Anti-apoptotic Protection Research Articles

Antioxidant Defenses, Oxidative Stress Responses, and Apoptosis Modulation in Spontaneous Abortion: An Immunohistochemistry Analysis of First-Trimester Chorionic Villi.

Oxidative stress (OS) and apoptosis are critical factors in placental development and function. Their interplay influences trophoblast proliferation, differentiation, and invasion, as well as vascular development. An imbalance between these processes can lead to pregnancy-related disorders such as preeclampsia, intrauterine growth restriction, and even spontaneous abortion. Our study seeks to elucidate the associations between preventive antioxidant/protective OS response factors-glutathione (GSH), MutT Homolog 1 (MTH1), and apoptotic regulation modulators-tumor protein p53 and B-cell lymphoma (Bcl-2) transcripts, in the context of spontaneous abortion (30 samples) versus elective termination of pregnancy (20 samples), using immunohistochemistry (IHC) to determine their proteomic expression in chorionic villi within abortive fetal placenta tissue samples. Herein, comparative statistical analyses revealed that both OS response factors, GSH and MTH1, were significantly under-expressed in spontaneous abortion cases as compared to elective. Conversely, for apoptotic regulators, p53 expression was significantly higher in spontaneous abortion cases, whereas Bcl-2 expression was significantly lower in spontaneous abortion cases. These findings suggest that a strong pro-apoptotic signal is prevalent within spontaneous abortion samples, alongside reduced anti-apoptotic protection, depleted antioxidant defenses and compromised oxidative DNA damage prevention/repair, as compared to elective abortion controls. Herein, our hypothesis that OS and apoptosis are closely linked processes contributing to placental dysfunction and spontaneous abortion was thus seemingly corroborated. Our results further highlight the importance of maintaining redox homeostasis and apoptotic regulation for a successful pregnancy. Understanding the mechanisms underlying this interplay is essential for developing potential therapies to manage OS, promote placentation, and avoid unwanted apoptosis, ultimately improving pregnancy outcomes. Antioxidant supplementation, modulation of p53 activity, and the enhancement of DNA repair mechanisms may represent potential approaches to mitigate OS and apoptosis in the placenta. Further research is needed to explore these strategies and their efficacy in preventing spontaneous abortion.

Abstract 1125: Percutaneous Optofluidic Catheter-guided Photo-triggered Nanogoldsomes Reduce Infarct Size And Mitigate Myocardial Dysfunction In A Swine Model Of Acute Myocardial Infarction

Introduction: S-nitrosoglutathione (GSNO) is an endogenous S-nitrosothiol that plays important roles in nitric oxide (NO) generation and signaling. We previously designed an Au-polymersomes/GSNO nanoparticle (nanogoldsome) that can be triggered by laser or ultrasound to accelerate the release of NO, which effectively dilates the arteries of the target organ and induces anti-apoptotic protection against ischemia-reperfusion injury. In this study we integrated an optofluidic coronary catheter with the photo-triggered nanogoldsomes, and investigate their roles in mitigating the infarct size in a swine model of acute myocardial infarction (AMI). Methods: In a swine AMI model of 90 min coronary artery (CA) occlusion, nanogoldsomes (0.145 mg Au and 3.66 μg GSNO/kg) were administered 5 min after balloon-off using a percutaneous coronary optofluidic catheter with 532 nm laser stimulation at the tip. The CA diameter was measured at baseline and at 0 and 15 min of reperfusion. Left ventricular (LV) function (max dp/dt) were measured at baseline, and 1 h and 7 days after reperfusion. The infarct area was determined at day 7 using triphenyltetrazolium chloride stain with the infarct fraction calculated in reference to the area at risk stained by Evan’s blue coronary infusion with balloon occlusion at the same site. Results: Compared to AMI group, the CA diameter showed significant dilatation 10 min after nanogoldsome/laser treatment (1.22 vs. 0.98 folds, P < 0.05), while systemic blood pressure showed no significant drop. The infarct fraction was significantly reduced (49% vs. 73%, P < 0.05) and LV function markedly better 7 days after AMI. By contrast, no significant differences were noted in the control groups of GSNO (3.66 μg/kg) and nanogoldsomes without laser stimulation. Conclusion: Percutaneous optofluidic catheter-guided infusion of nanogoldsomes at coronary orifice with simultaneous tip laser stimulation in the first minutes of AMI reperfusion magnified NO release in the most critical reperfusion period. This instantly dilated coronary arteries, and reduced infarct area and mitigated LV dysfunction 7 days after AMI. With large animal experiments showing pathophysiological benefits, further translation to pre-clinical studies is encouraged.

