Apoptotic effects of dipyrido [3,2-a:2′,3′-c] phenazine (dppz) Au(III) complex against diethylnitrosamine/phenobarbital induced experimental hepatocarcinogenesis in rats

We evaluated the effects of dipyrido [3,2-a:2′,3′-c] phenazine (dppz) Au(III) complex ([Au(dppz)Cl₂]Cl) on apoptosis during chemically induced hepatocellular carcinoma. 48 male Spraque-Dawley rats were divided into six groups; group I (control), group II [Dimethyl sulfoxide (DMSO)], group III ([Au(dppz)Cl₂]Cl), group IV [diethylnitrosamine + Phenobabital (DEN + PB)], group V (DEN + PB + [Au(dppz)Cl₂]Cl (2nd week)), and group VI (DEN + PB + [Au(dppz)Cl₂]Cl (7th week). The rats in groups IV through VI were administrated with DEN in a single dose of intraperitoneal 175 mg/kg. After 2 weeks of DEN administration, these groups of rats were given daily PB in a dose of 500 ppm. In group V, after two weeks of DEN administration, [Au(dppz)Cl₂]Cl complex (2 mg/kg) was given once a week by intraperitoneal injection. In the group VI, the rats were given a dose of 2 mg/kg [Au(dppz)Cl₂]Cl complex once a week, 7 weeks after DEN administration. At the end of the study, blood and tissue samples were collected from the rats to determine levels of serum AST, ALT, and LDH, and caspase 3, p53, Bax, Bcl-2 and DNA fragmentation in liver. AST, ALT, LDH, and Bcl-2 levels were higher in group IV, compared to group I, but caspase 3 and p53 levels were lower. In group V, caspase 3, p53, Bax, and DNA fragmentation levels were higher than those of group IV. Caspase 3 and p53 levels increased in group VI compared with group IV. In conclusion, [Au(dppz)Cl₂]Cl complex induced apoptosis by elevating levels of caspase 3, p53, Bax, and DNA fragmentation.     

Intrathecal Epigallocatechin Gallate Treatment Improves Functional Recovery After Spinal Cord Injury by Upregulating the Expression of BDNF and GDNF

This study aimed to investigate the therapeutic effects of epigallocatechin-3-gallate (EGCG) administered by subarachnoid injection following spinal cord injury (SCI) in rats and to explore the underlying mechanism. Sprague–Dawley rats were randomly divided into four groups of 12 as follows: a sham group (laminectomy only); a control group; a 10 mg/kg EGCG-treated group; and a 20 mg/kg EGCG-treated group. SCI was induced in the rats using the modified weight-drop method (10 g × 4 cm) at the T10 (10th thoracic vertebral) level. EGCG (10 or 20 mg/kg) or vehicle as control was administered by subarachnoid injection at lumbar level 4 immediately after SCI. Locomotor functional recovery was assessed during the four weeks post-operation using open-field locomotor tests and inclined-plane tests. At the end of the study, the segments of spinal cord encompassing the injury site were removed for histopathological analysis. Immunohistochemical and Western blot analyses were performed to observe the expression of: the B cell CLL/lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax), brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF). The results showed that the EGCG-treated animals had significantly better recovery of locomotor function, less myelin loss, greater Bcl-2 expression and attenuated Bax expression. In addition, the EGCG treatment significantly increased the expression of BDNF and GDNF after SCI. These findings suggest that EGCG treatment can significantly improve locomotor recovery, and this neuroprotective effect may be related to the up-regulation of BDNF and GDNF, and the inhibition of apoptosis-related proteins. Therefore, EGCG may be a promising therapeutic agent for SCI.     

