OBJECTIVE: To investigate whether CaN-NFAT3 pathway mediates the protective effects of aldehyde dehydrogenase (ALDH) 2 in high glucose-treated neonatal rat ventricular myocytes. METHODS: The ventricular myocytes were isolated from the heart of neonatal (within 3 days) SD rats by enzyme digestion and cultured in the presence of 5-Brdu. After reaching confluence, the cultured ventricular myocytes were identified using immunofluorescence assay for -SA protein. The cells were then cultured in either normal (5 mmol/L) or high glucose (30 mmol/L) medium in the presence of ALDH2 agonist Alda-1, ALDH 2 inhibitor Daidzin, or Alda-1 and NFAT3 inhibitor (11R-VIVIT). Fluorescent probe and ELISA were used to detect intracellular Ca concentration and CaN content, respectively; ALDH2, CaN and NFAT3 protein expressions in the cells were detected using Western blotting. RESULTS: Compared with cells cultured in normal glucose, the cells exposed to high glucose showed a significantly decreased expression of ALDH2 protein ( < 0.05) and increased expressions of CaN ( < 0.05) and NFAT3 proteins with also increased intracellular CaN and Ca concentrations ( < 0.01). Alda-1 treatment significantly lowered Ca concentration ( < 0.05), intracellular CaN content ( < 0.01), and CaN and NFAT3 protein expressions ( < 0.05), and increased ALDH2 protein expression ( < 0.05) in high glucose- exposed cells; Daidzin treatment significantly increased Ca concentration ( < 0.01) and intracellular CaN content ( < 0.05) in the exposed cells. Compared with Alda-1 alone, treatment of the high glucose-exposed cells with both Alda-1 and 11R-VIVIT did not produce significant changes in the expression of ALDH2 protein (>0.05) but significantly reduced the expression of NFAT3 protein ( < 0.05). CONCLUSIONS Mitochondrial ALDH2 protects neonatal rat cardiomyocytes against high glucose-induced injury possibly by negatively regulating Ca-CaN-NFAT3 signaling pathway.
Aldehyde Dehydrogenase, Mitochondrial ; antagonists & inhibitors ; metabolism ; Animals ; Animals, Newborn ; Benzamides ; pharmacology ; Benzodioxoles ; pharmacology ; Calcium ; metabolism ; Cells, Cultured ; Culture Media ; Enzyme Inhibitors ; pharmacology ; Glucose ; administration & dosage ; pharmacology ; Isoflavones ; pharmacology ; Mitochondria, Heart ; enzymology ; Myocytes, Cardiac ; drug effects ; metabolism ; NFATC Transcription Factors ; metabolism ; Nuclear Pore Complex Proteins ; metabolism ; Rats ; Rats, Sprague-Dawley

The aims of the present study were to evaluate the effects of puerarin on angiotensin II-induced cardiac fibroblast proliferation and to explore the molecular mechanisms of action. Considering the role of HO in nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation, we hypothesized that modulating catalase activity would be a potential target in regulating the redox-sensitive pathways. Our results showed that the activation of Rac1 was dependent on the levels of intracellular HO. Puerarin blocked the phosphorylation of extracellular regulated protein kinases (ERK)1/2, abolished activator protein (AP)-1 binding activity, and eventually attenuated cardiac fibroblast proliferation through the inhibition of HO-dependent Rac1 activation. Further studies revealed that angiotensin II treatment resulted in decreased catalase protein expression and enzyme activity, which was disrupted by puerarin via the upregulation of catalase protein expression at the transcriptional level and the prolonged protein degradation. These findings indicated that the anti-proliferation mechanism of puerarin was mainly through blocking angiontensin II-triggered downregulation of catalase expression and HO-dependent Rac1 activation.
Angiotensin II ; pharmacology ; Angiotensin II Type 1 Receptor Blockers ; pharmacology ; Animals ; Animals, Newborn ; Catalase ; genetics ; metabolism ; Cell Proliferation ; drug effects ; Cells, Cultured ; Extracellular Signal-Regulated MAP Kinases ; antagonists & inhibitors ; metabolism ; Fibroblasts ; Gene Expression Regulation ; drug effects ; Heart ; drug effects ; Hydrogen Peroxide ; metabolism ; pharmacology ; Isoflavones ; pharmacology ; Mice ; Myocardium ; cytology ; enzymology ; metabolism ; NADPH Oxidases ; antagonists & inhibitors ; metabolism ; Neuropeptides ; metabolism ; Signal Transduction ; drug effects ; Transcription Factor AP-1 ; antagonists & inhibitors ; metabolism ; Transcriptional Activation ; drug effects ; rac1 GTP-Binding Protein ; metabolism