Efficacy of glucagon-like peptide-1 and estrogen dual agonist in pancreatic islets protection and pre-clinical models of insulin-deficient diabetes

SummaryWe study the efficacy of a glucagon-like peptide-1 (GLP-1) and estrogen dual agonist (GLP1-E2) in pancreatic islet protection. GLP1-E2 provides superior protection from insulin-deficient diabetes induced by multiple low-dose streptozotocin (MLD-STZ-diabetes) and by the Akita mutation in mice than a GLP-1 monoagonist. GLP1-E2 does not protect from MLD-STZ-diabetes in estrogen receptor-α (ERα)-deficient mice and fails to prevent diabetes in Akita mice following GLP-1 receptor (GLP-1R) antagonism, demonstrating the requirement of GLP-1R and ERα for GLP1-E2 antidiabetic actions. In the MIN6 β cell model, GLP1-E2 activates estrogen action following clathrin-dependent, GLP-1R-mediated internalization and lysosomal acidification. In cultured human islet, proteomic bioinformatic analysis reveals that GLP1-E2 amplifies the antiapoptotic pathways activated by monoagonists. However, in cultured mouse islets, GLP1-E2 provides antiapoptotic protection similar to monoagonists. Thus, GLP1-E2 promotes GLP-1 and E2 antiapoptotic signals in cultured islets, but in vivo, additional GLP1-E2 actions in non-islet cells expressing GLP-1R are instrumental to prevent diabetes.

Experimental hyperthermia: expression of proteins involved in the regulation of ovarian corpus luteum apoptosis in the acute and recovery periods

Background.Heat shock effects can initiate apoptosis of oocytes and corpus luteum cells in mammalian ovaries. During folliculogenesis, follicular apoptosis is regulated by Bcl-2 and BAX proteins which are key effectors of granular cell death. Mechanisms of disruption of the ovarian corpus luteum development under heat stress remain largely unclear.Aim of the research: to identify the expression features of anti-apoptotic Bad and proapoptotic Bcl-2 proteins in the rat ovarian luteocytes in the acute (by day 3) and recovery (by days 7 and 14) periods after a single exposure of experimental hyperthermia (EH) (rectal temperature 43.5 °C).Materials and methods. The expression of Bad and Bcl-2 was determined immunohistochemically using an indirect two-stage streptavidin-biotin method.Results. On day 3 after EH, the expression areas of both Bad and Bcl-2 increased 2-fold, but the ratio of Bcl-2/Bad areas did not change, indicating that the intensity of apoptosis along the mitochondrial pathway in luteocytes in the acute period was maintained within physiological values. On day 7, the Bad and Bcl-2 expression areas remained at the level of day 3, but the Bcl-2/Bad index decreased, indicating the activation of the apoptosis internal pathway in the ovarian corpus luteum cells. By day 14, the protein expression areas decreased (Bad – by 1.7 times, Bcl-2 – by 3.2 times) compared to the acute period, and the Bcl-2/Bad index decreased by 2 times compared to the control and the acute period group.Conclusion. The observed predominance of proapoptotic Bad protein over antiapoptotic Bcl-2 in luteocytes on day 14 after EH indicates the anti-apoptotic protection violation, which leads to the apoptosis mitochondrial pathway activation of the latter. A decrease in Bcl-2 expression can be regarded as a manifestation of the defective luteocytes removal mechanism and the body’s desire to normalize the ovarian-uterine cycle disrupted by high temperature exposure.

Chinese Herbal Medicine Formula Guizhi Li-Zhong Tang as an Alternative to Antibiotic Feed Additives for Preventing Pneumonia in Piglets through Antioxidant, Anti-Inflammatory, and Antiapoptotic Protection.

Weanling piglets often develop respiratory diseases such as pneumonia because they encounter substantial environmental stress. This study investigated an alternative herbal feed additive, Guizhi Li-Zhong Tang (GLZ), for preventing pneumonia in weanling piglets. An in vitro experiment demonstrated that GLZ has high antioxidant capacity and low cytotoxicity toward Kupffer cells. In addition, GLZ treatment can alleviate lipopolysaccharide (LPS)-induced damage in Kupffer cells. A total of 94 4-week-old piglets were randomly divided into three groups, which received sham treatment, 0.2% Tilmicosin antibiotic (TAB) treatment, or 0.2% GLZ treatment. Piglets receiving the GLZ treatment had a higher survival rate and higher immunoglobulin G levels but lower allergy-related eosinophil levels and cough incidence than did piglets receiving the sham or 0.2% TAB treatments. Through immunohistochemistry and Western blot analysis, we discovered that piglets receiving the 0.2% GLZ treatment had significantly higher expression of antioxidant-related SOD2 and lower expression of oxidative-stress-related 3-NT (p < 0.01), inflammation-related TNF-α (p < 0.01) and NF-κB (p < 0.05), and apoptosis-related caspase-3 (p < 0.01) in lung tissue than did piglets receiving the sham or 0.2% TAB treatment. Therefore, GLZ treatment is promising as an alternative to antibiotic medicine for weanling piglets because of its protective antioxidative, anti-inflammatory, and antiapoptotic effects in lung tissue.

