MAPKs and NF-κB-mediated acrylamide-induced neuropathy in rat striatum and human neuroblastoma cells SY5Y

Acrylamide (ACR) is a potent neurotoxin that can be produced during high-temperature food processing, but the underlying toxicological mechanism remains unclear. In this study, the detrimental effects of ACR on the striatal dopaminergic neurons and the roles of mitogen-activated protein kinases (MAPKs) and nuclear factor κB (NF-κB) in ACR-induced neuronal apoptosis were investigated. Acute ACR exposure caused dopaminergic neurons loss and apoptosis as revealed by decreased tyrosine hydroxylase (TH)-positive cells and TH protein level and increased terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive cells in the striatum. ACR-decreased glutathione content, increased levels of malondialdehyde, proinflammatory cytokines tumor necrosis factor α, and interleukin 6. In addition, nuclear NF-κB and MAPKs signaling pathway with c-Jun N-terminal kinase (JNK) and p38 were activated by ACR. Specific inhibitors were used to explore the roles of MAPKs and NF-κB pathways in ACR-induced apoptosis in SH-SY5Y cells. Pretreatment with JNK-specific inhibitors SP600125 markedly upregulated the reduced B-cell lymphoma 2 (Bcl-2) content and downregulated the increased Bcl-2-associated X protein (Bax) level and thereby eventually reduced the proportions of early and late apoptotic cells induced by ACR, while p38 suppression by SB202190 only reversed the decrease in Bcl-2 expression. Inhibition of NF-κB by BAY 11-7082 markedly upregulated Bax level and decreased Bcl-2 expression, and eventually increasing the proportions of neuronal apoptosis compared with that in ACR alone. These results suggested that JNK contributed to ACR-induced apoptosis, while NF-κB acted as a protective regulator in response to ACR-induced neuropathy. This study helps to offer a deeper insight into the mechanism of ACR-induced neuropathy.     

Resveratrol ameliorates oxidative DNA damage and protects against acrylamide-induced oxidative stress in rats

Acrylamide (ACR), used in many fields from industrial manufacturing to laboratory personnel work is also formed during the heating process through interactions of amino acids. Therefore ACR poses a significant risk to human health. This study aimed to elucidate whether resveratrol (RVT) treatment could modulate ACR-induced oxidative DNA damage and oxidative changes in rat brain, lung, liver, kidney and testes tissues. Rats were divided into four groups as control (C); RVT (30 mg/kg i.p. dissolved in 0.9% NaCl), ACR (40 mg/kg i.p.) and RVT + ACR groups. After 10 days rats were decapitated and tissues were excised. 8-hydroxydeoxyguanosine (8-OHdG) is a biomarker of oxidative DNA damage. 8-OHdG content in the extracted DNA solution was determined by enzyme-linked immunosorbent assay method. Malondialdehyde (MDA), glutathione (GSH) levels and myeloperoxidase activity (MPO) were determined in tissues, while oxidant-induced tissue fibrosis was determined by collagen contents. Serum enzyme activities, cytokine levels, leukocyte apoptosis were assayed in plasma. As an indicator of oxidative DNA damage, 8-OHdG levels significantly increased in ACR group and this was reversed significantly by RVT treatment. In ACR group, GSH levels decreased significantly while the MDA levels, MPO activity and collagen content increased in the tissues suggesting oxidative organ damage. In RVT-treated ACR group, oxidant responses reversed significantly. Serum enzyme activities, cytokine levels and leukocyte late apoptosis which increased following ACR administration, decreased with RVT treatment. Therefore supplementing with RVT can be useful in individuals at risk of ACR toxicity.     

Hsp25-induced radioresistance is associated with reduction of death by apoptosis: involvement of Bcl2 and the cell cycle

We previously demonstrated the protective effect of the small heat-shock protein against oxidative damage induced by tumor necrosis factor alpha. Here we have extended our studies of the possible role of Hsp25 in ionizing radiation-induced damage. For these studies, we transfected murine fibroblast L929 cells with the Hsp25 gene and selected three stably transfected clones. Hsp25 overexpression conferred radioresistance as detected by clonogenic survival and induction of apoptosis. Interestingly, the Hsp25-transfected cells showed an increase in the level of the anti-apoptosis molecule Bcl2. We also observed alterations of cell growth in the Hsp25-transfected cells. The cell cycle time of Hsp25-transfected cells was 3-4 h slower than that of vector-transfected control cells. Flow cytometry analysis of synchronized cells at late G(1) phase by mimosine treatment also showed the growth delay in Hsp25-overexpressing cells. In addition, reduced cyclin D1, cyclin A and Cdc2 levels and increased levels of Cdkn1a (also known as p21(Waf)) were observed in Hsp25-transfected cells, which probably caused the reduction in cell growth. In addition, synchronization by mimosine treatment only partially altered radioresistance in the Hsp25-transfected cells. Taken together, these data suggest that Hsp25-induced radioresistance is associated with growth delay as well as induction of Bcl2.     