OBJECTIVETo explore the effects of Panax Quinquefolium Saponin (PQS) on phosphatidylinositol 3-kinase/serine threonine kinase (PI3K/Akt) pathway of neonatal rat myocardial cells subjected to hypoxia.

METHODSNeonatal rat myocardial cells were cultured in vitro. After the myocardial cell injury was induced by hypoxia, the cells were randomized into 5 groups: the normal group, the model group, the positive control group (Ciclosporin A, 2 µ mol/L), the low-dose PQS group (PQSL, 25mg/L), and the high-dose PQS group (PQSH, 50 mg/L). Morphology and behavior of myocardial cells were observed under an inverted microscope. Apoptosis rate and lactate dehydrogenase (LDH) leakage rate of myocardial cells were determined by colorimetry. Mitochondrial transmembrane potential was assessed using a fluorexon laser. Phospho-glycogen synthase kinase (GSK)-3β and phospho-Akt as well as cytochrome C were determined by Western blot

RESULTSLDH leakage in the Ciclosporin A group, PQSH group and PQSL group reduced progressively compared with the model group (P<0.05). Akt and GSK-3β was strongly phosphorylated after treatment with Ciclosporin A and PQS compared with the model group (P<0.05, P<0.01). Compared with the model group (16.41±1.74; 35.28±6.30), both the integrated optical density of mitochondrial permeability transition pore (MPTP) and the mitochondrial transmembrane potential significantly increased in the PQSH group (42.74±2.12; 71.36±6.54) and the PQSL group (39.58±1.49; 66.99±5.45; P<0.05, P<0.01). However, the protein of cytochrome C outside the mitochondrion decreased in the PQSH group (273.66±14.61) and the PQSL group (259.62±17.31) compared with the model group (502.41±17.76; P<0.05).

CONCLUSIONThrough activation of the PI3K/Akt pathway and inhibition of the MPTP, PQS might protect the heart against ischemia injury and apoptosis of myocardial cells.

Animals ; Animals, Newborn ; Cell Hypoxia ; drug effects ; Cell Shape ; drug effects ; Cell Survival ; drug effects ; Cells, Cultured ; Glycogen Synthase Kinase 3 ; metabolism ; Glycogen Synthase Kinase 3 beta ; L-Lactate Dehydrogenase ; metabolism ; Membrane Potential, Mitochondrial ; drug effects ; Mitochondria ; drug effects ; metabolism ; Mitochondrial Membrane Transport Proteins ; metabolism ; Myocytes, Cardiac ; cytology ; drug effects ; enzymology ; Phosphatidylinositol 3-Kinases ; metabolism ; Phosphorylation ; drug effects ; Protein-Serine-Threonine Kinases ; metabolism ; Rats, Sprague-Dawley ; Saponins ; pharmacology ; Signal Transduction ; drug effects

OBJECTIVETo observe the long-term changes in anxiety-like behavior and tyrosine hydroxylase (TH) expression in the substantia nigra (SN) after hypoxic-ischemic brain damage (HIBD) in a neonatal rat model and to further explore the relationship between dopamine (DA) level and long-term anxiety-like behavior using the DA receptor (DAR) antagonist.

