The protective effect of aspirin during exposure to heat stress in broiler chickens was investigated. We assayed pathological damage, expression and distribution of Hsp90 protein and hsp90 mRNA expression in chicken heart tissues after oral administration of aspirin following exposure to high temperature for varying times. Heat stress induced increases in plasma aspartate aminotransferase, creatine kinase and lactate dehydrogenase activities while causing severe heart damage, which was characterized by granular and vacuolar degeneration, nuclear shrinkage and even myocardium fragmentation in cardiac muscle fibers. After aspirin administration, myocardial cells showed fewer pathological lesions than broilers treated with heat alone. A high positive Hsp90 signal was always detected in the nuclei of myocardial cells from broilers treated with aspirin, while in myocardial cells treated with heat alone, Hsp90 in the nuclei decreased, as did that in the cytoplasm. Aspirin induced rapid and significant synthesis of Hsp90 before and at the initial phase of heat stress, and significant expression of hsp90 mRNA was stimulated throughout the experiment when compared with cells exposed to heat stress alone. Thus, specific pre-induction of Hsp90 in cardiovascular tissue was useful for resisting heat stress damage because it produced stable damage-related enzymes and fewer pathologic changes.
Animals ; Anti-Inflammatory Agents, Non-Steroidal/pharmacology ; Aspirin/*pharmacology ; Cell Nucleus/genetics ; Chickens ; Gene Expression Regulation/*drug effects ; HSP90 Heat-Shock Proteins/*genetics ; Hot Temperature ; Myocytes, Cardiac/*drug effects/enzymology/pathology ; Stress, Physiological/*drug effects

Recent studies have documented that Janus-activated kinase (JAK)-signal transducer and activator of transcription (STAT) pathway can modulate the apoptotic program in a myocardial ischemia/reperfusion (I/R) model. To date, however, limited studies have examined the role of JAK3 on myocardial I/R injury. Here, we investigated the potential effects of pharmacological JAK3 inhibition with JANEX-1 in a myocardial I/R model. Mice were subjected to 45 min of ischemia followed by varying periods of reperfusion. JANEX-1 was injected 1 h before ischemia by intraperitoneal injection. Treatment with JANEX-1 significantly decreased plasma creatine kinase and lactate dehydrogenase activities, reduced infarct size, reversed I/R-induced functional deterioration of the myocardium and reduced myocardial apoptosis. Histological analysis revealed an increase in neutrophil and macrophage infiltration within the infarcted area, which was markedly reduced by JANEX-1 treatment. In parallel, in in vitro studies where neutrophils and macrophages were treated with JANEX-1 or isolated from JAK3 knockout mice, there was an impairment in the migration potential toward interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1), respectively. Of note, however, JANEX-1 did not affect the expression of IL-8 and MCP-1 in the myocardium. The pharmacological inhibition of JAK3 might represent an effective approach to reduce inflammation-mediated apoptotic damage initiated by myocardial I/R injury.
Animals ; Apoptosis/drug effects ; Cell Movement/drug effects ; Chemokines/pharmacology ; Heart Function Tests/drug effects ; Inflammation/pathology ; Janus Kinase 3/*antagonists & inhibitors/metabolism ; Macrophages/drug effects/metabolism/pathology ; Male ; Mice ; Mice, Inbred C57BL ; Myocardial Reperfusion Injury/drug therapy/*enzymology/physiopathology/*prevention & control ; Myocardium/enzymology/pathology ; Myocytes, Cardiac/drug effects/metabolism/pathology ; Neutrophils/drug effects/metabolism/pathology ; Quinazolines/pharmacology/therapeutic use

OBJECTIVETo determine the effect of berberine (Ber) on norepinephrine (NE)-induced apoptosis in neonatal rat cardiomyocytes.

METHODSThe cultured neonatal rat cardiomyocytes were treated with NE in the presence or absence of Ber. The activity of lactate dehydrogenase (LDH) in the culture medium was examined, and apoptosis of cardiomyocytes was assessed by Hoechst 33258, isothiocyanate (FITC)-conjugated annexin-V, and propidine iodide (PI) staining. In addition, the activities of caspases-2 and-3 were measured by a fluorescent assay kit. The level of secreted tumor necrosis factor α (TNF-α) and production of intracellular reactive oxygen species (ROS) were also determined.

