Influence of tanshinone on rabbit malignant arrhythmias and calmodulin signal transduction pathway after myocardial infarction / 心血管康复医学杂志

Objective: To explore influence of tanshinone on rabbit malignant arrhythmias and calmodulin signal transduction pathway after myocardial infarction. Methods: A total of 30 rabbits were randomly and equally divided into sham operation group (sham group), model group and tanshinone group. Acute myocardial infarction model was established through ligating left anterior descending branch of coronary. After modeling, the rabbits were randomly and equally divided into model group and tanshinone group. Incidence rate of malignant arrhythmias, action potential duration (APD) of three myocardial layers (namely endocardium layer, myocardium layer and epicardium layer), transmural dispersion of repolarization, Ca2+ concentration, compared among three groups. Results: Incidence rate of malignant arrhythmias in tanshinone group was significantly lower than that of model group (20.0% vs. 70.0%, P<0.01); compared with sham group, there were significant rise in transmural dispersion of repolarization, 90% APD, Ca2+ concentration, expression levels of calmodulin and calmodulin kinase Ⅱ of three layers in model group and tanshinone group (P<0.01 all) ; compared with model group, there were significant reductions in transmural dispersion of repolarization[(46.2±10.9) ms vs.(35.5±8.8) ms], 90% APD [epicardium layer, (231.5±17.4) ms vs.(211.0±16.3) ms], Ca2+ concentration [epicardium layer, (132.0±12.3) mmol/L vs.(102.3±10.3) mmol/L], expression levels of calmodulin [epicardium layer, (0.724±0.014) vs. (0.563±0.014)] and calmodulin kinase Ⅱ[epicardium layer, (0.759±0.019) vs. (0.589±0.017)] in tanshinone group, (P<0.05～0.01). Conclusion: Tanshinone possesses anti-malignant arrhythmias effect after myocardial infarction. Its mechanism may be regulating calmodulin and calmodulin kinase Ⅱ signal transduction pathway.

The expression level of serum IL-7 and its influence on prognosis in patients with acute coronary syndrome / 心血管康复医学杂志

Objective: To explore the expression level of serum interleukin (IL)-7 in patients with acute coronary syndrome (ACS) and analyze the relationship between IL-7 level and prognosis. Methods: A total of 130 ACS patients [ACS group, including 70 cases with acute myocardial infarction (AMI) and 60 cases with unstable angina pectoris (UAP)], 33 cases with stable angina pectoris (SAP，SAP group) and 89 healthy subjects (healthy control group) were selected. IL-7 level was measured using enzyme linked immunosorbent assay (ELISA) and compared among all groups. The 130 ACS patients were followed up, and Logistic regression analysis was used to analyze the relationship between IL-7 level and prognosis. Results: Compared with healthy control group and SAP group, there was significant rise in IL-7 level in UAP group and AMI group [(1.84±0.47) pg/ml, (2.11±0.63) pg/ml vs. (4.87±0.52) pg/ml, (5.15±0.71) pg/ml, P0.05 both); Logistic regression analysis indicated that expression level of serum IL-7 was an independent risk factor for adverse cardiovascular events in ACS patients (OR=1.212, 95%CI:1.061-1.418). Conclusion: Interleukin-7, as an important inflammatory cytokines, its serum level abnormally elevated in patients with acute coronary syndrome, it may have important prognostic value.

Inhibitory effect of tanshinone II A on TGF II-β1-induced cardiac fibrosis / 华中科技大学学报（医学）（英德文版）