METHODSSeven-day-old (P7) neonatal Sprague-Dawley (SD) rats were randomized into normal control, sham-operated, HIBD and HIBD+DAR antagonist groups. HIBD model was prepared by ligating the right common carotid artery and 8% hypoxia exposure. The rats in the sham-operated group were sham-operated and were not subjected to right common carotid artery ligation and hypoxia exposure. The DAR antagonist was injected intraperitoneally before and after inducing HIBD. The same amount of normal saline was given to the other three groups as a control. Anxiety-like behavior was evaluated by elevated plus maze test, and TH expression in the SN was measured by immunohistochemistry on P14, P21, and P28.

RESULTSOn P21 and P28, the time spent in the open arms and the percentage of open arms entries in the HIBD group were significantly increased compared with those in the normal control, sham-operated and HIBD+DAR antagonist groups (P<0.05); in addition, the HIBD+DAR antagonist group showed a significantly longer time spent in the open arms than the normal control group (P<0.05). On P14, P21, and P28, TH expression in the HIBD and HIBD+DAR antagonist groups was significantly lower than that in the normal control and sham-operated groups, and TH level in the HIBD group was significantly lower than that in the HIBD+DAR antagonist group (P<0.05).

CONCLUSIONSDAR antagonist allows the restoration of anxiety-like behavior and alleviates the damage to dopaminergic neurons in SD rats after HIBD.

Animals ; Animals, Newborn ; Anxiety ; etiology ; prevention & control ; Dopamine Antagonists ; therapeutic use ; Hypoxia-Ischemia, Brain ; complications ; Maze Learning ; Rats ; Rats, Sprague-Dawley ; Receptors, Dopamine ; physiology ; Substantia Nigra ; enzymology ; Tyrosine 3-Monooxygenase ; analysis

OBJECTIVETo study the roles of type II 11β-hydroxysteroid dehydrogenase (11β-HSD2) and it's signaling factors in the lung tissue in pathogenesis of persistent pulmonary hypertension (PPH) in neonatal rats.

METHODSSix Sprague-Dawley rats on the 19th day of pregnancy were randomly divided into PPH and control groups (n=3 each). The PPH group was intraperitoneally injected with indomethacin (0.5 mg/kg) twice daily and exposed in 12% oxygen for three days, in order to prepare a fetal rat model of PPH. The control group was intraperitoneally injected with an equal volume of normal saline and exposed to air. Neonatal rats were born by caesarean section from both groups on the 22nd day of pregnancy. In each group, 15 neonatal rats were randomly selected and sacrificed. 11β-HSD2 expression in the lung tissue of neonatal rats were observed by Confocal laser technology, and serum cortisol levels and prostacyclin, renin, angiotensin and aldosterone in the lung tissue of both groups were measured using ELISA.

RESULTS11β-HSD2 protein was widely expressed in the lung tissue of the control and PPH groups. The levels of 11β-HSD2 and prostacyclin in the lung tissue were lower in the PPH group than in the control group, while serum cortisol levels and renin, angiotensin and aldosterone in the lung tissue were higher in the PPH group than in the control group (P<0.05).

CONCLUSIONS11β-HSD2 and it's signaling factors play roles in pathogenesis of PPH in neonatal rats.

11-beta-Hydroxysteroid Dehydrogenase Type 2 ; analysis ; physiology ; Animals ; Animals, Newborn ; Female ; Hypertension, Pulmonary ; enzymology ; etiology ; Male ; Rats ; Rats, Sprague-Dawley ; Signal Transduction

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 investigate the effects of Matrigel on expression of focal adhesion kinase and on proliferation and apoptosis of alveolar epithelial cell II of premature rat exposed to hyperoxia.