Protective Effect of Fish Oil on Changes in the Activities of Membrane-Bound ATPases and Mineral Status in Experimentally Induced Myocardial Infarction in Wistar Rats

The present study evaluated the protective effect of fish oil in isoproterenol-induced myocardial infarction in rats. The results of the present study indicate that the IPH administration decreases the activities of membrane-bound ATPases compared to control animals. Fish oil pretreatment brought about significant increase in the activity of these membrane-bound ATPases in IPH (isoproterenol hydrochloride)-treated animals. Significant increase in serum potassium level with concomitant decrease in the values of sodium, magnesium, and calcium were observed in IPH-treated rats compared to control rats, fish oil pretreatment reversed these changes to near normal. Significant elevation of sodium and calcium levels with concomitant decrease in the levels of potassium and magnesium were observed in the myocardial tissue of IPH-administered rats compared to control rats, fish oil pretreatment followed by IPH administration brought these levels to near normal. The levels of lipid peroxidation (LPO) in both serum and tissue were increased in IPH-treated rats compared with control rats, whereas pretreatment with fish oil in IPH-treated rats maintained near-normal LPO levels. The results of the present study reveals that the pretreatment of fish maintains the activities of membrane-bound ATPases and the mineral levels at near normal by the inhibition of lipid peroxidation     

Neuroprotective Efficacy of Eugenol and Isoeugenol in Acrylamide-Induced Neuropathy in rats: Behavioral and Biochemical evidence

The primary objective of this investigation was to assess the neuroprotective efficacy of spice active principles namely Eugenol (Eug) and isoeugenol (IE) in an acrylamide (ACR) neuropathy model in rats. In the present study, ACR administration (50 mg/kg bw, i.p. 3 times/week) for 5 weeks to growing rats caused typical symptoms of neuropathy. We found that treatment of ACR rats with spice active principles (10 mg/kg bw, for 5 weeks) caused marked improvement in gait score and responses in a battery of behavioral tests. Terminally, both spice active principles markedly attenuated ACR-induced markers of oxidative stress viz., reactive oxygen species (ROS), malondialdehyde (MDA) and nitric oxide (NO) in sciatic nerve (SN) as well as brain regions (cortex Ct, cerebellum Cb). Treatment with Eug restored the reduced glutathione levels in SN and brain regions. Interestingly, both spice active principles effectively diminished ACR-induced elevation in cytosolic calcium levels and acetylcholinesterase activity in SN and Ct. Further, the diminished activity of ATPase among ACR rats was enhanced in SN and restored in brain regions. Furthermore, Eug treatment significantly offset ACR-induced depletion in dopamine levels in brain regions. Collectively our findings suggest the propensity of these spice active principles to attenuate ACR-induced neuropathy. Further studies are necessary to understand the precise molecular mechanism/s by which these spice active principles attenuate neuropathy. Nevertheless, our data clearly demonstrate the beneficial effects of spice active principles in ACR-induced neuropathy in rats and suggest their possible therapeutic usage as an adjuvant in the management of other forms of neuropathy in humans.     

Neuroprotective effect of naringin, a dietary flavonoid against 3-nitropropionic acid-induced neuronal apoptosis