RESULTSNE at a concentration of 50 μ mol/L induced an obvious increase in the activity of LDH in the culture medium (P<0.05), which was inhibited by coincubation with 0.5, 1.0, or 2.0 μ mol/L Ber (P<0.05). Ber also significantly attenuated NE-induced apoptosis in a dose-dependent manner (P<0.01). Moreover, Ber at a dose of 2 μ mol/L markedly decreased the ROS and TNF-α productions (P <0.05) and inhibited the activation of caspases-2 and -3 in cardiomyocytes exposed to NE (P<0.05)h.

CONCLUSIONThe present study suggested that Ber could reduce NE-induced apoptosis in neonatal rat cardiomyocytes through inhibiting the ROS-TNF-α-caspase signaling pathway.

Animals ; Animals, Newborn ; Annexin A5 ; metabolism ; Apoptosis ; drug effects ; Berberine ; pharmacology ; Caspase 2 ; metabolism ; Caspase 3 ; metabolism ; Caspases ; metabolism ; Cell Nucleus ; drug effects ; metabolism ; Cell Shape ; drug effects ; DNA ; metabolism ; Enzyme Activation ; drug effects ; Flow Cytometry ; Fluorescein-5-isothiocyanate ; metabolism ; Immunohistochemistry ; L-Lactate Dehydrogenase ; metabolism ; Myocytes, Cardiac ; drug effects ; enzymology ; pathology ; Norepinephrine ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Reactive Oxygen Species ; metabolism ; Signal Transduction ; Tumor Necrosis Factor-alpha ; metabolism

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

OBJECTIVETo investigate the role of the TLR4-NFκB-TNFα inflammation pathway on: lipopolysaccharide (LPS)-induced neonatal rat cardiomyocyte injury and the possible protective effects of salvianolic acid B (Sal B).

METHODSWistar rat (1-2 days old) cardiomyocytes were isolated and cultured. Sal B 10(-5)mol/L, 10(-6)mol/L and 10(-7)mol/L were pre-treated for 6 h in the culture medium. LPS (1 μg/mL) was added to mol/the culture medium and kept for 6 h to induce inflammation injury. The concentration of lactate dehydrogenase (LDH) in the supernatant was detected by spectrophotometry. The concentrations of tumor necrosis factor α (TNFα) and heat shock protein 70 (HSP70) in the supernatant were detected by enzyme linked immunosorbent assay. The protein expressions of toll, such as receptor 4 (TLR4) and nuclear factor kappa B (NFκB) were detected by immunohistochemistry. The mRNA expressions of TLR4 and NFκB were detected by real-realtime reverse transcription polymerase chain reaction (RT-PCR).

RESULTS(1) The concentrations of LDH and: TNFα in the LPS control group were significantly higher than those in the control group (561.41±67.39 U/L and 77.94±15.08 pg/mL, versus 292.13±26.02 U/L and 25.39±16.53 pg/mL, respectively, P<0.01, P<0.05). Compared with the LPS control group, the concentrations of LDH and TNFα were significantly decreased in the Sal B 10(-5)mol/L pre-treated group (451.76±83.96 U/L and 34.00±10.38 pg/mL, respectively, P<0.05). (2) The TLR4 and NFκB protein expression area in the LPS control group were significantly higher than those in the control group (1712.41±410.12 μm(2) and 2378.15±175.29 μm(2), versus 418.62±24.42 μm(2) and 1721.74±202.87 μm(2), respectively, P<0.01). The TLR4 and NFκB protein expression internal optical density (IOD) values in the LPS control group were also significantly higher than those in the control group (3.06±0.33 and 7.20±1.04, versus 0.91±0.21 and 4.24±0.48, respectively, P<0.05 and P<0.01). Compared with the LPS control group, the TLR4 and NFκB protein expression areas were significantly decreased in the Sal B 10(-5)mol/L pre-treated group (1251.54±133.82 μm(2) and 1996.37±256.67 μm(2), respectively, P<0.05), the TLR4 and NFκB protein expression IOD values were also significantly decreased in the Sal B 10(-5)mol/L pre- mol/pretreated group (1.92±0.28 and 5.17±0.77, respectively, treated P<0.05). (3) The TLR4 and NFκB mRNA expressions (2(-ΔΔ)CT value) in the LPS control group were significantly higher than those in the control group (3.16±0.38 and 5.03±0.43 versus 1.04±0.19 and 1.08±0.21, respectively, P<0.01). Compared with the LPS control group, the TLR4 and NFκB mRNA expressions (2(-ΔΔ) -CT value) were significantly decreased in the Sal B 10(-5)mol/L pre- mol/pretreated group (1.34±0.22 and 1.74±0.26, respectively, treated P<0.05). The concentration of HSP70 did not show any 0.05).