METHODSThe primary premature rat AECII (gestation 19 d) were cultured in vitro. For establishing hyperoxia-exposed cell model, purified AECII were cultured for 12 hours after culture flasks were filled with 95% oxygen-5% CO2 at 5 L/min, and then sealed for 12 hours. DNA content, phosphor and total protein of FAK were detected by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blotting respectively after 12 hours of air or hyperoxia exposure in the presence or absence of Matrigel. To investigate the relationship between FAK activated and proliferation or apoptosis of type II alveolar epithelial cells, levels of proliferation and apoptosis of AECII were measured by immunohistochemical assay of proliferating cell nuclear antigen (PCNA) and TUNEL method respectively.

RESULTSFAK and FAK-Tyr(397) activity of AECII on Matrigel-coated substrate increased: compared with air group, the expression of PCNA decreased and apoptotic index increased markedly in hyperoxia group (0.1498 ± 0.009 vs. 0.0953 ± 0.006, P < 0.05; 1.232 ± 0.6 vs. 13.40 ± 3.2, P < 0.01), but the expression of PCNA of AECII on Matrigel-coated substrate increased significantly (0.1498 ± 0.009 vs. 0.1921 ± 0.008, P < 0.01) and apoptotic index did not change. The expression of PCNA increased significantly (0.0953 ± 0.006 vs. 0.1125 ± 0.012, P < 0.05) and apoptotic index decreased markedly in hyperoxia + Matrigel group as compared with hyperoxia group (13.40 ± 3.2 vs. 7.641 ± 1.6, P < 0.05).

CONCLUSIONHyperoxia decreased the level of FAK and FAK-Tyr(397) in AECII, which may be a contributory mechanism of impaired proliferation and apoptosis of AECII in hyperoxia induced lung injury in premature rat. Matrigel could inhibit apoptosis and promote proliferation of AECII resulted from hyperoxia in vitro. Matrigel may play a protective role in hyperoxia-induced lung injury partly due to activated FAK.

Alveolar Epithelial Cells ; Animals ; Animals, Newborn ; Apoptosis ; Cell Proliferation ; Cells, Cultured ; Collagen ; pharmacology ; Drug Combinations ; Epithelial Cells ; drug effects ; enzymology ; Focal Adhesion Protein-Tyrosine Kinases ; metabolism ; Hyperoxia ; Laminin ; pharmacology ; Male ; Proteoglycans ; pharmacology ; Pulmonary Alveoli ; cytology ; enzymology ; pathology ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo study the expression of extracellular signal regulated protein kinase (ERK) 1/2 in lung tissues of newborn rats with chronic lung disease (CLD) caused by hyperoxia.

METHODSForty-eight full-term newborn rats were randomly divided into two groups: hyperoxia and control. The two groups were exposed to a hyperoxic gas mixture (0.90 O(2)) for an induction of CLD and room air within 12 hrs after birth, respectively. The levels of ERK1/2 protein and mRNA in lung tissues were measured using immunohistochemistry, Western blot and real-time PCR methods on postnatal days 3, 7 and 14. The severity of pulmonary fibrosis was evaluated.

RESULTSThe expression of p-ERK protein in lung tissues in the hyperoxia group was significantly higher than that in the control group on postnatal days 7 and 14 (P<0.01). There were no significant differences in the levels of total ERK1/2 protein and ERK1/2 mRNA.

CONCLUSIONSThe activation of phosphorated ERK1/2 may lead to lung fibrosis caused by hyperoxia in newborn rats.

Animals ; Animals, Newborn ; Chronic Disease ; Female ; Hyperoxia ; complications ; Lung ; enzymology ; pathology ; Lung Diseases ; etiology ; metabolism ; pathology ; Male ; Mitogen-Activated Protein Kinase 1 ; analysis ; Mitogen-Activated Protein Kinase 3 ; analysis ; Phosphorylation ; Pulmonary Fibrosis ; etiology ; Rats ; Rats, Wistar

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