The apoptosis of peripheral blood lymphocytes promoted by hyperbaric oxygen treatment contributes to attenuate the severity of early stage acute pancreatitis in rats

The aim of this study was to investigate the immunoregulatory effects of hyperbaric oxygen (HBO) via promoting the apoptosis of peripheral blood lymphocytes (PBLs) to attenuate the severity of early stage acute pancreatitis (AP) in rats. Additionally, the persistence of the HBO treatment effects was evaluated. One hundred and twenty male Wistar rats were randomized into four groups: sham, AP, AP + normobaric oxygen (NBO), and AP + HBO. Each group consisted of 30 rats. Four hours after the induction of AP, the 30 rats in the AP + NBO group were given normobaric oxygen treatment with 100 % oxygen at 1 atm for 90 min. The 30 rats in the AP + HBO group received 100 % oxygen at 2.5 atm for 90 min, with a compression/decompression time of 15 min. The 30 rats in the AP group remained untreated. At 6, 12, and 24 h after the induction of AP, surviving rats from each group were sacrificed, and the blood and tissue samples were collected for the following measurements: the partial pressure of oxygen (PaO₂) and oxygen saturation (SaO₂) of the arterial blood, the levels of serum amylase, lipase, interleukin-2 (IL-2), interferon-γ (IFN-γ), interleukin-10 (IL-10), hepatocyte growth factor (HGF), and reactive oxygen species (ROS), and the mitochondrial membrane potential (?Ψm) of the PBLs. The expression levels of procaspase-3, caspase-3, procaspase-9, and caspase-9 were also evaluated in the PBLs. Additionally, the apoptosis of PBLs was assessed, and the pancreatic tissues were subjected to a histopathological analysis by pathological grading and scoring. The histopathology of the lung, liver, kidney, duodenum, and heart was also analyzed at 12 h after the induction of AP. Significant differences were found at 6 and 12 h after AP induction. The HBO treatment significantly elevated the PaO₂ and SaO₂ levels, and the ROS levels in the PBLs. Additionally, HBO downregulated the levels of amylase and lipase. The HBO treatment also reduced the ?Ψm levels, upregulated the expression of caspase-3 and caspase-9, and increased the apoptosis rate of the PBLs. Moreover, the HBO treatment decreased the serum concentrations of IL-2, IFN-γ and HGF, and reduced the pathological scores of the pancreatic tissue. The histopathological changes of the lung, liver, kidney, duodenum, and heart were also improved. A significant elevation of IL-10 occurred only at the 12-h time point. However, no obvious differences were found at the 24-h time point. This study demonstrated that the HBO treatment can promote the apoptosis of PBLs via a mitochondrial-dependent pathway and inhibit the inflammatory response. These immunoregulatory effects may play an important therapeutic role in attenuating the severity of early stage AP. The repeated administration of HBO or the use of HBO in combination with other approaches may further improve outcomes.     

Inhibition of hydrogen sulfide synthesis provides protection for severe acute pancreatitis rats via apoptosis pathway

We aimed to investigate the relationship between the synthesis of hydrogen sulfide (H₂S) and the pancreatic acinar cell apoptosis in severe acute pancreatitis (SAP) rats, as well as analyse the potential apoptotic pathway involved in this process. Sixty rats had been equally divided into four groups: sham, SAP, SAP + sodium hydrosulfide (NaHS) and SAP + DL-propargylglycine (PAG). 24 h after SAP induction, all surviving animals of each group were sacrificed to collect blood and tissue samples for the following measurements: the level of serum H₂S as well as the levels of tumor necrosis factor-alpha (TNF-α), interleukin-10 (IL-10), H₂S synthesizing activity, CSE mRNA and protein expression, maleic dialdehyde (MDA) and myeloperoxidase (MPO) activity, the expression of Bax, Bcl-2, caspase-3, -8 and -9, the release of cytochrome c and the activation of nuclear factor-kappa B (NF-κB), ERK1/2, JNK1/2 and p38 in pancreas. Furthermore, in situ detection of cell apoptosis was examined and the severity of pancreatic damage was analyzed by pathological grading and scoring. Results Significant differences in every index except IL-10 had been found between the SAP, NaHS and PAG groups (P < 0.05). Treatment with PAG obviously induced the pancreatic acinar cell apoptosis as well as improved all the pathological changes and inflammatory parameters. In contrast, administration of NaHS significantly attenuated apoptosis in the pancreas and aggravated the severity of pancreatic damage. Moreover, the expressions of caspase-3, -8, -9 and the release of cytochrome c were all increased in the apoptotic cells, and the activity of NF-κB as well as the phosphorylation of ERK1/2, JNK1/2 and p38 decreased accompanying with the reduction of the serum H₂S level. H₂S plays a pivotal role in the regulation of pancreatic acinar cell apoptosis in SAP rats. The present results showed that inhibition of H₂S synthesis provided protection for SAP rats via inducing acinar cell apoptosis. This process acted through both extrinsic and intrinsic apoptotic pathways, and may be regulated by reducing the activity of NF-κB.     

