Satyendra K. Rajput

Reactive oxygen species: friend or foe?

Reactive oxygen species (ROS) are an inevitable by-product of cellular metabolism. ROS generation can be associated with the interaction of ionizing radiation with biological molecules, and devoted enzymes in phagocytic cells (NADPH oxidase and myeloperoxidase) or may be the result of an imbalance between radical generating and scavenging systems. Typically ROS have been consider as Pandora’s box, they have several innovative physiological roles in the body. ROS serve as signalling messengers for the activation of transcription factors from cytokine–receptor interactions. This facilitates the evolution and membrane fusion of spermatozoon and oocyte during fertilization. NADPH oxidase enzyme and nitric oxide (NO) function as potent vasodilators and immunity boosters. ROS have been suggested as prevalent regulators of several nuclear factors, including erythroid 2-related factor 2 (Nrf2), nuclear factor kappa-B (NFκB) cells, mitogen-activated protein kinase (MAPK) and p53, which are further associated with several signalling cascades. Under physiological conditions the amount of ROS generated in the body can be counterbalanced by natural antioxidants in the body. However, aberrant augmented levels of ROS predominantly lead to various defined disorders comprising myocardial infarction, autoimmune diseases, atherosclerosis, Alzheimer’s and Parkinson’s diseases and emphysema. Ordinarily, it is observed that the physiological roles of ROS are insubstantial compared with their pathological action. But there is a need to clearly define the line between pathological and physiological functions of ROS. Of particular worth is to reveal the beneficial responsibilities of ROS in different cellular pathways and metabolic functions, over its injurious consequences.

Antiepileptic potential and behavioral profile of L-pGlu-(2-propyl)-L-His-L-ProNH2, a newer thyrotropin-releasing hormone analog.

Thyrotropin-releasing hormone (TRH) and its analogs have a number of neurobiological functions and therapeutic uses in disorders of the central nervous system. In this study, the newly synthesized TRH analogs were evaluated for central nervous system activity in pentobarbital-induced sleeping in mice. The most potent TRH analog (L-pGlu-(2-propyl)-L-His-L-ProNH(2) coded as NP-647) was evaluated for its antiepileptic potential in various seizure models in mice in comparison with TRH. Intravenous pretreatment with NP-647 (10 and 20 micromol/kg body wt) significantly delayed the onset and reduced the frequency of convulsions in the pentylenetetrazole model, but not in the maximum electroshock seizure model. Also, it was found to be protective against picrotoxin- and kainic acid-induced seizures. However, NP-647 did not significantly affect theophylline-induced seizures. Further study of the effect of NP-647 on locomotor activity and a functional observational battery revealed that it did not significantly exhibit any undesirable effects as compared with vehicle and TRH. NP-647 did not significantly affect cerebral blood flow, whereas the native peptide TRH markedly increased cerebral blood flow. Furthermore, NP-647 exerted antiepileptic activity without significantly altering plasma thyroid-stimulating hormone levels and mean arterial blood pressure. This suggests that NP-647 is more selective for central nervous system activity and devoid of hormonal and cerebrovascular system effects. In contrast, TRH exhibited cardiac and endocrine effects as marked by significant elevation in mean arterial blood pressure and plasma thyroid-stimulating hormone levels. This study demonstrates that NP-647 has potential antiepileptic activity devoid of undesirable effects and, thus, can be exploited for the prevention and treatment of epilepsy.

Pharmacoresistant Epilepsy: A Current Update on Non-Conventional Pharmacological and Non-Pharmacological Interventions

Uncontrolled seizure or epilepsy is intricately related with an increase risk of pharmacoresistant epilepsy. The failure to achieve seizure control with the first or second drug trial of an anticonvulsant medication given at the appropriate daily dosage is termed as pharmacoresistance, despite the fact that these drugs possess different modes of action. It is one of the devastating neurological disorders act as major culprit of mortality in developed as well as developing countries with towering prevalence. Indeed, the presence of several anti-epileptic drug including carbamazepine, phenytoin, valproate, gabapentin etc. But no promising therapeutic remedies available to manage pharmacoresistance in the present clinical scenario. Hence, utility of alternative strategies in management of resistance epilepsy is increased which further possible by continuing developing of promising therapeutic interventions to manage this insidious condition adequately. Strategies include add on therapy with adenosine, verapamil etc or ketogenic diet, vagus nerve stimulation, focal cooling or standard drugs in combinations have shown some promising results. In this review we will shed light on the current pharmacological and non pharmacological mediator with their potential pleiotropic action on pharmacoresistant epilepsy.