Proteolysis Targeting Chimeras for BTK Efficiently Inhibit B-Cell Receptor Signaling and Can Overcome Ibrutinib Resistance in CLL Cells.

Proteolysis targeting chimeras (PROTACs) are small molecules that form ternary complexes between their target and E3 ligase, resulting in ubiquitination and proteasomal degradation of the target protein. Using our own designed Bruton’s tyrosine kinase (BTK) PROTAC compounds, we show herein efficient BTK degradation in chronic lymphocytic leukemia (CLL) cells. The reversible non-covalent compound (NC-1) was the most potent and therefore we focused on this PROTAC to investigate its subsequent effects on the BCR pathway. NC-1 decreased baseline BTK phosphorylation as well as activation of BTK and other signaling molecules downstream of the BCR pathway, following IgM engagement. These effects were also obtained in samples from CLL patients with clinical resistance to ibrutinib and mutations at C481. NC-1 treatment further decreased baseline CD69 surface levels, completely abrogated its upregulation following IgM activation, decreased CLL cells migration toward SDF-1 and overcame stromal anti-apoptotic protection. In conclusion, our results indicate that targeting BTK using the PROTAC strategy could be a potential novel therapeutic approach for CLL.

The expression of apoptosis-regulating proteins Bcl-2 and Bad in liver cells of C57Bl/6 mice under light-induced functional pinealectomy and after correction with melatonin.

The presence of humans and animals under long-term continuous lighting leads to a suppression of melatonin synthesis, that is, to light-induced functional pinealectomy (LIFP), and the development of desynchronosis. To create LIFP, C57Bl/6 mice were kept under 24-hour lighting (24hL) for 14 days. The animals in the control group were kept under standard lighting conditions. In the next series of experiments, mice with LIFP received daily intragastrically either melatonin (1 mg/kg body weight in 200 μl of distilled water) or 200 μl of water as a placebo. The comparison group consisted of intact animals that received placebo under standard lighting conditions. Immunohistochemical analysis (using an indirect avidin-biotin peroxidase method) revealed the expression of the antiapoptotic protein Bcl-2 and the proapoptotic protein Bad in sinusoid liver cells (a heterogeneous population consisting of the endotheliocytes, Kupffer cells, Ito cells, and Pit cells) and in individual hepatocytes. The Bad expression area in the liver of LIFP mice increased 4 times against a background of the unchanged Bcl-2 expression area. Changes in the brightness (a parameter inversely proportional to the marker concentration) of Bad and Bcl-2 areas did not reach significance. Our results indicate a weakening of the antiapoptotic protection of liver cells of LIFP animals, which creates conditions for activation of the “mitochondrial branch” of apoptosis. Melatonin treatment of LIFP mice resulted in a 3.3-fold increase in Bcl-2 expression area and a 2.7 % decrease in Bcl-2 region brightness compared with the experimental untreated group. Bad protein parameters were unreliable. Thus, melatonin treatment of animals cancels the effect of LIFP, restoring the Bcl-2 expression area and increasing this protein concentration, which indicates an increase in antiapoptotic protection and creates conditions for blocking the development of the “mitochondrial branch” of apoptosis in liver cells.

Abstract P750: 3K3A-APC Restores Oligodendrocyte Pools in Models of White Matter Stroke via PAR1 Signaling

A recombinant variant of activated protein C, 3K3A-APC, was recently tested in a successful phase II clinical trial for ischemic stroke (RHAPSODY), which demonstrated that the treatment may reduce hemorrhages and is safe in patients that were treated with tPA and/or thrombectomy. Animal models of large ischemic stroke (middle cerebral artery occlusion) and neurodegeneration have shown that the effects of 3K3A-APC are mediated by preserved APC cell signaling activities (anti-apoptotic neuronal protection, protection of blood-brain barrier (BBB) integrity and anti-inflammatory activities). Because recent studies have suggested a role of protease activated receptor 1 (PAR1) in oligodendrocyte recovery after white matter damage, we wanted to test whether biased PAR1 signaling mediated by 3K3A-APC would protect oligodendrocytes in in vivo and ex vivo models of ischemic white matter stroke. First, we used a mouse model of white matter stroke induced by injecting a vasoconstrictor N 5 -(1-Iminoethyl)-L-ornithine (L-NIO) into the corpus callosum. Our results show that treatment with recombinant murine 3K3A-APC (0.2 mg/kg, starting 4h after stroke) protected oligodendrocytes (OLS, Olig2+CNPase+) and oligodendrocyte precursor cells (OPCs, Olig2+PDGFRα+) from cell death at 1 and 7 days after stroke. In vitro , 3K3A-APC protected primary mouse oligodendrocytes from apoptosis when exposed to OGD conditions at nanomolar concentrations (EC50 concentration was 5.2 nM). Improved oligodendrocyte viability in vivo was associated by about ~67% reduction in overall cell death (cresyl violet staining, T2w and diffusion tensor MRI) and reduced BBB leakage (fibrin staining and DCE-MRI) as well as improved functional recovery (adhesive removal and grid walking tests). Protective effects of 3K3A-APC were abolished by silencing of PAR1 with small interfering RNA (siRNA) in vivo and in vitro . In conclusion, PAR1 mediated signaling initiated by 3K3A-APC protects oligodendrocyte pools in models of ischemic white matter damage with preservation of neurovascular unit integrity.

