Amount Of Lactate Dehydrogenase Research Articles

Comparative evaluation of the serum biochemical and lipid profile changes in spayed male and female rats

Background and aims: This study investigated the effects of gonadal removal in male and female rats on changes in serum biochemical parameters. Methods: Twenty-eight adult male and female rats were divided into four groups of 7 animals for a period of 9 weeks. The first and second groups of intact male and female rats, as well as the third and fourth groups of male and female rats, were gonadectomized, respectively. At the end of the ninth week of the study, the rats were anesthetized with chloroform, and the amount of glucose, some lipid parameters in serum, and the activity of a number of serum enzymes were measured after taking blood from the heart. Results: Alanine aminotransferase, and alkaline phosphatase (ALP) levels were higher in intact male rats than in intact female rats, respectively (P=0.03, P=0.015). The amounts of glucose and cholesterol of low-density lipoprotein (LDL) in intact female rats were higher than in the ovariectomized rats, respectively (P=0.07, P=0.039, P=0.03). The amount of lactate dehydrogenase (LDH) in spayed female rats demonstrated a significant increase compared to other groups (P=0.001). However, there was a significant decrease in calcium levels in gonadectomized rat groups in comparison to intact female rats (P=0.02). Finally, a significant increase was found in phosphorous levels in the intact male rats group compared to other groups (P=0.002). Conclusion: Decreased sex hormones in gonadectomized rats compared with intact rats could lead to increased serum levels of LDH, ALP, and LDL cholesterol that may result in the development of metabolic and atherogenesis syndromes and acute liver failure.

Pyroptosis induced by TLR9 ligand, ODN1826, requires ROS production and caspase-11 activation in Raw264.7 cells.

Toll-like receptor 9 (TLR9), located in the endosomal compartment, is known to play a role in inflammation by recognizing oligonucleotides that contain CpG motive (CpG-ODN). Signaling by TLR9 leads to the production of proinflammatory cytokines and can trigger cell death. This study aims to investigate the molecular mechanism of pyroptosis induced by ODN1826 in the mouse macrophage cell line (Raw264.7). The protein expression and the amount of lactate dehydrogenase (LDH) of ODN1826-treated cells were determined by immunoblotting and LDH assay, respectively. In addition, the level of cytokine production was observed by ELISA assay and the ROS production was determined by flow cytometry. Our results showed that ODN1826 induced pyroptosis as judged by LDH releases. Furthermore, caspase-11 and gasdermin D activation, which are the key molecules in pyroptosis, were also observed in ODN1826-activated cells. Moreover, we also demonstrated that Reactive Oxygen Species (ROS) production by ODN1826 is essential for caspase-11 activation and gasdermin D release, which leads to pyroptosis. ODN1826 induces pyroptosis in Raw264.7 cells via caspase-11 and GSDMD activation. Moreover, the production of ROS by this ligand plays an essential role in the regulation of caspase-11 and GSDMD activation, which then controls pyroptosis in TLR9 activation.

Hydrogen Peroxide Induced Toxicity Is Reversed by the Macrocyclic IRAP-Inhibitor HA08 in Primary Hippocampal Cell Cultures.

Angiotensin IV (Ang IV), a metabolite of Angiotensin II, is a bioactive hexapeptide that inhibits the insulin-regulated aminopeptidase (IRAP). This transmembrane zinc metallopeptidase with many biological functions has in recent years emerged as a new pharmacological target. IRAP is expressed in a variety of tissues and can be found in high density in the hippocampus and neocortex, brain regions associated with cognition. Ang IV is known to improve memory tasks in experimental animals. One of the most potent IRAP inhibitors known today is the macrocyclic compound HA08 that is significantly more stable than the endogenous Ang IV. HA08 combines structural elements from Ang IV and the physiological substrates oxytocin and vasopressin, and binds to the catalytic site of IRAP. In the present study we evaluate whether HA08 can restore cell viability in rat primary cells submitted to hydrogen peroxide damage. After damaging the cells with hydrogen peroxide and subsequently treating them with HA08, the conceivable restoring effects of the IRAP inhibitor were assessed. The cellular viability was determined by measuring mitochondrial activity and lactate dehydrogenase (LDH) release. The mitochondrial activity was significantly higher in primary hippocampal cells, whereas the amount of LDH was unaffected. We conclude that the cell viability can be restored in this cell type by blocking IRAP with the potent macrocyclic inhibitor HA08, although the mechanism by which HA08 exerts its effects remains unclear.