Therapeutical Effects and Mechanism of Salubrinal Combined with Ulinastatin on Treating Paraquat Poisoning

To explore therapeutic effects and underlying mechanism of Salubrinal combined with Ulinastatin (UTI) on acute Paraquat (PQ) poisoning. Four hundred rats were randomly allocated into UTI group, SAL group, SAL + UTI and control group according to random number table with 100 rats in each group. Acute PQ poisoning models were established, and all rats received UTI, Salubrinal, SAL + UTI and normal saline injection, respectively. Afterward, we analyzed the change of lung tissue and explored the mechanism. Acute PQ poisoning caused significantly damage in rat lung tissue structure, and UTI could effectively repair lung tissue damage. Salubrinal suppressed hemorrhage and fibrosis, but promoted inflammatory infiltration. In contrast, UTI + Salubrinal suppressed hemorrhage, fibrosis and inflammatory infiltration, but could not improve lung tissue damage. Expression of LC3 and Bcl-2 showed statistically significant difference among different groups (p < 0.05). LC3 and Bcl-2 levels in UTI group were much higher than in the other groups, and LC3 and Bcl-2 levels in UTI + SAL group was second higher. LC expression in SAL group was lower than in UTI group and UTI + SAL group with Bcl-2 in control group significantly lower than in the other groups (p < 0.05). Expression of Caspase-3 and Bcl-2/Bax in lung tissue in different groups had statistically significant difference (p < 0.05). Caspase-3 in UTI group was lower than in the other groups; however, Bcl-2/Bax in UTI group was higher than in the other groups (p < 0.05). Acute PQ poisoning can cause endoplasmic reticulum stress–autophagy in rat, and UTI can increase Bcl-2 expression, decrease Caspase-3, which can inhibit progress of lung injury by suppressing apoptosis and exert good therapeutic effects. Although salubrinal has marked effects on protecting lung tissue, it can increase Bcl-2 expression, which is not beneficial to lung tissue protection. The underlying mechanism still needs further exploration.     

Effect of sevoflurane and halothane anesthesia on cognitive function and immune function in young rats

In the current study, we scrutinized the effect of sevoflurane and halothane on cognitive and immune function in young rats. The rats were divided into following groups: sevoflurane, halothane and sevoflurane + halothane groups, respectively. The rats were regularly treated with the pre-determined treatment. We also scrutinized the serum proinflammatory cytokines including IL-10, IL-4 and IL-2; brain level IL-1β; hippocampal neuronal apoptosis concentration were estimated. The water maze test was performed in rats for the estimation of cognitive ability. During the water maze test, on the 1st day the sevoflurane group showed the latency; sevoflurane and sevoflurane + halothane group demonstrated the declined latency gradually as compared to the control group rats after the 3 days. The latency of the control, halothane, sevoflurane + halothane group rats showed the reduced latency and also showed the reduced crossing circle times. The hippocampal neuron apoptosis was significantly increased in halothane and sevoflurane + halothane group as compared to control group rats, respectively. Control group rats demonstrated the increased neuron apoptosis. The proinflammatory cytokines including IL-10 and IL-4 was significantly higher in sevoflurane, halothane and sevoflurane + halothane group rats after anesthesia and the whole brain IL-1β was significantly decrease in the sevoflurane, halothane and sevoflurane + halothane as compared to control group. Sevoflurane can inhibit the anesthesia effect of halothane on the immune and cognitive function of rats.     

