PF2401-SF, standardized fraction of Salvia miltiorrhiza and its constituents, tanshinone I, tanshinone IIA, and cryptotanshinone, protect primary cultured rat hepatocytes from bile acid-induced apoptosis by inhibiting JNK phosphorylation.

Bile acid-induced hepatocyte apoptosis plays an important role in cholestatic liver disease, and the role of apoptosis may be of therapeutic interest in preventing liver disease. The dried root of Salvia miltiorrhiza Bunge (Labiatae) has been used traditionally to treat liver diseases. We investigated the antiapoptotic effects of a standardized fraction of S. miltiorrhiza (PF2401-SF) and its components, tanshinone I, tanshinone IIA, and cryptotanshinone, in primary cultured rat hepatocytes. PF2401-SF was enriched with tanshinone I (11.5%), tanshinone IIA (41.0%), and cryptotanshinone (19.1%). Glycochenodeoxycholic acid (GCDC)-induced apoptosis, as shown by DNA fragmentation, poly(ADP-ribose) polymerase cleavage, and activation of caspases-8, -9, and -3. PF2401-SF and its components, tanshinone I, tanshinone IIA, and cryptotanshinone showed antiapoptotic activity. Treatment with PF2401-SF or with its components significantly inhibited the generation of intracellular reactive oxygen species. Hydrophobic bile acids activate c-Jun-NH(2)-terminal kinase (JNK), p38 mitogen-activated protein kinases (MAPK), and extracellular signal-regulated kinase 1/2, and PF2401-SF inhibited the phosphorylation of JNK and p38. All three components of PF2401-SF inhibited JNK phosphorylation. Addition of inhibitors of MAPK showed that inhibition of JNK decreased apoptosis. These data indicate that PF2401-SF and its components protect hepatocytes from GCDC-induced apoptosis in vitro by inhibiting JNK.     

丹参酮IIA磺酸钠对血管紧张素II诱导的心肌肥大及p-ERK表达的影响

目的:观察丹参酮IIA磺酸钠(STS)对血管紧张素II(Ang II)诱导的心肌肥大及p-ERK表达的影响。方法:培养新生大鼠心肌细胞,考马斯亮蓝法测定心肌细胞蛋白含量、[3H]-亮氨酸掺入法测定蛋白合成速率作为心肌肥大指标;用West-ern-blot测定p-ERK表达。结果:STS能显著降低Ang II诱导的心肌细胞总蛋白含量、蛋白合成速率上升,同时对p-ERK表达具有剂量、时间依赖性抑制作用。结论:STS可以抑制Ang II诱导的心肌肥大,机制与抑制p-ERK表达有关。     

Tanshinone IIA protects cardiac myocytes against oxidative stress-triggered damage and apoptosis

Tanshinone IIA (tan), a derivative of phenanthrenequinone, is one of the key components of Salvia miltiorrhiza Bunge. Previous reports showed that tan inhibited the apoptosis of cultured PC12 cells induced by serum withdrawal or ethanol. However, whether tan has a cardioprotective effect against apoptosis remains unknown. In this study, we investigated the effects of tan on cardiac myocyte apoptosis induced both by in vitro incubation of neonatal rat ventricular myocytes with H(2)O(2) and by in vivo occlusion followed by reperfusion of the left anterior descending coronary artery in adult rats. In vitro, as revealed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium (MTT) assay, treatment with tan prior to H(2)O(2) exposure significantly increased cell viability. Tan also markedly inhibited H(2)O(2)-induced cardiomyocyte apoptosis, as detected by ladder-pattern fragmentation of genomic DNA, chromatin condensation, and hypodioloid DNA content. In vivo, tan significantly inhibited ischemia/reperfusion-induced cardiomyocyte apoptosis by attenuating morphological changes and reducing the percentage of terminal transferase dUTP nick end-labeling (TUNEL)-positive myocytes and caspase-3 cleavage. These effects of tan were associated with an increased ratio of Bcl-2 to Bax protein in cardiomyocytes, an elevation of serum superoxide dismutase (SOD) activity and a decrease in serum malondialdehyde (MDA) level. Taken together, these data for the first time provide convincing evidence that tan protects cardiac myocytes against oxidative stress-induced apoptosis. The in vivo protection is mediated by increased scavenging of oxygen free radicals, prevention of lipid peroxidation and upregulation of the Bcl-2/Bax ratio.     

