Mahendra Kashyap

Influence of cytotoxic doses of 4-hydroxynonenal on selected neurotransmitter receptors in PC-12 cells.

Effect of 4-hydroxynonenal (HNE), a long-chain alpha, beta unsaturated aldehyde product, generated by the oxidation of omega-6 polyunsaturated fatty acids on the sensitivity of selected neurotransmitter receptors was studied in PC-12 cells. Cytotoxicity profiling was carried out at varying concentrations of HNE (0.1-50microM) for 30min to 24h. Trypan blue dye exclusion, MTT, LDH release and neutral red uptake (NRU) assays were carried out to assess the cytotoxicity of HNE. Cytotoxic response was found to be significant at 2h of exposure. Cytotoxicity of HNE at 50microM was exerted even at 90min. HNE 10-50microM was found to be cytotoxic, whereas, 2-5microM causes physiological stress only and 1-0.1microM non-cytotoxic. Effect on dopamine, cholinergic, serotonin and benzodiazepine receptors was studied at varying concentrations of HNE (1, 10, 25 and 50microM for 1-8h). A significant decrease in binding of 3H-QNB, 3H-Fluinitrazepam and 3H-Ketanserin, known to label cholinergic (muscarinic), benzodiazepine and serotonin (5HT(2A)) receptors respectively was observed at 1h exposure of PC-12 cells to HNE at 25 and 50microM concentrations. The decrease in the binding of (3)H-Spiperone, known to label dopamine (DA-D2) receptors was evident at 4h of exposure of PC-12 cells to HNE. The decrease in the binding with DA-D2 receptors continued till 8h. Effect on the binding of (3)H-Fluinitrazepam and 3H-Ketanserin appeared to be maximum at 25 and 50microM concentrations of HNE for 4h and 8h. The PC-12 cells appear to be vulnerable to cytotoxic concentrations of HNE. Experimental HNE exposure provides an intriguing model of toxicant-cell interactions involving neurotransmitter receptors in HNE neurotoxicity.

Down-Regulation of Nerve Growth Factor Expression in the Bladder by Antisense Oligonucleotides as New Treatment for Overactive Bladder

PURPOSE Nerve growth factor over expression in the bladder has a role in overactive bladder symptoms via the mediation of functional changes in bladder afferent pathways. We studied whether blocking nerve growth factor over expression in bladder urothelium by a sequence specific gene silencing mechanism would suppress bladder overactivity and chemokine expression induced by acetic acid. MATERIALS AND METHODS Female Sprague-Dawley® rats anesthetized with isoflurane were instilled with 0.5 ml saline, scrambled or TYE™ 563 labeled antisense oligonucleotide targeting nerve growth factor (12 μM) alone or complexed with cationic liposomes for 30 minutes. The efficacy of nerve growth factor antisense treatments for acetic acid induced bladder overactivity was assessed by cystometry. Bladder nerve growth factor expression levels and cellular distribution were quantified by immunofluorescence staining and enzyme-linked immunosorbent assay. Effects on bladder chemokine expression were measured by Luminex® xMAP® analysis. RESULTS Liposomes were needed for bladder uptake of oligonucleotide, as seen by the absence of bright red TYE 563 fluorescence in rats instilled with oligonucleotide alone. At 24 hours after liposome-oligonucleotide treatment baseline bladder activity during saline infusion was indistinct in the sham and antisense treated groups with a mean ± SEM intercontraction interval of 348 ± 55 and 390 ± 120 seconds, respectively. Acetic acid induced bladder overactivity was shown by a decrease in the intercontraction interval to a mean of 33.2% ± 4.0% of baseline in sham treated rats. However, the reduction was blunted to a mean of 75.8% ± 3.4% of baseline in rats treated with liposomal antisense oligonucleotide (p <0.05). Acetic acid induced increased nerve growth factor in the urothelium of sham treated rats, which was decreased by antisense treatment, as shown by enzyme-linked immunosorbent assay and reduced nerve growth factor immunoreactivity in the urothelium. Increased nerve growth factor in bladder tissue was associated with sICAM-1, sE-selectin, CXCL-10 and 1, leptin, MCP-1 and vascular endothelial growth factor over expression, which was significantly decreased by nerve growth factor antisense treatment (p <0.01). CONCLUSIONS Acetic acid induced bladder overactivity is associated with nerve growth factor over expression in the urothelium and with chemokine up-regulation. Treatment with liposomal antisense suppresses bladder overactivity, and nerve growth factor and chemokine expression. Local suppression of nerve growth factor in the bladder could be an attractive approach for overactive bladder. It would avoid the systemic side effects that may be associated with nonspecific blockade of nerve growth factor expression.