Protective effects of l-pGlu-(2-propyl)-l-His-l-ProNH2, a newer thyrotropin releasing hormone analog in in vitro and in vivo models of cerebral ischemia

In the present study, the newly synthesized TRH analog (L-pGlu-(2-propyl)-L-His-l-ProNH(2); NP-647) was evaluated for its effects in in vitro (oxygen glucose deprivation (OGD)-, glutamate- and H(2)O(2)-induced injury in PC-12 cells) and in vivo (transient global ischemia) models of cerebral ischemic injury. PC-12 cells were subjected to oxygen and glucose deprivation for 6h. Exposure of NP-647 was given before and during OGD. In glutamate and H(2)O(2) induced injury, exposure of NP-647 was given 1, 6 and 24h prior to exposure of glutamate and H(2)O(2) exposure. NP-647, per se found to be non-toxic in 1-100μM concentrations. NP-647 showed protection against OGD at the 1 and 10μM. The concentration-dependent protection was observed in H(2)O(2)- and glutamate-induced cellular injury. In in vivo studies, NP-647 treatment showed protection of hippocampal (CA1) neuronal damage in transient global ischemia in mice and subsequent improvement in memory retention was observed using passive avoidance retention test. Moreover, administration of NP-647 resulted in decrease in inflammatory cytokines TNF-α and IL-6 as well as lipid peroxidation. These results suggest potential of NP-647 in the treatment of cerebral ischemia and its neuroprotective effect may be attributed to reduction of excitotoxicity, oxidative stress and inflammation.

Synthesis, Receptor Binding, and CNS Pharmacological Studies of New Thyrotropin-Releasing Hormone (TRH) Analogues

As part of our search for selective and CNS‐active thyrotropin‐releasing hormone (TRH) analogues, we synthesized a set of 44 new analogues in which His and pGlu residues were modified or replaced. The analogues were evaluated as agonists at TRH‐R1 and TRH‐R2 in cells in vitro, and in vivo in mice for analeptic and anticonvulsant activities. Several analogues bound to TRH‐R1 and TRH‐R2 with good to moderate affinities, and are full agonists at both receptor subtypes. Specifically, analogue 21 a (R=CH3) exhibited binding affinities (Ki values) of 0.17 μM for TRH‐R1 and 0.016 μM for TRH‐R2; it is 10‐fold less potent than TRH in binding to TRH‐R1 and equipotent with TRH in binding to TRH‐R2. Compound 21 a, the most selective agonist, activated TRH‐R2 with a potency (EC50 value) of 0.0021 μM, but activated TRH‐R1 at EC50=0.05 μM, and exhibited 24‐fold selectivity for TRH‐R2 over TRH‐R1. The newly synthesized TRH analogues were also evaluated in vivo to assess their potencies in antagonism of barbiturate‐induced sleeping time, and several analogues displayed potent analeptic activity. Specifically, analogues 21 a,b and 22 a,b decreased sleeping time by nearly 50 % more than TRH. These analogues also displayed potent anticonvulsant activity and provided significant protection against PTZ‐induced seizures, but failed to provide any protection in MES‐induced seizures at 10 μmol kg−1. The results of this study provide evidence that TRH analogues that show selectivity for TRH‐R2 over TRH‐R1 possess potent CNS activity.

Neuropharmacological profile of L-pGlu-(1-benzyl)-L-His-L-ProNH2, a newer thyrotropin-releasing hormone analog: effects on seizure models, sodium current, cerebral blood flow and behavioral parameters.

In the present study, L-pGlu-(1-benzyl)-L-His-L-ProNH(2) (NP-355), a newer CNS active thyrotropin-releasing hormone (TRH) analog was evaluated for its antiepileptic potential. NP-355 (5, 10 and 20 micromol/kg; i.v.) pretreatment significantly delayed onset and reduced the frequency of convulsions in pentylenetetrazole-induced seizures. NP-355 was also found to be protective against picrotoxin- and kainic acid-induced seizures. Maximum electroshock-induced seizures were not protected even at 20 micromol/kg in mice. Effects of NP-355 on functional observation battery did not exhibit any undesirable effects. Moreover, the antiepileptic activity produced by NP-355 was observed without significantly altering mean arterial blood pressure. NP-355 significantly increases the CBF to 17+/-3% as compared to saline (6+/-2%). NP-355 (100, 300 and 1000 microM) produces a concentration-dependent depression (16%, 63% and 77%, respectively) of the peak sodium current. NP-355 did not alter neurobehavioral parameters. This study demonstrates that NP-355 has potential antiepileptic activity and devoid of undesirable effects.

Synthesis and preliminary therapeutic evaluation of copper nanoparticles against diabetes mellitus and -induced micro- (renal) and macro-vascular (vascular endothelial and cardiovascular) abnormalities in rats

The current study synthesized and investigated the effect of low-dose copper nanoparticles (CuNPs) against diabetes mellitus (streptozotocin, 50 mg kg−1, i.p., once) and -induced experimental micro- (nephropathy) and macro-vascular (cardio and endothelium) complications. Diabetes mellitus (DM)-induced vascular abnormalities were revealed by the reduction in acetylcholine-induced endothelium-dependent relaxation, the decrease in aortic and serum nitrite/nitrate concentration, increased CKMB, LDH, SGOT/SGPT, serum creatinine, and blood urea nitrogen, and the induction of proteinuria and oxidative stress. However, treatment with low-dose CuNPs (1 mg kg−1, p.o. 4 weeks) after streptozotocin administration reduced serum glucose concentration. Moreover, CuNPs had shown a partial but significant prevention of cardio-vascular structural and functional abnormalities in diabetic rats. Increased bioavailability of NO in the endothelium and reduction in oxidative stress might be the possible mechanisms involved for the protective role of CuNPs against diabetes-induced micro- and macro-complications.