The aim of this study was to investigate the protective effect of naringin, a flavonoid on 3-Nitropropionic acid (3-NP)-induced neurodegeneration through the modulation of intrinsic apoptotic cascade in Wistar rats. 3-NP is an irreversible inhibitor of complex II in the mitochondria. 3-NP-induced neurodegeneration has been widely used as an animal model of Huntington’s disease (HD). Increased oxidative stress is one of the major deleterious events in 3-NP-induced neuronal apoptosis. Rats administered with 3-NP showed increase in the levels of lipid peroxidation and protein carbonyl, which was significantly decreased upon naringin treatment (80 mg/kg body weight). 3-NP-induced rats showed decrease in the activities of enzymic antioxidants and reduced levels of non-enzymic antioxidants. Naringin treatment ameliorated the antioxidant status by increasing the activities of enzymic antioxidants and the levels of non-enzymatic antioxidants. 3-NP-induced rats showed decrease in the activities of ATPases in striatum, which was restored to normal level upon naringin treatment. Histopathological observation of the striatal tissue showed protective role of naringin in 3-NP-induced rats. Naringin also reduced the 3-NP-induced apoptosis via decrease in the cytochrome c release from mitochondria and caspase 3 activation as revealed by Western blot. Naringin treatment also decreased the expressions of pro-apoptotic markers like Bad and Bax. Further, naringin antagonized 3-NP-induced decrease in Bcl-2 mRNA expression. The results of this study show evidence on the neuroprotective effect of naringin against 3-NP-induced neuronal apoptosis through its antioxidant and anti-apoptotic effects.     

Neuroprotective Effect of Crocin on Acrylamide-induced Cytotoxicity in PC12 cells

Acrylamide (ACR) is a potent neurotoxic in human and animal models. In this study, the effect of crocin, main constituent of Crocus sativus L. (Saffron) on ACR-induced cytotoxicity was evaluated using PC12 cells as a suitable in vitro model. The exposure of PC12 cells to ACR reduced cell viability, increased DNA fragmented cells and phosphatidylserine exposure, and elevated Bax/Bcl-2 ratio. Results showed that ACR increased intracellular reactive oxygen species (ROS) in cells and ROS played an important role in ACR cytotoxicity. The pretreatment of cells with 10–50 μM crocin before ACR treatment significantly attenuated ACR cytotoxicity in a dose-dependent manner. Crocin inhibited the downregulation of Bcl-2 and the upregulation of Bax and decreased apoptosis in treated cells. Also, crocin inhibited ROS generation in cells exposed to ACR. In conclusion, our results indicated that pretreatment with crocin protected cells from ACR-induced apoptosis partly by inhibition of intracellular ROS production.     

Fish oil up-regulates hepatic autophagy in rats with chronic ethanol consumption

In this study, we examined the regulation of autophagy by fish oil in rats under ethanol-containing diets. Thirty male Wistar rats (8-week-old) were divided into six groups and fed a control diet or an ethanol-containing diet, which was adjusted with fish oil to replace 25% or 57% of the olive oil. After 8 weeks, rats in the E (ethanol diet) group showed the significantly higher plasma aspartate transaminase (AST) and alanine transaminase (ALT) activities, protein expression of cytochrome P450 2E1 (CYP2E1), and levels of hepatic inflammatory cytokines. However, all of those items had significantly decreased in the EF25 (ethanol with 25% fish oil) and EF57 (ethanol with 57% fish oil) groups. As to autophagic indicators, protein expressions of mammalian target of rapamycin (mTOR), Unc-51-like autophagy activating kinase 1 (ULK1) and p62 were significantly increased in the E group. Conversely, the protein expressions of light chain 3II (LC3II)/LC3I and Beclin1 were significantly decreased in the E group. On the other hand, protein expressions of phosphorylated Akt, mTOR, ULK1, and p62 were down-regulated, protein expressions of LC3II/LC3I and Beclin1 were conversely up-regulated in the EF25 and EF57 groups. Fish oil activated hepatic autophagy via inhibiting the Akt signaling pathway, which exerted protective effects against ethanol-induced liver injury in rats.     

Low ambient temperature reveals distinct mechanisms for MDMA-induced serotonergic toxicity and astroglial Hsp27 heat shock response in rat brain

3,4-Methylenedioxymethamphetamine (MDMA, ‘ecstasy’) is a widely used recreational drug known to cause selective long-term serotonergic damage. In our recent paper we described region-specific, dose-dependent increase in the protein expression of astroglial Hsp27 and neuronal Hsp72 molecular chaperones after MDMA administration of rats. Here, we examined the possible interaction of elevated Hsp27 protein level to hyperthermic responses after MDMA administration and its separation from drug-induced serotonergic neurotoxicity. For this, 7-8 week old male Dark Agouti rats were treated with 15 mg/kg i.p. MDMA. Treatment at an ambient temperature of 22 ± 1 °C caused a significant elevation of the rectal temperature, an increase of Hsp27 immunoreactive protoplasmic astrocytes in the hippocampus, the parietal and cingulate cortices, and a significant decrease in the density of tryptophan hydroxylase immunoreactive fibers in the same brain regions, 8 h as well as 24 h after drug administrations. In addition, serotonergic axons exhibited numerous swollen varicosities and fragmented morphology. MDMA treatment at low ambient temperature (10 ± 2 °C) almost completely abolished the elevation of body temperature and the increased astroglial Hsp27 expression but failed to alter - or just slightly attenuated - the depletion in the density of tryptophan hydroxylase immunoreactive fibers. These results suggest that the increased astroglial Hsp27 protein expression is rather related to the hyperthermic response after the drug administration and it could be separated from the serotonergic neurotoxicity caused by MDMA. In addition, the induction of Hsp27 per se is uneffective to protect serotonergic fibers after MDMA administration. Our results also suggest that Tph immunohistochemistry is an early and sensitive method to demonstrate MDMA-caused vulnerability.     

