Mustoori Sairam

Neuroprotective effect of N-acetyl cysteine on hypoxia-induced oxidative stress in primary hippocampal culture

Hippocampus has received a considerable attention in the recent past due to its role in a number of important functions such as learning and memory. The effect of hypoxia on neuronal cell injury especially on hippocampal cells is not well known. The aim of the present study was to characterize the biochemical changes in primary cultured hippocampal neurons during hypoxic exposure and the protective effect of N-acetyl cysteine on hypoxia-induced cytotoxicity. The hippocampal culture grown in 24-well plates was exposed to hypoxia for 3 h in a dessicator in 95% N(2), 5% CO(2) atmosphere at 37 degrees C. Later, the cells were allowed to recover for 1 h under normoxia. It was observed that there is an appreciable increase in cytotoxicity in cells exposed to hypoxia. Further, there was a significant decrease in mitochondrial membrane potential and appreciable increase in reactive oxygen species and single-strand DNA breaks in cells exposed to hypoxia compared to control. There is a significant fall in glutathione peroxidase, glutathione reductase, reduced glutathione levels, and nitric oxide in the cells exposed to hypoxia. Significant elevation in the intracellular calcium level in the cells on exposure to hypoxia was observed. Supplementation with NAC (50 microM) resulted in a significant cytoprotection, fall in ROS generation, and higher antioxidant levels similar to that of control cells. NAC also inhibited DNA strand breaks induced by hypoxia. The study indicates that NAC has significant neuroprotective activity during hypoxia in primary hippocampal culture.

Neuroprotective effect of cobalt chloride on hypobaric hypoxia-induced oxidative stress

Hypobaric hypoxia, characteristic of high altitude is known to increase the formation of reactive oxygen and nitrogen species (RONS), and decrease effectiveness of antioxidant enzymes. RONS are involved and may even play a causative role in high altitude related ailments. Brain is highly susceptible to hypoxic stress and is involved in physiological responses that follow. Exposure of rats to hypobaric hypoxia (7619 m) resulted in increased oxidation of lipids and proteins due to increased RONS and decreased reduced to oxidized glutathione (GSH/GSSG) ratio. Further, there was a significant increase in superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione-S-transferase (GST) levels. Increase in heme oxygenase 1 (HO-1) and heat shock protein 70 (HSP70) was also noticed along with metallothionein (MT) II and III. Administration of cobalt appreciably attenuated the RONS generation, oxidation of lipids and proteins and maintained GSH/GSSH ratio similar to that of control cells via induction of HO-1 and MT offering efficient neuroprotection. It can be concluded that cobalt reduces hypoxia oxidative stress by maintaining higher cellular HO-1 and MT levels via hypoxia inducible factor 1alpha (HIF-1alpha) signaling mechanisms. These findings provide a basis for possible use of cobalt for prevention of hypoxia-induced oxidative stress.

Hypoxic preconditioning with cobalt attenuates hypobaric hypoxia-induced oxidative damage in rat lungs.

Shukla, Dhananjay, Saurabh Saxena, Purushotman Jayamurthy, Mustoori Sairam, Mrinalini, Singh, Swatantra Kumar Jain, Anju Bansal, and Govindaswamy Ilavazaghan. High Alt. Med. Biol. 10:57-69, 2009.-Hypoxic preco759nditioning (HPC) provides robust protection against injury from subsequent prolonged hypobaric hypoxia, which is a characteristic of high altitude and is known to induce oxidative injury in lung by increasing the generation of reactive oxygen species (ROS) and decreasing the effectiveness of the antioxidant defense system. We hypothesize that HPC with cobalt might protect the lung from subsequent hypobaric hypoxia-induced lung injury. HPC with cobalt can be achieved by oral feeding of CoCl(2) (12.5 mg kg(-1)) in rats for 7 days. Nonpreconditioned rats responded to hypobaric hypoxia (7619 m) by increased reactive oxygen species (ROS) generation and a decreased GSH/GSSG ratio. They also showed a marked increase in lipid peroxidation, heat-shock proteins (HSP32, HSP70), metallothionins (MT), levels of inflammatory cytokines (TNF-alpha, IFN-gamma, MCP-1), and SOD, GPx, and GST enzyme activity. In contrast, rats preconditioned with cobalt were far less impaired by severe hypobaric hypoxia, as observed by decreased ROS generation, lipid peroxidation, and inflammatory cytokine release and an inceased GSH/GSSG ratio. Increased expression of antioxidative proeins Nrf-1, HSP-32, and MT was also observed in cobalt- preconditioned animals. A marked increase in the protein expression and DNA binding activity of hypoxia-inducible transcriptional factor (HIF-1alpha) and its regulated genes, such as erythropoietin (EPO) and glucose transporter-1 (glut-1), was observed after HPC with cobalt. We conclude that HPC with cobalt enhances antioxidant status in the lung and protects from subsequent hypobaric hypoxia-induced oxidative stress.

