Free Fatty Acid Induces Endoplasmic Reticulum Stress and Apoptosis of β-cells by Ca2+/Calpain-2 Pathways

Dysfunction of β-cells is a major characteristic in the pathogenesis of type 2 diabetes mellitus (T2DM). The combination of obesity and T2DM is associated with elevated plasma free fatty acids (FFAs). However, molecular mechanisms linking FFAs to β-cell dysfunction remain poorly understood. In the present study, we identified that the major endoplasmic reticulum stress (ERS) marker, Grp78 and ERS-induced apoptotic factor, CHOP, were time-dependently increased by exposure of β-TC3 cells to FFA. The expression of ATF6 and the phosphorylation levels of PERK and IRE1, which trigger ERS signaling, markedly increased after FFA treatments. FFA treatments increased cell apoptosis by inducing ERS in β-TC3 cells. We also found that FFA-induced ERS was mediated by the store-operated Ca²⁺ entry through promoting the association of STIM1 and Orai1. Moreover, calpain-2 was required for FFA-induced expression of CHOP and activation of caspase-12 and caspase-3, thus promoting cell apoptosis in β-TC3 cells. Together, these results reveal pivotal roles for Ca²⁺/calpain-2 pathways in modulating FFA-induced β-TC3 cell ERS and apoptosis.     

Effect of Zinc and Melatonin on Oxidative Stress and Serum Inhibin-B Levels in a Rat Testicular Torsion–Detorsion Model

The present study was aimed to examine the effects of 3-week zinc and melatonin administration on testicular tissue injury and serum Inhibin-B levels caused by unilateral testicular torsion–detorsion in rats. The study was performed on 60 Wistar Albino-type adult male rats. The animals were allocated to 6 groups in equal numbers. 1. Control; 2. Sham; 3. Ischemia–reperfusion; 4. Zinc + ischemia–reperfusion; 5. Melatonin + ischemia–reperfusion; 6. Zinc + melatonin + ischemia–reperfusion. Zinc and melatonin were administered before ischemia–reperfusion at doses of 5 and 3 mg/kg respectively, by intraperitoneal route for a period of 3 weeks. Testicular torsion–detorsion procedures consisted of ischemia for 1 h and then reperfusion for another hour of the left testis. Blood and testicular tissue samples were collected to analyze erythrocyte and tissue GSH and plasma and tissue MDA, Inhibin-B levels. The highest erythrocyte and testis GSH values were found in zinc, melatonin, and zinc + melatonin groups (p < 0.001). Torsion–detorsion group has significantly lower erythrocyte GSH levels and higher plasma MDA values (p < 0.001). Serum inhibin-B and spermatogenic activity levels in the torsion–detorsion group were also significantly lower than those in the other groups (p < 0.001). However, zinc-, melatonin-, and melatonin + zinc-supplemented groups have higher inhibin-B and spermatogenetic activity (p < 0.001). The results of the study show that zinc, melatonin, and melatonin + zinc administration partially restores the increased oxidative stress, as well as the reduced inhibin-B and spermatogenic activity levels in testes ischemia–reperfusion in rats. Suppressed inhibin-B levels in the testicular tissue may be a marker of oxidative stress.     

Endoplasmic Reticulum Oxidoreductin-1α (Ero1α) Improves Folding and Secretion of Mutant Proinsulin and Limits Mutant Proinsulin-induced Endoplasmic Reticulum Stress

Upon chronic up-regulation of proinsulin synthesis, misfolded proinsulin can accumulate in the endoplasmic reticulum (ER) of pancreatic β-cells, promoting ER stress and type 2 diabetes mellitus. In Mutant Ins-gene-induced Diabetes of Youth (MIDY), misfolded mutant proinsulin impairs ER exit of co-expressed wild-type proinsulin, limiting insulin production and leading to eventual β-cell death. In this study we have investigated the hypothesis that increased expression of ER oxidoreductin-1α (Ero1α), despite its established role in the generation of H₂O₂, might nevertheless be beneficial in limiting proinsulin misfolding and its adverse downstream consequences. Increased Ero1α expression is effective in promoting wild-type proinsulin export from cells co-expressing misfolded mutant proinsulin. In addition, we find that upon increased Ero1α expression, some of the MIDY mutants themselves are directly rescued from ER retention. Secretory rescue of proinsulin-G(B23)V is correlated with improved oxidative folding of mutant proinsulin. Indeed, using three different variants of Ero1α, we find that expression of either wild-type or an Ero1α variant lacking regulatory disulfides can rescue mutant proinsulin-G(B23)V, in parallel with its ability to provide an oxidizing environment in the ER lumen, whereas beneficial effects were less apparent for a redox-inactive form of Ero1. Increased expression of protein disulfide isomerase antagonizes the rescue provided by oxidatively active Ero1. Importantly, ER stress induced by misfolded proinsulin was limited by increased expression of Ero1α, suggesting that enhancing the oxidative folding of proinsulin may be a viable therapeutic strategy in the treatment of type 2 diabetes.     

