Arttatrana Pal

Hyperglycemia-induced oxidative stress induces apoptosis by inhibiting PI3-kinase/Akt and ERK1/2 MAPK mediated signaling pathway causing downregulation of 8-oxoG-DNA glycosylase levels in glial cells.

Glial cells are very important for normal brain function and alterations in their activity due to hyperglycemia, could contribute to diabetes-related cognitive dysfunction. Oxidative insults often cause rapid changes in almost all cells including glial cells. However, pathophysiologic mechanisms that lead to diabetic complications are not completely elucidated. Therefore, we examined whether elevated glucose levels directly or indirectly disrupt antioxidant defense mechanisms causing alterations in signaling pathways, cell cycle dysregulation, and reactive oxygen/nitrogen species-mediated apoptosis in glial cells. Findings of this study demonstrated that exposure of glial cells to high glucose markedly induces cellular and molecular injuries, as evidenced by elevated levels of reactive oxygen/nitrogen species, biomolecules damage, cell cycle dysregulation, decrease in antioxidant enzymes, and decrease in cell viability. Pretreatment of cells with N-acetyl-L-cysteine reduced high glucose-induced cytotoxicity by increasing the levels of antioxidant enzymes, and decreasing the number of apoptotic cells. Further, at molecular level high glucose treatment resulted in a significant increase in phosphorylation of Akt, MAPKs, tuberin, down regulation of 8-oxoG-DNA glycosylase and increase in 8-hydroxydeoxyguanosine accumulations. Pretreatment of cells with N-acetyl-L-cysteine, phosphatidylinositol3-kinase/Akt and ERK1/2 inhibitors completely abolished the apoptotic effects of high glucose. Moreover, N-acetyl-L-cysteine significantly inhibited reactive oxygen/nitrogen species generation, elevated antioxidants levels, inhibited Akt, ERK1/2, tuberin phosphorylation, decreased 8-hydroxydeoxyguanosine accumulation and upregulated 8-oxoG-DNA glycosylase expression. Our results demonstrate that high glucose induces apoptosis and inhibits proliferation of glial cells, which may be mediated by the phosphorylation of tuberin, down regulation of 8-oxoG-DNA glycosylase and 8-hydroxydeoxyguanosine accumulation via activation of Akt and ERK1/2MAPK pathways.

Hyperglycemia-Induced Oxidative-Nitrosative Stress Induces Inflammation and Neurodegeneration via Augmented Tuberous Sclerosis Complex-2 (TSC-2) Activation in Neuronal Cells.

Diabetes is a systemic disease mainly characterized by chronic hyperglycemia and with extensive and long-lasting spiteful complications in central nervous systems (CNS). Astrocytes play an important role in the defense mechanism of CNS, with great ability of withstanding accumulation of toxic substances. Apart from functional disorders, hyperglycemia leads to slow progressive structural abnormalities in the CNS through oxidative stress pathways. However, the molecular mechanism by which neurons die under oxidative stress induced by high glucose (HG) remains largely unclear. Here, we report that HG-induced inflammation and neurodegeneration in brain tissues, brain astrocytes (C6), and pheochromocytoma (PC-12) cells are cultured in HG conditions. Our results show that the increases in phosphorylation of Akt and ERK1/2MAPK are associated with increased accumulations of reactive oxygen species (ROS) in neuronal cells, which simultaneously enhanced phosphorylations of tuberous sclerosis complex-2 (TSC-2) and mammalian target of rapamycin (mTOR) in the diabetic brain and in HG-exposed neuronal cells. Pharmacologic inhibition of Akt or ERK1/2 or siRNA-mediated gene silencing of TSC-2 suppressed the strong downregulation of TSC-2-mTOR activation. Findings of this study also demonstrate that HG resulted in phosphorylation of NF-κB, coinciding with the increased production of inflammatory mediators and activation of neurodegenerative markers. Pretreatment of cells with antioxidants, phosphoinositide3-kinase (PI3-K)/Akt, and ERK1/2 inhibitors significantly reduced HG-induced TSC-2 phosphorylation and restored NF-κB protein expression leading to decreased production of inflammatory mediators and neurodegenerative markers. These results illustrate that ROS functions as a key signaling component in the regulatory pathway induced by elevated glucose in neuronal cell activation leading to inflammation and neurodegeneration.