PDZ Scaffold Protein CAL Couples with Metabotropic Glutamate Receptor 5 to Protect Against Cell Apoptosis and Is a Potential Target in the Treatment of Parkinson's Disease.

Targeting mGluR5 has been an attractive strategy to modulate glutamate excitotoxicity for neuroprotection. Although human clinical trials using mGluR5 negative allosteric modulators (NAMs) have included some disappointments, recent investigations have added several more attractive small molecules to this field, providing a promise that the identification of more additional strategies to modulate mGluR5 activity might be potentially beneficial for the advancement of PD treatment. Here, we determined the role of the interacting partner CAL (cystic fibrosis transmembrane conductance regulator-associated ligand) in mGluR5-mediated protection in vitro and in vivo. In astroglial C6 cells, CAL deficiency blocked (S)-3, 5-dihydroxyphenylglycine (DHPG)-elicited p-AKT and p-ERK1/2, subsequently prevented group I mGluRs-mediated anti-apoptotic protection, which was blocked by receptor antagonist 1-aminoindan-1, 5-dicarboxylic acid (AIDA), and PI3K or MEK inhibitor LY294002 or U0126. In rotenone-treated MN9D cells, both CAL and mGluR5 expressions were decreased in a time- and dose-dependent manner, and the correlation between these 2 proteins was confirmed by lentivirus-delivered CAL overexpression and knockdown. Moreover, CAL coupled with mGluR5 upregulated mGluR5 protein expression by inhibition of ubiquitin-proteasome-dependent degradation to suppress mGluR5-mediated p-JNK and to protect against cell apoptosis. Additionally, CAL also inhibited rotenone-induced glutamate release to modulate mGluR5 activity. Furthermore, in the rotenone-induced rat model of PD, AAV-delivered CAL overexpression attenuated behavioral deficits and dopaminergic neuronal death, while CAL deficiency aggravated rotenone toxicity. On the other hand, the protective effect of the mGluR5 antagonist MPEP was weakened by knocking down CAL. In vivo experiments also confirmed that CAL inhibited ubiquitination-proteasome-dependent degradation to modulate mGluR5 expression and JNK phosphorylation. Our findings show that CAL protects against cell apoptosis via modulating mGluR5 activity, and may be a new molecular target for an effective therapeutic strategy for PD.

Abstract 16: Spatiotemporally-Controlled Ultrasound-Triggered Release of Nitric Oxide Using Nano Au-Polymersomes/S-Nitrosoglutathione Mitigates Post-Resuscitation Cerebral Vasoconstriction and Neuronal Apoptosis via Reciprocating Akt-eNOS-NO Signaling

Introduction: Cerebral vasoconstriction in the post-resuscitation phase worsens neurological outcome. Nitric oxide (NO) plays important roles mediating vasodilatation and anti-apoptotic protection. We therefore designed an Au-polymersomes/S-nitrosoglutathione (Au-PLGA/GSNO) nanoparticle that can be triggered by ultrasound (US) to release NO, and investigated its roles in mitigating cerebral vasoconstriction and neuronal apoptosis post-CPR. Hypothesis: Spatiotemporally controlled, US-triggered NO release by Au-PLGA/GSNO improves post-CPR cerebral perfusion and confers anti-apoptotic neuroprotection. Methods: Using an established rat model of asphyxia cardiac arrest and CPR, Au-PLGA/GSNO (7500 PPM, 0.4 ml) was infused with simultaneous US (1 MHz) stimulation at the brain 10 min after ROSC. Brain tissue perfusion was continuously recorded by OxyFLO probe and cerebral vasculature videoed by CytoCam. The blood was sampled 2 h post-CPR for measurement of nitrate/nitrite, and the brain harvested for measurement of casepase-3, endothelial NO synthase (eNOS) and protein kinase B (Akt). In a subgroup the brain was harvested at 24 h for TUNEL stain. Results: After CPR, marked cerebral vasoconstriction was noted on CytoCam while brain perfusion significantly reduced to ~0.5 folds that of baseline. After Au-PLGA/GSNO infusion and US stimulation, cerebral vasoconstriction was ameliorated and the brain perfusion significantly enhanced ( P &lt; 0.05 vs. CPR control). The plasma NO indicated by nitrate/nitrite 2 h post-CPR was significantly increased ( P &lt; 0.01) while cleaved caspase-3/caspase-3 of the brain markedly reduced ( P &lt; 0.001). TUNEL stain of the hippocampus CA1 and CA3 regions were also remarkably abrogated, suggesting anti-apoptotic neuroprotection. Specifically, the phosphorylated (p)-eNOS/eNOS and p-Akt/Akt were also increased ( P &lt; 0.01 and 0.001, respectively), indicating reciprocating activation of Akt-eNOS signaling upstream of NO. Conclusion: Spatiotemporally controlled US-triggered NO release by Au-PLGA/GSNO mitigates cerebral vasoconstriction, improves brain perfusion and confers anti-apoptotic neuroprotection post-CPR via reciprocating Akt-eNOS-NO signaling.

