how salvianolic acid B influences growth characteristics of neural stem cells (NSCs).The purpose of the present study was to evaluate the effects of salvianolic acid B on proliferation,neurite outgrowth and differentiation of NSCs derived from the cerebral cortex of embryonic mice using MTT,flow cytometry,immunofluorescence and RT-PCR.It was found that 20 μg mL·1 and 40 μg mL·1 salvianolic acid B had similar effects on proliferation of NSCs,and a suitable concentration of salvianolic acid B increased the number of NSCs and their derivative neurospheres.The growth-promoting activity of salvianolic acid B was dependent on and associated with an accumulation in the G2/S-phase cell population.Salvianolic acid B also promoted the neurite outgrowth of NSCs and their differentiation into neurons.The mRNA for tau,GFAP and nestin were present in differentiating neurospheres induced by salvianolic acid B.However,high-level expression of tau mRNA and low-level expression of GFAP mRNA was detected in differentiated cells,in contrast to the control conditions.This collective evidence indicates that exogenous salvianolic acid B is capable of promoting proliferation of neurospheres and differentiation towards the neuronal lineage in vitro and may act in the proliferation of NSCs and may promote NSC differentiation into neuronal cells.     

MEK/ERK pathway mediates cytoprotection of salvianolic acid B against oxidative stress‐induced apoptosis in rat bone marrow stem cells

To improve the survival and/or differentiation of grafted BMSCs (bone marrow stem cells) represents one of the challenges for the promising cell‐based therapy. Considerable reports have implicated Sal B (salvianolic acid B), a potent aqueous extract of Salvia miltiorrhiza, in enhancing the survival of cells under various conditions. In this study, we investigated the effect of Sal B on H₂O₂‐induced apoptosis in rat BMSCs, focusing on the survival signalling pathways. Results indicated that the MEK [MAPK (mitogen‐activated protein kinase)/ERK (extracellular‐signal‐regulated kinase) kinase] inhibitor (PD98059) and 10 μM Sal B remarkably prevented BMSCs from H₂O₂‐induced apoptosis through attenuating caspase‐3 activation, which is accompanied by the significant up‐regulation of Bcl‐2. In addition, the ROS (reactive oxygen species) accumulation was also reduced after Sal B treatment. Furthermore, Sal B inhibited the ERK1/2 phosphorylations stimulated by H₂O₂. Taken together, our results showed that H₂O₂‐induced apoptosis in BMSCs via the ROS/MEK/ERK1/2 pathway and Sal B may exert its cytoprotection through mediating the pathway.     

Clarifying the signal network of salvianolic acid B using proteomic assay and bioinformatic analysis

Salvianolic acid B (SB) is a natural compound with protective effect against ischemia-reperfusion heart injury. However, the signal network of SB including both direct target proteins and downstream signal-related proteins has not been clarified. In the present study, epidermal growth factor receptor (EGFR) was predicted to be the most possible direct protein target of SB by INVDOCK, a ligand-protein inverse-docking algorithm. Possible signal-related proteins of SB in H9C2 cells, including both under normal condition and under ischemia-reperfusion injury, were searched using 2-DE analysis. Totally, 14 signal-related proteins were found. Finally, signal network from EGFR to the signal-related proteins was established using bioinformatic analysis. Interestingly, 9 of the 14 signal-related proteins could be included in a network together with EGFR through direct interaction or only one intermediate partner. The signal cascade from EGFR to heat shock protein 27 (HSP27) and mitofilin (IMMT, inner membrane mitochondrial protein) might be the most important cascade. The signal network was certified by measuring the binding affinity of SB to EGFR in vitro, the effect of SB on internalization and phosphorylation of EGFR, the effect of SB on viability and proliferation of H9C2 cells, and the expression of inner membrane mitochondrial protein in the presence of EGFR inhibitor AG 1478.     