Effect of melatonin on torsion and reperfusion induced pathogenesis of rat uterus

ABSTRACT

We investigated the use of melatonin to improve fertility and reduce uterine damage caused by torsion of the uterus in pregnant rats. We used 35 pregnant rats at gestational age 18 days. The animals were randomized into five groups. Group 1 was anesthetized only. Group 2 was subjected to experimental uterine torsion of 360° and the torsion was corrected after 6 h. Group 3 was subjected to uterine torsion of 360°, the torsion was corrected after 6 h and melatonin was administered at the time of correction. Group 4 rats were subjected to 360º uterine torsion and melatonin was administered 6 h later at the time of correction. Group 5 was administered melatonin followed by uterine torsion of 360 degrees followed by correction of torsion 6 h later. Samples were obtained from the uterine horns on the day 1 postpartum. We used Bax, Bcl-2 and caspase 3 staining to measure apoptosis in the uterine tissues. The mRNA levels of Rho-associated, coiled-coil containing protein kinases 1 (ROCK1), homeobox D10 (Hox4 HoxD10), TLR4, NFκB1, caveolin 1 (Cav1) heat shock protein 90 alpha (cytosolic), class B member 1 (Hsp90ab1) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were determined using quantitative real-time polymerase chain reaction analysis (qRT-PCR). Bax, Bcl-2 and caspase 3 were detected using immunohistochemistry. No difference was observed among groups with respect to abortion, neonatal mortality or congenital abnormalities. Compared to the control group, the mRNA levels of Rock1, Hox4, TLR4, NFκB1, Cav1 and Hsp90 genes were decreased significantly in the study groups; the decrease was greater in groups 3 and 4, which were treated with melatonin. The greatest amount of Bax staining was found in group 1 and the least amount of Bcl-2 staining was found in groups 4 and 5; the greatest amount of caspase 3 staining was found in group 2. Our findings indicate that melatonin reduced uterine torsion related tissue damage and that its application during torsion was more effective than application following removal of torsion.     

Upregulation of Excitatory Amino Acid Transporters by Coexpression of Janus Kinase 3

Janus kinase 3 (JAK3) contributes to cytokine receptor signaling, confers cell survival and stimulates cell proliferation. The gain of function mutation JAK3^A572V is found in acute megakaryoplastic leukemia. Replacement of ATP coordinating lysine by alanine yields inactive JAK3^K855A. Most recent observations revealed the capacity of JAK3 to regulate ion transport. This study thus explored whether JAK3 regulates glutamate transporters EAAT1-4, carriers accomplishing transport of glutamate and aspartate in a variety of cells including intestinal cells, renal cells, glial cells, and neurons. To this end, EAAT1, 2, 3, or 4 were expressed in Xenopus oocytes with or without additional expression of mouse wild-type JAK3, constitutively active JAK3^A568V or inactive JAK3^K851A, and electrogenic glutamate transport was determined by dual electrode voltage clamp. Moreover, Ussing chamber was employed to determine electrogenic glutamate transport in intestine from mice lacking functional JAK3 (jak3 ^?/?) and from corresponding wild-type mice (jak3 ^+/+). As a result, in EAAT1, 2, 3, or 4 expressing oocytes, but not in oocytes injected with water, addition of glutamate to extracellular bath generated an inward current (I _g), which was significantly increased following coexpression of JAK3. I _g in oocytes expressing EAAT3 was further increased by JAK3^A568V but not by JAK3^K851A. I _g in EAAT3 + JAK3 expressing oocytes was significantly decreased by JAK3 inhibitor WHI-P154 (22 µM). Kinetic analysis revealed that JAK3 increased maximal I _g and significantly reduced the glutamate concentration required for half maximal I _g (K _m). Intestinal electrogenic glutamate transport was significantly lower in jak3 ^?/? than in jak3 ^+/+ mice. In conclusion, JAK3 is a powerful regulator of excitatory amino acid transporter isoforms.     