丹参酮ⅡA对心室重构作用的研究进展

丹参酮ⅡA（TanⅡA）是中药丹参的脂溶性成分之一,其磺化剂丹参酮ⅡA磺酸钠（STS）现已广泛用于心绞痛、脑卒中、肿瘤、肝纤维化等多种疾病的临床治疗。 TanⅡA通过改善心肌肥大,减少心肌细胞凋亡,抑制心肌纤维化及改善心脏神经重构和电生理异常等机制,发挥抗心室重构作用。 TanⅡA对各种心血管疾病所致心室重构具有较好的临床应用前景。     

Sodium tanshinone IIA sulfonate derived from Slavia miltiorrhiza Bunge up-regulate the expression of prolactin releasing peptide (PrRP) in the medulla oblongata in ovariectomized rats

Sodium tanshinone IIA sulfonate (STS), a derivative of tanshinone IIA, is isolated from the root of Salvia miltiorrhiza known as "Danshen". Although injection of S. miltiorrhiza extract and STS is used successfully in clinics in China for treating postmenopausal syndrome, the exact mechanism for its therapeutic basis is poorly understood. The present study was undertaken to characterize the effect of STS on the expression of prolactin releasing peptide (PrRP) in the medulla oblongata in ovariectomized rats. In addition, estrogen (E2) levels were detected in OVX rats treated with STS. The results showed that STS might significantly increase the blood level of E2 and PrRP cell number in the medulla oblongata of ovariectomized rats. The number of PrRP immunoreactivity (ir) neurons was higher in the group ovariectomized with STS than that in the ovariectomized group. The numbers of PrRP-ir neurons in Sham and Sham+STS were not significantly different between the two groups. These results suggest that the mechanism that STS improved postmenopausal symptoms induced by ovariectomy in rats might be related to the modulation of the blood E2 level and the expression of PrRP in medulla oblongata of ovariectomized rats.     

Cytotoxicity of major tanshinones isolated from Danshen (Salvia miltiorrhiza) on HepG2 cells in relation to glutathione perturbation.

Tanshinones are abietane type-diterpene quinones isolated from the roots of Radix Salvia miltiorrhiza (Danshen), a well-known traditional Chinese medicine in the treatment of cardiovascular diseases. Among the major diterpenes isolated, including cryptotanshinone, tanshinone I, tanshinone IIA and dihydrotanshinone, tanshinone IIA had been shown to posses various pharmacological activities including antioxidant, protection/prevention from angina pectoris and myocardial infarction, and anticancer properties. Tanshinone IIA, usually the most abundant tanshinone present in the herb, has been the focus of studies in its clinical potential, among which its ability to inhibit the proliferation of cancer cell lines. The aim of this study was to study the cytotoxicity of the tanshinones on human HepG2 cells in vitro in relation to intracellular glutathione perturbation (reduced glutathione, GSH and oxidized glutathione, GSSG). Studies using MTT assay showed that all tanshinones decreased cell viability of HepG2 cells in a concentration-dependent manner, with the cell viability decreased to 60% and 35% after 24 h and 48 h treatment, respectively. Assessment of apoptotic cells with fragmented DNA by flow cytometry indicated that only tanshinone IIA (12.5 and 25 microM) induced apoptosis in the cancer cells. Tanshinone IIA and cryptotanshinone caused significant decreases in G(1) cells by 23% and 13%, respectively, after 24 h treatment. The declines in G(1) cells were compensated by increases in G(2)/M (15% for tanshinone IIA) and S cells (8% and 13% for tanshinone IIA and cryptotanshinone, respectively). All the tanshinones studied, except tanshinone IIA, elevated GSH/GSSG ratio at low concentrations (1.56 and 3.13 microM), but the ratio decreased, indicating oxidative stress at high concentrations (6.25-25 microM). Taken together, tanshinone IIA caused HepG2 cytotoxicity through apoptosis without influencing oxidative stress, while the other tanshinones showed lower efficacy in inducing apoptosis in the HepG2 cells.     