Electrochemically synthesized highly crystalline nitrogen doped graphene nanosheets with exceptional biocompatibility

This work reports first electrochemical preparation of exceptionally biocompatible, highly crystalline, and well exfoliated nitrogen doped graphene nanosheets (eNGS) from carbon nanosheets for the development of mighty platforms in the field of modern biosensing and other biological applications for human welfare. eNGS displayed exceptional biocompatibility. Administration of the as-synthesized eNGS to rat models did not lead to any significant deviation or inimical consequences in its functional observation battery (FOB) tests, GSH levels or the histology of the vital organs of the rat models. The pictomicrographs of myocytes nuclei and myofibrillar for heart, hippocampus (CA1) section for brain, central vein, and hepatocytes for liver and parenchyma, tubules and glomeruli for kidney also remained unaffected. Moreover, the resultant nanoelectrocatalyst displayed enhanced electrochemical performance towards real-time sensing of dopamine (DA) from human urine sample in the presence of interferences, such as ascorbic acid (AA) and uric acid (UA).

Hydrothermally functionalized biocompatible nitrogen doped graphene nanosheet based biomimetic platforms for nitric oxide detection

Hydrothermal synthesis of nanocomposites is of significant importance, as it affords facile, biocompatible, nontoxic, and economic fabrication. Herein, we report a hitherto unexplored cytocompatible and reusable biomimetic electrochemical sensor based on pyridyl porphyrin functionalized nitrogen doped graphene nanosheets. The porphyrin functionalized nitrogen doped graphene nanosheets (PFNGS) were prepared by a low temperature hydrothermal method via non-covalent strategies with a minimal impact on their physicochemical properties. Owing to their exceptional attributes like operational ease, low cost, portability, and sensitivity, the as-synthesized PFNGS, formed by π-π interactions, were employed for sensing nitric oxide (NO), which is a key regulator of diverse biological processes. Compared to porphyrin and nitrogen doped graphene nanosheets alone, PFNGS exhibited exceptional sensitivity (3.6191 μA μM-1) and remarkable electrocatalytic properties (0.61 V). This clearly outperforms the previously reported modified electrode materials for the electrochemical detection of NO. Cyclic voltammetry (CV) data also suggested that the PFNGS modified electrode possessed an increased reactive surface area, which results in an increase in the number of reactive sites and low charge transfer resistance. These results also demonstrated that the PFNGS modified electrode showed high stability and reproducibility, the limit of detection (LOD) (S/N = 3) of which was estimated to be 1 nM. Our PFNGS were found to be highly biocompatible and could also detect NO released from macrophage cells. This blend of biocompatibility, electrode stability, electrocatalytic activity along with enhanced sensitivity and selectivity makes PFNGS a powerful and reliable nanomaterial for various biomedical applications in complex biological systems.

Combined and individual strategy of exercise generated preconditioning and low dose copper nanoparticles serve as superlative approach to ameliorate ISO-induced myocardial infarction in rats

BACKGROUND Myocardial infarction (MI) is a solitary fatal condition with towering prevalence of mortality worldwide. Our previous study reports that low-dose copper nanoparticles (CuNP) can halt the progression of diabetes-induced cardiotoxicity as copper has anti-inflammatory, anti-proliferative and anti-oxidant potential. In addition, exercise training has also been considered a hallmark for cardiac health. METHOD Cardioprotective potential of CuNP (1mg/kg/day, po, 4 weeks) and exercise (swimming, 90min, 5days/4 weeks) either alone or in combination was estimated by measuring the surge in serum nitrite/nitrate concentration and reduction in creatine kinase MB (CKMB), lactate dehydrogenase (LDH), cardiac troponin I (cTnI), lipid profile, oxidative stress, structural abnormalities against isproterenol (ISO)-induced MI. RESULTS ISO significantly increased CKMB, LDH, cTnI, lipid alteration, oxidative stress, structural abnormalities and decrease nitrite/nitrate concentration in serum. Quantitative estimation of total and phosphorylated Akt(SER-473)/GSK-3b(SER-9) indicated the significant reduction in pAkt and pGSK-3b in ISO treated animal. Individual and combined treatment of CuNP and exercise significantly reduce ISO -induced CKMB, cTnI, LDH, and improve nitrite/nitrate concentration and lipid profile. Attenuation of myocardial oxidative stress and serum TBARS revealed the associated preconditioning effect of exercise and CuNP against oxidative stress. Exercise and CuNP also showed the protective potential against structural abnormalities. However, the cardioprotective effect of individual and combined strategy of exercise and CuNP was vanished by wortmannin and also avoid the downregulation of pGSK-3b. CONCLUSION Low-dose CuNP and exercise training significantly prevents ISO-induced MI through preconditioning and GSK-3b inhibition. Ability to upsurge the NO level, lipid profile and reduced oxidative stress improve the potency of combined strategy.