Impact of the inspiratory oxygen fraction on the cardiac output during jugulo-femoral venoarterial extracorporeal membrane oxygenation in the rat

BackgroundVenoarterial extracorporeal membrane oxygenation (V-A ECMO) with femoral access has gained wide acceptance in the treatment of critically ill patients. Since the patient´s cardiac output (CO) can compete with the retrograde aortic ECMO-flow, the aim of this study was to examine the impact of the inspiratory oxygen fraction on the cardiac function during V-A ECMO therapy.MethodsEighteen male Lewis rats (350–400 g) received V-A ECMO therapy. The inspiratory oxygen fraction on the ventilator was randomly set to 0.5 (group A), 0.21 (group B), or 0 in order to simulate apnea (group C), respectively. Each group consisted of six animals. Arterial blood pressure, central venous saturation (ScvO2), CO, stroke volume, left ventricular ejection fraction (LVEF), end diastolic volume, and pressure were measured. Cardiac injury was determined by analyzing the amount of lactate dehydrogenase (LDH).ResultsDuring anoxic ventilation the systolic, mean and diastolic arterial pressure, CO, stroke volume, LVEF and ScvO2 were significantly impaired compared to group A and B. The course of LDH values revealed no significant differences between the groups.ConclusionAnoxic ventilation during V-A ECMO with femoral cannulation leads to cardiogenic shock in rats. Therefore, awake V-A ECMO patients might be at risk for hypoxia-induced complications.

Mechanistic insight of cell anti-proliferative activity of fluoroquinolone drug-based Cu(II) complexes.

Pefloxacin-based mixed ligand Cu(II) complexes with substituted isatin of type [Cu(Isatin)(Pefloxacin)Cl] were synthesized, and characterized by EPR, mass, FT-IR, electronic spectrometry, metal content, magnetic moment, and conductance measurement. The g factors g [Formula: see text] > g [Formula: see text] > 2.0023 observed in EPR suggest a square-pyramidal environment of ligands around the copper metal. The compounds were screened for diverse biological activities. The compounds inhibit efficiently the cell proliferation of HCT 116 cancer cells. To take the insight of anticancer activity mechanism, we investigated compound-1 for further cellular assay-based biological activities like trypan blue assay, cell morphological alteration assay, colony formation assay, cell apoptosis, and cell necrosis assay. The compound-1 induced distinct morphological alteration in cells, inhibits cell viability, decreases % plating efficiency, and decreases the clonogenic ability of the HCT 116 cells. The cell death mechanism was confirmed by annexin V-FITC / PI assay and LDH release assay. The positive annexin V/PI stained cells in presence of compound-1 and the absence of a significant amount of lactate dehydrogenase suggest cell apoptosis mechanism for anticancer activity of compounds. We also screened compounds for in vitro antibacterial and cytotoxic activities. Synthesis, characterization, antibacterial, anticancer, and cytotoxicity activities of pefloxacin based Cu(II) complexes were studied. The compound -1 is more potent than standard anticancer drugs and it induced apoptosis to the HCT 116 cells.

Role of long non-coding RNA MEG3 in hyperglycose-induced neurocyte damage in rats: relationship with mitochondrion-dependent apoptosis

Objective To evaluate the role of long non-coding RNA maternally expressed gene 3 (MEG3) in hyperglycose-induced neurocyte damage and the relationship with mitochondrion-dependent apoptosis in rats. Methods Normally cultured PC12 cells were divided into 5 groups (n=18 each) using a random number table method: normal concentration of glucose control group (C group), normal concentration of glucose plus MEG3 group (C+ MEG3 group), high-concentration glucose group (HG group), high-concentration glucose plus MEG3 group(HG+ MEG3 group), and high-concentration glucose plus negative lentiviral vector (LV-NC) group (HG+ NC group). PC12 cells were cultured in DMEM medium with 25 mmol/L glucose in group C. PC12 cells were cultured in DMEM medium with 25 mmol/L glucose after being transfected with MEG3 lentiviral vector (LV-MEG3) in C+ MEG3 group.PC12 cells were cultured in DMEM medium with 250 mmol/L glucose in HG group.PC12 cells were incubated in DMEM medium containing 250 mmol/L glucose after being transfected with LV-MEG3 in HG+ MEG3 group.PC12 cells were incubated in DMEM medium containing 250 mmol/L glucose after being transfected with LV-NC in HG+ NC group.After the cells were cultured or incubated for 1 day, the cell viability was measured by CCK8 assay, the apoptosis rate and level of reactive oxygen species (ROS) were determined by flow cytometry, and the amount of lactic dehydrogenase (LDH) released was measured by DCFH-DA, the expression of Cyt c, caspase-3, caspase-9, Bcl-2, Bax and Apaf-1 was determined by Western blot, and the opening of mitochondrial permeability transition pore (mPTP) was determined by fluorescent method.Blc-2/Bax ratio was calculated. Results Compared with group C, the cell viability was significantly decreased, the amount of LDH released, ROS level and apoptosis rate were increased, the opening of mPTP was increased, and the expression of caspase-3, caspase-9, Cyt c, Bax, Bcl-2 and Apaf-1 was up-regulated in HG, HG+ MEG3 and HG+ NC groups, and Bcl-2/Bax ratio was increased in HG+ MEG3 group and decreased in HG and HG+ NC groups (P<0.05). Compared with HG group and HG+ NC group, the cell activity was significantly increased, the amount of LDH released, ROS level and apoptosis rate were decreased, the opening of mPTP was decreased, the expression of caspase-3, caspase-9, Cyt c, Bax, and Apaf-1 was down-regulated, the expression of Bcl-2 was up-regulated, and Bcl-2/Bax ratio was increased in HG+ MEG3 group (P<0.01). Conclusion MEG3 may be involved in the endogenous protective mechanism during hyperglycose-induced neurocyte damage by inhibiting mitochondrion-dependent apoptosis in rats. Key words: RNA, long noncoding; Diabetes mellitus; Neurons; Apoptosis