Incidence of cystoid macular oedema in diabetic patients after phacoemulsification and free radical link to its pathogenesis

Purpose Postoperative onset of cystoid macular oedema (CME) in diabetic patients after cataract surgery is a frequent problem in working-age adults worldwide. Here, we investigate the postoperative development of CME in diabetic patients after undergoing phacoemulsification with other ailing factors associated with CME. Methods This prospective study included 65 Type 2 diabetic patients with no diabetic retinopathy (DR), mild to moderate DR, moderate to severe DR and proliferative DR who underwent phacoemulsification surgery. Indirect ophthalmoscopy, fluorescein angiograms and optical coherence tomography were taken for a period of 8 weeks postoperatively to determine visual outcome and development of CME. Serum samples were collected for the measurement of antioxidants and reactive oxygen species (ROS) levels. Results Our data showed that CME occurred postoperatively in 47% without pre-existing DR and 55% of eyes with pre-existing DR (p<005). Positive association was noticed between morbid conditions, like hypertension (p<0.01) and diabetic nephropathy (p<0.05), and postoperative incidence of CME. The activity of antioxidant enzymes in patients with DR was found to be lower as compared with diabetic (D) patients, but catalase activity was recorded the maximum in these patients. The ROS activity was recorded highest in the serum samples of DR becoming CME positive. Conclusions The present results suggest that after phacoemulsification, the chance of development of CME in DR is more as compared to D. Moreover, the development of CME is significantly associated with decrease in antioxidant levels, increased ROS activities, hypertension, diabetic nephropathy, and hyperlipidaemia.

Lactobacillus acidophilus attenuates Aeromonas hydrophila induced cytotoxicity in catla thymus macrophages by modulating oxidative stress and inflammation.

The pathogenesis of Aeromonas hydrophila, a potent fish pathogen, is attributed to its ability to cause motile aeromonad septicaemia leading to apoptosis in a myriad of fish species, including freshwater carp Catla catla. However, the underlying mechanism of antagonistic activity of probiotics against A. hydrophila induced apoptosis is not elucidated due to lack of appropriate in-vitro models. This study reported that the exposure of catla thymus macrophages (CTM) to A. hydrophila markedly induced cellular injuries as evidenced by elevated levels of reactive oxygen species (ROS), reactive nitrogen species (RNS), increased apoptosis, DNA damage and decreased cellular viability. Flow cytometry analysis and Annexin-V/propidium iodide assay further confirmed increased ROS positive cells leading to cell death after infection. The quantitative real-time PCR analysis, also revealed upregulation of inducible nitric-oxide synthase (iNOS), pro-inflammatory cytokine (TNFα), cyclooxygenase2 (COX-2) and downregulation of anti-inflammatory cytokine (IL-10). Pretreatment of cells with probiotic, Lactobacillus acidophilus attenuated A. hydrophila induced apoptosis as evident from the decrease in the levels of ROS, RNS and DNA damage. Significant increase (P≤0.05) in expression of TNFα and IL-10 and decrease in iNOS and COX-2 was observed on probiotic stimulation. In-vivo study using catla fingerlings confirmed similar pattern of ROS, iNOS, NO production and cytokine expression in thymus. This study provides a comprehensive insight into the mechanistic basis of L. acidophilus induced macrophage mediated inflammatory response against A. hydrophila in CTM cells. Further, it speculates the possibility of using cost-effective in-vitro models for screening probiotic candidates of therapeutic potential in aquaculture industry.

Seasonal changes in hematological parameters of Catla catla (Hamilton 1822)

Hematological parameters are repeatedly used as an essential diagnostic tool to assess the health condition of fish. The purpose of this study was to assess the reference values of some hematological parameters of Catla, Catla catla collected from a freshwater pond in a tropical climate of India from July 2008 to June 2010. Variations in hematological parameters such as hemoglobin, erythrocyte count, total leucocyte count, hematocrit, mean corpuscular volume (MCV), and mean corpuscular hemoglobin concentration (MCHC) of fish were compared according to sex and seasonal differences. Analysis of variance showed that there were significant differences between sexes and the results indicated that blood parameters levels between the sexes in summer were significantly different than that measure in other seasons except mean corpuscular hemoglobin (MCH) and MCHC value (P < 0.05). The number of total leukocyte levels was found to be higher in female fish especially in reproductive seasons, but the levels of hemoglobin, hematocrit, and MCV values were high in male fish in an annual period. However, there was no difference in MCH and MCHC values between the sexes and seasons throughout the study period. These may be related to season of sampling and changing physiological cycles during these months. The correlations between measured hematological parameters were also determined.