Bladder afferent hyperexcitability in bladder pain syndrome/interstitial cystitis

Bladder pain syndrome/interstitial cystitis is a disease with lower urinary tract symptoms, such as bladder pain and urinary frequency, which results in seriously impaired quality of life of patients. The extreme pain and urinary frequency are often difficult to treat. Although the etiology of bladder pain syndrome/interstitial cystitis is still not known, there is increasing evidence showing that afferent hyperexcitability as a result of neurogenic bladder inflammation and urothelial dysfunction is important to the pathophysiological basis of symptom development. Further investigation of the pathophysiology will lead to the effective treatment of patients with bladder pain syndrome/interstitial cystitis.

Depolymerized chitosans functionalized with bPEI as carriers of nucleic acids and tuftsin-tethered conjugate for macrophage targeting.

Development of efficient and safe nucleic acid carriers (vectors) is one of the essential requirements for the success of gene therapy. Here, we have evaluated the gene transfer capability of chitosan-PEI (CP) conjugates prepared by conjugating low molecular weight branched polyethylenimine (LMWP) with depolymerized chitosans (7 and 10 kDa) via their terminal aldehyde/keto groups. The CP conjugates interacted efficiently with nucleic acids and also showed higher cellular uptake. These conjugates on complexation with DNA yielded nanoparticles in the size range of 100-130 nm (in case of C7P) and 115-160 nm (in case of C10P), which exhibited significantly higher transfection efficiency (~2-42 folds) in vitro compared to chitosans (high and low mol. wt.) and the commercially available transfection reagents retaining cell viability almost comparable to the native chitosan. Of the two CP conjugates, chitosan 7 kDa-LMWP (C7P) displayed higher gene transfer ability in the presence and absence of serum. Luciferase reporter gene analysis in male Balb/c mice receiving intravenous administration of C7P3/DNA polyplex showed the maximum expression in their spleen. Further, tuftsin, a known macrophage targeting molecule, was tethered to C7P3 and the resulting complex, i.e., C7P3-T/DNA, exhibited significantly higher gene expression in cultured mouse peritoneal macrophages as compared to unmodified C7P3/DNA complex without any cytotoxicity demonstrating the suitability of the conjugate for targeted applications. Conclusively, the study demonstrates the potential of the projected conjugates for gene delivery for wider biomedical applications.

Kolaviron protects apoptotic cell death in PC12 cells exposed to Atrazine

Abstract Kolaviron (KV), a natural biflavonoid obtained from the seeds of Garcinia kola, has been documented for its wide pharmacological window, including anti-apoptotic activities. However, the underlying mechanisms are poorly understood at the cellular level. This study investigates the anti-apoptotic activity of KV in PC12 cells, a rat pheochromocytoma, exposed to endocrine disruptor-atrazine (ATZ). KV (60 μM) treatment for 24 h shows significant anti-apoptotic responses in PC12 cells exposed to ATZ (232 μM) for 24 h. KV treatment recovers the ATZ-induced levels of malondialdehyde, reactive oxygen species (ROS), caspase-3 activity and depleted levels of glutathione and catalase activity. However, KV was found to be ineffective to restore the ATZ-induced expression (mRNA) and activity of glutathione-peroxidase (GSH-Px) and glutathione reductase (GR). KV treatment also demonstrates significant restoration in ATZ-induced alterations in the expression of apoptosis markers viz., p53, Bax, Bcl2, caspase-3, caspase-9, cyclooxygenase-2 (COX-2), c-Jun and c-fos. Flow cytometric analysis confirms the involvement of ROS in the mediation of ATZ-induced apoptosis in PC12 cells. Together, these data suggest that KV has the therapeutic potential against chemical-induced apoptotic cell death in the neuronal system.