Conversion of salvianolic acid B into salvianolic acid A in tissues of Radix Salviae Miltiorrhizae using high temperature,high pressure and high humidity

Salvianolic acid A (Sal A), an important constituent of Radix Salviae Miltiorrhizae (RSM), is effective for the treatment of myocardial infarction (MI) and coronary heart disease due to its potential in the improvement of acute myocardial ischemia. However, its content is very low in RSM. So it is obvious to find a rich source of Sal A or to improve its content by conversion of other related components into Sal A modifying reaction conditions. In this research we focused on the conversion of Sal B into Sal A in aqueous solutions of RSM by using different reaction conditions including pH, temperature, pressure and humidity. During the reactions, the contents of Sal A, Sal B and danshensu in the RSM were analyzed by high-performance liquid chromatography (HPLC) and liquid chromatography–mass spectrometry (LCMS). The results indicated that the conversion of Sal B into Sal A in RSM tissues under the conditions of a high temperature, high pressure and high humidity was efficient and thereby, was readily utilized to prepare rich Sal A materials in practice.     

Salvianolic acid B inhibits ototoxic drug–induced ototoxicity by suppression of the mitochondrial apoptosis pathway

It has been claimed that salvianolic acid B (Sal B), a natural bioactive antioxidant, exerts protective effects in various types of cells. This study aims to evaluate the antioxidant and anti‐apoptosis effects of Sal B in a cultured HEI‐OC1 cell line and in transgenic zebrafish (Brn3C: EGFP). A CCK‐8 assay, Annexin V Apoptosis Detection Kit, TUNEL and caspase‐3/7 staining, respectively, examined apoptosis and cell viability. The levels of reactive oxygen species (ROS) were evaluated by CellROX and MitoSOX Red staining. JC‐1 staining was employed to detect the mitochondrial membrane potential (ΔΨm). Western blotting was used to assess expressions of Bax and Bcl‐2. The expression pattern of p‐PI3K and p‐Akt was determined by immunofluorescent staining. We found that Sal B protected against neomycin‐ and cisplatin‐induced apoptotic features, enhanced cell viability and accompanied with decreased caspase‐3 activity in the HEI‐OC1 cells. Supplementary experiments determined that Sal B reduced ROS production (increased ΔΨm), promoted Bcl‐2 expression and down‐regulated the expression of Bax, as well as activated PI3K/AKT signalling pathways in neomycin‐ and cisplatin‐injured HEI‐OC1 cells. Moreover, Sal B markedly decreased the TUNEL signal and protected against neomycin‐ and cisplatin‐induced neuromast HC loss in the transgenic zebrafish. These results unravel a novel role for Sal B as an otoprotective agent against ototoxic drug–induced HC apoptosis, offering a potential use in the treatment of hearing loss.     

Salvianolic acid B attenuates VCAM-1 and ICAM-1 expression in TNF-alpha-treated human aortic endothelial cells

Attachment to, and migration of leukocytes into the vessel wall is an early event in atherogenesis. Expression of cell adhesion molecules by the arterial endothelium may play a major role in atherosclerosis. It has been suggested that antioxidants inhibit the expression of adhesion molecules and may thus attenuate the processes leading to atherosclerosis. In the present study, the effects of a potent water-soluble antioxidant, salvianolic acid B (Sal B), and an aqueous ethanolic extract (SME), both derived from a Chinese herb, Salvia miltiorrhiza, on the expression of endothelial-leukocyte adhesion molecules by tumor necrosis factor-alpha (TNF-alpha)-treated human aortic endothelial cells (HAECs) were investigated. When pretreated with SME (50 and 100 microg/ml), the TNF-alpha-induced expression of vascular adhesion molecule-1 (VCAM-1) was notably attenuated (77.2 +/- 3.2% and 80.0 +/- 2.2%, respectively); and with Sal B (1, 2.5, 5, 10, and 20 microg/ml), 84.5 +/- 1.9%, 78.8 +/- 1.2%, 58.9 +/- 0.4%, 58.7 +/- 0.9%, and 57.4 +/- 0.3%, respectively. Dose-dependent lowering of expression of intercellular cell adhesion molecule-1 (ICAM-1) was also seen with SME or Sal B. In contrast, the expression of endothelial cell selectin (E-selectin) was not affected. SME (50 microg/ml) or Sal B (5 microg/ml) significantly reduced the binding of the human monocytic cell line, U937, to TNF-alpha-stimulated HAECs (45.7 +/- 2.5% and 55.8 +/- 1.2%, respectively). SME or Sal B significantly inhibited TNF-alpha-induced activation of nuclear factor kappa B (NF-kappaB) in HAECs (0.36- and 0.48-fold, respectively). These results demonstrate that SME and Sal B have anti-inflammatory properties and may explain their anti-atherosclerotic properties. This new mechanism of action of Sal B and SME, in addition to their previously reported inhibition of LDL, may help explain their efficacy in the treatment of atherosclerosis.     