Sodium tanshinone IIA silate inhibits oxygen-glucose deprivation/recovery-induced cardiomyocyte apoptosis via suppression of the NF-κB/TNF-α pathway

Background and Purpose

Inhibition of apoptosis may attenuate the irreversible injury associated with reperfusion. In the current study, we focused on the cytoprotective effects and the underlying mechanism of sodium tanshinone IIA silate (STS) against damage induced by oxygen-glucose deprivation/recovery (OGD/R). in H9c2 cardiomyocytes and the underlying mechanisms.

Experimental Approach

We used a model of cardiac ischaemia/reperfusion, OGD/R in H9c2 cardiomyocytes, to assess the cardioprotective effects of STS. Apoptosis of cells was measured with Hoechst 33342-based fluorescence microscopy, and annexin V-FITC-based flow cytometry. Caspase-3 and caspase-8 activities and mitochondrial membrane potential were also measured using commercial kits. TNF-α in the cell culture supernatant fractions were measured with sandwich elisa, and protein levels assayed using Western blot.

Key Results

STS inhibited OGD/R-induced apoptosis by suppressing JNK-mediated activation of NF-κB, TNF-α expression, activation of caspase-3 and caspase-8 and the Bax/Bcl-2 ratio. Additionally, positive feedback between NF-κB and TNF-α and amplification of TNF-α were inhibited, suggesting that STS plays a protective role against apoptosis in cardiomyocytes, even upon activation of pro-inflammatory cytokines. Interestingly, the cytoprotective effects of STS on OGD/R-induced apoptosis and promotion of cell survival were attenuated after inhibition of PI3K.

Conclusion and Implications

The inhibitory effects of STS on TNF-α and positive feedback signalling of the NF-κB/TNF-α pathways may play important roles in myocardial protection against ischaemia/reperfusion. These protective effects of STS are mediated by suppressing JNK activity through activation of the PI3K-Akt pathway.     

Growth inhibition and apoptosis induction by tanshinone IIA in human colon adenocarcinoma cells

Tanshinone IIA is the most abundant diterpene quinone in Danshen, Salviae miltiorrhizae Radix, a widely prescribed traditional herbal medicine that is used to treat cardiovascular and inflammatory diseases. Recently, tanshinone IIA was demonstrated to induce cell death and apoptosis in a variety of tumors. However, the effect of tanshinone IIA on human colon cancer cells is not clearly understood yet. In this study, the antigrowth and apoptosis-eliciting effects of tanshinone IIA, as well as its cellular mechanisms of actions, were investigated in Colo-205 human colon cancer cells. Tanshinone IIA reduced cell growth in a concentration-dependent manner, inducing apoptosis accompanied by an increase in TUNEL staining and by an increased percentage of cells in the sub-G1 fraction. The expression of p53 and p21 and mitochondrial cytochrome c release were increased in tanshinone IIA-treated cells. In addition, the expression of Fas proteins was up-regulated by tanshinone IIA. Tanshinone IIA-induced catalytic activation of caspases was confirmed by cleavage of caspase-8 and caspase-3. These findings suggest that tanshinone IIA induces apoptosis in Colo-205 cells through both mitochondrial-mediated intrinsic and Fas-mediated extrinsic caspase cell-death pathways. Accordingly, the chemotherapeutic potential of tanshinone IIA for colon cancer warrants further study.     

Irciniastatin A induces JNK activation that is involved in caspase-8-dependent apoptosis via the mitochondrial pathway

Irciniastatin A (ISA)/psymberin, a pederin-type natural product isolated from marine sponge, exhibits extremely potent and selective cytotoxicity against certain human cancer cell lines, but its molecular target and cytotoxic mechanisms are still unknown. Here we show that ISA is a potent inhibitor of protein translation, and induces apoptosis accompanied with activation of the stress-activated protein kinases via the mitochondrial pathway in human leukemia Jurkat cells. ISA potently inhibited protein translation, and induced a slow but prolonged activation of the stress-activated protein kinases, JNK and p38, at between 1h and 6h after treatment. In Bcl-x(L)-transfected cells, the activation of JNK and p38 by ISA was shortened. The same results were obtained in the cells treated with N-acetyl-L-cysteine, suggesting that the prolonged activation of JNK and p38 by ISA is mediated by reactive oxygen species generated from mitochondria. ISA strongly induced apoptosis, which was partially suppressed by the JNK inhibitor SP600125, but not by the p38 inhibitor SB202190. Apoptosis induction by ISA was partially reduced, but not suppressed by SP600125 in caspase-8-deficient Jurkat cells. These results suggest that ISA activates stress-activated kinases by a mitochondria-mediated mechanism, and that activation of JNK is required for caspase-8-dependent apoptosis.     