Neuroprotection Through Rapamycin-Induced Activation of Autophagy and PI3K/Akt1/mTOR/CREB Signaling Against Amyloid-β-Induced Oxidative Stress, Synaptic/Neurotransmission Dysfunction, and Neurodegeneration in Adult Rats

Autophagy is a catabolic process involved in the continuous removal of toxic protein aggregates and cellular organelles to maintain the homeostasis and functional integrity of cells. The mechanistic understanding of autophagy mediated neuroprotection during the development of neurodegenerative disorders remains elusive. Here, we investigated the potential role of rapamycin-induced activation of autophagy and PI3K/Akt1/mTOR/CREB pathway(s) in the neuroprotection of amyloid-beta (Aβ1-42)-insulted hippocampal neurons in rat model of Alzheimer’s disease (AD) like phenotypes. A single intra-hippocampal injection of Aβ1-42 impaired redox balance and markedly induced synaptic dysfunction, neurotransmission dysfunction, and cognitive deficit, and suppressed pro-survival signaling in the adult rats. Rapamycin administration caused a significant reduction of mTOR complex 1 phosphorylation at Ser2481 and a significant increase in levels of autophagy markers such as microtubule-associated protein-1 light chain-3 (LC3), beclin-1, sequestosome-1/p62, unc-51-like kinase 1 (ULK1). In addition, rapamycin induced the activation of autophagy that further activated p-PI3K, p-Akt1 (Ser473), and p-CREB (Ser183) expression in Aβ1-42-treated rats. The activated autophagy markedly reversed Aβ1-42-induced impaired redox homeostasis by decreasing the levels of prooxidants—ROS generation, intracellular Ca2+ flux and LPO, and increasing the levels of antioxidants—SOD, catalase, and GSH. The activated autophagy also provided significant neuroprotection against Aβ1-42-induced synaptic dysfunction by increasing the expression of synapsin-I, synaptophysin, and PSD95; and neurotransmission dysfunction by increasing the levels of CHRM2, DAD2 receptor, NMDA receptor, and AMPA receptor; and ultimately improved cognitive ability in rats. Wortmannin administration significantly reduced the expression of autophagy markers, p-PI3K, p-Akt1, and p-CREB, as well as the autophagy mediated neuroprotective effect. Our study demonstrate that autophagy can be an integrated part of pro-survival (PI3K/Akt1/mTOR/CREB) signaling and autophagic activation restores the oxidative defense mechanism(s), neurodegenerative damages, and maintains the integrity of synapse and neurotransmission in rat model of AD.

Oxygen Glucose Deprivation Model of Cerebral Stroke in PC-12 Cells: Glucose as a Limiting Factor

Optimum time points for oxygen-glucose deprivation (OGD) and re-oxygenation have been identified to suggest the suitability of PC-12 cells as rapid and sensitive in vitro model of cerebral stroke. Further, the precise role of glucose as one of the limiting factors was ascertained. PC-12 cells were subjected to receive OGD of 1–8 h followed by re-oxygenation for 6 to 96 h in medium having glucose 0–10 mg/ml. Loss of cell viability was assessed using trypan blue dye exclusion and MTT assays. The significant (p < 0.05) reduction in percent viable cell count was started at 2 h of OGD (80.7 ± 2.0) and continued in further OGD periods (3, 4, 5, 6, 7, and 8 h), i.e. 65.7 ± 3.5, 59.7 ± 4.6, 54.3 ± 3.2, 44.7 ± 2.9, 20.3 ± 4.3, 5.7 ± 2.0 of counted cells, respectively. Cells growing in glucose-free medium have shown a gradual (p < 0.001) decrease in cell viability throughout the re-oxygenation. Re-oxygenation of 24 h was found to be first statistically significant time point for all the glucose concentrations. Glucose concentration during re-oxygenation was found to be one of the key factors involved in the growth and proliferation in PC-12 cells. The OGD of 6 h followed by a re-oxygenation period of 24 h with 4–6 mg/ml glucose concentration could be recorded as optimum conditions under our experimental conditions.

Expression and Inducibility of Cytochrome P450s (CYP1A1, 2B6, 2E1, 3A4) in Human Cord Blood CD34+ Stem Cell–Derived Differentiating Neuronal Cells