Neuroprotective Effect of Atorvastatin Involves Suppression of TNF-α and Upregulation of IL-10 in a Rat Model of Intracerebral Hemorrhage

We evaluated the neuroprotective effects of atorvastatin (2, 5, and 10 mg/kg) on experimentally induced intracerebral hemorrhage (ICH) in adult rats; controls were administered PBS. Plasma TNF-α and IL-10 levels before and after ICH were analyzed at various time points by enzyme-linked immunosorbent assay (ELISA) and neurological behavior of rats was assessed by climbing scores. At 3-days postoperatively, brain water contents and TNF-α/IL-10 expression in brain tissue were determined. Histopathological changes and microglial cells in the brain tissue were evaluated by light-microscopy. Post-ICH neurological deficits differed significantly between sham-operated group A and experimental-ICH group B (P < 0.05). Brain water contents were significantly less in group A than in group B (P < 0.05). Significant differences (P < 0.05) between two groups were observed regarding activated microglia, TNF-α and IL-10 levels. Compared with group B, neurological deficits, brain water contents, pathological changes, and activated microglia were reduced (P < 0.05) in groups C (Experimental-ICH + atorvastatin 2 mg/kg), D (Experimental-ICH + atorvastatin 5 mg/kg) and E (Experimental-ICH + atorvastatin 10 mg/kg). Atorvastatin-induced a dose-dependent reduction of TNF-α and increase of IL-10 levels (P < 0.05). Therefore, it was concluded that atorvastatin improved neurofunctional rehabilitation in rats through the suppression of cytokines-mediated inflammatory response and attenuation of brain damage following intracerebral hemorrhage.     

Effects of Chronic Stress and Phenytoin on the Long-term Potentiation （LTP） in Rat Hippocampal CA1 Region

Stress is the response to stimulation from inside andoutside with complicated effects on organisms. Appropri-ate stressful reactions are helpful in resisting diseases byactivating unspecific modulation system, while severe orprolonged stresses are harmful and even induce mentaland physical disorders such as recurrent depression, post-traumatic stress disorder (PTSD), Alzheimer’s disease andepilepsy [1]. Hippocampus, a main brain region of keyimportance for learning, memory and emotion, is t…     

Improvement of Spatial Memory Disorder and Hippocampal Damage by Exposure to Electromagnetic Fields in an Alzheimer’s Disease Rat Model

Although some epidemiological investigations showed a potential association between long-term exposure of extremely low frequency electromagnetic fields (ELF-EMF) and Alzheimer’s disease (AD), no reasonable mechanism can explain this association, and the related animal experiments are rare. In this study, ELF-EMF exposure (50Hz 400µT 60d) combined with D-galactose intraperitoneal (50mg/kg, q.d., 42d) and Aβ_25–35 hippocampal (5μl/unilateral, bilateral, single-dose) injection was implemented to establish a complex rat model. Then the effects of ELF-EMF exposure on AD development was studied by using the Morris water maze, pathological analysis, and comparative proteomics. The results showed that ELF-EMF exposure delayed the weight gain of rats, and partially improved cognitive and clinicopathologic symptoms of AD rats. The differential proteomic analysis results suggest that synaptic transmission, oxidative stress, protein degradation, energy metabolism, Tau aggregation, and inflammation involved in the effects mentioned above. Therefore, our findings indicate that certain conditions of ELF-EMF exposure could delay the development of AD in rats.     