Diterpene quinone tanshinone IIA selectively inhibits mouse and human cytochrome P4501A2

1. Tanshinone IIA is the main active diterpene quinone in the herbal medicine Salvia miltiorrhiza. In untreated mouse liver microsomes, tanshinone IIA selectively inhibited 7-ethoxyresorufin O -deethylation (EROD) and 7-methoxyresorufin O -demethylation (MROD) activities without affecting the oxidation of benzo(a)pyrene, tolbutamide, N -nitrosodimethylamine and nifedipine. Tanshinone IIA was a competitive inhibitor of MROD activity with a K i of 7.2 ± 0.7 nM. 2. In 3-methylcholanthrene-treated mouse liver microsomes, tanshinone IIA and two minor tanshinones, tanshinone I and cryptotanshinone, inhibited liver microsomal MROD activity without affecting EROD and benzo(a)pyrene hydroxylation activities at the concentrations up to 1 µ M. Tanshinone IIA induced a type I binding spectrum with a spectral dissociation constant K?s of 2.3 ± 0.8 µ M without cooperativity. 3. In human liver microsomes, tanshinone IIA decreased EROD and MROD activities without affecting the oxidation of benzo(a)pyrene, tolbutamide, chlorzoxazone and nifedipine. 4. In Escherichia coli membranes expressing bicistronic human CYP1A enzymes, tanshinone IIA inhibited EROD activity of CYP1A1 with an IC 50 48 times higher than that for CYP1A2. Tanshinone I and cryptotanshinone had the same IC 50 ratio (1A1/1A2) of 4. 5. The results indicate that tanshinone represents a new group of CYP1A inhibitors, and tanshinone IIA had the highest selectivity in inhibition of CYP1A2.     

Sodium tanshinone IIA sulfonate and its interactions with human CYP450s

1.Sodium tanshinone IIA sulfonate (STS) is a water-soluble derivative of tanshinone IIA, a famous Chinese medicine used for many years to treat cardiovascular disorders. However, the role of cytochrome P450 (CYP) enzymes in the metabolism of STS was unclear. In this study, we screened the main CYPs for the metabolism of STS and studied their interactions in vitro.

2.Seven CYPs were screened for the metabolism of STS by human liver microsomes (HLMs) or recombinant CYP isoforms. To determine the potential of STS to affect CYP-mediated phase I metabolism in humans, phenacetin (CYP1A2), coumarin (CYP2A6), tolbutamide (CYP2C9), metoprolol (CYP2D6), chlorzoxazone (CYP2E1), S-Mephenytoin (CYP2C19), and midazolam (CYP3A4) were used as the respective probe substrates. Enzyme kinetic studies were performed to investigate the mode of inhibition of the enzyme–substrate interactions.

3.STS inhibited the activity of CYP3A4 in a dose-dependent manner in the HLMs and CYP3A4 isoform. Other CYP isoforms, including CYP1A2, CYP2A6, CYP2C9, CYP2D6, CYP2E1, and CYP2C19, showed minimal or no effect on the metabolism of STS.

4.The results suggested that STS primarily inhibits the activities of CYP3A4 in vitro, and STS has the potential to perpetrate drug–drug interactions with other CYP3A4 substrates.     