Neuronal preservation and reactive gliosis attenuation following neonatal sciatic nerve axotomy by a fluorinated cannabidiol derivative

Immature peripheral nervous system damage, such as the transection of a peripheral nerve, results in the extensive degeneration of motoneurons and dorsal root ganglia (DRG) sensory neurons, mostly due to apoptotic events. We have previously shown that cannabidiol (CBD), the most abundant non-psychotropic molecule present in the Cannabis sativa plant, exhibits neuroprotective action when administered daily at a dose of 15 mg/kg. This study shows that use of the fluorinated synthetic version of CBD (4′-fluoro-cannabidiol, HUF-101) significantly improves neuronal survival by 2-fold compared to that achieved with traditional CBD at one-third the dose. Furthermore, we show that HUF-101 administration significantly upregulates anti-apoptotic genes and blocks the expression of pro-apoptotic nuclear factors. Two-day-old Wistar rats were subjected to unilateral sectioning of the sciatic nerve and treated daily with HUF-101 (1, 2.5, 5 mg/kg/day, i.p.) or a vehicle solution for five days. The results were evaluated by Nissl staining, immunohistochemistry, and qRT-PCR. Neuronal counting revealed a 47% rescue of spinal motoneurons and a 79% rescue of DRG neurons (HUF-101, 5 mg/kg). Survival was associated with complete depletion of p53 and a 60-fold elevation in BCL2-like 1 gene expression. Additionally, peroxisome proliferator-activated receptor gamma (PPAR-gamma) gene expression was downregulated by 80%. Neuronal preservation was coupled with a high preservation of synaptic coverage and a reduction in astroglial and microglial reactions that were evaluated in nearby spinal motoneurons present in the ventral horn of the lumbar intumescence. Overall, these data strongly indicate that HUF-101 exerts potent neuroprotective effects that are related to anti-apoptotic protection and the reduction of glial reactivity.

Molecular Genetics of Lidocaine-Containing Cardioplegia in the Human Heart During Cardiac Surgery

During cardiac surgery with cardiopulmonary bypass, delivery of cardioplegia solution to achieve electromechanical cardiac quiescence is obligatory. The addition of lidocaine to cardioplegia has advantages, although its consequences at a molecular level remain unclear. We performed whole-genome RNA sequencing of the human left ventricular (LV) myocardium to elucidate the differences between whole-blood (WB) cardioplegia with and without addition of lidocaine (LC) on gene expression. We prospectively enrolled 130 patients undergoing aortic valve replacement surgery. Patients received high-potassium blood cardioplegia either with (n= 37) or without (n= 93) lidocaine. The LVapex was biopsied at baseline, and after an average of 74 minutes of cold cardioplegic arrest. We performed differential gene expression analysis for 18,258 genes between these 2 groups. Clinical and demographic variables were adjusted in the model. Gene ontology (GO) and network enrichment analysis of the retained genes were performed using g:Profiler and Cytoscape. A total of 1,298 genes were differentially expressed between cardioplegic treatments. Compared with the WB group, genes upregulated in the LC group were identified by network enrichment to play a protective role in ischemic injury by inhibiting apoptosis, increasing transferrin endocytosis, and increasing cell viability. Downregulated genes in the LC group were identified to play a role in inflammatory diseases, oxygen transport, and neutrophil aggregation. The addition of lidocaine to cardioplegia had pronounced effects on a molecular level with genes responsible for decreased inflammation, reduced intracellular calcium binding, enhanced antiapoptotic protection, augmented oxygen accessibility through transferrins, and increased cell viability showing measurable differences.