The Protective Effect of Na+/Ca2+ Exchange Blocker KB-R7943 on Myocardial Ischemia–Reperfusion Injury in Hypercholesterolemic Rat

KB-R7943 reduces lethal reperfusion injury under normal conditions, but its effectiveness under certain pathological states is in dispute. In the present study, we sought to determine the effect of KB-R7943 in hyperlipidemic animals and assess if the K _ATP ⁺ are involved in the protective mechanisms. In group 1 (G1), isolated rat hearts underwent 25 min global ischemia (GI) and 120 min reperfusion (R). In group 2 (G2), G1 was repeated but the animals were subjected to a 1.5 % cholesterol-enriched diet during 6 weeks (hypercholesterolemic animals). In group 3 (G3), G2 was repeated but 1 μM KB-R7943 was added to the perfusate for 10 min from the start of reperfusion. In group 4 (G4), G3 was repeated, and glibenclamide (K _ATP ⁺ , blocker, 0.3 μM) was administered. The infarct size was measured by triphenyltetrazolium. The infarct size was 35 ± 5.0 % in G1 and 46 ± 8.7 % in G2 (P < 0.05); KB-R7943 reduced the infarct size (28.6 ± 3.3 % in G3 vs. G2, P < 0.05). In addition, KB-R7943 attenuated apoptotic cell (G3 vs. G2, P < 0.05), but glibenclamide abolished the effect reached by KB-R7943. Thus, diet-induced hypercholesterolemia enhances myocardial injury; KB-R7943 reduces infarct size and apoptosis in hyperlipidemic animals through the activation of K⁺ATP channels.     

Post-tetanic Potentiation and Depression in Hippocampal Neurons in a Rat Model of Alzheimer’s Disease: Effects of Teucrium Polium Extract

Neurophysiology - The plant Teucrium polium (T.p.) possesses a wide range of pharmacological activities due to the presence, in particular, of different phytochemicals, phenols and flavonoids. We...     

Involvement of Endoplasmic Reticulum Stress in Inflammatory Bowel Disease: A Different Implication for Colonic and Ileal Disease?

Background

Endoplasmic reticulum (ER) stress has been suggested to play a role in inflammatory bowel disease (IBD). The three branches (ATF6, IRE1 and PERK) of the unfolded protein response (UPR) have different roles and are not necessarily activated simultaneously.

Methodology/Principal Findings

Expression of UPR-related genes was investigated in colonic and ileal biopsies of 23 controls, 15 ulcerative colitis (UC) and 54 Crohn''s disease (CD) patients. This expression was confirmed at protein level in colonic and ileal samples of five controls, UC and CD patients. HSPA5, PDIA4 and XBP1s were significantly increased in colonic IBD at mRNA and/or protein levels, indicating activation of the ATF6 and IRE1 branch. Colonic IBD was associated with increased phosphorylation of EIF2A suggesting the activation of the PERK branch, but subsequent induction of GADD34 was not observed. In ileal CD, no differential expression of the UPR-related genes was observed, but our data suggested a higher basal activation of the UPR in the ileal mucosa of controls. This was confirmed by the increased expression of 16 UPR-related genes as 12 of them were significantly more expressed in ileal controls compared to colonic controls. Tunicamycin stimulation of colonic and ileal samples of healthy individuals revealed that although the ileal mucosa is exhibiting this higher basal UPR activation, it is still responsive to ER stress, even more than colonic mucosa.

Conclusions/Significance

Activation of the three UPR-related arms is seen in colonic IBD-associated inflammation. However, despite EIF2A activation, inflamed colonic tissue did not increase GADD34 expression, which is usually involved in re-establishment of ER homeostasis. This study also implies the presence of a constitutive UPR activation in healthy ileal mucosa, with no further activation during inflammation. Therefore, engagement of the UPR differs between colon and ileum and this could be a factor in the development of ileal or colonic disease.     

Bioprotective and Functional Effect of Carnosine on Sepsis Induced Renal Damage in Male Albino Rat Model through Targeting IL-1β and TNF-α Production

Doklady Biochemistry and Biophysics - Acute kidney injury (AKI), one of the frequently diagnosed and serious sepsis induced complication has high morbidity and mortality. The present study...     

Protective Effect of Lycopene on Oxidative Stress and Cognitive Decline in Rotenone Induced Model of Parkinson’s Disease