Down-Regulation of the Epithelial Na+ Channel ENaC by Janus kinase 2

Janus kinase-2 (JAK2), a signaling molecule mediating effects of various hormones including leptin and growth hormone, has previously been shown to modify the activity of several channels and carriers. Leptin is known to inhibit and growth hormone to stimulate epithelial Na⁺ transport, effects at least partially involving regulation of the epithelial Na⁺ channel ENaC. However, no published evidence is available regarding an influence of JAK2 on the activity of the epithelial Na⁺ channel ENaC. In order to test whether JAK2 participates in the regulation of ENaC, cRNA encoding ENaC was injected into Xenopus oocytes with or without additional injection of cRNA encoding wild type JAK2, gain-of-function ^V617FJAK2 or inactive ^K882EJAK2. Moreover, ENaC was expressed with or without the ENaC regulating ubiquitin ligase Nedd4-2 with or without JAK2, ^V617FJAK2 or ^K882EJAK2. ENaC was determined from amiloride (50 μM)-sensitive current (I _amil) in dual electrode voltage clamp. Moreover, I _amil was determined in colonic tissue utilizing Ussing chambers. As a result, the I _amil in ENaC-expressing oocytes was significantly decreased following coexpression of JAK2 or ^V617FJAK2, but not by coexpression of ^K882EJAK2. Coexpression of JAK2 and Nedd4-2 decreased I _amil in ENaC-expressing oocytes to a larger extent than coexpression of Nedd4-2 alone. Exposure of ENaC- and JAK2-expressing oocytes to JAK2 inhibitor AG490 (40 μM) significantly increased I _amil. In colonic epithelium, I _amil was significantly enhanced by AG490 pretreatment (40 μM, 1 h). In conclusion, JAK2 is a powerful inhibitor of ENaC.     

Ghrelin is a Regulator of Cellular Apoptosis and Proliferation in the Rat Ovary

Apoptosis and proliferation are the common and essential events of reproductive function and development in the ovary, especially during follicular growth and atresia or luteal regression. Therefore, this study was set to investigate the influence of ghrelin treatment on apoptosis and proliferation specific indices in the rat ovary. Twenty-eight adult female Wistar rats were randomly allocated into control and treatment groups. Treatment group (n = 14) received 3 nmol of ghrelin as subcutaneous injection for 14 consecutive days or vehicle (normal saline) to the control rats. The animals from each group were equally sacrificed on days 9 and 14 after onset of ghrelin treatment and their ovaries were taken for immunohistochemical evaluation and caspase-3 assay. Accumulation of apoptosis-associated peptide Bax was significantly reduced following ghrelin treatment particularly in granulosa and luteal cells on day 14 (P < 0.01). In contrast, immunoreactivity against anti-apoptotic protein Bcl-2 was significantly elevated in ghrelin-exposed animals in granulosa, theca and luteal cells (P < 0.05). However, ghrelin administration was not able to change caspase-3 activity prominently, so that the means of enzyme activity were not statistically significant between groups (P > 0.05). Moreover, significant up-regulation of proliferation-associated peptide PCNA was also seen in the granulosa, theca and luteal cells of ghrelin-treated rats by day 14 (P < 0.05), but not on day 9. These findings indicate the first evidence of ghrelin involvement in the control of key gonadal functions, apoptosis and proliferation in the rat ovary, which is mainly mediated through decrease in Bax/Bcl-2 ratio consistent with upstream of PCNA level, however not depends on the reduction of caspase-3 activation. This may have potential implications that ghrelin can be considered as an apoptotic modulator of some ovarian disorders.     