丹酚酸B对大鼠离体工作心脏血流动力学的影响

目的 研究丹酚酸B对离体大鼠工作心脏血流动力学的影响.方法 采用Langendorff离体心脏灌流的方法,以左室内压( LVSP)、左室舒末压(LVEDP)、室内压最大上升速率(+dp/dtmax)、室内压最大下降速率(- dp/dtmax)、心率(HR)等血流动力学参数为指标,观察丹酚酸B对心肌收缩性能的影响.结果 不同剂量(10、5、2.5 mg/L)的丹酚酸B可使LVSP、±dp/dtmax明显升高,同时使HR减慢,并呈剂量依赖性,但对LVEDP无明显作用.结论 丹酚酸B对离体工作心脏有剂量依赖性正性肌力作用.     

Salvianolic acid B attenuates cyclooxygenase-2 expression in vitro in LPS-treated human aortic smooth muscle cells and in vivo in the apolipoprotein-E-deficient mouse aorta

Inflammation plays an essential role in atherosclerosis and post-angioplasty restenosis and the synthesis and release of inflammatory cytokines from vascular smooth muscle cells is an important contributor to these pathologies. It is assumed that drugs that prevent the overproduction of inflammatory cytokines may inhibit cardiovascular disorders. In the present study, the effects of a water-soluble antioxidant, salvianolic acid B (Sal B), derived from a Chinese herb, on the expression of cyclooxygenase (COX) in lipopolysaccharide (LPS)-treated human aortic smooth muscle cells (HASMCs) and in the aortas of cholesterol-fed apoE deficient mice were investigated. In unstimulated HASMCs, COX-2 mRNA and protein were almost undetectable, but were strongly upregulated in response to LPS. In contrast, HASMCs with or without LPS treatment showed constitutive expression of COX-1 mRNA and protein. The activation of COX-2 protein synthesis in LPS-stimulated HASMCs was shown to involve the activation of the extracellular-signal-regulated kinase 1/2 (ERK1/2), c-Jun NH(2)-terminal kinase (JNK), and p38 mitogen-activated protein kinase pathway. Incubation of HASMCs with Sal B before LPS stimulation resulted in pronounced downregulation of COX-2 expression. Sal B treatment suppressed ERK1/2 and JNK phosphorylation and attenuated the increase in prostaglandin E(2) production and NADPH oxidase activity in LPS-treated HASMCs. When apoE-deficient mice were fed a 0.15% cholesterol diet with or without supplementation with 0.3% Sal B for 12 weeks, the intima/media area ratio in the thoracic aortas was significantly reduced in the Sal B group (0.010 +/- 0.009%) compared to the apoE-deficient group (0.114 +/- 0.043%) and there was a significant reduction in COX-2 protein expression in the thickened intima. These results demonstrate that Sal B has anti-inflammatory properties and may explain its anti-atherosclerotic properties. This new mechanism of action of Sal B, in addition to its previously reported inhibition of LDL oxidation, may help explain its efficacy in the treatment of atherosclerosis.     

丹参水溶性成分的地域分布特点分析

分析全国不同产地丹参中水溶性成分的含量变化情况及其地域分布特点。采用HPLC法测定不同产地丹参水溶性成分的含量,运用SAS统计软件分析丹参水溶性成分相关性及其地域分布特点。回归分析表明,丹参素与丹酚酸A含量呈负相关,原儿茶醛与迷迭香酸含量呈负相关,咖啡酸与丹参素含量呈负相关,丹酚酸B与丹酚酸A、丹参素含量呈正相关。聚类分析表明,三个野生药材与山东和河南大部分产地、四川中江栽培药材水溶性成分较高聚为一类。建议丹参药材中丹参素≥0.14%,原儿茶醛≥0.01%,咖啡酸≥0.01%,迷迭香酸≥0.17%,丹酚酸B≥3.0%,丹酚酸A≥0.01%。丹参的水溶性成分呈现地域性差异,传统道地产区的含量较高。     