Expression of Bcl-2 Family Proteins in the Ovarian Follicular Apparatus in the Acute Period after Experimental Hyperthermia.

The expression of apoptosis regulators (proapoptotic protein Bad and anti-apoptotic protein Bcl-2) was analyzed and Bcl-2/Bad ratio in the follicular apparatus of the rat ovary was determined on day 3 after hyperthermia (rectal temperature 43.5°C). Hyperthermia in the catabolic phase leads to different degrees of activation of the molecular "switches" of apoptosis in cells of ovarian follicular epithelium. This was seen from increased intensity of immunohistochemical staining for Bad protein against the background of more pronounced expression of Bcl-2 protein. On day 3 after exposure to hyperthermia, Bcl-2/Bad ratio increased, which reflects antiapoptotic protection of cells and conditions for blockade of mitochondrial pathway of apoptosis in the follicular apparatus of the ovaries during the acute period after hyperthermia.

Hepatoprotective effect of melatonin via activation of Nrf2 and anti-apoptotic proteins in burn rats

Abstract Objective: Burn- induced acute hepatic injury due to increased production of lipid peroxides and increased cellular apoptosis. The nuclear factor erythroid 2-related factor 2 (Nrf2) pathway is essential for cytoprotection against oxidative stress. We hypothesized that melatonin by activation of Nrf2 may shift Bax/Bcl-2 ratio to protect rat hepatocytes against apoptosis and progressive liver injury. The aim of this experimental study was to investigate the protective effects of melatonin against burn-induced apoptotic injury and the relationship between lipid peroxides expression of transcription factor Nrf2 and apoptotic protein in burn rat model. Methods: Melatonin was applied immediately after the burn. The expression of hepatic 4-hydroxynonenal (4-HNE), as a marker of liver peroxidative injury, Nrf2, as a marker of antioxidant defense and apoptosis-related genes Bcl-2 and Bax were evaluated using light immunоhistochemistry. Results: Burns caused an increased expression of 4-HNE, Bax and Bax/Bcl-2 ratio and induced apoptosis of sinusoidal endothelial cells (SECs) in liver tissue. Melatonin treatment augmented the increase in Nrf2 expression, decreased both burn-induced peroxidative damage and hepatic apoptosis as evidenced by reduced expression of Bax, enhanced expression of Bcl-2. In conclusion, our data suggest that activation of transcription factor Nrf2 by melatonin is protective against oxidative stress, apoptosis and hepatic injury in burns. The available information by melatonin`s effect on the redox sensing transcription factor Nrf2, as a regulator of antioxidant enzymes, antioxidants and antioxidant protection of the liver is limited. Melatonin activates the Nrf2/signaling pathway and acts as a natural inducer of anti-apoptotic and antioxidant protection under condition of burn-induced oxidative stress. This is a new cellular mechanism for protection against progressive burn-induced liver damage not only in animals but also in humans.

Effect of Oral Minocycline on Clinical Recovery Process in Patients with Acute Ischemic Stroke: A Randomized Clinical Trial

Background: Acute ischemic stroke is one of the most common causes of death worldwide with one new case being diagnosed every five seconds. The mortality rate and permanent disability are very high and the current treatment still needs to improve to a large extent. Minocycline drug, a derivative of tetracycline, is an anti-inflammatory and anti-apoptotic protection of neurons, the role of which has been studied recently in recovery from nerve degenerative diseases, especially stroke. This study aimed at evaluating the effect of minocycline in the recovery of patients with a history of stroke. Methods: In this randomized clinical trial, 42 patients with ischemic stroke were divided to 2 groups: receiving minocycline 200 mg for 5 days and receiving the placebo. Aspirin was prescribed to all patients. Clinical assessment before and 90 days after the intervention was performed by the National institutes of health stroke scale score (NIHSS). Results: A total of 36 patients completed the study. The number of females in the case and control groups was 55.5% and 51.1%, respectively. In the case group, NIHSS decreased from 9.55 to 6.1 and in the control group, it decreased from 10.2 to 7.33, which was statistically significant. Although the NIHSS decreased in patients taking minocycline more than the control group, this difference was not statistically significant. Conclusions: According to the findings of this study, it seems that minocycline could be used as a complementary therapy in patients with ischemic stroke. However, these results need to be confirmed by further studies in this field.