THE EFFECTS OF VITAMIN C ON GLYCIDAMIDE-INDUCED CELLULAR DAMAGE AND APOPTOSIS IN MOUSE LEYDIG CELLS

The aim of this study was to elucidate the role of vitamin C on glycidamide-induced cytotoxicity, oxidative damage and cell death in Leydig (TM3) cells. Leydig cells were exposed to glycidamide (1, 10, 100 and 1000 μM) and/or vitamin C (50 μM) for 24 h. After completion of the exposure time, cell viability, amount of lactate dehydrogenase enzyme, apoptosis-necrosis rates, levels of oxidative stress parameters such as hydroxyl radical, hydrogen peroxide and lipid peroxidation were determined in Leydig cells. The results showed that glycidamide administration decreased Leydig cell viability and increased cytotoxicity significantly at high concentration (1000 μM). In addition, glycidamide generated oxidative damage by significantly increasing the production of reactive oxygen species and lipid peroxidation. Exposure to glycidamide increased the formation of early apoptosis, apoptosis and necrosis in Leydig cells. Consequently, glycidamide has been shown to cause apoptosis due to lipid peroxidation and formation of reactive oxygen species in Leydig cells, and vitamin C has a therapeutic role against toxicity caused by glycidamide.

Relationship between SIRT1-ERK1/2 pathway and chikusetsu saponin IVa-induced reduction of isoflurane-elicited neurotoxicity in fetal rats: an in vitro experiment

Objective To evaluate the relationship between silent information regulator 1 (SIRT1)-extracellular-regulated kinase1/2 (ERK1/2) pathway and chikusetsu saponin IVa-induced reduction of isoflurane-elicited neurotoxicity in fetal rats in an in vitro experiment. Methods The hippocampal neurons isolated from rats at 16-18 days of gestation were primarily cultured for 7 days and divided into 3 groups (n = 6 each) using a random number table method: control group (Con group), isoflurane group (Iso group) and chikusetsu saponin IVa plus isoflurane group (ChIV+ Iso group). Hippocampal neurons were cultured routinely for 6 h in Con group.Hippocampal neurons were exposed to 1.8% isoflurane for 6 h in an incubator in Iso group.Chikusetsu saponin IVa 25 μg/ml was added to the culture medium, and hippocampal neurons were incubated for 6 h and then exposed to 1.8% isoflurane for 6 h in an incubator in ChIV+ Iso group.The supernatant was collected for determination of the amount of lactic dehydrogenase (LDH) released, neuronal viability (by CCK-8) and expression of SIRT1, ERK1/2 and phosphorylated ERK1/2 (p-ERK1/2) (by Western blot). Results Compared with Con group, the neuronal viability was significantly decreased, the amount of LDH released was increased, and the expression of SIRT1 and p-ERK1/2 was down-regulated in Iso group (P<0.05). Compared with Iso group, the neuronal viability was significantly increased, the amount of LDH released was decreased, and the expression of SIRT1 and p-ERK1/2 was up-regulated in ChIV+ Iso group (P<0.05). Conclusion The mechanism by which chikusetsu saponin IVa reduces isoflurane-elicited neurotoxicity is related to activating SIRT1-ERK1/2 pathway in fetal rats in an in vitro experiment. Key words: Isoflurane; Sirtuins; Extracellular signal-regulated MAP kinases; Drug toxicity; Neurons

Diethyl citrate and sodium citrate reduce the cytotoxic effects of nanosized hydroxyapatite crystals on mouse vascular smooth muscle cells.