Salvianolic acid B inhibits the amyloid formation of human islet amyloid polypeptideand protects pancreatic beta‐cells against cytotoxicity

The misfolding of human islet amyloid polypeptide (hIAPP) is regarded as one of the causative factors of type 2 diabetes mellitus (T2DM). Salvia miltiorrhiza (Danshen), one of the most commonly used of traditional Chinese medicines, is often used in Compound Recipes for treating diabetes, however with unclear mechanisms. Since salvianolic acid B (SalB) is the most abundant bioactive ingredient of salvia miltiorrhiza water‐extract. In this study, we tested whether SalB has any effect on the amyloidogenicity of hIAPP. Our results clearly suggest that SalB can significantly inhibit the formation of hIAPP amyloid and disaggregate hIAPP fibrils. Furthermore, photo‐crosslinking based oligomerization studies suggest SalB significantly suppresses the toxic oligomerization of hIAPP monomers. Cytotoxicity protection effects on pancreatic INS‐1 cells by SalB were also observed using MTT‐based assays, potentially due to the inhibition on the membrane disruption effects and attenuated mitochondria impairment induced by hIAPP. These results provide evidence that SalB may further be studied on the possible pharmacological treatment for T2DM. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Discovery of 1,3-diphenyl-1H-pyrazole derivatives containing rhodanine-3-alkanoic acid groups as potential PTP1B inhibitors

Two series of 1,3-diphenyl-1H-pyrazole derivatives containing rhodanine-3-alkanoic acid groups were identified as competitive protein tyrosine phosphatase 1B (PTP1B) inhibitors. Among the compounds studied, IIIv was found to have the best in vitro inhibition activity against PTP1B (IC₅₀?=?0.67?±?0.09?µM) and the best selectivity (9-fold) between PTP1B and T-cell protein tyrosine phosphatase (TCPTP). Molecular docking studies demonstrated that compounds IIIm, IIIv and IVg could occupy simultaneously at both the catalytic site and the adjacent pTyr binding site. These results provide novel lead compounds for the design of inhibitors of PTP1B as well as other PTPs.     

Studies on composition and stability of a large membered bacterial consortium degrading phenol

A ten member microbial consortium (AS) consisting of eight phenol-degrading and two non-phenol-degrading strains of bacteria was developed and maintained in a fed-batch reactor by feeding 500 mg l⁻¹ phenol for four years at 28 ± 3 °C. The consortium could degrade 99% of 500 mg l⁻¹ phenol after 24 hours incubation with a biomass increase of 2.6 × 10⁷ to 4 × 10¹² CFU ml⁻¹. Characterization of the members revealed that it consisted of 4 principal genera, Bacillus, Pseudomonas, Rhodococcus, Streptomyces and an unidentified bacterium. Phenol degradation by the mixed culture and Bacillus subtilis, an isolate from the consortium was compared using a range of phenol concentrations (400 to 700 mg l⁻¹) and by mixing with either 160 mg l⁻¹ glucose or 50 mg l⁻¹ of 2,4-dichlorophenol in the medium. Simultaneous utilization of unrelated mixed substrates (glucose/2,4-dichlorophenol) by the consortium and Bacillus subtilis, indicated the diauxic growth pattern of the organisms. A unique characteristic of the members of the consortia was their ability to oxidize chloro aromatic compounds via meta pathway and methyl aromatic compounds via ortho cleavage pathway. The ability of a large membered microbial consortia to maintain its stability with respect to its composition and effectiveness in phenol degradation indicated its suitability for bioremediation applications.     