This study examined the effect of tanshinone II A (TSN II A) on the cardiac fibrosis induced by transforming growth factor β1 (TGF-β1) and the possible mechanisms. Cardiac fibroblasts were isolated from cardiac tissues of neonatal Sprague-Dawley (SD) rats by the trypsin digestion and differential adhesion method. The cells were treated with 5 ng/mL TGF-β1 alone or pretreated with TSN II A at different concentrations (10(-5) mol/L, 10(-4) mol/L). Immunocytochemistry was used for cell identification, RT-PCR for detection of the mRNA expression of connective tissue growth factor (CTGF) and collagen type I (COL I), Western blotting for detection of the protein expression of Smad7 and Smad3, and immunohistochemistry and immunofluorescence staining for detection of the protein expression of phosphorylated Smad3 (p-Smad3), CTGF and COLI. The results showed that TGF-β1 induced the expression of CTGF, COL I, p-Smad3 and Smad7 in a time-dependent manner. The mRNA expression of CTGF and COL I was significantly increased 24 h after TGF-β1 stimulation (P<0.01 for all). The protein expression of p-Smad3 and Smad7 reached a peak 1 h after TGF-β1 stimulation, much higher than the baseline level (P<0.01 for all). Pretreatment with high concentration of TSN A resulted in a decrease in the expression of p-Smad3, CTGF and COL I (P<0.01). The protein expression of Smad7 was substantially upregulated after pretreatment with two concentrations of TSN II A as compared with that at 2 h post TGF-β1 stimulation (P<0.05 for low concentration of TSN I IA; P<0.01 for high concentration of TSN II A). It was concluded that TSN II A may exert an inhibitory effect on cardiac fibrosis by upregulating the expression of Smad7, suppressing the TGF-β1-induced phosphorylation of Smad3 and partially blocking the TGF-β1-Smads signaling pathway.

Inhibitory effect of tanshinone II A on TGF II-β1-induced cardiac fibrosis / 华中科技大学学报（医学）（英德文版）

This study examined the effect of tanshinone II A (TSN II A) on the cardiac fibrosis induced by transforming growth factor β1 (TGF-β1) and the possible mechanisms. Cardiac fibroblasts were isolated from cardiac tissues of neonatal Sprague-Dawley (SD) rats by the trypsin digestion and differential adhesion method. The cells were treated with 5 ng/mL TGF-β1 alone or pretreated with TSN II A at different concentrations (10(-5) mol/L, 10(-4) mol/L). Immunocytochemistry was used for cell identification, RT-PCR for detection of the mRNA expression of connective tissue growth factor (CTGF) and collagen type I (COL I), Western blotting for detection of the protein expression of Smad7 and Smad3, and immunohistochemistry and immunofluorescence staining for detection of the protein expression of phosphorylated Smad3 (p-Smad3), CTGF and COLI. The results showed that TGF-β1 induced the expression of CTGF, COL I, p-Smad3 and Smad7 in a time-dependent manner. The mRNA expression of CTGF and COL I was significantly increased 24 h after TGF-β1 stimulation (P<0.01 for all). The protein expression of p-Smad3 and Smad7 reached a peak 1 h after TGF-β1 stimulation, much higher than the baseline level (P<0.01 for all). Pretreatment with high concentration of TSN A resulted in a decrease in the expression of p-Smad3, CTGF and COL I (P<0.01). The protein expression of Smad7 was substantially upregulated after pretreatment with two concentrations of TSN II A as compared with that at 2 h post TGF-β1 stimulation (P<0.05 for low concentration of TSN I IA; P<0.01 for high concentration of TSN II A). It was concluded that TSN II A may exert an inhibitory effect on cardiac fibrosis by upregulating the expression of Smad7, suppressing the TGF-β1-induced phosphorylation of Smad3 and partially blocking the TGF-β1-Smads signaling pathway.

Effects of tanshinone ⅡA on transforming growth factor beta 1/Smads signaling pathway in cardiac fibroblasts / 中国组织工程研究

