Ikram Ullah

Neuroprotection with metformin and thymoquinone against ethanol-induced apoptotic neurodegeneration in prenatal rat cortical neurons.

BackgroundExposure to ethanol during early development triggers severe neuronal death by activating multiple stress pathways and causes neurological disorders, such as fetal alcohol effects or fetal alcohol syndrome. This study investigated the effect of ethanol on intracellular events that predispose developing neurons for apoptosis via calcium-mediated signaling. Although the underlying molecular mechanisms of ethanol neurotoxicity are not completely determined, mitochondrial dysfunction, altered calcium homeostasis and apoptosis-related proteins have been implicated in ethanol neurotoxicity. The present study was designed to evaluate the neuroprotective mechanisms of metformin (Met) and thymoquinone (TQ) during ethanol toxicity in rat prenatal cortical neurons at gestational day (GD) 17.5.ResultsWe found that Met and TQ, separately and synergistically, increased cell viability after ethanol (100 mM) exposure for 12 hours and attenuated the elevation of cytosolic free calcium [Ca2+]c. Furthermore, Met and TQ maintained normal physiological mitochondrial transmembrane potential (ΔψM), which is typically lowered by ethanol exposure. Increased cytosolic free [Ca2+]c and lowered mitochondrial transmembrane potential after ethanol exposure significantly decreased the expression of a key anti-apoptotic protein (Bcl-2), increased expression of Bax, and stimulated the release of cytochrome-c from mitochondria. Met and TQ treatment inhibited the apoptotic cascade by increasing Bcl-2 expression. These compounds also repressed the activation of caspase-9 and caspase-3 and reduced the cleavage of PARP-1. Morphological conformation of cell death was assessed by TUNEL, Fluoro-Jade-B, and PI staining. These staining methods demonstrated more cell death after ethanol treatment, while Met, TQ or Met plus TQ prevented ethanol-induced apoptotic cell death.ConclusionThese findings suggested that Met and TQ are strong protective agents against ethanol-induced neuronal apoptosis in primary rat cortical neurons. The collective data demonstrated that Met and TQ have the potential to ameliorate ethanol neurotoxicity and revealed a possible protective target mechanism for the damaging effects of ethanol during early brain development.

Anthocyanins Protect Against Ethanol-Induced Neuronal Apoptosis via GABAB1 Receptors Intracellular Signaling in Prenatal Rat Hippocampal Neurons

Here, we investigated the possible involvement of gamma-aminobutyric acid B1 receptor (GABAB1R) in mediating the protective effects of black soybean anthocyanins against ethanol-induced apoptosis in prenatal hippocampal neurons because GABARs are known to play an important role in the development of central nervous system. Treatments were performed on primary cultures of prenatal rat hippocampal neurons transfected with or without GABAB1R small interfering RNA (siRNA). The results showed that, when ethanol treatment was followed by anthocyanins treatment, cellular levels of proapoptotic proteins such as Bax, activated caspase-3, and cleaved poly (ADP-ribose) polymerase 1 (PARP-1) were decreased, and the cellular level of the antiapoptotic protein Bcl-2 was increased compared to treatment with ethanol alone. Furthermore, the effects of ethanol on cellular levels of GABAB1R and its downstream signaling molecules such as protein kinase A, calcium/calmodulin-dependent protein kinase II (CaMKII), and phosphorylated cAMP response element binding protein were diminished or reversed by anthocyanins treatment. The ability of anthocyanins to reverse the effects of ethanol on cellular levels of Bax, Bcl-2, active caspase-3, cleaved PARP-1, GABAB1R, and CaMKII were abrogated in cells transfected with GABAB1R siRNA. In a GABAB1R-dependent manner, anthocyanins also inhibited the ability of ethanol to elevate intracellular free Ca2+ level and increase the proportion of cells with low mitochondrial membrane potential in the population. Cell apoptosis assay and morphological studies also confirmed the neuroprotective effect of anthocyanins against ethanol via GABAB1R. Our data suggest that GABAB1R plays an important role in the neuroprotective effects of anthocyanins against ethanol.

Vitamin C protects against ethanol and PTZ‐induced apoptotic neurodegeneration in prenatal rat hippocampal neurons