Possible Involvement of PTEN Signaling Pathway in the Anti-apoptotic Effect of Electroacupuncture Following Ischemic Stroke in Rats

As a traditional therapeutic method, electroacupuncture (EA) has been adopted as an alternative therapy for stroke recovery. Here, we aimed to evaluate whether EA therapy at points of Quchi (LI11) and Zusanli (ST36) alleviated neuronal apoptosis by PTEN signaling pathway after ischemic stroke. A total of 72 male Sprague–Dawley rats were randomized into three groups, including sham group, MCAO group, and EA group. EA was initiated after 24 h of reperfusion for 3 consecutive days. At 72 h following ischemia/reperfusion, neurological deficits, infarct volumes, and TUNEL staining were evaluated and the PTEN pathway-related proteins together with apoptosis-related proteins were detected. The results indicated that EA treatment significantly decreased cerebral infarct volume, neurological deficits and alleviated proportion of apoptotic cells in cerebral ischemic rats. Furthermore, EA significantly up-regulated the phosphorylation levels of PDK1, Akt(Thr308), GSK-3β, and down-regulated the phosphorylation levels of PTEN, Akt(Ser473) in the peri-infarct cortex. EA treatment significantly reduced the up-regulation of caspase-3, cleaved-caspase-3, Bim, and reversed the reduction of Bcl-2 induced by the ischemic stroke. These findings suggest that EA treatment at points of Quchi (LI11)- and Zusanli (ST36)-induced neuroprotection might involve inhibition of apoptosis via PTEN pathway.     

Kinetics of Apoptosis and Expression of Apoptosis-Related Proteins in Rat CA3 Hippocampus Cells After Experimental Diffuse Brain Injury

The present study examined kinetics of apoptosis and expression of apoptosis-related proteins Bcl-2, Bax, and caspase-3 in the CA3 hippocampus cells after diffuse brain injury (DBI) induced experimentally in rats. Percentage of apoptotic cells and expressions of above proteins were examined by flow cytometry and immunohistochemistry. Substantial neuronal apoptosis was documented in the CA3 hippocampus cells after DBI (22.26 ± 2.97 % at 72 h after DBI vs. 2.92 ± 0.88 % in sham-operated animals). Expression of Bc1-2 decreased, while expression of Bax and caspase-3 increased after DBI, with caspase-3 expression peaking after that of Bax (72 vs. 48 h, respectively). Further, the Bc1-2/Bax expression ratio decreased prior to increase of caspase-3 expression. In conclusion, cell apoptosis and altered expressions of Bcl-2, Bax, and caspase-3 are present in the CA3 region of hippocampus after experimental DBI. Changes in the Bc1-2/Bax expression ratio may facilitate activation of caspase-3 and aggravate neuronal apoptosis after brain injury.     

Neuroprotection of Interleukin-6 Against NMDA-induced Neurotoxicity is Mediated by JAK/STAT3, MAPK/ERK, and PI3K/AKT Signaling Pathways

We have previously shown that interleukin-6 (IL-6) has neuroprotective effect against N-methyl-d-aspartate (NMDA)-induced excitotoxicity. The current study aimed to reveal signal transduction pathways involved in the IL-6 neuroprotection. Cerebellar granule neurons (CGNs) from postnatal 8-day infant rats were exposed to IL-6 (120 ng/ml) for 8 days and stimulated with NMDA (100 μM) for 15 or 30 min. Dynamic intracellular Ca²⁺ fluorescence intensity, cytosolic Ca²⁺-dependent phospholipase A2 (cPLA2) expression, and apoptosis and necrosis in cultured CGNs were measured by laser scanning confocal microscope, real-time PCR and Western blot, and annexin V-FITC/propidium iodide staining, respectively. NMDA stimulation of neurons evoked an intracellular Ca²⁺ overload, an upregulated expression of cPLA2, and an increase in cell death. Chronic IL-6 exposure prevented the NMDA-evoked neuronal Ca²⁺ overload, cPLA2 expression upregulation, and apoptosis and necrosis. Anti-gp130 monoclonal antibody (mAb), a blocker of gp130 that is a 130-kDa signal-transducing β-subunit of IL-6 receptor complex, blocked these effects of IL-6 preventing NMDA neurotoxicity. AG490, PD98059, or LY294002, inhibitors specific for the intracellular signals, JAK, MAPK, and PI3K, respectively, partially blocked these IL-6 neuroprotective effects. Phosphorylation levels of STAT3, ERK1/2, and AKT, the downstream proteins for these enzymes of JAK, MAPK, and PI3K, respectively, were elevated by IL-6 pretreatment. The enhanced activation of STAT3, ERK1/2, and AKT by IL-6 was abolished by AG490, PD98059, and LY294002, respectively. Anti-gp130 mAb attenuated the activation of all the three detected signaling molecules. The present findings suggest that IL-6 neuroprotection is jointly mediated by the cellular signal transduction pathways, gp130-JAK-STAT3, gp130-MAPK-ERK, and gp130-PI3K-AKT.     