CONCLUSIONSThe TLR4-NFκB-TNFα pathway was quickly activated: and was independent of HSP70 in the early phase of neonatal cardiomyocyte injury induced by LPS. The protective effects of Sal B may be through inhibiting the TLR4-NFκB-TNFα pathway and are dose-dependent.

Animals ; Animals, Newborn ; Benzofurans ; chemistry ; pharmacology ; Gene Expression Regulation ; drug effects ; HSP70 Heat-Shock Proteins ; metabolism ; L-Lactate Dehydrogenase ; metabolism ; Lipopolysaccharides ; pharmacology ; Myocytes, Cardiac ; drug effects ; metabolism ; pathology ; NF-kappa B ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Wistar ; Signal Transduction ; drug effects ; Subcellular Fractions ; drug effects ; enzymology ; Toll-Like Receptor 4 ; genetics ; metabolism ; Transcription, Genetic ; drug effects ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVETo investigate whether the administration of the ultra-filtration extract from Danggui Buxue Decoction (EDBD) was able to protect cardiomyocytes from oxidative injury of rats induced by hydrogen peroxide (H(2)O(2)) and its potential mechanism.

METHODSMyocardial cells from 1- to 2-day-old neonatal rats were cultured in Dulbecco's modified Eagle's medium low-glucose and Ham's F12 medium (1:1), and the cellular injury was induced by H(2)O(2). The ultra-filtration extract mixture from Angelica sinensis and Hedysarum polybotrys was given in three doses of 3.75, 7.5, and 15 mg/mL. Morphological changes of cardiomyocytes were observed by microscope. Survival rate of myocardial cells was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The cardiomyocyte damages were estimated by detecting lactate dehydrogenase (LDH) and creatine kinase (CK) releases in the medium, superoxide dismutase (SOD) activities, and intracellular malondialdehyde (MDA) and myeloperoxidase (MPO) contents. The levels of caspase-3 and heat shock protein 70 (hsp70) mRNA expression in cardiomyocytes were measured by reverse transcription polymerase chain reaction.

RESULTSThe EDBD could protect the cardiomyocytes from H(2)O(2) injury in a dosedependent manner (3.75, 7.50, and 15.00 mg/mL). The EDBD could significantly decrease LDH and CK leakages and intracellular MDA and MPO contents, increase SOD activity, up-regulate hsp70 expression, and down-regulate caspase-3 expression.

CONCLUSIONThe EDBD has protection on cardiomyocytes injured by H(2)O(2) through improving cell antioxidant ability, up-regulating hsp70 expression, and inhibiting caspase-3 activity.

Animals ; Animals, Newborn ; Caspase 3 ; genetics ; metabolism ; Cell Survival ; drug effects ; Creatine Kinase ; metabolism ; Cytoprotection ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Gene Expression Regulation ; drug effects ; HSP70 Heat-Shock Proteins ; genetics ; metabolism ; L-Lactate Dehydrogenase ; metabolism ; Malondialdehyde ; metabolism ; Mice ; Myocardium ; pathology ; ultrastructure ; Myocytes, Cardiac ; drug effects ; enzymology ; pathology ; Oxidative Stress ; drug effects ; Peroxidase ; metabolism ; Phytotherapy ; Plant Extracts ; pharmacology ; RNA, Messenger ; genetics ; metabolism ; Rats ; Reverse Transcriptase Polymerase Chain Reaction ; Superoxide Dismutase ; metabolism ; Ultrafiltration

OBJECTIVETo observe the effect of pioglitazone on hypoxia/reoxygenation injury and the expression of protein kinase C (PKC) in neonatal rat cardiomyocytes.

