Dinakar Sasmal

Pharmacological evaluation of glutamate transporter 1 (GLT-1) mediated neuroprotection following cerebral ischemia/reperfusion injury ☆

Recently glutamate transporters have emerged as a potential therapeutic target in a wide range of acute and chronic neurological disorders, owing to their novel mode of action. The modulation of GLT-1, a major glutamate transporter has been shown to exert neuroprotection in various models of ischemic injury and motoneuron degeneration. Therefore, an attempt was made to explore its neuroprotective potential in cerebral ischemia/reperfusion injury using ceftriaxone, a GLT-1 modulator. Pre-treatment with ceftriaxone (100mg/kg. i.v) for five days resulted in a significant reduction (P<0.01) in neurological deficit as well as cerebral infarct volume after 1h of ischemia followed by 24h of reperfusion injury. It also caused a significant (P<0.05) upregulation of GLT-1 mRNA, protein and glutamine synthetase (GS) activity. Furthermore, inhibition of ceftriaxone-mediated increased glutamine synthetase activity by dihydrokainate (DHK), a GLT-1 specific inhibitor, confirms the specific effect of ceftriaxone on GLT-1 activity. In addition, ceftriaxone also induced a significant (P<0.01) increase in [(3)H]-glutamate uptake, mediated by GLT-1 in glial enriched preparation, as evidenced by use of DHK and DL-threo-beta-benzyloxyaspartate (DL-TBOA). Thus, the present study provides overwhelming evidence that modulation of GLT-1 protein expression and activity confers neuroprotection in cerebral ischemia/reperfusion injury.

Development of selective and reversible pyrazoline based MAO-B inhibitors: virtual screening, synthesis and biological evaluation.

In an effort to develop selective MAO (monoamine oxidase) B inhibitors, structure based virtual screening was initiated on an in-house library. Top 10 HITS were synthesized and evaluated for MAO (A and B) inhibitory activity, both against human and rat enzymes. All the compounds were found selective, reversible and active in nM range (100 times more potent than selegeline) towards MAO-B. Outstanding co-relation between predicted and experimental K(i) values were observed.

Deltamethrin induced an apoptogenic signalling pathway in murine thymocytes : exploring the molecular mechanism.

Deltamethrin (DLM) is a well‐known pyrethroid insecticide; however, the immunotoxic effects of DLM on the mammalian system and its mechanism is still unclear. This study has been designed to first observe the binding affinity of DLM to immune cell receptors and its effects on the immune system. The docking score revealed that DLM has a strong binding affinity towards the CD4 and CD8 receptors. DLM induces apoptosis in murine thymocytes in a concentration‐dependent manner. The ear\ly markers of apoptosis such as enhanced reactive oxygen species (ROS) and caspase‐3 activation are evident as early as 1 h by 25 and 50 μM DLM. Glutathione (GSH) depletion has also been observed at 1 h by 50 μM DLM concentration. In cell‐cycle studies using flow cytometry, the fraction of hypodiploid cells has gradually increased with all the concentrations of DLM at 18 h. The Annexin V binding assay measures the effect of DLM on apoptotic and necrotic cells. The apoptotic cells raised from 18.6% to 35.21% (10–50 μM DLM) at 18 h. N‐acetyl cysteine (NAC) effectively reduced the percentage of apoptotic cells which is increased by DLM. In contrast, buthionine sulfoximine (BSO) caused an elevation in the percentage of apoptotic cells. These results demonstrate that caspase activation, ROS activation and GSH act as critical mediators in a DLM‐induced apoptogenic signalling pathway in murine thymocytes. In the presence of caspase inhibitor, the percentage of apoptotic cells is partially decreased. Thus, there may be the possibility of some other caspase‐independent pathways in DLM‐induced apoptosis. Copyright

Evaluation of immunomodulatory activity of Glycyrhiza glabra L roots in combination with zing

Objective To determine the immunomodulatory properties of roots of Glycyrrhiza glabra (G. glabra) L. (Liquorice), and also to check whether significant potentiation in immunomodulation occurs or not with the combination of zinc.