Evidence from clinical and experimental studies indicate that oxidative stress is involved in pathogenesis of Parkinson’s disease. The present study was designed to investigate the neuroprotective potential of lycopene on oxidative stress and neurobehavioral abnormalities in rotenone induced PD. Rats were treated with rotenone (3 mg/kg body weight, intraperitoneally) for 30 days. NADH dehydrogenase a marker of rotenone action was observed to be significantly inhibited (35%) in striatum of treated animals. However, lycopene administration (10 mg/kg, orally) to the rotenone treated animals for 30 days increased the activity by 39% when compared to rotenone treated animals. Rotenone administration increased the MDA levels (75.15%) in striatum, whereas, lycopene administration to rotenone treated animals decreased the levels by 24.33%. Along with this, significant decrease in GSH levels (42.69%) was observed in rotenone treated animals. Lycopene supplementation on the other hand, increased the levels of GSH by 75.35% when compared with rotenone treated group. The activity of SOD was inhibited by 69% in rotenone treated animals and on lycopene supplementation; the activity increased by 12% when compared to controls. This was accompanied by cognitive and motor deficits in rotenone administered animals, which were reversed on lycopene treatment. Lycopene treatment also prevented release of cytochrome c from mitochondria. Collectively, these observations suggest that lycopene supplementation along with rotenone for 30 days prevented rotenone-induced alterations in antioxidants along with the prevention of rotenone induced oxidative stress and neurobehavioral deficits. The results provide an evidence for beneficial effect of lycopene supplementation in rotenone-induced PD and suggest therapeutic potential in neurodegenerative diseases involving accentuated oxidative stress.     

α-Melanocyte-Stimulating Hormone Protects Retinal Vascular Endothelial Cells from Oxidative Stress and Apoptosis in a Rat Model of Diabetes

Aims

Oxidative stress and apoptosis are among the earliest lesions of diabetic retinopathy. This study sought to examine the anti-oxidative and anti-apoptotic effects of α-melanocyte-stimulating hormone (α-MSH) in early diabetic retinas and to explore the underlying mechanisms in retinal vascular endothelial cells.

Methods

Sprague-Dawley rats were injected intravenously with streptozocin to induce diabetes. The diabetic rats were injected intravitreally with α-MSH or saline. At week 5 after diabetes, the retinas were analyzed for reactive oxygen species (ROS) and gene expression. One week later, the retinas were processed for terminal deoxynucleotidyl transferase dUTP nick-end labeling staining and transmission electron microscopy. Retinal vascular endothelial cells were stimulated by high glucose (HG) with or without α-MSH. The expression of Forkhead box O genes (Foxos) was examined through real-time PCR. The Foxo4 gene was overexpressed in endothelial cells by transient transfection prior to α-MSH or HG treatment, and oxidative stress and apoptosis were analyzed through CM-H2DCFDA and annexin-V assays, respectively.

Results

In diabetic retinas, the levels of H₂O₂ and ROS and the total anti-oxidant capacity were normalized, the apoptotic cell number was reduced, and the ultrastructural injuries were ameliorated by α-MSH. Treatment with α-MSH also corrected the aberrant changes in eNOS, iNOS, ICAM-1, and TNF-α expression levels in diabetic retinas. Furthermore, α-MSH inhibited Foxo4 up-regulation in diabetic retinas and in endothelial cells exposed to HG, whereas Foxo4 overexpression abrogated the anti-oxidative and anti-apoptotic effects of α-MSH in HG-stimulated retinal vascular endothelial cells.

Conclusions

α-MSH normalized oxidative stress, reduced apoptosis and ultrastructural injuries, and corrected gene expression levels in early diabetic retinas. The protective effects of α-MSH in retinal vascular endothelial cells may be mediated through the inhibition of Foxo4 up-regulation induced by HG. This study suggests an α-MSH-mediated potential intervention approach to early diabetic retinopathy and a novel regulatory mechanism involving Foxo4.     

Effect of prostaglandins E2 and F2α on in vitro development and hatching of caprine blastocysts

Prostaglandin E₂ has been shown to increase the ovine embryo hatching rate, and PGF_2α to reduce the development of rabbit, bovine, and rat embryos. The objective was to determine the effects of PGE₂ and PGF_2α on development of caprine embryos. Estrus was synchronized in does (n = 25) with medroxyprogesterone acetate (MAP) intravaginal sponges for 12 days, and superovulated with 20 units of FSH. On day 6 following estrus, embryos were flushed (n = 128) and incubated individually per well in 25 μl droplets of TCM-199 and BSA (8 mg/ml) for 6 days at 38.5 °C in a 5% CO₂: air with one of the following treatments: (1) control (0.0002% EtOH), (2) PGE₂ (7 ng/ml), (3) PGF_2α (7 ng/ml), (4) low PGE₂:high PGF_2α (3.5 ng/ml:14 ng/ml), (5) balanced PGE₂:PGF_2α (7 ng/ml:7 ng/ml), or (6) high PGE₂:low PGF_2α (14 ng/ml:3.5 ng/ml). Treatment with PGE₂ alone reduced (P < 0.05) the hatching rate (1/15; 7%). The hatching rate of embryos treated with PGF_2α alone (9/18; 50%), low PGE₂:high PGF_2α (8/16; 50%), and balanced PGE₂:PGF_2α (11/16; 69%) were similar to control (6/18; 33%). In contrast, the hatching rate was non-significantly increased (13/18; 72%) with the high PGE₂:low PGF_2α treatment. None of the treatments affected development from the morula to blastocyst stage. From the current data, it can be concluded that PGE₂ alone reduced hatching rate, and PGF_2α alone had no effect on the development of caprine embryos. High concentrations of PGE₂ with PGF_2α improved the hatching rates. Thus, uterine concentrations of PGE₂ may need to reach a threshold level to improve embryo hatching, as previously reported, while increased uterine concentrations of PGF_2α during early pregnancy would not affect development of the embryo.     