METHODSNeonatal Sprague-Dawley rat cardiomyocytes in primary culture were treated with pioglitazone or GW9662 for 24 h prior to hypoxia/reoxygenation injury. Cardiomyocyte apoptosis was evaluated with Hoechst33258 staining and the expression of PKC was detected using Western blotting.

RESULTSIn the early stage of hypoxia/reoxygenation injury, the apoptosis rates of the cardiomyocytes increased significantly from (0.20∓0.03)% of the control level to (12.22∓1.45)% (P<0.05). Pretreatment with pioglitazone significantly lowered the apoptosis rate of the cardiomyocytes with hypoxia/reoxygenation injury to (8.32∓0.89)%, and this effect was antagonized by GW9662, a specific blocker of peroxisome proliferators activated receptors γ (PPARγ). Pioglitazone did not cause increased expression of PKC in the cardiomyocytes.

CONCLUSIONPioglitazone can ameliorate neonatal rat cardiomyocyte injury induced by hypoxia/reoxygenation partially by activating PPARγ and does not increase the expression of PKC in the cells.

Animals ; Animals, Newborn ; Apoptosis ; drug effects ; Cell Hypoxia ; physiology ; Female ; Ischemic Preconditioning, Myocardial ; methods ; Male ; Myocardial Reperfusion Injury ; physiopathology ; prevention & control ; Myocytes, Cardiac ; enzymology ; pathology ; PPAR gamma ; metabolism ; Potassium Channels ; metabolism ; Primary Cell Culture ; Protein Kinase C ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Thiazolidinediones ; pharmacology

OBJECTIVETo explore the possible mechanism of lipopolysaccharide (LPS)-induced cardiomyocyte hypertrophy in rats.

METHODSNeonatal rat cardiomyocytes cultured in vitro were stimulated with 100 µg/L LPS for 1, 4 or 8 h and scanned by atomic force microscopy (AFM) for measurement of the two-dimensional area, three-dimensional surface area and volume of each cell. The total proteins and Na(+)-K(+)-ATPase activity in the cardiomyocytes were determined. The same measurements were also carried out in neonatal rat cardiomyocyte cultures stimulated by 0.5 µmol/L ouabain for 8 h and the total protein levels were measured.

RESULTSFollowing a 8-hour stimulation with LPS, the two-dimensional area, three-dimensional surface area and volume of the single cardiomyocyte became enlarged and the total cellular proteins increased significantly as compared with those in the normal control cells (P < 0.05). LPS treatment for 4 and 8 h resulted in significantly decreased activity of Na(+)-K(+)-ATPase in the cardiomyocytes (P < 0.05). In the cells treated with ouabain for 8 h, the two-dimensional area, three-dimensional surface area, volume of the single cardiomyocyte and the total cellular proteins increased significantly in comparison with the normal control group (P < 0.05).

CONCLUSIONLPS can result in cardiomyocyte hypertrophy in rats possibly in relation to lowered Na(+)-K(+)-ATPase activity in the cardiomyocytes after LPS exposure.

Animals ; Animals, Newborn ; Cell Enlargement ; drug effects ; Cells, Cultured ; Hypertrophy ; chemically induced ; Lipopolysaccharides ; Myocytes, Cardiac ; enzymology ; pathology ; Rats ; Rats, Wistar ; Sodium-Potassium-Exchanging ATPase ; metabolism

OBJECTIVETo test whether postconditioning could inhibit the expression of phospho-JNK (P-JNK) mitogen activated protein kinase (MAPK) and study its relation to apoptosis of cardiocyte.