Deltamethrin-induced oxidative stress and mitochondrial caspase-dependent signaling pathways in murine splenocytes

Deltamethrin (DLM) is a well‐known pyrethroid insecticide used extensively in pest control. Exposure to DLM has been demonstrated to cause apoptosis in various cells. However, the immunotoxic effects of DLM on mammalian system and its mechanism is still an open question to be explored. To explore these effects, this study has been designed to first observe the interactions of DLM to immune cell receptors and its effects on the immune system. The docking score revealed that DLM has strong binding affinity toward the CD45 and CD28 receptors. In vitro study revealed that DLM induces apoptosis in murine splenocytes in a concentration‐dependent manner. The earliest markers of apoptosis such as enhanced reactive oxygen species and caspase 3 activation are evident as early as 1 h by 25 and 50 µM DLM. Western blot analysis demonstrated that p38 MAP kinase and Bax expression is increased in a concentration‐dependent manner, whereas Bcl 2 expression is significantly reduced after 3 h of DLM treatment. Glutathione depletion has been also observed at 3 and 6 h by 25 and 50 µM concentration of DLM. Flow cytometry results imply that the fraction of hypodiploid cells has gradually increased with all the concentrations of DLM at 18 h. N‐acetyl cysteine effectively reduces the percentage of apoptotic cells, which is increased by DLM. In contrast, buthionine sulfoxamine causes an elevation in the percentage of apoptotic cells. Phenotyping data imply the effect of DLM toxicity in murine splenocytes. In brief, the study demonstrates that DLM causes apoptosis through its interaction with CD45 and CD28 receptors, leading to oxidative stress and activation of the mitochondrial caspase‐dependent pathways which ultimately affects the immune functions. This study provides mechanistic information by which DLM causes toxicity in murine splenocytes.

Aqueous extract of Boerhaavia diffusa root ameliorates ethylene glycol-induced hyperoxaluric oxidative stress and renal injury in rat kidney.

Introduction: Boerhaavia diffusa Linn. (Nyctaginaceae) is widely used in traditional Indian medicines against renal afflictions including calcium oxalate (CaOx) urolithiasis and is known for antioxidant activity. Objective: The present study was designed to investigate the ameliorating effect of aqueous extract of B. diffusa roots (BDE) in hyperoxaluric oxidative stress and renal cell injury. Material and methods: In vitro antioxidant activity of BDE was estimated in terms of total phenolic content and 1,1-diphenyl-2-picryl hydrazyl free radical scavenging activity. Wistar albino rats were given 0.75% v/v ethylene glycol in drinking water to induce chronic hyperoxaluria and simultaneously BDE was given to nephrolithiasic treated rats at the dose of 100 and 200 mg/kg b.w. orally for 28 days. Urinary volume, oxalate, serum creatinine, blood urea nitrogen (BUN), malondialdehyde (MDA) and antioxidant enzyme (SOD, CAT, GST, GPx) were evaluated. Results and discussion: BDE extract was found to posses a high total phenolic content and exhibited significant free radicals scavenging activity. Oxalate excretion significantly increased in hyperoxaluric animals as compared to control which was protected in BDE-treated animals. BDE treatment significantly reduced level of MDA and improved the activity of antioxidant enzymes followed by reduction in BUN and serum creatinine. In addition, BDE reduced the number of CaOx monohydrate crystals in the urine. Histological analysis depicted that BDE treatment inhibited deposition of CaOx crystal and renal cell damage. Conclusion: The present study reveals that antioxidant activity of BDE significantly protects against hyperoxaluric oxidative stress and renal cell injury in urolithiasis.

Immunomodulatory role of piperine in deltamethrin induced thymic apoptosis and altered immune functions.

Deltamethrin (DLM), a well-known pyrethroid insecticide, is a potent immunotoxicant. In rodents, it is primarily characterized by marked thymic apoptosis. Mechanism of DLM induced thymic apoptosis in primary murine thymocytes has been recently explored. Oxidative stress and activation of caspase dependent pathways appear to be involved in the DLM induced thymic injury. Thus, for the amelioration of its effect, this study has been designed to first observe the binding affinity of piperine to immune cell receptors and its protective effects on the DLM induced immunotoxicity under in vitro condition. The docking results demonstrated that piperine has good binding affinity towards CD4 and CD8 receptors. In vitro study results have shown that piperine (1, 10 and 50 μg/ml) increased cell viability in a concentration dependent manner. The early activated markers of apoptosis such as enhanced reactive oxygen species (ROS) and caspase-3 activation by DLM was significantly reduced by piperine treatment. GSH depletion induced by DLM has been also restored by piperine treatment. At 18 h, all concentration of piperine (1, 10 and 50 μg/ml) significantly ameliorated the DLM induced apoptosis. Further, DLM induced phenotypic changes were mitigated by the piperine. In addition, piperine also restored the cytokine levels, which were suppressed by DLM treatment. These findings strongly indicate the anti-oxidative, anti-apoptotic and chemo-protective ability of piperine in the DLM induced thymic apoptosis.