The status of xenobiotic metabolism in developing human brain cells is not known. The reason is nonavailability of developing human fetal brain. We investigate the applicability of the plasticity potential of human umbilical cord blood stem cells for the purpose. Characterized hematopoietic stem cells are converted into neuronal subtypes in eight days. The expression and substrate-specific catalytic activity of the cytochrome P450s (CYPs) CYP1A1 and 3A4 increased gradually till day 8 of differentiation, whereas CYP2B6 and CYP2E1 showed highest expression and activity at day 4. There was no significant increase in the expression of CYP regulators, namely, aryl hydrocarbon receptor (AHR), constitutive androstane receptor (CAR), pregnane X receptor (PXR), and glutathione-S-transferase (GSTP1-1) during differentiation. Differentiating cells showed significant induction in the expression of CYP1A1, 2B6, 2E1, 3A4, AHR, CAR, PXR, and GSTP1-1 when exposed to rifampin, a known universal inducer of CYPs. The xenobiotic-metabolizing capabilities of these differentiating cells were confirmed by exposing them to the organophosphate pesticide monocrotophos (MCP), a known developmental neurotoxicant, in the presence and absence of a universal inhibitor of CYPs-cimetidine. Early-differentiating cells (day 2) were found to be more vulnerable to xenobiotics than mature well-differentiated cells. For the first time, we report significant expression and catalytic activity of selected CYPs in human cord blood hematopoietic stem cell-derived neuronal cells at various stages of maturity. We also confirm significant induction in the expression and catalytic activity of selected CYPs in human cord blood stem cell-derived differentiating neuronal cells exposed to known CYP inducers and MCP.

The Anti-Inflammatory and Antibacterial Basis of Human Omental Defense: Selective Expression of Cytokines and Antimicrobial Peptides

Background The wound healing properties of the human omentum are well known and have extensively been exploited clinically. However, the underlying mechanisms of these effects are not well understood. We hypothesize that the omentum tissue promotes wound healing via modulation of anti-inflammatory pathways, and because the omentum is rich in adipocytes, the adipocytes may modulate the anti-inflammatory response. Factors released by human omentum may affect healing, inflammation and immune defense. Methodology Six human omentum tissues (non obese, free from malignancy, and any other systemic disorder) were obtained during diagnostic laparoscopies having a negative outcome. Healthy oral mucosa (obtained from routine oral biopsies) was used as control. Cultured adipocytes derived from human omentum were exposed to lipopolysaccharide (LPS) (1–50 ng/mL) for 12–72 hours to identify the non-cytotoxic doses. Levels of expression (mRNA and protein) were carried out for genes associated with pro- and anti-inflammatory cytokine responses and antibacterial/antimicrobial activity using qRT-PCR, western blotting, and cell-based ELISA assays. Results The study shows significant higher levels of expression (mRNA and protein) of several specific cytokines, and antibacterial peptides in the omentum tissues when compared to oral sub-mucosal tissues. In the validation studies, primary cultures of adipocytes, derived from human omentum were exposed to LPS (5 and 10 ng/mL) for 24 and 48 h. The altered expressions were more pronounced in cultured adipocytes cells when exposed to LPS as compared to the omentum tissue. Conclusions/Significance Perhaps, this is the first report that provides evidence of expressional changes in pro- and anti-inflammatory cytokines and antibacterial peptides in the normal human omentum tissue as well as adipocytes cultured from this tissue. The study provides new insights on the molecular and cellular mechanisms of healing and defense by the omentum, and suggests the potential applicability of cultured adipocytes derived from the omentum for future therapeutic applications.

Short-term exposure of 4-hydroxynonenal induces mitochondria-mediated apoptosis in PC12 cells

4-Hydroxynonenal (4-HNE) is one of the most reactive aldehydic by-products of lipid peroxidation. The role of 4-HNE in the etiology of various neurodegenerative disorders including cerebral ischemia/reperfusion, Alzheimer’s disease, Parkinson’s disease, etc. has been documented. We and others have reported that long-term toxic insults of 4-HNE triggers apoptotic signals and oxidative stress in various cells. However, the status of apoptosis following short-term exposure and underlying mechanisms has not been explored so far. We studied the apoptotic changes in PC12 cells receiving short-term exposure of 4-HNE. A significant dose-dependent induction in reactive oxygen species (ROS) and early response markers (c-Fos, c-Jun, and GAP-43) were observed in cells exposed to 4-HNE (10, 25, and 50 µM) for 1h. Following the exposure of PC12 cells to 4-HNE, the levels of protein and messenger RNA expressions of P53, Bax, and caspase 3 were significantly upregulated, whereas the levels of Bcl2 was downregulated. We could record the apoptotic signals and ROS generation in PC12 cells receiving 4-HNE exposure for such a short period of time. Induction in the expression and activity of caspase 3 has also indicated the mitochondrial mediation in the apoptosis induction.