Hyperglycemia-induced inflammation caused down-regulation of 8-oxoG-DNA glycosylase levels in murine macrophages is mediated by oxidative-nitrosative stress-dependent pathways

High glucose-induced increase in production of reactive oxygen/nitrogen species (ROS/RNS) is recognized as a major cause of the clinical complications associated with diabetes. ROS/RNS apart from being redox agents, cause an unwanted severe physiological load to cells, also act as cellular messengers, and play a key role in activation of circulating macrophages. However, the molecular mechanisms of activation of macrophages by hyperglycemic conditions are currently unclear. In the present study, we report that high glucose (HG) causes a dramatic increase in the production of inflammatory cytokines and chemokines, at least in part through enhanced mRNA transcription. The increase in levels of inflammatory cytokines/chemokines corresponds to increased levels of ROS/RNS, which is accompanied by increased activities of Akt, ERK1/2, tuberin, down regulation of 8-oxoG-DNA glycosylase (OGG1), and increase in 8-hydroxydeoxyguanosine (8-OHdG) accumulation in DNA. Elevated levels of ROS/RNS are triggering alteration in antioxidants level, biomolecules damage, cell cycle dysregulation, and apoptosis in macrophage cells. Pretreatment of antioxidants caused decrease in the levels of ROS/RNS leads to an increase in the levels of antioxidants, decrease in biomolecules damage, alterations in Akt, ERK1/2, tuberin, upregulation of OGG1, and decrease in 8-OHdG accumulations in DNA. Further, antioxidants treatments inhibit the effects of HG on the transcriptional activity of cytokines and chemokines. Our results demonstrate that intracellular signaling pathways mediated by ROS/RNS are linked to each other by elevated glucose in macrophages activation leading to inflammation. These findings provide a mechanistic explanation of how ROS/RNS cooperate to conduct inflammatory intracellular signals in macrophages related complications in hyperglycemic conditions.

Effects of CEES and LPS synergistically stimulate oxidative stress inactivates OGG1 signaling in macrophage cells.

2-chloroethyl ethyl sulphide (CEES), a monofunctional analogue of sulfur mustard, is a strong vesicant and an alkylating chemical warfare agent. We studied the molecular mechanism of oxidative stress triggered signaling cascades in murine macrophages exposed to CEES with lipopolysaccharide (LPS). Exposure of CEES with specific dose of LPS stimulates oxidative stress caused increasing level of intracellular ROS and RNS, decreased antioxidant enzymes, increasing bimolecular damage, reduced cell viability, and cell cycle arrest. Synergistic exposure of CEES and LPS provoked significant increase in phosphorylation of MAPKs, Akt, tuberin, that down regulate OGG1 expression and 8-OHdG accumulations. Treatment with Akt and ERK1/2 inhibitors, the cells with constitutively active inhibiting activity of Akt and ERK1/2MAPK significant reduce CEES and LPS challenge tuberin but not the OGG1. In addition, the N-acetylcysteine inhibited ROS/RNS generation, elevation of antioxidants level, expression of ERK1/2, Akt, tuberin phosphorylation, resulted in deceased 8-OHdG accumulation and upregulation of OGG1 protein expression suggesting no involvement of Akt and ERK1/2MAPK pathways after CEES and LPS challenge. Collectively, our results indicate that exposure of CEES and LPS induces oxidative stress and the activation of tuberin, and 8-OHdG accumulation via upstream signaling pathways including Akt and ERK1/2MAPK pathway in macrophages but not the down regulation of OGG1.

Hematological and plasma biochemistry in Cirrhinus mrigala (Hamilton 1822)

Hematological and plasma chemistry indices are important parameters for the evaluation of fish physiological status. In this study, we determined the results of selected hematological and plasma biochemical profiles in Cirrhinus mrigala captured from the wild in a tropical climate of India. Blood was analyzed using standard techniques, and differences in hematological parameters including hemoglobin concentration, erythrocyte count, total leukocyte count, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) of fish were compared according to sex and different seasons. Analysis of variance showed that there were significant differences (p < 0.05) between sexes, and the results indicated that blood parameters levels between the sexes in summer were significantly different than that measure in other seasons except MCH and MCHC value. The number of total leukocyte levels was found to be higher in female fish especially in reproductive seasons (summer), but the levels of hemoglobin, hematocrit, and MCV values were high in male fish in an annual period. However, there was no difference in MCH and MCHC values between the sexes and seasons throughout the study period. These may be related to season of sampling and changing physiological cycles during these months. Differences were noted in plasma lipid, cholesterol, and glucose level in the summer season, but the higher amount of plasma protein was found in spring and winter in male and female fish, respectively. In conclusion, monitoring fish hematological and biochemical parameters essentially can be a way to evaluate the physiological and health status of their populations, which may be a useful indicator of the environmental status.