ObjectiveThis study aimed to investigate the damage mechanism of nanosized hydroxyapatite (nano-HAp) on mouse aortic smooth muscle cells (MOVASs) and the injury-inhibiting effects of diethyl citrate (Et2Cit) and sodium citrate (Na3Cit) to develop new drugs that can simultaneously induce anticoagulation and inhibit vascular calcification.MethodsThe change in cell viability was evaluated using a cell proliferation assay kit, and the amount of lactate dehydrogenase (LDH) released was measured using an LDH kit. Intracellular reactive oxygen species (ROS) and mitochondrial damage were detected by DCFH-DA staining and JC-1 staining. Cell apoptosis and necrosis were detected by Annexin V staining. Intracellular calcium concentration and lysosomal integrity were measured using Fluo-4/AM and acridine orange, respectively.ResultsNano-HAp decreased cell viability and damaged the cell membrane, resulting in the release of a large amount of LDH. Nano-HAp entered the cells and damaged the mitochondria, and then induced cell apoptosis by producing a large amount of ROS. In addition, nano-HAp increased the intracellular Ca2+ concentration, leading to lysosomal rupture and cell necrosis. On addition of the anticoagulant Et2Cit or Na3Cit, cell viability and mitochondrial membrane potential increased, whereas the amount of LDH released, ROS, and apoptosis rate decreased. Et2 Cit and Na3Cit could also chelate with Ca+ to inhibit the intracellular Ca2+ elevations induced by nano-HAp, prevent lysosomal rupture, and reduce cell necrosis. High concentrations of Et2Cit and Na3Cit exhibited strong inhibitory effects. The inhibitory capacity of Na3Cit was stronger than that of Et2Cit at similar concentrations.ConclusionBoth Et2Cit and Na3Cit significantly reduced the cytotoxicity of nano-HAp on MOVASs and inhibited the apoptosis and necrosis induced by nano-HAp crystals. The chelating function of citrate resulted in both anticoagulation and binding to HAp. Et2Cit and Na3Cit may play a role as anticoagulants in reducing injury to the vascular wall caused by nano-HAp.

Role of PTEN in diabetes mellitus-induced reduction of hypoxic postconditioning-induced protection of cardiomyocytes: the relationship with GSK-3β-mediated mitochondrial apoptotic pathway

Objective To evaluate the role of phosphatase and tensin homologue deleted on chromosome 10(PTEN)in diabetes mellitus-induced reduction of hypoxic postconditioning(HPO)-induced protection of cardiomyocytes and the relationship with glycogen synthase kinase-3β(GSK-3β)-mediated mitochondrial apoptotic pathway. Methods H9c2 cells incubated in high-glucose(30 mmol/L)medium for 24 h were divided into 6 groups(n=5 each)using a random number table: normoxia group(group N), hypoxia-reoxygenation(H/R)group, group HPO, PTEN gene silencing normoxia group(group P-N), PTEN gene silencing H/R group(group P-H/R), and PTEN gene silencing HPO group(group P-HPO). H9c2 cells were exposed to 95% N2-5% CO2 for 4 h followed by 2 h reoxygenation with 90% O2-10% CO2.HPO was induced by 3 cycles of 5 min reoxygenation followed by 5 min hypoxia before reoxygenation.At the end of reoxygenation, the level of lactate dehydrogenase(LDH)in the supernatant was detected by enzyme-linked immunosorbent assay, the changes in mitochondrial membrane potential(MMP)were assessed by JC-1 fluorescence assay, the cell apoptosis was detected by AnnexinV-FITC/PI flow cytometry, and the expression of PTEN and phosphorylated GSK-3β(p-GSK-3β)was determined by Western blot.The JC-1 monomer/polymer ratio and apoptosis rate were calculated. Results Compared with group N, the amount of LDH released, JC-1 monomer/polymer ratio and apoptosis rate were significantly increased, and the expression of PTEN was up-regulated in H/R and HPO groups(P 0.05). Compared with group HPO, the amount of LDH released, JC-1 monomer/polymer ratio and apoptosis rate were significantly decreased, PTEN expression was down-regulated, and the expression of p-GSK-3β was up-regulated in group P-HPO(P 0.05). Compared with group H/R, the expression of PTEN was significantly down-regulated, and no significant changes were found in the other parameters mentioned above in group P-H/R(P>0.05). Conclusion PTEN is involved in diabetes mellitus-induced reduction of HPO-induced protection of cardiomyocytes, and the mechanism is associated with PTEN-induced activation of GSK-3β-modulated mitochondrial apoptotic pathway. Key words: Glycogen synthase kinase 3; Anoxia; Diabetes mellitus; Myocytes, cardiac; Reperfusion injury; Post-conditioning; PTEN

(−)-Epigallocatechin-3-gallate alleviates doxorubicin-induced cardiotoxicity in sarcoma 180 tumor-bearing mice