Antioxidative role of selenoprotein W in oxidant-induced chicken splenic lymphocyte death

To verify the antioxidative role of SelW in oxidant-induced chicken splenic lymphocyte, in this report, the influence of selenite supplementation and SelW gene silence on H₂O₂-mediated cell viability and cell apoptosis in cultured splenic lymphocyte derived from spleen of chicken were examined. The cultured cells were treated with sodium selenite and H₂O₂, or knocked down SelW with small interfering RNAs (siRNAs). The lymphocytes were examined for cell viability, cell apoptosis and mRNA expression levels of SelW and apoptosis-related genes (Bcl-2, Bax, Bak-1, caspase-3 and p53). The results show that the mRNA expression of SelW were effectively increased after treatment with sodium selenite, and H₂O₂-induced cell apoptosis was significantly decreased and cell viability was significantly increased. 20 μM H₂O₂ was found to induce cell apoptosis and decrease cell viability, which was alleviated obviously when cells were pretreated with sodium selenite before exposure to 20 μM H₂O₂. Meanwhile, H₂O₂ induced a significantly up-regulation of the Bax/Bcl-2 ratio, Bax, Bak-1, caspase-3 and p53 and down-regulation of Bcl-2 (P < 0.05). When lymphocytes were pretreated with Se before treated with H₂O₂, the Bax/Bcl-2 ratio and mRNA expression of those genes were significantly decreased, and Bcl-2 was increased (P < 0.05). SelW siRNA-transfected cells were more sensitive to the oxidative stress induced by treatment of H₂O₂ than control cells. Silencing of the lymphocyte SelW gene decreased their cell viability, and increased their apoptosis rate and susceptibility to H₂O₂. Silencing of SelW significantly up-regulated the Bax/Bcl-2 ratio, Bax, Bak-1, caspase-3 and p53 and down-regulated Bcl-2 (P < 0.05). The present study demonstrates that SelW plays an important role in protection of splenic lymphocyte of birds from oxidative stress.     

The changes of technetium-99m-labeled annexin-V in delayed anesthetic preconditioning during myocardial ischemia/reperfusion

This study was designed to use real-time imaging to test the hypothesis that delayed cardiac protection induced by volatile anesthetics inhibits apoptosis. Rats were divided into two groups. One group was exposed to 120 min of 33 % O₂ [control group (CON group)] and the other group was exposed to 2.5 % sevoflurane in 33 % O₂ for 120 min [sevoflurane group (SEVO group)]. Both groups were allowed to return to their cages for 24 h. After 24 h recovery, all rats underwent 30 min myocardial ischemia by occluding coronary artery followed by 2 h of reperfusion. After reperfusion, technetium-99m-labeled annexin-V was administered intravenously to identify apoptosis. Left ventricular samples were obtained to measure infarct size and radionuclide imaging and caspase-3. Radionuclide imaging indicated that apoptosis was reduced in SEVO group (0.78 % ± 0.82) when compared with the CON group (1.15 % ± 0.61), and the infarct size was also decreased in the SEVO group (40 % ± 7). The transferase dUTP nick end labeling (TUNEL)-positive cardiomyocytes in the SEVO group (16 % ± 6) were significantly decreased in the peri-infarct zone when compared with the CON group (28 % ± 4). After reperfusion, caspase-3 expression was significantly blunted in the SEVO group than in CON group (50 % ± 11 vs. 68 % ± 10, p < 0.05). This study used technetium-99m-labeled annexin-V of real-time imaging to detect cardiomyocyte apoptosis and the results were confirmed by the TUNEL assay and caspase-3 expression. We concluded that delayed volatile anesthetic preconditioning (APC) protects against I/R in vivo. The method of technetium-99m-labeled annexin-V of real-time imaging can be used to detect cardiomyocyte apoptosis in delayed APC during ischemia/reperfusion.     