BACKGROUND: One of important mechanisms underlying myocardial fibrosis is that transforming growth factor β1(TGF-β1) stimulates the proliferation and differentiation of cardiac fibroblasts via Smads signaling pathway.Previous studies have confirmed that tanshinone ⅡA can effectively inhibit myocardial fibrosis.But whether blockage of TGF-β1/Smads signaling pathway is involved in this process remains unclear. OBJECTIVE: To investigate the effects of tanshinone ⅡA on TGF-β1 signal transduction in rat cardiac fibroblasts. METHODS: Neonatal rat cardiac fibroblasts were harvested by trypsin digestion and differential attachment and treated with 5 μg/L TGF-βI and different concentrations of tanshinone Ⅱ A(106,10-5 and 10-4 mol/L).At 6,12,and 24 hours after TGF-β1 application,fibronectin expression was detected by reverse transcription-polymerase chain reaction and Western blot analysis.At 15,30,60,and 120 minutes after TGF-β1 application,Smads protein expression was determined by Western blot analysis. RESULTS AND CONCLUSION: Fibronectin mRNA and protein expression began to increase at 6 hours after TGF-β1 application and was 1.3 and 1.8 times higher than initial level,respectively(P < 0.01),at 24 hours after TGF-β1 application.Phosphorylated Smad2/3 protein expression began to increase at 15 minutes after TGF-β1 application,peaked at 1 hour,decreased at 2 hours,but it was still 3.9 times higher than initial level(P < 0.01).Tanshinone ⅡA(10-5 and 10-4 mol/L)pretreatment downregulated fibronectin and phosphorylated Smad2/3 expression(P < 0.05 or P < 0.01)in a dose-dependent manner.These findings demonstrate that TGF-β1 induced fibronectin protein and mRNA expression and Smad2/3 protein expression in a time-dependent manner.Tanshinone ⅡA against myocardial fibrosis was likely related to its inhibition of TGF-β1-induced Smad2/3 phosphorylation and blockage of TGF-β1/Smads signaling pathways within cardiac fibroblasts.

Sodium tanshinone IIA sulfonate depresses angiotensin II-induced cardiomyocyte hypertrophy through MEK/ERK pathway

Cardiomyocyte hypertrophy is a major cause of morbidity and mortality worldwide. The aim of this study is to determine the effects of sodium tanshinone IIA sulfonate (STS) on cardiomyocyte hypertrophy induced by angiotensin II (Ang II) in vivo and in vitro. In long-term treatment, adult Wistar rats were infused with Ang II for three weeks via osmotic mini-pumps and some of them were given intragastrically of STS. Left ventricle was isolated; the ratio of left ventricular weight to body weight and systolic blood pressure (SBP) were determined and heart morphometry was assessed after hematoxylin and eosin staining. Results indicated STS inhibited Ang II-induced increases in myocyte diameter and decreased the LVW/BW ratio independent of decreasing systolic blood pressure. In vitro, treatment of cultured cardiomyocytes with STS inhibited Ang II-induced increase in cell size, protein synthesis, ANP expression, activation of extracellular signal-regulated kinase (ERK) and ERK kinase (MEK). Then we reexamined the mechanism of STS-induced anti-hypertrophic effects. Results revealed MEK inhibitor U0126 (20 microM) markedly enhanced STS-induced depressions in [3H]leucine incorporation and ANP expression. In conclusion, MEK/ERK pathway plays a significant role in the anti-hypertrophic effects of STS.

Inhibitory effect of tanshinone on the activation of peripheral blood mononuclear cells in patients with essential hypertension / 中国组织工程研究