Exposure to alcohol during brain development may cause a neurological syndrome called fetal alcohol syndrome, characterized by pre‐ and postnatal growth deficiencies, craniofacial anomalies, and evidence of CNS dysfunction. The objective of this study was to evaluate pentylenetetrazol (PTZ) and ethanol effects on Bax, Bcl‐2 expression, which further induced activation of caspase‐3, release of cytochrome‐c from mitochondria, and to observe the protective effects of vitamin C (vit‐C) against PTZ and ethanol‐induced apoptotic neurodegeneration in primary‐cultured neuronal cells at gestational day 17.5. Apoptotic neurodegeneration and neuroprotective effect of vit‐C were measured by using 3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenly tetrazolium bromide assay, Western blot analysis, which further conformed by the measurement of mitochondrial membrane potential using JC‐1 detection kit and immunofluorescence analysis. The results showed that PTZ and ethanol produced extensive Bax‐dependent caspase‐9 and caspase‐3 activation and caused neuronal apoptosis. Furthermore, the cotreatment of vit‐C along with ethanol and PTZ showed significantly decreased expression of Bax, caspase‐9, caspase‐3, cytochrome‐c, and significantly increased expression of antiapoptotic Bcl‐2 protein when compared with control group. Our findings indicate that PTZ and ethanol activate an intrinsic apoptotic death program in neurons that is likely to contribute to the neuropathologic effects in fetal alcohol exposure, and vit‐C can prevent some of the deleterious effects of PTZ and ethanol on the developing brain. The available experimental evidence and the safety of vit‐C in pregnancy suggest the experimental use of ascorbic acid as a new and effective protective agent ethanol and PTZ‐induced apoptotic neurodegeneration. Synapse, 2011.

Protective effect of pyruvate against ethanol-induced apoptotic neurodegeneration in the developing rat brain

Exposure to alcohol during the early stages of brain development can lead to neurological disorders in the CNS. Apoptotic neurodegeneration due to ethanol exposure is a main feature of alcoholism. Exposure of developing animals to alcohol (during the growth spurt period in particular) elicits apoptotic neuronal death and causes fetal alcohol effects (FAE) or fetal alcohol syndrome (FAS). A single episode of ethanol intoxication (at 5 g/kg) in a seven-day-old developing rat can activate the apoptotic cascade, leading to widespread neuronal death in the brain. In the present study, we investigated the potential protective effect of pyruvate against ethanol-induced neuroapoptosis. After 4h, a single dose of ethanol induced upregulation of Bax, release of mitochondrial cytochrome-c into the cytosol, activation of caspase-3 and cleavage of poly (ADP-ribose) polymerase (PARP-1), all of which promote apoptosis. These effects were all reversed by co-treatment with pyruvate at a well-tolerated dosage (1000 mg/kg). Histopathology performed at 24 and 48 h with Fluoro-Jade-B and cresyl violet stains showed that pyruvate significantly reduced the number of dead cells in the cerebral cortex, hippocampus and thalamus. Immunohistochemical analysis at 24h confirmed that ethanol-induced cell death is both apoptotic and inhibited by pyruvate. These findings suggest that pyruvate treatment attenuates ethanol-induced neuronal cell loss in the developing rat brain and holds promise as a safe therapeutic and neuroprotective agent in the treatment of neurodegenerative disorders in newborns and infants.

Anthocyanins attenuate body weight gain via modulating neuropeptide Y and GABAB1 receptor in rats hypothalamus

Anthocyanins in a variety of plant species have been identified and are known for its hypolipidemic and anti-obesity effects. The effect of anthocyanins extracted from black soybean on body weight and daily food intake in adult rats raised on normal diet were studied. Male Sprague-Dawley rats were daily intra-gastric administered water or anthocyanins 6 mg/kg and 24 mg/kg for 40 days. During this period daily food intake and body weight were measured prior to anthocyanins treatment. These findings showed that anthocyanins treatment resulted in significantly lowered body weight and food intake compared with water treated rats. In addition, anthocyanins dose dependently reduced the adipose tissue size compared with control group. Western blot analysis showed that high dose of anthocyanins treatment significantly reduced the expression of neuropeptide Y (NPY) and increased γ-amino butyric acid receptor (GABAB1R) in hypothalamus. Furthermore, these events were followed by a decreased in expression of GABAB1R downstream signaling molecules protein kinase A-α (PKA) and phosphorylated cAMP-response element binding protein (p-CREB) in hypothalamus. These data support the concept that anthocyanins even in normal circumstances have the capability to reduce body weight and food intake through its modulatory effect on NPY and GABAB1R in hypothalamus. These results suggest that anthocyanins from black soybean seed coat might have a novel role in preventing obesity in rats on normal diet.

Anthocyanins Protect against Kainic Acid‐induced Excitotoxicity and Apoptosis via ROS‐activated AMPK Pathway in Hippocampal Neurons

Excitotoxicity is an important mechanism involved in neurodegeneration. Kainic acid (KA)‐induced excitotoxicity results an unfavorable stress, and we investigated the signaling pathways activated in such conditions.

Ethanol and PTZ effects on siRNA-mediated GABAB1 receptor: Down regulation of intracellular signaling pathway in prenatal rat cortical and hippocampal neurons

GABAB receptors (R) are widely expressed and distributed in the nervous system, and have been implicated in variety of neurodegenerative and pathophysiological disorders. However, the exact molecular mechanism regarding responsibility of GABAB1R in downstream signaling pathway is not well understood. The present study was undertaken to explore the downstream signaling and role of GABAB1R upon acute ethanol and pentylenetetrazol (PTZ) exposure for (20 min) in cortical and hippocampal neuronal cell cultures by using GABAB1R RNA interference (i) (30 nM, 48 h) at gestational days 17.5. The results showed that GABAB1R and protein kinase A‐α (PKA) showed decreased expression upon ethanol and PTZ exposure in cortical and hippocampal neurons during transfected and nontransfected conditions, whereas these effects could lead to significant changes in phosphorylation of cAMP‐response element binding protein (p‐CREB) expression where GABAB1R was knocked down. Furthermore, intracellular Ca+2 concentrations were also reduced in some groups after transfection with GABAB1R RNAi. These results showed a critical role of GABAB1R upon ethanol and PTZ exposure by modulating downstream signaling pathway. Finally, these findings suggested that inhibition of GABAB1R results in the modulation of PKA, p‐CREB pathway may play a role in long‐term changes in the nervous system, and may be an underlying cause of ethanols effects. Synapse 64:181–190, 2010.