Modulation of heat shock protein response in SH-SY5Y by mobile phone microwaves

AIM: To investigate putative biological damage caused by GSM mobile phone frequencies by assessing electromagnetic fields during mobile phone working. METHODS: Neuron-like cells, obtained by retinoicacid-induced differentiation of human neuroblastoma SH-SY5Y cells, were exposed for 2 h and 4 h to microwaves at 1800 MHz frequency bands. RESULTS: Cell stress response was evaluated by MTT assay as well as changes in the heat shock protein expression (Hsp20, Hsp27 and Hsp70) and caspase-3 activity levels, as biomarkers of apoptotic pathway. Under our experimental conditions, neither cell viability nor Hsp27 expression nor caspase-3 activity was significantly changed. Interestingly, a significant decrease in Hsp20 expression was observed at both times of exposure, whereas Hsp70 levels were significantly increased only after 4 h exposure. CONCLUSION: The modulation of the expression of Hsps in neuronal cells can be an early response to radiofrequency microwaves.     

Evidence for a role of Hsp70 in the neuroprotection induced by heat shock pre-treatment against 3,4-methylenedioxymethamphetamine toxicity in rat brain

3,4-Methylenedioxymethamphetamine (MDMA, 'ecstasy') produces acute hyperthermia which increases the severity of the selective serotoninergic neurotoxicity produced by the drug in rats. Heat shock protein 70 (Hsp70) is a major inducible cellular protein expressed in stress conditions and which is thought to exert protective functions. MDMA (12.5 mg/kg, i.p.), given to rats housed at 22 degrees C, produced an immediate hyperthermia and increased Hsp70 in frontal cortex between 3 h and 7 days after administration. MDMA, given to rats housed at low ambient temperature (4 degrees C) produced transient hypothermia followed by mild hyperthermia but no increase in Hsp70 expression, while rats treated at elevated room temperature (30 degrees C) showed enhanced hyperthermia and similar expression of Hsp70 to that seen in rats housed at 22 degrees C. Fluoxetine-induced inhibition of 5-HT release and hydroxyl radical formation did not modify MDMA-induced Hsp70 expression 3 h later. Four- or 8-day heat shock (elevation of basal rectal temperature by 1.5 degrees C for 1 h) or geldanamycin pre-treatment induced Hsp70 expression and protected against MDMA-induced serotoninergic neurotoxicity without affecting drug-induced hyperthermia. Thus, MDMA-induced Hsp70 expression depends on the drug-induced hyperthermic response and not on 5-HT release or hydroxyl radical formation and pre-induction of Hsp70 protects against the long-term serotoninergic damage produced by MDMA.     

Blood coagulation and fibrinolysis of rats fed fish oil: reduced coagulation factors especially involved in intrinsic pathway and increased activity of plasminogen activator inhibitor

Differences in the coagulation and fibrinolytic system of rats fed a fish oil based diet (fish oil diet) and fed a soybean oil based diet (control diet) were determined. Concentrations of plasma lipids were depressed in rats fed the fish oil diet. Prothrombin time (PT) and activated partial thromboplastin time (APTT) of rats fed the fish oil diet were longer than for the rats fed the control diet. Fish oil intake lowered the activities of most of the blood coagulation factors, and strongly depressed the factors involved in the intrinsic pathway. Fish oil also affected the fibrinolysis of rats. Plasminogen activator inhibitor (PAI) activity was elevated in rats fed the fish oil diet. In this study, both blood coagulation and fibrinolysis were down-regulated by feeding the fish oil diet.     