A-Raf and C-Raf differentially regulate mechanobiological response of osteoblasts to guide mechanical stress-induced differentiation

Regulation of osteoblast activity by mechanical stress is important for bone remodeling. However, the precise mechanotransduction mechanism that triggers the anabolic reaction of osteoblasts is largely unknown. In this study, we performed RNA interference (RNAi) screening to identify the signaling molecules upstream of ERK, which was responsible for osteogenesis. Of twenty-two mitogen-activated protein kinase (MAPK) kinase kinases (MAP3Ks), we identified A-Raf and C-Raf as upstream MAP3Ks of the mechanical stretch-activated ERK pathway. Subsequently we screened the mechanosensitive cation channel, and identified P2X7 as an upstream molecule of the ERK pathway. Intriguingly, P2X7 functioned as an upstream activator of A-Raf but not of C-Raf. Furthermore, A-Raf contributed to mechanical stretch-induced osteoblast differentiation. In contrast, C-Raf but not A-Raf protected osteoblasts from mechanical stretch-induced apoptosis. These results suggested that A-Raf and C-Raf were involved in mechanobiological osteogenesis in a distinct way: A-Raf was responsible for osteogenesis while C-Raf for anti-apoptotic protection and promotion.

A Futile Redox Cycle Involving Neuroglobin Observed at Physiological Temperature.

Previous studies identifying the potential anti-apoptotic role of neuroglobin raise the question as to how cells might employ neuroglobin to avoid the apoptotic impact of acute hypoxia whilst also avoiding chronic enhancement of tumour formation. We show that under likely physiological conditions neuroglobin can take part in a futile redox cycle. Determination of the rate constants for each of the steps in the cycle allows us to mathematically model the steady state concentration of the active anti-apoptotic ferrous form of neuroglobin under various conditions. Under likely normal physiological conditions neuroglobin is shown to be present in the ferrous state at approximately 30% of its total cellular concentration. Under hypoxic conditions this rapidly rises to approximately 80%. Temporal analysis of this model indicates that the transition from low concentrations to high concentration of ferrous neuroglobin occurs on the seconds time scale. These findings indicate a potential control model for the anti-apoptotic activity of neuroglobin, under likely physiological conditions, whereby, in normoxic conditions, the anti-apoptotic activity of neuroglobin is maintained at a low level, whilst immediately a transition occurs to a hypoxic situation, as might arise during stroke, the anti-apoptotic activity is drastically increased. In this way the cell avoids unwanted increased oncogenic potential under normal conditions, but the rapid activation of neuroglobin provides anti-apoptotic protection in times of acute hypoxia.

Analysis of expression patterns of IGF-1, caspase-3 and HSP-70 in developing human tooth germs

AimsTo analyze expression patterns of IGF-1, caspase-3 and HSP-70 in human incisor and canine tooth germs during the late bud, cap and bell stages of odontogenesis. Materials and methodsHead areas or parts of jaw containing teeth from 10 human fetuses aged between 9th and 20th developmental weeks were immunohistochemically analyzed using IGF-1, active caspase-3 and HSP-70 markers. Semi-quantitative analysis of each marker’s expression pattern was also performed. ResultsDuring the analyzed period, IGF-1 and HSP-70 were mostly expressed in enamel organ. As development progressed, expression of IGF-1 and HSP-70 became more confined to differentiating tissues in the future cusp tip area, as well as in highly proliferating cervical loops. Few apoptotic bodies highly positive to active caspase-3 were observed in enamel organ and dental papilla from the cap stage onward. However, both enamel epithelia moderately expressed active caspase-3 throughout the investigated period. ConclusionsExpression patterns of IGF-1, active caspase-3 and HSP-70 imply importance of these factors for early human tooth development. IGF-1 and HSP-70 have versatile functions in control of proliferation, differentiation and anti-apoptotic protection of epithelial parts of human enamel organ. Active caspase-3 is partially involved in formation and apoptotic removal of primary enamel knot, although present findings might reflect its ability to perform other non-death functions such as differentiation of hard dental tissues secreting cells and guidance of ingrowth of proliferating cervical loops.

Epigallocatechin-3-gallate and zinc provide anti-apoptotic protection against hypoxia/reoxygenation injury in H9c2 rat cardiac myoblast cells.

It has previously been demonstrated that phospha-tidylinositol-3-kinase (PI3K)/Akt and cleaved caspase-3 serve critical roles in the apoptosis of cardiac myocytes following ischemia/reperfusion injury. Epigallocatechin-3-gallate (EGCG), the predominant catechin component of green tea, has been reported to have potential cardioprotective effects in primary cultures of cardiac myocytes exposed to I/R injury, mediated through inhibition of signal transducers and activators of transcription-1 activity. In addition, it is also known that the biological behavior of EGCG may be influenced by metal ions, for example the hepatoprotective activity of EGCG has been reported to be enhanced by zinc. In the present study, the protective effects of EGCG with zinc were assessed on cultures of rat cardiac myoblasts exposed to hypoxia/reoxygenation (H/R) injury. H9c2 cells were subjected to 3-h hypoxia, followed by 1-h reperfusion. EGCG and/or zinc were perfused prior to induced hypoxic stress. It was demonstrated that when EGCG interacted with zinc, the anti-apoptotic activity was significantly enhanced. To the best of our knowledge, the current study was the first to demonstrate that EGCG + Zn2+ protects H9c2 cells against H/R injury through activation of the PI3K/Akt pathway, as determined by western blotting. Since EGCG + Zn2+ may, at least in part, protect cardiac myocytes against H/R-induced apoptotic cell death, the PI3K/Akt pathway of EGCG may be enhanced by its interactions with zinc during H/R injury. Furthermore, it was suggested that a similar procedure may be implemented in a clinical setting, in order to maximize PI3K/Akt activation levels in patients with acute coronary artery disease. EGCG and zinc may therefore represent effective agents for use in the prevention of I/R injury in clinical practice.

