OBJECTIVETo evaluate the in vitro anti-hypertrophic effect of total Glycosides of Ranunculus Japonius (TGRJ).

METHODSNeonatal rat cardiomyocytes were cultured and hypertrophy was induced by administrating isoproterenol (ISO, 10 µmol/L) or angiotensin 2 (Ang 2, 1 µmol/L) for 48 hours. In the treatment groups, cells were pretreated with TGRJ (0.3 g/L) for 30 minutes prior to hypertrophic stimuli. The anti-hypertrophic effects of TGRJ were examined by measuring cell size, total protein content, and protein synthesis. Intracellular free Ca(2+) concentration ([Ca(2+)]i) was evaluated using fluorescence dye Fura-2/AM. Sacroplasmic/endoplasmic reticulum Ca(2+) ATPase 2a (SERCA2a), atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), and beta-myosin heavy chain (β-MHC) protein expression levels were measured by Western blotting . SERCA2a activity was assayed by p-nitrophenal phosphate disodium salt hexahydrate method.

RESULTSIncreased cell size, total protein content, and protein synthesis following ISO or Ang 2 stimulation were significantly inhibited by pretreatment with TGRJ (all P<0.05). This anti-hypertrophic effect of TGRJ was confirmed by its suppressing effect on elevated expression of the three hypertrophic related genetic markers, ANP, BNP, and β-MHC. In addition, TGRJ inhibited ISO or Ang 2 induced up-regulation of [Ca(2+)]i under chronic but not acute conditions. And ISO or Ang 2 induced down-regulation of SERCA2a expression and activity was also effectively rectified by TGRJ pretreatment.

CONCLUSIONSThe results of present study suggested that TGRJ could prevent ISO or Ang 2 induced cardiac hypertrophy through improving chronic [Ca(2+)]i disorder, might via normalizing SERCA2a expression and activity.

Animals ; Animals, Newborn ; Calcium ; metabolism ; Cells, Cultured ; Glycosides ; analysis ; pharmacology ; Myocytes, Cardiac ; drug effects ; metabolism ; pathology ; Ranunculus ; chemistry ; Rats

AIMTo study the effect of taurine (Tau) on rabbit cardiomyocyte apoptosis during ischemia/reperfusion (I/R) injury.

METHODSRabbit heart I/R injury was induced by occluding the left anterior descending coronary artery for 45 min and reperfusion for 180 min. taurine (200 mg/kg) was intravenously injected 5 min before heart ischemia. Cardiomyocyte apoptosis was measured by using terminal deoxynucleotidyl transferase--mediated dUTP nick end labeling method (TUNEL), flow cytometry (FCM) and DNA agarose gel electrophoresis.

RESULTSDNA ladder pattern of DNA in myocardium was revealed by agarose gel electrophoresis in I/R group while was not found in Tau + I/R group. Apoptotic cardiomyocytes were sparse within ischemic myocardium at risk in Tau + I/ R group as compared with that in I/R group (TUNEL stain). Apoptosis rate in ischemic myocardium from I/R and Tau + I/R groups detected by flow cytometry was 17.66% +/- 1.54% and 4.86% +/- 1.23%, respectively. Fas and Bax protein expressions in ischemic myocardium of I/R group were higher than that in nonischemic myocardium group (P < 0.01), Bcl-2/Bax ratio in I/R group was lower than that in nonischemic myocardium (P < 0.01); while in Tau + I/R group, Fas and Bax protein expressions were lower than that in I/R group (P < 0.01), the Bcl-2/Bax ratio was higher than that in I/R group (P < 0.01).

CONCLUSIONTaurine reduced apoptosis of myocytes in I/R rabbit heart; its mechanism may involve Fas, Bax and Bcl-2 proteins expression.

Animals ; Apoptosis ; drug effects ; Apoptosis Regulatory Proteins ; metabolism ; Male ; Myocardial Ischemia ; metabolism ; pathology ; Myocytes, Cardiac ; cytology ; drug effects ; Rabbits ; Reperfusion Injury ; metabolism ; pathology ; Taurine ; pharmacology

OBJECTIVETo investigate the protective effect of reactive oxygen species (ROS) scavenger, N-acetyl-L-cysteine (NAC), against H9c2 cardiomyocytes from injuries induced by chemical hypoxia.