Protective effects and potential mechanism of salvianolic acid B on sodium laurate-induced thromboangiitis obliterans in rats

BackgroundThe root of Salvia miltiorrhiza f. alba (RSMA) (Lamiaceae) is used for the treatment of patients with thromboangiitis obliterans (TAO) in traditional Chinese medicine. Previously, a mixture of phenolic acids extracted from RSMA has shown significant protective effects on TAO rats.PurposeThis study investigates the inhibitory effects of salvianolic acid B on TAO induced by sodium laurate injection in rats to explore the effective constituents of RSMA in TAO treatment.MethodsTAO rats were developed using injected sodium laurate. After treatment with ligustrazine hydrochloride (15 mg/kg) and various doses of salvianolic acid B (10, 20, 40 mg/kg) by tail intravenous injection, levels of thromboxane B₂ (TXB₂), 6-keto-prostaglandin F_1α (6-keto-PGF_1α) and endothelin-1 (ET-1) in plasma were determined using enzyme-linked immunosorbent assay. The right femoral arteries were studied by hematoxylin and eosin staining and immunohistochemical analysis to determine pathological changes and overexpression of tumor necrosis factor (TNF)–α and inducible nitric oxide synthase (iNOS) in the femoral artery walls of TAO rats.ResultsSalvianolic acid B significantly decreased the expressions of TXB₂ and ET-1 and increased the expression of 6-keto-PGF_1α in plasma, and significantly inhibited the overexpression of TNF-α and iNOS in the femoral artery walls of TAO rats at medium and high doses.ConclusionSalvianolic acid B has a protective effect on TAO rats. The mechanism may involve inhibition of thrombosis and TAO-associated inflammatory responses, which may explain the success of RSMA treatment of TAO in humans in traditional Chinese medical practice. Hence, it may be a potential drug for TAO treatment in conventional medicine.     

Salvianolic acid B attenuates MMP-2 and MMP-9 expression in vivo in apolipoprotein-E-deficient mouse aorta and in vitro in LPS-treated human aortic smooth muscle cells

Salvianolic acid B (Sal B), a water-soluble antioxidant derived from a Chinese medicinal herb, is believed to have multiple therapeutic and preventive against human vascular diseases, including atherosclerosis and restenosis. To elucidate the underlying cellular mechanisms, we produced hypercholesterolemia by feeding apo-E-deficient mice a 0.15% cholesterol diet and inflammation in human aortic smooth muscle cells (HASMCs) with the endotoxin lipopolysaccharide (LPS), focusing on the metallopreteinases MMP-2 and MMP-9, the relevant signal transduction pathways and the effects of Sal B. Immunohistochemical analyses indicated apo-E-deficient mice fed a 0.15% cholesterol diet for 12 weeks exhibited thickened intima and elevated levels of MMP-2 and MMP-9 in aortic sections, both of which were attenuated by 0.3% Sal B dietary supplement. Western blotting and zymography analyses indicated that unstimulated HASMCs exhibited basal levels of protein and activity levels for MMP-2 and barely detectable levels for MMP-9, both of which were markedly upregulated by LPS, which also induced cell migration. Sal B significantly attenuated upregulations of both MMPs as well as the LPS-induced cell migration through the inactivation of MMP-2 and MMP-9 protein synthesis as well as the downregulation of the extracellular-signal-regulated kinase 1/2 (ERK1/2) and c-Jun NH(2)-terminal kinase (JNK). These results demonstrate that Sal B has anti-migration properties on smooth muscle cells and may explain its anti-atherosclerotic properties. This novel mechanism of action of Sal B, in addition to its previously reported inhibition of LDL oxidation, may help explain its efficacy in the treatment of atherosclerosis.     

丹酚酸B对高糖日粮小鼠消化系统自由基水平的影响

本文研究了丹酚酸B(salvianolic acid B,Sal B)对灌注及长期摄食高糖小鼠活性氧自由基(reactive oxygen species,ROS)的影响.结果表明:灌胃高糖使小鼠全血ROS于1.5 h达到最大值,灌胃葡萄糖的ROS释放量显著高于蔗糖(P＜0.05);灌胃高糖使小鼠血糖浓度于0.5 h达到最高,灌胃葡萄糖的小鼠血糖浓度有高于灌胃蔗糖的小鼠血糖浓度的趋势(P＞0.05);添加Sal B后并没有改变灌胃高糖小鼠的全血ROS释放和血糖浓度变化趋势,但有降低血糖趋势(P＞0.05),显著减少了ROS的释放量(P＜0.05);添加Sal B后显著降低长期高糖膳食小鼠的全血、肝脏、胰腺、胃、十二指肠、空肠和回肠的ROS释放量.