Pleiotropic functions of TIMP-1 in patients with chronic kidney disease

Sir, We have read with great interest a recent in-depth review by Christian Ries [1], giving a picture of multiple activities of a tissue inhibitor of metalloproteinases (TIMP)-1 and revealing its complex cytokine impact on various cell functions. The pleiotropic role of TIMP-1 results from both metalloproteinase (MMP)-dependent and MMP-independent actions, creating a tool for the control of such diverse phenomena as apoptosis, cell differentiation or uncontrolled cell migration, resulting in metastases. In addition to a detailed theoretical background, the author has described the clinical implications of TIMP-1 activity, including cancer and cardiovascular disease. In our opinion, chronic kidney disease (CKD) is also worth mentioning among those examples. CKD, with its irreversible progressive clinical course and multiple complications, seems an ideal example of TIMP-1 multifaceted activity, influencing chronic inflammation, impaired immunity, enhanced apoptosis and cardiovascular comorbidity. Our recent investigation has concentrated on various aspects of MMP and the role of TIMP-1 in the course of CKD in the pediatric population. We have shown that the increase of serum TIMP-1 concentrations in CKD children appears only in the late stages of renal failure and is delayed in comparison to the early rise of MMP values, being an anti-fibrotic response to extracellular matrix accumulation [2]. Moreover, TIMP-1 was the best predictor of TGFbeta1, a hallmark of fibrosis, a fact that is concordant with the tight TIMP–MMP–TGFbeta1 connections described by Ries [1]. Another interesting aspect of TIMP-1 activity is its anti-apoptotic function, realized by both direct, receptor-mediated, and indirect, by MMP inhibition, pathways [1]. Enhanced apoptosis is one of the key elements in the course of chronic kidney disease, additionally aggravated by the commencement of dialysis. Indeed, our investigation has revealed increased MMP/TIMP concentrations in chronically dialyzed children versus controls [3]. The additional discrepancy between dialysis modalities, most evident in the case of TIMP-1, has strengthened the suggestion that hemodialysis triggers apoptosis and reciprocal anti-apoptotic protection to greater extent than peritoneal dialysis. We have also noticed the statistically significant correlations between markers of matrix turnover and apoptosis [3]. In addition, regression analysis has shown that TIMP-1 was more accurate than TIMP-2, MMP-2, or MMP-9 in the prediction of the well-established apoptotic markers, sFas and sFasL [3]. The abovementioned MMP overactivity and MMP/TIMP imbalance disrupts the integrity of the extracellular matrix in the course of CKD. The subsequent tissue remodeling, together with cell damage and matrix accumulation, gives way to atherosclerosis, renal fibrosis and aggravated cell migration to sites of inflammation. In particular, cells losing their anchorage due to proteolytic overactivity, if not eliminated by anoikis, gain the ability for uncontrolled migration and further metastatic spread. The hallmark of anoikis activity is E-cadherin, released in a circulating form from destroyed cell–cell junctions, and easing epithelial-to-mesenchymal transition (EMT). We have shown for the first time significant correlations between this marker and MMP/TIMP in CKD children, particularly in those on chronic dialysis [4]. Moreover, on regression analysis, TIMP-1 has turned out to be the best predictor of E-cadherin among examined MMPs, TIMPs, and apoptotic indexes [4]. The last intriguing aspect of TIMP-1 activity in the CKD population is its functional similarity with heat shock proteins (HSP). HSP are classified as damage-associated molecular patterns (DAMP), highly conserved in their structure throughout species. Likewise, TIMPs appear in all mammals [1]. They all influence the immune system by activating (HSP) or inhibiting (TIMP-1) nuclear factor (NF)κB. We have revealed for the first time the significant correlations between HSP and MMP/TIMP both in pre-dialysis children and in those on chronic dialysis [2, 5]. Moreover, we discovered that TIMP-1 was the most valuable predictor of various HSP (Hsp27, Hsp90alpha, anti-Hsp60), suggesting that they share some common features, and that TIMP-1 may, under the circumstances, behave like a stress protein. However, this hypothesis requires further in-depth investigation.