Aims(−)-Epigallocatechin-3-gallate (EGCG), a major green tea polyphenol compound, plays an important role in the prevention of cardiovascular disease and cancer. The present study aimed to investigate the effects of EGCG on doxorubicin (DOX)-induced cardiotoxicity in Sarcoma 180 (S180) tumor-bearing mice. Main methodsS180 tumor-bearing mice were established by subcutaneous inoculation of S180 cells attached to the axillary region. The extent of myocardial injury was accessed by the amount of lactate dehydrogenase (LDH) released in serum. Heart tissue was morphologically studied with transmission electron microscopy. Apoptosis, reactive oxygen species (ROS) generation, mitochondrial membrane potential (ΔѰm) as well as calcium concentration were measured by flow cytometric analysis. Expression levels of manganese superoxide dismutase (MnSOD) were analyzed by Western blot. Key findingsResults showed that the combination with EGCG and DOX significantly inhibited tumor growth and enhanced induction of apoptosis compared with DOX alone. Moreover, administration of EGCG could suppress DOX-induced cardiotoxicity as evidenced by alleviating LDH release and apoptosis in cardiomyocyte. EGCG-evoked cardioprotection was in association with the increase of ΔѰm and MnSOD expression. EGCG was also found to attenuate ROS generation and myocardial calcium overload in Sarcoma 180 tumor-bearing mice subjected to DOX. SignificanceEGCG alleviated DOX-induced cardiotoxicity possibly in part mediated by increasing of MnSOD and Ѱm, reducing myocardial calcium overload and subsequently attenuating the apoptosis and LDH release. Our findings suggest that co-administration of EGCG and DOX have potential as a feasible strategy to mitigate cardiotoxicity of DOX without compromising its chemotherapeutic value.

Relationship between TDAG8 and endogenous neuron-protective mechanism against oxygen-glucose deprivation and restoration-induced apoptosis in rat neurons

Objective To investigate the relationship between T-cell death-associated gene 8 (TDAG8) and endogenous neuron-protective mechanism against oxygen-glucose deprivation and restoration (OGD/R)-induced apoptosis in rat neurons. Methods The primary cortical neurons obtained from fetal rats were seeded in 6-well plates at a density of 1×105 cells/ml and divided into 5 groups using a random number table: control group (group C, n=24), group OGD/R (n=48), TDAG8 agonist BTB09089 group (group BTB, n=24), TDAG8-siRNA group (group siRNA, n=24), and blank vehicle group (group V, n=24). The medium was replaced with glucose- and serum-free Locke′s buffer, and the neurons were exposed to 95% N2-5% CO2 in an air-tight incubator at 37 ℃ for 60 min followed by routine culture to establish the model of OGD/R.In BTB, siRNA and V groups, 20 μmol/L TDAG8 agonist BTB09089, 200 pmol/L TDAG8-siRNA, and 6 μl/200 μl transfection reagent were added, respectively, at 24 h before oxygen-glucose restoration.At 6 h of oxygen-glucose restoration, the neuronal viability and amount of lactic dehydrogenase (LDH) released were measured, and the expression of TDAG8 and caspase-3 mRNA in neurons was detected by fluorescent quantitative real-time polymerase chain reaction.In group OGD/R, the expression of TDAG8 and caspase-3 was measured by Western blot at 0, 3, 6, 12 and 24 h of oxygen-glucose restoration.In C, OGD/R, BTB, siRNA and V groups, the expression of TDAG8, caspase-3 and p-Akt was detected at 6 h of oxygen-glucose restoration. Results In group OGD/R, the expression of TDAG8 was gradually up-regulated after oxygen-glucose restoration, and the expression of caspase-3 peaked at 6 h of oxygen-glucose restoration.Compared with group C, the neuronal viability was significantly decreased, the amount of LDH released was significantly increased, and the expression of TDAG8 and caspase-3 protein and mRNA and p-Akt was significantly up-regulated in OGD/R, V and siRNA groups (P 0.05). Conclusion TDAG8 is partially involved in the endogenous neuron-protective mechanism against OGD/R-induced apoptosis in rat neurons, which may be related to activation of Akt signaling pathway. Key words: Receptors, G-protein-coupled; Apoptosis; Neurons; Reperfusion injury

Newly Developed Neutralized pH Icodextrin Dialysis Fluid: Nonclinical Evaluation.