METHODSH9c2 cells were treated with cobalt chloride (CoCl2), a chemical hypoxia-mimetic agent, to establish the chemical hypoxia-induced cardiomyocyte injury model. NAC was added into the cell medium 60 min prior to CoCl2 exposure. The cell viability was evaluated using cell counter kit (CCK-8), and the intercellular ROS level was measured by 2', 7'- dichlorfluorescein-diacetate (DCFH-DA) staining and photofluorography. Mitochondrial membrane potential (MMP) of the cells was observed by Rhodamine123 (Rh123) staining and photofluorography, and the ratio of GSSG/ (GSSG+GSH) was calculated according to detection results of the GSSG kit.

RESULTSExposure of H9c2 cardiomyocytes to 600 micromol/L CoCl2 for 36 h resulted in significantly reduced cell viability. Pretreatment with NAC at the concentrations ranging from 500 to 2000 micromol/L 60 min before CoCl2 exposure dose-dependently inhibited CoCl2-induced H9c2 cell injuries, and obviously increased the cell viability. NAC at 2000 micromol/L obviously inhibited the oxidative stress induced by CoCl2, decreased the ratio of GSSG/(GSSG+GSH), increased ROS level, and antagonized CoCl2-induced inhibition on MMP.

CONCLUSIONNAC offers obvious protective effect on H9c2 cardiomyocytes against injuries induced by chemical hypoxia by decreasing in the ratio of GSSG/(GSSG+GSH) and ROS level and ameliorating MMP.

Animals ; Cell Hypoxia ; drug effects ; Cells, Cultured ; Embryo, Mammalian ; Free Radical Scavengers ; pharmacology ; Myocytes, Cardiac ; drug effects ; metabolism ; pathology ; Oxidative Stress ; drug effects ; Rats ; Reactive Oxygen Species ; metabolism

The function for cardiac vascular system of taurine is extensive, and the mechanism is complicated. Taurine protects the cells from the cell injury caused by ischemia etc. Through repressing apoptosis, prevents endothelial dysfunction caused by hyperglycemia, hypercholesterolemia, smoking and homocysteine; suppresses the proliferation and calcification in vascular smooth muscle cells, promotes metabolization and excretion of cholesterol in the animal models of hyperlipemia, and confers the resistance to an oxidant, hypochlorous acid, produced by neutrophil on cells, and taurine chrolamine to inhibit activation of NF-kappaB, which might be associated with anti-atherosclerotic effect. Taurine mainly acts inside the cell. However, taurine transport system becomes aberrant in pathological myocardial and vascular tissue. In addition, taurine improves cardiovascular function in fructose-induced hypertension and an iron-overload murine animal models.
Animals ; Antioxidants ; pharmacology ; Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; Humans ; Lipid Metabolism ; drug effects ; Materia Medica ; pharmacology ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Cardiac ; pathology ; Taurine ; pharmacology

We investigated the effect of phenylephrine (PE)- and isoproterenol (ISO)-induced cardiac hypertrophy on subcellular localization and expression of caveolin-3 and STAT3 in H9c2 cardiomyoblast cells. Caveolin-3 localization to plasma membrane was attenuated and localization of caveolin-3 to caveolae in the plasma membrane was 24.3% reduced by the catecholamine-induced hypertrophy. STAT3 and phospho-STAT3 were up-regulated but verapamil and cyclosporin A synergistically decreased the STAT3 and phospho-STAT3 levels in PE- and ISO-induced hypertrophic cells. Both expression and activation of STAT3 were increased in the nucleus by the hypertrophy. Immunofluorescence analysis revealed that the catecholamine-induced hypertrophy promoted nuclear localization of pY705-STAT3. Of interest, phosphorylation of pS727-STAT3 in mitochondria was significantly reduced by catecholamine-induced hypertrophy. In addition, mitochondrial complexes II and III were greatly down-regulated in the hypertrophic cells. Our data suggest that the alterations in nuclear and mitochondrial activation of STAT3 and caveolae localization of caveolin-3 are related to the development of the catecholamine-induced cardiac hypertrophy.
Animals ; Catecholamines/*pharmacology ; Caveolae/*metabolism ; Caveolin 3/*metabolism ; Cell Line ; Hypertrophy/metabolism ; Mitochondria/*metabolism ; Myocardium/cytology/*pathology ; Myocytes, Cardiac/cytology/*drug effects/metabolism ; Rats ; STAT3 Transcription Factor/*metabolism

OBJECTIVETo study the myocardial protection on septic rats by Tongguan Capsule (TC).