Antioxidant and DNA damage protective properties of anthocyanin-rich extracts from Hibiscus and Ocimum: a comparative study

Anthocyanin extracts (AEs) from Ocimum tenuiflorum (leaf), Hibiscus rosa-sinensis (petal) and Hibiscus sabdariffa (calyx) were investigated and compared for in vitro antioxidant activity and DNA damage protective property. Total phenolic content (TPC) and total anthocyanin content (TAC) of the AEs were determined and the major anthocyanins were characterised. In vitro antioxidant activities were assessed by ferric-reducing antioxidant power (FRAP) assay, 2,2-diphenyl-1-picryl hydrazyl (DPPH) radical-scavenging activity, 2-deoxy-d-ribose degradation assay and lipid peroxidation assay. The protective property of the AEs was also examined against oxidative DNA damage by H2O2 and UV using pUC19 plasmid. All the AEs particularly those from O. tenuiflorum demonstrated efficient antioxidant activity and protected DNA from damage. Strong correlation between antioxidant capacity and TPC and TAC was observed. Significant correlation between antioxidant capacity and TPC and TAC ascertained that phenolics and anthocyanins were the major contributors of antioxidant activity.

Evaluation of CNS Depressant Activity of Different Plant parts of Nyctanthes arbortristis Linn.

The present study was carried out with the water-soluble portion of the ethanol extracts of flowers, barks, seeds and leaves of Nyctanthes arbortristis Linn. to confirm their CNS depressant activity. The ethanol extracts of the plant parts were obtained by soxhlet extraction. After performing the gross behavioral study, the CNS depressant activity was evaluated by observing the prolongation of sleeping time induced by pentobarbital sodium in mice. Attempts have been made to explore the possible mechanism behind this activity by determining their effect on brain monoamine neurotransmitters like dopamine and serotonin. The gross behavioral study showed that ethanol extracts of the leaves, flowers and seeds possess significant CNS depressant activity. The leaves, flowers, seeds and barks (600 mg/kg) showed significant and dose-dependent prolongation of onset and duration of sleep and so found to cause decrease dopamine and increase serotonin level. From which it can be concluded that the CNS depressant activity of the ethanol extracts of seeds, leaves and flowers may be due to the decrease in dopamine and increase in serotonin level.

Neuroprotection by rosiglitazone in transient focal cerebral ischemia might not be mediated by glutamate transporter-1.

Glutamate transport represents a key mechanism for maintaining low level of glutamate in the extracellular milieu to restrict the excitotoxic action of glutamate released during ischemia/reperfusion (I/R) injury. Recently, it has been reported that glutamate transporter‐1 (GLT‐1) is a novel target for peroxisome proliferator‐activated receptor‐γ (PPARγ) agonist, which shows neuroprotection following oxygen glucose deprivation (OGD) in neuronal–astrocytic cocultures. Hence, the present study was undertaken to investigate the role of rosiglitazone in neuroprotection mediated by GLT‐1 following focal cerebral I/R injury in rat. We found that rosiglitazone (2 mg/kg i.p) administered pre‐ or post‐I/R injury significantly improved behavioral outcome and decreased cerebral infarct volume. However, no significant changes were observed in GLT‐1 mRNA and protein expression in rosiglitazone‐treated rats following 1 hr of ischemia/24 hr of reperfusion (1/24 hr I/R) injury. Interestingly, bioinformatics analysis also does not reveal any PPAR response element on the GLT‐1/EAAT2 promoter region. Further rosiglitazone neither increased [3H]glutamate uptake in glia‐enriched preparations nor caused any change in glutamine synthetase activity. On the other hand, there was a significant (P < 0.05) downregulation in tumor necrosis factor‐α and interleukin‐1β gene expression, which were more pronounced in the posttreatment group. The posttreatment with rosiglitazone also significantly reduced the increase in prostaglandin E2 level in the ischemic brain. Therefore, the present findings suggest that the neuroprotective effect of rosiglitazone does not seem to be mediated by modulation of GLT‐1 protein expression/activity in a focal cerebral ischemia model. However, the results do provide increasing evidence that the neuroprotective effect may be mediated by its antiinflammatory action.