BACKGROUND: Preactivation of peripheral blood mononuclear cells (PBMCS) is one of the most important eerly events and facilitating factor for the formation of atherosclerosis. Tanshinone is a lipolytic component extracted from traditional Chinese medicine of denshen, it has definite anti-atherosclerotic effect.OBJECTTVE: To analyze whether PBMCS preactivation existed at early essential hypertension, and investigate the effects of tanshinone on inhibiting the PBMCS activation cultured in vitro by detecting the adhesion and excretory activities of PBMCS.DESTGN: A case-controlled analysis.SETTING: Department of Emergency and Research Room of Traditional Chinese Medicine, Tongji Hospital affiliated to Tongji Medical College, Huazhong University of Science and Technology.PARTTCTPANTS: Thirty patients with untreated essential hypertension or with withdrawal from antihypertensives for at least 2 weeks were selected from the Department of Cardiology, Tongji Hospital affiliated to Tongji Medical College,Huazhong University of Science and Technology from January 2003 to October 2004, including 16 males and 14 females, aged (44.6±7.4) years, body mass index of (26.2±4.5) kg/m2, average disease course of (38.5±16.9) months.Informed contents were obtained from all the subjects. Their hypertension was grade Ⅰ-Ⅱ according to the diagnostic standards for hypertension by WHO/ISH in 1999. Secondary hypertension, organic heart disease, hyperglyceridemia,diabetes mellitus, liver and kidney dysfunction, heart, brain, kidney, vessel and other target damaged induced by infection and other clinical conditions and hypertension were excluded by history, physical examination and assistant examination.Another 30 healthy physical examinees with normal blood pressure were enrolled as the normal control group. Human umbilical vein endothelial cells (Species Reserving Center of Wuhan University); Tanshinone injection (Yaan Sanjiu Pharmaceutical, Co., Ltd., batch number: 020724);METHODS: ① Venous blood samples (4.0-5.0 mL) were drawn from all the subjects, and mononuclear cells were separated by means of Ficoll-Hypaque density gradient centrifugation and plastic adhesion, then incubated at 37 ℃ for 40-60 minutes, and the adherent cells were the PBMCS. These cells (viability ＞ 95%, Trypan blue staining) had the characteristics of mononuclear cells (Wright staining). The newly separated adherent PBMCS were resuspended, and then inoculated to the 24-well plate (4×107 L-1). There were 3 wells for each sample: the first was for basic excretion, the second for angiotensin Ⅱ stimulation, and the third for tanshinone pretreatment. The PBMCS were co-incubated with tanshinone for 30 minutes before angiotensin Ⅱ stimulation. The terminal concentration was 1×10-8 mol/L and 1×10-8 g/L for angiotensin Ⅱ andtanshinone respectively, and that of PBMCS was 2×107 L-1. The cells were cultured in the incubator (CO2 of 0.05 in volume fraction) at 37 ℃ for 24 hours, then the supernatant and cell ingredients were collected respectively. ② The PBMCS suspension was preparedl, and the cellular density was adjusted to 2.5×109 L-1. The human umbilical vein endothelial cells were cultured on the 24-well plate with M199 medium containing fetal bovine serum (0.1 in volume fraction), and spread to monolayer after the cells entered the logarithm phase. Each well was added with PBMCS suspension (100 μL), incubated at 37 ℃ for 2 and 4 hours respectively. The unadherent cells were removed, and the adherent ones were counted after fixed with 20 g/L glutaral, 40 visual sights were counted for each well under high power microscope, and the average value was used. ③ The double-antibody sandwich enzyme-linked immunoabsorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR) were used to determine the concentrations of tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β) and interleukin-6 (IL-6) in supernatant of PBMCS, and the expressions of their mRNA.MATN OUTCOME MEASURES: ① Changes of PBMCS adhesion activity; ② Concentrations of cytokines and their mRNA expressions in supernatant of PBMCS.RESULTS: ① At 2 and 4 hours of inoculation, the numbers of PBMCS adhered to endothelial cells under basic conditions were similar between the hypertension group and normal control group (t =1.153-1.577, P ＞ 0.05); After angiotensin Ⅱ stimulation, the adherent cells were obviously more in the hypertension group than in the normal control group (t =3.842-4.536, P ＜ 0.01); The numbers of the adherent cells were decreased to the same levels after tanshinone pretreatment (t =0.855-1.702, P ＞ 0.05). ②Under basic state, the concentrations of TNF-α, IL-1β and IL-6 in supernatant were all lower in both groups (t =0.981-1.829, P ＞ 0.05); The concentrations of the cytokines after angiotensin Ⅱ stimulation were obviously higher in the hypertension group than in the normal control group (t = 2.442, 5.075, P ＜ 0.01,0.01, 0.05); The concentrations of the cytokines after tanshinone pretreatment were all decreased to different extent, and there were no significant differences (t =1.227-1.940, P ＞ 0.05). Similar changes were observed in the mRNA expressions of the cytokines in PBMCS in the two groups.CONCLUSTON: ① The number of PBMCS adhered to endothelial cells, the concentrations and mRNA expressions of the cytokines under basic state in patients with essential hypertension were at the levels of normal subjects, and they were significantly increased after angiotensin Ⅱ stimulation, suggesting that the PBMCS were at preactivation at early essential hypertension. ② Tanshinone could decrease the adhesion and excretory activities of PBMCS in patients with essential hypertension to the normal levels, it is proved that tanshinone can inhibit the further activation of the preactivated PBMCS, and can prevent the occurrence of atherosclerosis to some extent.