METHODSTotally 120 SD rats were used to prepare the septic rat model using cecal, ligation and puncture (CLP). After modeling they were divided into three groups, i.e., the sham-operation group, the model group, the treatment group (treated by TC), 40 in each group. Each group was further divided into 5 subgroups according to different time points, i.e., 12 h, 24 h, 48 h, 72 h, and 7 days after CLP, 8 in each subgroup. CLP was performed in rats of the model group and the treatment group. The abdominal cavity of rats in the sham-operation group was opened. The incision of the abdominal wall was then sutured after their cecum was slightly flipped without puncture. Normal saline (at the daily dose of 10 mL/kg) was given by gastrogavage to rats in the model group and the sham-operation group. TC (at the daily dose of 600 mg/kg) was given to rats in the treatment group by gastrogavage, once daily, for 7 successive days. The hemodynamic changes such as left ventricular systolic pressure (LVSP), left ventricular pressure maximal rate of fall (- dp/dtmax), left ventricular pressure maximal rate of rise ( +dp/dtmax), inflammatory factors [such as tumor necrosis factor-alpha (TNF-alpha) and, IL-6, troponin I (CTn I )] were observed in all groups at each time point. The morphological changes of myocardial cells were observed under light and electron microscope 3 days later.

RESULTSCompared with the sham-operation group, the LVSP and +dp/dtmax decreased, and -dp/dtmax, CTn I , TNF-alpha, and IL-6 increased 24 -72 h after surgery in the model group, showing statistical difference (P<0.01). Compared with the model group, the LVSP and +dp/dtmax increased, and -dp/dtmax, CTn I , TNF-alpha, and IL-6 decreased 24 -72 h after surgery in the treatment group, showing statistical difference (P<0.05, P<0.01). Images under light microscope showed granular degeneration of myocardial cells could be occasionally seen in the treatment group. The transverse striation of myocardial cells was quite clear. The coronary artery was dilated and congested, with mild inflammation of interstitium. Images under electron microscope showed that the nucleus of myocardial cells was complete with quite clear Z-line in the treatment group. The number of the mitochondria increased. The mitochondria was swollen, but with more clear structure of cristae and mild fibrosis of interstitium.

CONCLUSIONTC could improve the depression state of myocardial cells of septic rats, thus improving the prognosis of sepsis.

Animals ; Drugs, Chinese Herbal ; pharmacology ; Interleukin-6 ; metabolism ; Male ; Myocardium ; pathology ; Myocytes, Cardiac ; drug effects ; metabolism ; Rats ; Rats, Sprague-Dawley ; Sepsis ; drug therapy ; pathology

The aim of the present study was to investigate the effects of adiponectin (APN) on the expression of T-cadherin in cultured Sprague-Dawley (SD) rat cardiomyocytes injured by hypoxia/reoxygenation (H/R). Primary myocardial cells from neonatal rats were obtained by enzymatic digestion. The cells were divided into control group, H/R group and H/R+APN (3, 10, 20 and 30 μg/mL) groups. The H/R group was incubated in anoxic environment (anoxic solution saturated with high concentration N2) for 3 h, and then in the reoxygenation environment (the reoxygenation solution saturated with pure oxygen) for 1 h. The H/R+APN group was pretreated with different concentrations of APN for 24 h prior to the initiation of H/R. The content of lactate dehydrogenase (LDH) was measured by chemistry chromatometry. Cellular apoptosis was analyzed by flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). The expression of T-cadherin was detected by RT-PCR and Western blotting. The results showed that, compared with control group, the apoptotic rate and release of LDH were significantly increased in the H/R group, whereas the expressions of T-cad mRNA and protein were decreased. Pretreating with APN significantly and dose-dependently decreased apoptotic rate and LDH release, and up-regulated T-cad mRNA and protein level in rat neonatal cardiomyocytes under H/R conditions. These results suggest that APN may protect cardiomyocytes against H/R-induced injury by up-regulating H/R-decreased T-cad expression.
Adiponectin ; pharmacology ; Animals ; Apoptosis ; Cadherins ; metabolism ; Cell Hypoxia ; L-Lactate Dehydrogenase ; metabolism ; Myocytes, Cardiac ; drug effects ; metabolism ; pathology ; Oxygen ; adverse effects ; Rats ; Rats, Sprague-Dawley ; Up-Regulation

OBJECTIVETo investigate whether calcineurin (CaN) contribute to tumor necrosis factor alpha (TNF-alpha)-induced cardiomyocyte hypertrophy.

METHODSThe protein content was assayed with lowry's method. The cardiomyocytes volumes were measured by computer photograph analysis system. The protein synthesis was assayed with [3H]-leucine incorporation method. [Ca2+]i transient was measured by Till image system by cell-loading Fura-2/AM. The expression of CaN was determined by Western blot.