Anti-allergic effects of salvianolic acid A and tanshinone IIA from Salvia miltiorrhiza determined using in vivo and in vitro experiments

Salvia miltiorrhiza root has been used in Asian traditional medicine for the treatment of cardiovascular diseases, asthma, and other conditions. Salvianolic acid B from S. miltiorrhiza extracts has been shown to improve airway hyperresponsiveness. We investigated the effects of salvianolic acid A, tanshinone I, and tanshinone IIA from S. miltiorrhiza in allergic asthma by using rat RBL-2H3 mast cells and female Balb/c mice. Antigen-induced degranulation was assessed by measuring β-hexosaminidase activity in vitro. In addition, a murine ovalbumin-induced allergic asthma model was used to test the in vivo efficacy of salvianolic acid A and tanshinone IIA. Tanshinone I and tanshinone IIA inhibited antigen-induced degranulation of mast cells, but salvianolic acid A did not. Administration of salvianolic acid A and tanshinone IIA decreased the number of immune cells, particularly eosinophils in allergic asthma-induced mice. Histological studies showed that salvianolic acid A and tanshinone IIA reduced mucin production and inflammation in the lungs. Administration of salvianolic acid A and tanshinone IIA reduced the expression and secretion of Th2 cytokines (IL-4 and IL-13) in the bronchoalveolar lavage fluid and lung tissues of mice with ovalbumin-induced allergic asthma. These findings provide evidence that salvianolic acid A and tanshinone IIA may be potential anti-allergic therapeutics.     

Sodium tanshinone IIA sulfonate protects against acute exacerbation of cigarette smoke-induced chronic obstructive pulmonary disease in mice

Exacerbation of chronic obstructive pulmonary disease (COPD) is characterized by acute airway inflammation and mucus hypersecretion, which is by far the most costly aspect of its management. Thus, it is essential to develop therapeutics with low side effects for CODP exacerbation. Sodium tanshinone IIA sulfonate (STS) is a water-soluble derivative of tanshinone IIA isolated as the major active component of Chinese herbal medicine Danshen. Although it possesses anti-inflammatory, anti-oxidative and anti-apoptotic properties, it remains unknown whether STS protects against COPD exacerbation. In this study, we challenged cigarette smoke (CS)-exposed mice with lipopolysaccharide (LPS), and then treated these mice with STS. We found that STS significantly ameliorated pulmonary inflammatory responses, mucus hypersecretion and lung function decline in CS-exposed mice challenged with LPS. STS treatment also significantly attenuated increased IL-6 and IL-8 releases from cigarette smoke extract (CSE)-treated human bronchial epithelial cells (16HBE) challenged with LPS. Mechanistically, STS reduced activation of ERK1/2 and NF-κB in lungs of CS-exposed mice and CSE-treated 16HBE cells challenged with LPS. Taken together, STS protects against acute exacerbation of CS-induced lung injury, which provides a promising and potential therapeutic avenue to halt acute exacerbation of COPD.     

Hydrogen peroxide inhibits the immune response to lipopolysaccharide by attenuating signaling through c-Jun N-terminal kinase and p38 associated with protein kinase C

This study examined the immunomodulatory effects of hydrogen peroxide (H(2)O(2)) in B6C3F1 mouse splenic lymphocytes. H(2)O(2) produced a marked and dose-related inhibition of both lipopolysaccharide (LPS)-induced B-cell proliferation and concanavalin A (Con A)-induced T-cell proliferation. Unexpectedly, little effect was observed with H(2)O(2) on the antibody-forming cell (AFC) response to the polyclonal B-cell activator, LPS. It was also observed that H(2)O(2) did not have any detectable effect on forskolin-stimulated adenylate cyclase, indicating that cyclic AMP (cAMP) is not a mediator of H(2)O(2)-induced suppression of the immune response. Rather, LPS-induced activation of protein kinase C (PKC) was completely inhibited when cells were pretreated with H(2)O(2) for 18 h, although PKC activity was increased approximately twofold following treatment with H(2)O(2) for 10 min. In addition, H(2)O(2) pretreatment blocked the phosphorylation of two stress-activated mitogen-activated protein kinases (MAPKs), c-Jun N-terminal kinase (JNK) and p38 by LPS in a concentration-dependent fashion. Therefore, these data suggest that H(2)O(2) suppresses immune response through the desensitization of PKC, which subsequently results in inhibition of JNK and p38.     