A two-compartment system (NICOPELIQ; NICO, Terumo Co., Tokyo, Japan) has recently been developed to neutralize icodextrin peritoneal dialysis fluid (PDF). In this study, a nonclinical evaluation of NICO was carried out to evaluate biocompatibility as well as water transport ability. Glucose degradation products (GDPs) in the icodextrin PDFs were analyzed via high-performance liquid chromatography (HPLC). The cell viability of human peritoneal mesothelial cells derived from peritoneal dialysis effluent (PDE-HPMCs) was evaluated as well as the amount of lactate dehydrogenase (LDH) released after exposure to different PDFs (NICO and EXTRANEAL [EX, Baxter Healthcare Corp., Chicago, IL, USA]) and neutralized pH glucose PDF MIDPELIQ 250 (M250, Terumo). The water transport ability of NICO, EX, and M250 was tested using dialysis tube membranes with various pore sizes: 1, 2, 6-8, and 12-16 kDa. Although cell viability decreased by 30% after 30 min exposure to NICO, it was maintained for 6 h while a significant decrease was observed after 6 h exposure to EX. However, following adjustment of the pH to the same pre-exposure pH value, there was no significant difference in cell viability within the same pH group despite a doubling of the difference in the total amount of GDPs (44.6 ± 8.6 µM in NICO and 91.9 ± 9.5 µM in EX, respectively). In contrast, a significant decrease in cell viability was observed when the pH decreased to less than pH 6. Levels of released LDH, a cytotoxic marker, were within 5% after a 6-h exposure of NICO to PDE-HPMCs. There was no significant difference in water transport ability represented as overall osmotic gradients between NICO and EX. In conclusion, neutralization of icodextrin PDF is beneficial for maintaining cell viability and minimizing LDH release while water transport ability is comparable to the conventional icodextrin PDF.

Effect of etomidate post-conditioning on mPTP in rat cortical neurons subjected to oxygen-glucose deprivation and restoration: the relationship with Robo receptors

Objective To evaluate the role of etomidate post-conditioning on mitochondrial permeability transition pore (mPTP) in the rat cortical neurons subjected to oxygen-glucose deprivation and restoration (OGD/R) and the relationship with Robo receptors. Methods The cortical neurons obtained from Sprague-Dawley rats (< 24 h after birth) were cultured in vitro and seeded in 6-well plates (2 ml/well). The neurons were divided into 4 groups (n=24 each) using a random number table: control group (group C), OGD/R group, etomidate post-conditioning group (group E), and etomidate post-conditioning + Robo receptor blocker group (group ER). The neurons were subjected to O2-glucose deprivation for 90 min followed by restoration of O2-glucose supply for 24 h. In E and ER groups, etomidate was added to the culture medium with the final concentration of 6 μmol/L immediately after onset of O2-glucose supply.In group ER, Robo blocker RoboN was added to the culture medium with the final concentration of 1 μg/ml at 6 h before O2-glucose deprivation.The neuronal apoptosis was detected using Hoechst/PI double staining, the viability of neurons was measured by MTT assay, and the amount of lactic dehydrogenase (LDH) released was measured using colorimetric method.The mitochondria were extracted, and mitochondrial permeability transition pore (mPTP) opening was detected. Results Compared with group C, the apoptosis rate, amount of LDH released, and mPTP opening were significantly increased, and the cell survival rate was decreased in OGD/R, E and ER groups (P<0.05). Compared with group OGD/R, the apoptosis rate, amount of LDH released, and mPTP opening were significantly decreased, and the cell survival rate was increased in group E, and the apoptosis and amount of LDH released were significantly decreased, and the cell survival rate was increased in group ER (P<0.05). Compared with group E, the apoptosis rate, amount of LDH released, and mPTP opening were significantly increased, and the cell survival rate was decreased in group ER (P<0.05). Conclusion Etomidate post-conditioning mitigates OGD/R-induced damage to the cortical neurons through activating Robo receptors and inhibiting mPTP opening in rats. Key words: Etomidate; Ischemic postconditioning; Cell hypoxia; Oxygen; Cerebral cortex; Neurons; Mitochondrial membrane transport proteins; Nerve tissue proteins; Receptors, immunologic

Apoptotic properties of polysaccharide isolated from fruiting bodies of medicinal mushroom Fomes fomentarius in human lung carcinoma cell line

Mushrooms are known to complement chemotherapy and radiation therapy by countering the side effects of cancer. Recently, there has been great interest in isolation of novel bioactive compounds from mushrooms due to their numerous health beneficial effects. Chemically water-extractable polysaccharide (MFKF-AP1β), with a molecular weight of 12kDa, was isolated from fruiting bodies of mushroom Fomes fomentarius. In this research, we investigated the anti-tumor effects of MFKF-AP1β on human lung carcinoma A549 cells. Results showed that MFKF-AP1β markedly inhibited A549 cell growth in a dose-dependent manner based on the amount of lactate dehydrogenase (LDH) released and morphological alterations. In addition, MFKF-AP1β induced cellular apoptosis by causing single-stranded DNA breakage, as evidenced by apoptosis assay. Furthermore, MFKF-AP1β (25–100μg/ml) significantly induced single-stranded DNA breakage in A549 cells, as shown by comet assay.Taken together, our results demonstrate that MFKF-AP1β has strong anti-tumor effects mediated through induction of apoptosis. Therefore, MFKF-AP1β could be useful in lung chemotherapy.