Nicotinamide Inhibits Alkylating Agent-Induced Apoptotic Neurodegeneration in the Developing Rat Brain

Background Exposure to the chemotherapeutic alkylating agent thiotepa during brain development leads to neurological complications arising from neurodegeneration and irreversible damage to the developing central nerve system (CNS). Administration of single dose of thiotepa in 7-d postnatal (P7) rat triggers activation of apoptotic cascade and widespread neuronal death. The present study was aimed to elucidate whether nicotinamide may prevent thiotepa-induced neurodegeneration in the developing rat brain. Methodology/Principal Findings Neuronal cell death induced by thiotepa was associated with the induction of Bax, release of cytochrome-c from mitochondria into the cytosol, activation of caspase-3 and cleavage of poly (ADP-ribose) polymerase (PARP-1). Post-treatment of developing rats with nicotinamide suppressed thiotepa-induced upregulation of Bax, reduced cytochrome-c release into the cytosol and reduced expression of activated caspase-3 and cleavage of PARP-1. Cresyl violet staining showed numerous dead cells in the cortex hippocampus and thalamus; post-treatment with nicotinamide reduced the number of dead cells in these brain regions. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) and immunohistochemical analysis of caspase-3 show that thiotepa-induced cell death is apoptotic and that it is inhibited by nicotinamide treatment. Conclusion Nicotinamide (Nic) treatment with thiotepa significantly improved neuronal survival and alleviated neuronal cell death in the developing rat. These data demonstrate that nicotinamide shows promise as a therapeutic and neuroprotective agent for the treatment of neurodegenerative disorders in newborns and infants.

Bacteria from marine sponges: A source of new drugs.

BACKGROUND Sponges are rich source of bioactive natural products synthesized by the symbiotic bacteria belonging to different phyla. Due to a competition for space and nutrients the marine bacteria associated with sponges could produce more antibiotic substances. To explore the proactive potential of marine microbes extensive research has been done. These bioactive metabolites have some unique properties that are pharmaceutically important. METHODS For this review, we have performed a non-systematic search of the available literature though various online search engines. This review provides an insight that how majority of active metabolites have been identified from marine invertebrates of which sponges predominate. RESULTS Sponges harbor abundant and diverse microorganisms, which are the sources of a range of marine bioactive metabolites. From sponges and their associated microorganisms, approximately 5,300 different natural compounds are known. Current research on sponge-microbe interaction and their active metabolites has become a focal point for many researchers. Various active metabolites derived from sponges are now known to be produced by their symbiotic microflora. CONCLUSION In this review, we attempt to report the latest studies regarding capability of bacteria from sponges as producers of bioactive metabolite. Moreover, these sponge associated bacteria are an important source of different enzymes of industrial significance. In present review, we will address some novel approaches for discovering marine metabolites from bacteria that have the greatest potential to be used in clinical treatments.

siRNA-mediated GABAB receptor at early fetal rat brain upon acute and chronic ethanol exposure: Down regulation of PKA and p-CREB expression

To observe the modulatory role of GABAB1R upon ethanols effect during early brain development, we studied the effects of chronic maternal (10% ethanol during pregnancy) and acute (in vitro) ethanol exposure on the neuronal protein kinase A (PKA‐α) and phosphorylation of cAMP‐response element binding protein (p‐CREB), using a system where GABAB1R were specifically knocked down in the primary cells cultured at gestational day (GD) 12.5. The results showed that upon acute and chronic ethanol treatment the GABAB1R expression was decreased and further decreased when GABAB1R was transfection with siRNA, while increased upon exposure of baclofen, and baclofen plus phaclofen treatment. PKA expression was also decreased with acute and chronic ethanol treatment, whereas it showed increase upon exposure of baclofen and baclofen with phaclofen. Furthermore, intracellular Ca2+ concentration was increased upon ethanol, baclofen, phaclofen exposure but showed decrease in GABAB1R siRNA group. Finally, these effects could lead to changes of p‐CREB expression, which showed same expression pattern as PKA. We speculate that GABABR activity upon ethanol exposure could modulate intracellular calcium homeostasis and the expressional changes of PKA and p‐CREB, which cause various negative effects on fetal brain development and modulation of GABABR upon ethanol exposure may underlying cause of ethanols effects. Synapse, 2011.