Addition of tanshinone IIA to UW solution decreases skeletal muscle ischemia-reperfusion injury

AIM: To investigate whether tanshinone IIA could improve the effect of UW solution for skeletal muscle preservation and to determine the dose range of tanshinone IIA providing optimal protection during ischemia and reperfusion. METHODS: Ischemic rat limbs were perfused with UW solution or UW plus tanshinone IIA (UW+T, 0.05, 0.1, or 0.2 mg/mL) for 0.5 h before reperfusion; controls (I/R) received no perfusion. Serum creatine phosphokinase (CPK), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH) were measured pre-ischemia and after reperfusion (2-h, 4-h, and 6-h). Muscle water content, superoxide dismutase (SOD), malondialdehyde (MDA), adenosine triphosphatase (ATPase) were assessed pre-reperfusion and after 6-h reperfusion. Intercellular adhesion molecule-1 (ICAM-1) and apoptosis were detected after 6-h reperfusion. Reperfusion blood flow was monitored during reperfusion period. RESULTS: UW and UW+T prevented luxury perfusion during reperfusion and inhibited ICAM-1 expression and apoptosis after 6-h reperfusion. Serum CPK, AST, and LDH levels in UW rats were significantly less than those in controls after 2-h reperfusion (no difference, 4-h or 6-h reperfusion). After 4-h ischemia, there were significant differences in water content, MDA, SOD, and ATPase between UW and controls, but no difference after 6-h reperfusion. All tests with UW+T rats were significantly different from control results at corresponding durations. Higher tanshinone doses improved results. CONCLUSION: UW plus tanshinone IIA increased protection against I/R injury, suggesting that tanshinone IIA has clinical value.     

Activation of CYP3A-mediated testosterone 6β-hydroxylation by tanshinone IIA and midazolam 1-hydroxylation by cryptotanshinone in human liver microsomes

This study evaluated the in vitro activation of CYP3A-mediated midazolam 1-hydroxylation and testosterone 6β-hydroxylation by tanshinone I, tanshinone IIA, and cryptotanshinone. The abilities of tanshinones to activate CYP3A-mediated midazolam 1-hydroxylation and testosterone 6β-hydroxylation in human liver microsomes (HLMs) were tested. Substrate- and effector-dependent activation of CYP3A by tanshinones were both observed. Cryptotanshinone was shown to activate CYP3A-mediated midazolam 1-hydroxylation in a concentration-dependent manner. In contrast, tanshinone IIA and tanshinone I did not activate this hydroxylation reaction. In addition, tanshinone IIA activated CYP3A-mediated testosterone 6β-hydroxylation, whereas cryptotanshinone and tanshinone I did not. The results from our study enhance the understanding of CYP3A activation by tanshinone IIA and cryptotanshinone in HLMs. Additionally, these data allow for an accurate prediction of the magnitude and likelihood of Danshen-drug interactions.     

咖啡因对LY294002诱导的小脑颗粒神经元凋亡的拮抗作用(英文)

目的:研究咖啡因对磷酸肌醇-3-激酶抑制剂诱导的小脑颗粒神经元凋亡的作用及机制。方法:神经元体外培养,凝胶电泳,SAPK/JNK分析盒测定JNK活性。结果:LY294002浓度依赖性地触发小脑颗粒神经元凋亡,但咖啡因具有浓度依赖性的保护作用。此作用不受ryanodine-敏感性钙释放阻断剂、L-型钙通道阻断剂和NMDA受体阻断剂的影响。而且,RP-cAMP,H89和KN62均不能抑制咖啡因的保护作用。c-Jun的磷酸化是LY294002诱导神经原凋亡所必需,咖啡因可直接抑制JNK的活性,降低神经元内磷酸化c-Jun的含量。结论:咖啡因通过直接抑制JNK活性而抑制小脑颗粒神经元的凋亡。     

Study of sodium tanshinone II A sulfonate tissue distribution in rat by liquid chromatography/tandem mass spectrometry