RESULTS(1) (CsA (0.2 micromol/L), a selective CaN inhibitor, significantly suppressed the increase of protein content, [3H]-leucine incorporation and cell size induced by TNF-alpha. (2) CsA (0.2 micromol/L) significantly suppressed the elevation of the amplitude of the spontaneous Ca2+ transients induced by TNF-alpha in cultured ventricular myocytes from the neonatal rat. (3) TNF-alpha significantly increased the expression of CaN.

CONCLUSIONCa(2+) -CaN signaling pathway are involved in cardiomyocyte hypertrophy induced by TNF-alpha in rats.

Animals ; Calcineurin ; metabolism ; Calcium Signaling ; Cardiomyopathy, Dilated ; metabolism ; pathology ; Cells, Cultured ; Female ; Male ; Myocytes, Cardiac ; drug effects ; metabolism ; pathology ; Rats ; Rats, Sprague-Dawley ; Tumor Necrosis Factor-alpha ; pharmacology

AIMTo discuss the effects of restrained stress on cardiac myocyte apoptosis of rats in the whole body, and the effect of anti-stress-induced cardiac myocyte injury treated by Chinese herbs yixinning.

METHODSAgarose gel electrophoresis and TUNEL are used to detect cardiac myocyte apoptosis.

RESULTS(1) When restrained stress 1,2,4 week, the DNA ladder of stress groups was negative, while in situ apoptosis of stress groups increased apparently compared with control group (P < 0.01). (3) The DNA ladder of yixinning groups was negative too, while in situ apoptosis of yixinning groups decreased compared with stress group (P < 0.05). (3) Whereas in distilled water group, the above indices had no statistical significance compared with stress model group (P > 0.05).

CONCLUSIONThe restrained stress can induce cardiac myocyte apoptosis in rats. Chinese herbs "yixnning" can inhibit cardiac myocyte apoptosis, and have functions of anti-stress injury.

Animals ; Apoptosis ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Male ; Myocytes, Cardiac ; drug effects ; metabolism ; pathology ; Rats ; Rats, Wistar ; Restraint, Physical ; Stress, Physiological

OBJECTIVETo explore changes of mitochondrial structure and functions, as well as the protection of ligustrazine in the process of myocardial hypertrophy.

METHODSNeonatal myocardial cells were isolated and cultured with angiotensin II (Ang II) for 72 or 96 h. The total protein content was detected using BCA method. The cell diameter was measured by inverted microscope, by which to reflect the proliferation situation of cardiomyocytes. The mitochondrial membrane potential (MMP) was measured by fluorescence microscope. The mitochondrial monoamine oxidase (MAO) activity was detected by spectrophotometer. The mitochondrial cytochrome oxidase (COX) activity and the mitochondrial damage percentage were detected by microplate reader, by which to reflect the damage of mitochondrial outer membrane's structure and the membranes' function. Also, cells were treated with ligustrazine and losartan and then the pharmacological effects on the mitochondrial structure and functions in the myocardial cells treated with Ang II were observed.

RESULTSAt 72 h and 96 h, when compared with the blank group, cells treated with Ang II had increased total protein content (P < 0.01) and enlarged diameter (P < 0.01). Treated with Ang II, the MAO activity and the outer membrane damage percentage of myocardial cells significantly increased (P < 0.01), and mitochondrial COX activity and the mitochondrial MMP significantly decreased (P < 0.01). Compared with the model group at the same time period, ligustrazine significantly reduced myocardial cells' total protein content and myocardial cell diameter, and significantly decreased myocardial cells' MAO activity, increased mitochondrial COX activity, improved the outer membrane damage percentage and inner membrane MMP at 72 and 96 h, all showing statistical difference (P < 0.01, P < 0.05).

CONCLUSIONSDuring the process of myocardial hypertrophy existed the damage to the mitochondrial structure and functions. Ligustrazine protected the mitochondrial structure and functions of the myocardial cells in reversing Ang II induced myocardial cell hypertrophy.

Angiotensin II ; adverse effects ; Animals ; Cardiomyopathy, Hypertrophic ; chemically induced ; metabolism ; pathology ; Cells, Cultured ; Electron Transport Complex IV ; metabolism ; Mitochondria, Heart ; drug effects ; enzymology ; Monoamine Oxidase ; metabolism ; Myocytes, Cardiac ; drug effects ; metabolism ; pathology ; Pyrazines ; pharmacology ; Rats ; Rats, Sprague-Dawley