Antitumor activity of a polysaccharide from longan seed on lung cancer cell line A549 in vitro and in vivo.

In this study, a water-soluble longan seed polysaccharide (WLSP), with a molecular weight of 57 kDa, was isolated from longan seed. Gas chromatography (GC) analysis showed that WLSP was composed mainly of rhamnose (Rha), mannose (Man), arabinose (Ara), galactose (Gal), and glucose (Glc), with molar ratios of 2.4:1.5:2.3:5.6:6.5. The result in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that WLSP showed a dose-dependent antiproliferative effect on the proliferation of A549 human lung cancer cells, which is consistent with the amount of lactate dehydrogenase (LDH) release from A549 cells. Prompted by this antiproliferative effect, we further examined its antiproliferative mechanism and in vivo anticancer effect. Our results showed that WLSP had the ability to cause cell cycle arrest in G1 phase, activation of caspases 3 and 9, and cleavage of poly[ADP (ribose)] polymerase (PARP) in A549 cells. The result of this in vivo study showed that WLSP could suppress the growth of xenograft A549 tumors and induce apoptosis. Taken together, these results indicate that WLSP exert an anticancer effect in vitro and in vivo and may be useful for the prevention of lung tumorigenesis.

Autophagy protects type II alveolar epithelial cells from Mycobacterium tuberculosis infection

This study was designed to investigate the protective effect of the autophagy signaling pathway against Mycobacterium tuberculosis infection in type II alveolar epithelial cells. An in vitro M. tuberculosis system was established using human A549 cells. Infection-induced changes in the expression of the autophagic marker LC3 were assessed by reverse transcription-PCR and Western blotting. Morphological changes in autophagosomes were detected by transmission electron microscopy (TEM). The function of the autophagy signaling pathway during infection was assessed by measuring the level of cell death and the amount of lactate dehydrogenase (LDH) released in the presence or absence of the inhibitor 3-methyladenine (3-MA). In addition, effects on LDH release were assessed after the siRNA-mediated knockdown of the essential autophagosomal structural membrane protein Atg5. LC3 mRNA expression was significantly reduced in M.tuberculosis-infected A549 cells (16888.76±1576.34 vs. uninfected: 12744.29±1089.37; P<0.05). TEM revealed M.tuberculosis bacilli-containing compartments that were surrounded by double membranes characteristic of the autophagic process. M.tuberculosis-infected A549 cells released more LDH (1.45±0.12 vs. uninfected: 0.45±0.04; P<0.05). The inhibition of autophagy signaling significantly enhanced M.tuberculosis-induced necrosis (3-MA: 75±5% vs. untreated: 15±1%; P<0.05) and LDH release (3-MA: 2.50±0.24 vs. untreated: 0.45±0.04; Atg5 knockdown: 3.19±0.29 vs. untreated: 1.28±0.11; P<0.05). Our results indicate that autophagy signaling pathway prevents apoptosis in type II alveolar epithelial cells infected with M.tuberculosis and may represent a molecular target for promoting cell survival during infection by respiratory pathogens.

Effect of sufentanil on anoxia/reoxygenation-induced injury to H9c2 cells with high-glucose incubation

Objective To investigate the effect of sufentanil on anoxia/rexogenation (A/R)-induced injury to H9c2 cells incubated in high-glucose culture medium. Methods The H9c2 cells were cultured in low-glucose DMEM/F12 culture medium supplemented with 10% fetal bovine serum. The cells were seeded in 96-well or 6-well plates and randomly divided into 5 groups ( n = 12 wells each): normal glucose control group (group NC),high-glucose control group (group HC), high-glucose + A/R group (group HA/R), high-glucose + sufentanil +A/R group (group HSA/R), high glucose + naloxone + A/R group (group HNA/R). The cells were exposed to 95 % N2-5 % CO2 in an incubator at 37 ℃ for 3 h followed by 3 h reoxygenation. In group NC, the cells were incubated in low-glucose culture medium for 48 h. In group HC, the cells were incubated in high-glucose culture medium for 48 h. In group HA/R, the cells were incubated in high-glucose culture medium for 48 h before anoxia.In group HSA/R, after the cells were incubated in high-glucose culture medium for48 h, sufentanil was added to the culture medium with the final concentration of 10-9 mol/L at 15 min before anoxia. In group HNA/R, after the cells were incubated inhigh-glucose culture medium for 48 h, naloxone was added to the culture medium with the final concentration of 10-6 mol/L, 10 min later sufentanil was added with the final concentration of 10-9 mol/L at 15 min before anoxia. The cell viability was measured by MTT assay. The amount of lactic dehydrogenase (LDH) released in the supernatant and superoxide dismutase (SOD) activity were measured. Results The cell viability and SOD activity were significantly lower, while the amount of LDH released was significantly higher in the other groups than in group NC, in groups HA/R and HNA/R than in group HC, and in group HNA/R than in group HSA/R (P ＜ 0.01 ). The cell viability and SOD activity were significantly higher, while the amount of LDH released was significantly lower in group HSA/R than in group HA/R ( P ＜ 0.01 ). There was no significant difference in the parameters mentioned above between group HNA/R and group HA/R ( P ＞ 0.05). Conclusion Sufentanil can attenuate A/R-induced injury to H9c2 cells with high-glucose incubation, and the mechanism may be related to the activation of opioid receptors. Key words: Sufentanil; Diabetes complications; Cell hypoxia