CX43 change in LPS preconditioning against apoptosis of mesenchymal stem cells induced by hypoxia and serum deprivation is associated with ERK signaling pathway

This study was designed to investigate the effect and mechanism of lipopolysaccharide (LPS) preconditioning on survival and connexin 43 (CX43) expression in rat bone marrow mesenchymal stem cells (bMSCs) under hypoxia and serum deprivation (Hypoxia/SD) conditions. Whole marrow cells were obtained from the femora and tibiae of SD rats, and bMSCs were isolated by density gradient centrifugation and attachment culture. Surface antigens were determined by FACS before the experiment using antibodies conjugated directly against anti-rat CD34, anti-CD45, anti-CD29, and anti-CD44. Passage 3 bMSCs were used for all experiments. The effect of LPS preconditioning on bMSCs apoptosis in response to Hypoxia/SD was investigated by an Annexin V-FITC/PI binding assay and a mitochondrial membrane potential (△Ψ_m) assay. Cyc-c released into the cytosol from mitochondria and CX43 in bMSCs was determined by Western blot before and after LPS preconditioning. Subsequently, extracellular signal-regulated kinase (ERK) was inhibited with PD98059 to analyze the role of ERK in modulating CX43 expression after LPS preconditioning. The bMSCs surface antigen profiles obtained by flow cytometry were positive for CD29 and CD44 and negative for CD34 and CD45. The Hypoxia/SD conditions induced significant apoptosis of bMSCs. Compared with the Hypoxia/SD group, cells treated with LPS prevented △Ψ_m from falling significantly. LPS inhibited Hypoxia/SD-induced Cyc-c release. These results were consistent with the total analysis of apoptosis of MSCs. Compared with the control group, the level of CX43 expression in the Hypoxia/SD group and LPS + Hypoxia/SD group decreased significantly at each time point. The level of CX43 expression in the Hypoxia/SD group was lower than that in the LPS + Hypoxia/SD group, while the difference was not significant between the PD98059 + LPS + Hypoxia/SD group and the PD98059 + Hypoxia/SD group (P > 0.05). Compared with the LPS + Hypoxia/SD group, CX43 level in the PD98059 + LPS + Hypoxia/SD group and PD98059 + Hypoxia/SD group decreased significantly (P < 0.05). These results demonstrated that Hypoxia/SD conditions could induce apoptosis of bMSCs markedly. Low-dose LPS preconditioning may preserve the mitochondrial function by maintaining the mitochondrial transmembrane potential and inhibiting Cyc-c release in Hypoxia/SD-induced bMSCs apoptosis. LPS preconditioning also had a stabilizing effect on the cell membrane by inhibiting the decrease of CX43, and this modulating mechanism may be related to the ERK signaling pathway.     

Passive Movement Improves the Learning and Memory Function of Rats with Cerebral Infarction by Inhibiting Neuron Cell Apoptosis

Passive movement has been found to improve evidently ischemic stroke patients’ impaired capacity of learning and memory, but the optimal time window of initiating the therapy and the underlying mechanism are not fully understood. In this study, the effect of passive movement at different time windows on learning and memory of rats with cerebral infarction was detected. The results showed that the expression of caspase-3 and escape latency in the passive movement group were all considerably lower than those in the model group (P?<?0.05), while the expression of Bcl-2 mRNA was significantly higher than those in the model group (P?<?0.05). Moreover, we found that there were most significant changes of escape latency and expressions of Bcl-2 mRNA and caspase-3 when the therapy started at 24 h after focal cerebral infarction. These results suggest that passive movement is able to contribute to the recovery of learning and memory of rats with cerebral infarction, which is partially mediated by inhibiting neuron cell apoptosis, and the optimal therapeutic time is at 24 h after cerebral infarction.