METHODSSixty rats were randomly divided into six groups: sham, reperfusion injury (R/I), postconditioning (Post), SP600125 (I_JNK), anisomycin and postconditioning (Ani+Post) and anisomycin (Ani) groups. After acute myocardial infarction was induced in rats, placebo solution (DMSO), SP600125 (6 mg/kg) or anisomycin (2 mg/kg) was injected through jugular vein 5 min before reperfusion; 6 h later 3 rats of each group were executed and the hearts were separated to measure the signaling molecules (phospho-JNK, TNF alpha, Caspase-8, Bcl-2/Bax, cytochrome-c). Twenty-two hours later hemodynamic data were measured in the left rats, and then blood samples were taken to determine serum markers of cardiac damage, and hearts were separated to measure the infarction area and cardiocyte apoptosis.

RESULTPostconditioning improved +/-DP/DTmax of left ventricle, limited infarct area, relieved apoptosis and necrosis of cardiocytes, and inhibited the expression of P-JNK (1.12 +/-0.21 Compared with 1.90 +/-0.32, P<0.05). At the same time the levels of TNFalpha Caspase-8, Bax and Cyt-c were lower in Post group than those in R/I group, but Bcl-2 expression levels were higher. I_JNK group presented the similar protection effect of postconditioning [TUNEL index: (6.23 +/-2.43)% Compared with (18.22 +/-5.10)%, P<0.05; Infarct area: (23.44 +/-6.34)% Compared with (42.31 +/-8.21)%, P<0.05]. On the other hand, Ani+Post group partially lost cardioprotection effect [TUNEL index: (14.12 +/-2.00)% Compared with (18.22 +/-5.10)%,P>0.05; Infarct area: (35.27 +/-5.28)% Compared with (42.31+/-8.21)%,P>0.05], because of the activation of JNK MAPK.

CONCLUSIONPostconditioning can inhibit phosphorylation of JNK MAPK, which attenuates cardiocyte apoptosis by both extrinsic and mitochondria pathway.

Animals ; Apoptosis ; drug effects ; Ischemic Preconditioning, Myocardial ; JNK Mitogen-Activated Protein Kinases ; metabolism ; pharmacology ; Male ; Myocardial Infarction ; enzymology ; pathology ; therapy ; Myocardial Reperfusion Injury ; prevention & control ; Myocytes, Cardiac ; enzymology ; pathology ; Random Allocation ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the role of focal adhesion kinase (FAK) in cardiac hypertrophy induced by hypertension.

METHODSUsing immunofluorescent labeling, confocal microscopy and Western blot, the expression and subcellular location of FAK-pSer722 and FAK-pSer910 were determined in cardiac myocytes of the left ventricles from 2, 6, 12, and 18 month-old spontaneously hypertensive heart failure (SHHF) rats and age-matched Wistar-Kyoto (WKY) control rats, respectively.

RESULTSThere was no obvious difference in FAK-pSer722 and FAK-pSer910 expression between 2 month-old SHHF and WKY rats. In contrast with the control groups, the expression of FAK-pSer722 and FAK-pSer910 significantly increased in cardiac myocytes of the left ventricle, from 6, 12 and 18 month-old SHHF rats. Both FAK-pSer722 and FAK-pSer910 were translocated and acummulated in nuclei of cardiac myocytes from 6, 12, and 18 month-old SHHF rats.

CONCLUSIONPhosphorylation and translocation of serine 722 and serine 910 of phosphorylated FAK play an important role in the de-compensatory cardiac hypertrophy.

Animals ; Cardiomegaly ; enzymology ; metabolism ; Cell Nucleus ; enzymology ; metabolism ; Focal Adhesion Kinase 1 ; metabolism ; Focal Adhesion Protein-Tyrosine Kinases ; metabolism ; physiology ; Heart Failure ; Heart Ventricles ; pathology ; Hypertension ; enzymology ; Hypertrophy ; enzymology ; Myocytes, Cardiac ; enzymology ; pathology ; Phosphorylation ; Protein Transport ; drug effects ; physiology ; Rats ; Rats, Inbred SHR ; Rats, Inbred WKY ; Serine ; metabolism ; Signal Transduction ; drug effects ; physiology