A rapid and sensitive method based on liquid chromatography/tandem mass spectrometry (LC-MS/MS) has been developed and fully validated for the quantitative determination of sodium tanshinone IIA sulfonate (STS, sodium (1,6,6-trimethyl-10,11-dioxo-7,8,9-trihydrophenanthro[1,2-b]furan)-yl-2-sulfonate) in rat biosamples including plasma and different tissues using sodium tanshinone I sulfonate (sodium (1,6-dimethyl-10,11-dioxo-phenanthro[1,2-b]furan)-yl-2-sulfonate) as internal standard. Simple protein precipitation by acetonitrile was utilized for extracting STS from the rat biosamples. Chromatographic separation of the sample matrix from the analyte and the internal standard was performed using a commercially available analytical column with a mobile phase consisting of methanol-5 mmoL/L ammonia acetate (70:30, v/v) at a flow rate of 0.2 mL/min. Detection was performed on a triple quadrupole tandem mass spectrometer equipped with an electrospray ionization source and operated in the negative-ion mode. The intra- and inter-day precisions (RSD%) and deviations of the assay accuracies were within 10.0% for STS. The extraction recovery of STS was more than 86.5%. The limit of detection (LOD) of STS was 1.0 ng/mL. The method was successfully applied to the tissue distribution study of STS intravenously administered to healthy Sprague-Dawley rats. The tissue distribution results showed that liver, kidney, lung, small intestine and duodenum were the major distribution tissues of STS in rats, and that STS had difficulty in crossing the blood-brain barrier. After 24 h, STS could be detected only in the kidney, stomach and small intestine, indicating that there was no long-term accumulation of STS in rat.     

丹参酮ⅡA磺酸钠对AngⅡ诱导的心肌细胞肥大反应中磷酸化细胞外信号调节激酶1/2的作用

目的从细胞外信号调节激酶1/2(ERK1/2)激活角度研究丹参酮ⅡA磺酸钠(sodium tanshinoneⅡ Asulfonate,STS)对血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)诱导的心肌肥大反应中的作用。方法培养新生大鼠心肌细胞,考马斯亮蓝法测定心肌细胞蛋白含量、[3H]-亮氨酸参入法测定蛋白合成速率作为心肌肥大指标;用免疫荧光标记法和Western blot测定磷酸化ERK1/2蛋白(p-ERK1/2)表达。结果(1)AngⅡ(1μmol·L-1)处理24h,心肌细胞[3H]-亮氨酸参入率、蛋白含量明显增加,STS能明显抑制AngⅡ介导心肌细胞[3H]-亮氨酸参入率、蛋白含量的增加;(2)AngⅡ刺激心肌细胞可见胞核内出现磷酸化ERK1/2荧光染色,丹参酮ⅡA可阻断AngⅡ引起的ERK1/2活化、入核过程;(3)用AngⅡ(1μmol·L-1)处理心肌细胞5min,磷酸化ERK1/2蛋白(p-ERK1/2)表达即开始增加,10min左右时最明显。以AngⅡ(1μmol·L-1)处理心肌细胞10min,磷酸化ERK1/2蛋白(p-ERK1/2)表达为标准,预先以STS(2,10,50μmol·L-1)处理心肌细胞30min,发现STS可明显抑制AngⅡ诱导的心肌细胞磷酸化ERK1/2蛋白表达;(4)预先以不同浓度STS处理心肌细胞30min,发现STS对AngⅡ诱导的心肌细胞磷酸化ERK1/2蛋白表达的抑制作用存在剂量依赖性。结论STS可以抑制AngⅡ诱导的心肌肥厚,其机制与抑制磷酸化ERK1/2表达有关。     

Tanshinone IIA suppresses inflammatory bone loss by inhibiting the synthesis of prostaglandin E2 in osteoblasts

Tanshinone IIA isolated from Danshen is widely used in Oriental medicine. However, the action of tanshinone IIA in inflammatory bone-resorptive diseases remains unknown. Here we examined the effect of tanshinone IIA in inflammation-mediated osteoclastic bone resorption. Tanshinone IIA inhibited osteoclast differentiation in cocultures of bone marrow cells and calvarial osteoblasts. Tanshinone IIA regulated the expression of receptor activator of NF-kappaB ligand and osteoprotegerin in osteoblasts treated with lipopolysaccharide (LPS). Also, tanshinone IIA inhibited prostaglandin E(2) (PGE(2)) synthesis by inhibiting Cyclooxygenase-2 (COX-2) expression induced by LPS. Furthermore, tanshinone IIA greatly suppressed bone loss in the mouse models of bone loss. Our findings suggest that tanshinone IIA inhibits osteoclast formation by inhibiting COX-2/PGE(2) signaling and by suppressing bone erosion in vivo. These results suggest that tanshinone IIA may be of therapeutic value as an anti-bone-resorptive drug in the treatment of bone-related disease.