Pro-apoptotic signaling in neuronal cells following iron and amyloid beta peptide neurotoxicity

In a previous report, we characterized several oxidative stress parameters during the course of amyloid beta (Abeta) peptide/Fe2+-induced apoptotic death in neuronal cells. In extending these findings, we now report a marked decrease in protein kinase C (PKC) isoforms, reduced Akt serine/threonine kinase activity, Bcl 2-associated death promoter (BAD) phosphorylation and enhanced p38 mitogen-activated protein kinase (MAPK) and caspase-9 and -3 activation, 12 h after addition of both 5 micro m Abeta and 5 micro m Fe2+. These activities reminiscent for a pro-apoptotic cellular course were blocked in the presence of the iron chelator deferroxamine. Abeta alone, increased PKC isoform levels between three- and four-fold after 12 h, enhanced Akt activity approximately eight-fold and Ser136 BAD phosphorylation two-fold, suggesting that by itself is not toxic. Fe2+ alone transiently enhanced p38 MAPK and caspase-9 and -3 enzymes indicative for cell damage, but was not sufficient to cause cell death as previously indicated. GF, a PKC inhibitor or wortmannin, a blocker of the Akt pathway enhanced Abeta/Fe2+-induced toxicity, while SB, a p38 MAPK inhibitor, prevented cell damage and apoptosis. These findings further support the hypothesis that metal ion chelation and inhibitors of pro-apoptotic kinase cascades may be beneficial for Alzheimer's disease therapy.     

Down‐regulation of miRNA‐27b‐3p suppresses keratinocytes apoptosis in oral lichen planus

Oral lichen planus (OLP) is considered a precancerous lesion with no known cure. Recent studies reported that abnormal regulation of apoptosis was involved in the pathogenesis of OLP. Next generation sequencing was used to screen the candidate microRNAs and genes in biopsies from patients with OLP and healthy mucosa. Human oral keratinocytes were transfected into the related oligonucleotides of miR‐27b‐3p/cyclophilin D and their control groups. Apoptosis was detected by TdT‐mediated dUTP nick end labelling and flow cytometry. The levels of mRNA and protein were detected by quantitative PCR, Western blots, and enzyme‐linked immunosorbent assays, respectively. Luciferase assays were performed to detect the luciferase activities of miR‐27b‐3p and cyclophilin D. Here, we showed that basal epithelium apoptosis was reduced and the miR‐27b‐3p levels were decreased in clinical OLP samples. We also found that down‐regulation of miR‐27b‐3p inhibited epithelial keratinocyte apoptosis by up‐regulating cyclophilin D expression. Moreover, cyclophilin D increased the protein stability of Bcl2 through direct binding, and Bcl2 suppressed caspase9/3 activation and cytochrome C release. Taken together, these data showed that miR‐27b‐3p regulated keratinocyte apoptosis through cyclophilin D/Bcl2 signalling, suggesting the miR‐27b‐3p regulated the pathogenesis of OLP.     

Oral Presentations OP01: Neurodegenerative Diseases. BCL‐W Plays a key neuroprotective role in Alzheimer's disease

The mechanisms underlying neuronal degeneration in Alzheimer's disease (AD) are very controversial and none more so than whether apoptosis plays a role. Although neurons in AD face a wide assortment of apoptogenic stimuli, the temporal dichotomy between the acuteness of apoptosis vs. the chronicity of AD suggests that apoptosis should be extremely rare in AD. In this regard, survival factor(s) must be involved. In this study, we investigated Bcl‐w, a pro‐survival member of the Bcl‐2 family. Although expressed at low levels in brains of control cases, Bcl‐w is significantly up‐regulated in AD as shown by both immunocytochemistry and immunoblot analysis. Astonishingly, increased Bcl‐w was found to be associated with neurofibrillary pathologies in AD, which was further demonstrated by an EM study. Since neuronal death in AD is thought to be triggered by increased production of amyloid‐β (Aβ), it was interesting to find that exposure of human M17 neuroblastoma cells to Aβ1–42 (1 nm ?10 μm ) dramatically up‐regulates Bcl‐w protein levels. Such increases may be a protective response that attenuates apoptotic processes. Consistent with this, transfected M17 cells overexpressing Bcl‐w were protected from both STS‐induced and Aβ‐induced apoptosis compared to vector‐transfected controls. Notably, both tau phosphorylation and p38 is inhibited in Bcl‐w transfected cells which may contribute to the neuroprotective role of Bcl‐w. Taken together, these set of in vitro and in vivo results suggest that Bcl‐w plays an important protective role in neurons in the AD brain.