The Suppressive Effect of Resveratrol on HIF-1α and VEGF Expression after Warm Ischemia and Reperfusion in Rat Liver

Background

Hypoxia-inducible factor-1α (HIF-1α) is overexpressed in many human tumors and their metastases, and is closely associated with a more aggressive tumor phenotype. The aim of the present study was to investigate the effect of resveratrol (RES) on the expression of ischemic-induced HIF-1α and vascular endothelial growth factor (VEGF) in rat liver.

Methods

Twenty-four rats were randomized into Sham, ischemia/reperfusion (I/R), and RES preconditioning groups. I/R was induced by portal pedicle clamping for 60 minutes followed by reperfusion for 60 minutes. The rats in RES group underwent the same surgical procedure as I/R group, and received 20 mg/kg resveratrol intravenously 30 min prior to ischemia. Blood and liver tissue samples were collected and subjected to biochemical assays, RT-PCR, and Western blot assays.

Results

I/R resulted in a significant (P<0.05) increase in liver HIF-1α and VEGF at both mRNA and protein levels 60 minutes after reperfusion. The mRNA and protein expressions of HIF-1α and VEGF decreased significantly in RES group when compared to I/R group (P<0.05).

Conclusion

The inhibiting effect of RES on the expressions of HIF-1α and VEGF induced by I/R in rat liver suggested that HIF-1α/VEGF could be a promising drug target for RES in the development of an effective anticancer therapy for the prevention of hepatic tumor growth and metastasis.     

Pivotal Role of the α2A-Adrenoceptor in Producing Inflammation and Organ Injury in a Rat Model of Sepsis

Background

Norepinephrine (NE) modulates the responsiveness of macrophages to proinflammatory stimuli through the activation of adrenergic receptors (ARs). Being part of the stress response, early increases of NE in sepsis sustain adverse systemic inflammatory responses. The intestine is an important source of NE release in the early stage of cecal ligation and puncture (CLP)-induced sepsis in rats, which then stimulates TNF-α production in Kupffer cells (KCs) through the activation of the α₂-AR. It is important to know which of the three α₂-AR subtypes (i.e., α_2A, α_2B or α_2C) is responsible for the upregulation of TNF-α production. The aim of this study was to determine the contribution of α_2A-AR in this process.

Methodology/Principal Findings

Adult male rats underwent CLP and KCs were isolated 2 h later. Gene expression of α_2A-AR was determined. In additional experiments, cultured KCs were incubated with NE with or without BRL-44408 maleate, a specific α_2A-AR antagonist, and intraportal infusion of NE for 2 h with or without BRL-44408 maleate was carried out in normal animals. Finally, the impact of α_2A-AR activation by NE was investigated under inflammatory conditions (i.e., endotoxemia and CLP). Gene expression of the α_2A-AR subtype was significantly upregulated after CLP. NE increased the release of TNF-α in cultured KCs, which was specifically inhibited by the α_2A-AR antagonist BRL-44408. Equally, intraportal NE infusion increased TNF-α gene expression in KCs and plasma TNF-α which was also abrogated by co-administration of BRL-44408. NE also potentiated LPS-induced TNF-α release via the α_2A-AR in vitro and in vivo. This potentiation of TNF-α release by NE was mediated through the α_2A-AR coupled Gαi protein and the activation of the p38 MAP kinase. Treatment of septic animals with BRL-44408 suppressed TNF-α, prevented multiple organ injury and significantly improved survival from 45% to 75%.

Conclusions/Significance

Our novel finding is that hyperresponsiveness to α₂-AR stimulation observed in sepsis is primarily due to an increase in α_2A-AR expression in KCs. This appears to be in part responsible for the increased proinflammatory response and ensuing organ injury in sepsis. These findings provide important feasibility information for further developing the α_2A-AR antagonist as a new therapy for sepsis.