Protective effects of anisodamine on brain mitochondrial damage after complete cerebral ischemia and reperfusion in rabbits / 中国组织工程研究

BACKGROUND: An isodamine, a kind of alkaloid, is extracted from Anisodus tanguticus (Maxim.) Pascher and is also a good protective agent of cell. However, functional change of mitochondrion is the most sensitive index reflecting cell injury.OBJECTIVE: To study the effects of anisodamine on brain mitochondrial damage following global cerebral ischemia and reperfusion in domestic rabbits and explore its mechanism.DESIGN: Totally randomized controlled trials.SETTING: Emergency Department of Tongji Hospital, Tongji Medical College of Huazhong University of Science and Technology.MATERIALS: The experiment was carried out in the laboratory of Emergency Department, Tongji Hospital Affiliated to Tongji Medical College of Huazhong University of Science and Technology, from September to December in 2002. Thirty healthy domestic rabbits of either sex were used and randomized into sham-operation group, ischemia-reperfusion group and anisodamine group with 10 rabbits in each group.METHODS: The models of complete cerebral ischemia and reperfusion injury in rabbits were established by ligation of bilateral common carotids and vertebral arteries with systemic hypotension, ischemia lasting for 20 minutes followed by 2-hour reperfusion. Anisodamine group was injected with anisodamine at a dose of 10 mg/kg body mass via femoral vein one minute before reperfusion, and lasted for 2 hours at a dose of 5 mg/hour by micro-pump. Ischemia-reperfusion group was treated with normal saline of the same volume. Sham-operation group only underwent used to determine mitochondrial respiratory functions, including respiratory control rate (RCR), the ratio of adenosine diphosphate to oxygen nicotinamide adenine dinucleotide hydrogenated (NADH) oxidase, succinate oxidase and cytochrome C oxidase were measured by the oxygenmethod of Yagi.drial calcium (Ca2+) and malondiadhyde (MDA) in cortex.reperfusion group and anisodamine group, RCR, ADP/O, OPR levels were lower than those in sham-operation group [nicotinamide adenine dinucleotide chain: RCR: 2.34±0.18,3.58±0.29,4.07±0.38,P ＜ 0.05-0.01;ADP/O: 1.77±0.10,2.23±0.14,2.41±0.17,P ＜ 0.05-0.01; OPR: (5.27±0.78),(8.03±1.30), (9.63±1.50)μkat/g, P ＜ 0.05-0.01; flavin adenine dinucleotide chain: RCR: 1.47±0.23,2.53±0.28,2.84±0.36,P ＜ 0.05-0.01;ADP/O: 0.88±0.09,1.58±0.11,1.73±0.17 ,P ＜ 0.05-0.01; OPR: (6.05±1.13),(7.47±1.40), (8.62±1.60)μkat/g,P ＜ 0.05-0.01], and those were higher in chemia-reperfusion group and anisodamine group, the activities of respiratory chain oxidase of NADH, succinate and cytochrome C were lower than those in sham-operation group [NADH: (2.62±0.35), (4.55±0.48), (5.07±0.60)μkat/g;succinate: (1.48±0.17), (1.83±0.22), (2.10±0.28)μkat/g; cytochrome C:(5.03±1.12), (7.62±1.23), (9.00±1.53)μkat/g, P ＜ 0.05-0.01], and those were higher in anisodamine group than in ischemia-reperfusion group, the content of mitochondrial Ca2+ [(2.36±0.23), (1.39±0.17),(1.22±0.12) mg/g] and MDA [(36.38±10.42), (22.69±9.56), (19.74±7.26)μmol/g,(P ＜ 0.05-0.01 )] was higher than that in sham-operation group, and it was lower in anisodamine group than in ischemia-reperfusion group (P ＜ 0.01).CONCLUSION: Anisodamine can protect the brain against ischemiareperfusion injury at the level of mitochondria by antagonism of Ca2+, inhibition of lipid peroxidation, stabilization of mitochondrial membrane, alleviation of mitochondrial damage, and improvement of motochondrial respiratory functions and the activities of enzymes of respiratory chain.