The experimental probe into the construction of oxygen glucose deprivation/reoxygenation model of hippocampal neurons of rat in vitro

Objective To establish the oxygen glucose deprivation (OGD)/reoxygenation experimental model of hippocatnpal neurons of rat in vitro, and to try to identify the length of time for producing optimum injury in this model. Method The primary hippocampal neurons of newborn Sprague-Dawley rats were cultured for 7 days and randomly (random number) divided into a control group and OGD groups. The OGD groups were assigned into 1 h, 2 h, 4 h, 6h, 8 h and 10 h subgroups in accordance with different lengths of time for oxygen glucose deprivation. The neurons of OGD groups were placed into a tri-gas incubator containing 0.5% oxygen and the culture medium was substituted with the glucose-free Earle' s balanced salt solution, simulating cerebral ischemia injury in vivo. The morphology of neurons was observed after reoxygenation for 24 hours. The MIT assay was used to determine the rate of survived cells derived from the value of optical density (OD) of cells. The lactate dehydro-genase (LDH) content in culture medium was detected to evaluate the neuron injury. The apoptotic rate of neurons was measured by using flow cytometry. Dunnett-test and Spearman correlation analysis were used to analyze the data with SPSS version 16.0 soft ware package. Results The morphological damage of neurons in OGD groups aggravated gradually, optical density and cell survival rate decreased (rs= -0.961 and rs = -0.966, P <0.01), and the amount of LDH increased (rs = 0.990, P <0.01) with longer duration of exposure to oxygen glucose deprivation, and the rate of neuron apoptosis increased obviously which was significantly statistical difference in com-parison with the control group (P < 0.05). Under the setting of oxygen glucose deprivation for 6 hours, the apop-tosis rate of neurons approximated to 50% . Conclusions The oxygen glucose deprivation/reoxygenation model of rat's hippocampal neurons in vitro was established successfully. From the findings of morphological changes and apoptosis rate of neurons, the oxygen glucose deprivation for 6 hours may be the suitable length of time for inducing neuron injury in this model. Key words: Hippocampal neurons; Oxygen glucose deprivation/reoxygenation; Apoptosis; Model

Morphine preconditioning reduces lipopolysaccharide and interferon-γ-induced mouse microglial cell injury via δ1 opioid receptor activation

Microglial cells play an important role in the inflammatory response of a broad range of brain diseases including stroke, brain infection and neurodegenerative diseases. However, there is very little information regarding how to protect microglial cells. Here, we showed that incubation of the C8-B4 mouse microglial cells with lipopolysaccharide (LPS) plus interferon-γ (IFNγ) induced cytotoxicity as assessed by the amount of lactate dehydrogenase (LDH) released from the cells. Preconditioning the cells with morphine for 30 min concentration-dependently reduced LPS plus IFNγ-induced cell injury. This morphine preconditioning effect was abolished by naloxone, a general opioid receptor antagonist, by naltrindole, a selective δ opioid receptor antagonist and by 7-benzylidenenaltrexone maleate, a selective δ1 opioid receptor antagonist. However, this protective effect was not affected by β-funaltrexamine, a selective μ opioid receptor antagonist, nor-binaltorphimine, a selective κ opioid receptor antagonist or naltriben, a selective δ2 opioid receptor antagonist. LPS plus IFNγ induced the expression of inducible nitric oxide synthase (iNOS), which was not affected by morphine preconditioning. Our results suggest that morphine induced a preconditioning effect in microglial cells. This effect may be mediated by δ1 opioid receptors and may not be through inhibiting the expression of iNOS, a potentially harmful protein.