Role of selenium in reducing hypoxia-induced oxidative stress: an in vivo study

At high altitudes, the reactive oxygen species are continuously generated as a consequence of low oxygen partial pressure (hypoxia), which causes tissue damage. The bodys defence system to combat the oxidative stress (e.g., anti-oxidant enzymes, free radical scavengers such as vitamin C, vitamin E, beta-carotene, reduced glutathione and minerals such as selenium, etc.) may diminish. In the present study, the antioxidant effect of selenium (Se) in reducing the hypoxia-induced oxidative stress was evaluated by exposing male albino rats to hypoxic stress in a decompression chamber. Exposure to hypoxia resulted in an increase in malondialdehyde (MDA) levels in plasma and tissues and a concurrent decrease in blood glutathione (GSH), glutathione peroxidase (GPx), plasma protein and plasma selenium content when compared with controls. Haemoglobin concentration (Hb%), red blood corpuscles (RBC) and white blood corpuscles (WBC) count were also increased in the hypoxia-exposed group. Selenium supplementation to animals reversed the trend. There was a significant decrease (P < 0.001) in MDA and subsequent increase in plasma and tissue GSH levels. Similarly the blood and tissue GPx and plasma protein also increased significantly in the Se supplemented animals compared with control animals. The Hb%, RBC and WBC counts showed no significant difference between Se-fed and control rats. These results suggest that selenium may help in reducing the lipid peroxidation during hypoxia.

Intraperitoneal immunization of recombinant HSP70 (DnaK) of Salmonella Typhi induces a predominant Th2 response and protective immunity in mice against lethal Salmonella infection.

Heat shock proteins serve as important antigens in defense against infectious diseases. Members of HSP70 family, particularly microbial HSP70s have acquired special significance in immunity. In the present study, we evaluated the immunogenicity and protective efficacy of HSP70 of Salmonella enterica serovar Typhi against lethal infection by Salmonella in mice with or without adjuvants. The HSP70 gene was cloned and expressed in Escherichia coli BL21 and purified by affinity chromatography. Immunization of mice with HSP70 either alone or in combination with complete Freunds adjuvant (CFA) resulted in a significant increase in antibody titers as compared to control. Antibody isotyping revealed that HSP70 immunization induces both IgG1 and IgG2a antibodies to a significant extent but a higher IgG1/IgG2a ratio indicates a predominant Th2 response. There was a significant increase in lymphocyte proliferation, and levels of both Th2 and Th1 cytokines in cells isolated from immunized mice as compared to control. Immunization of mice with recombinant HSP70 either alone or in combination with CFA conferred 70-90% protection against lethal infections by Salmonella Typhi Ty2 or Salmonella Typhimurium. However, passive immunization with anti-HSP70 sera induced only partial protection in the immunized mice.

Sub-chronic oral toxicity study in Sprague-Dawley rats with hypoxia mimetic cobalt chloride towards the development of promising neutraceutical for oxygen deprivation.

The present study evaluates the toxicity from sub-chronic administration of CoCl(2) (12.5mg cobalt kg(-1) BW for 7 days) to male Sprague-Dawley rats in view of the beneficial effects of CoCl(2) in animals and for developing efficacious therapeutic regimen in humans. 32 rats weighing 200+/-25 g were used for all experiments. Blood was collected for hematological and biochemical analysis and various organs were dissected after perfusion of animals under anesthesia for other analyses. Mean feed consumption and feed conversion efficiency values were comparable across all study groups; however, hematological analysis depicted a significant increase in hemoglobin, hematocrit and RBC in the entire cobalt-supplemented groups, which are a component of its beneficial effect. There was a significant increase in monocytes, granulocytes and WBC after 1 and 24h, which were comparable with control after 7 days. Other biochemical analyses also showed no change with respect to control. Though the metal content increased significantly in liver initially (1 and 24h) after treatment, it was equivalent to control after 7 days. Moreover, histopathological analysis revealed no evidence of changes that could be attributed to cobalt pretreatment. It is therefore reasonable to conclude that the present study supports further use of the present dose of CoCl(2), which was found to be nontoxic.

Cytoprotective and immunomodulatory properties of Geriforte, a herbomineral preparation, in lymphocytes.

The study was designed to determine the cytoprotective and immunomodulatory properties of Geriforte, an indigenous herbomineral compound, using lymphocytes as a model system. The possible involvement of free radicals and the ability of Geriforte to inhibit the oxidative process induced by tert-butylhydroperoxide (tert-BHP) was also investigated. The production of free radicals (evaluated by fluorescent probe fluorescein-diacetate), level of malondialdehyde (MDA, as index of lipid peroxidation), and levels of anti-oxidants--reduced glutathione (GSH) and glutathione peroxidase (GPx) were determined. There was an increase in cytotoxicity and apoptosis significantly in the presence of tert-BHP (100 microM) over control. Addition of tert-BHP resulted in a marked increase in free radical production and MDA level with a concomitant decrease in GSH level in lymphocytes. Geriforte supplementation reduced cytotoxicity and apoptosis induced by tert-BHP. Further, Geriforte inhibited tert-BHP induced lipid peroxidation and maintained higher anti-oxidant levels. tert-BHP significantly inhibited the lymphocyte proliferation stimulated by mitogens (Lipopolysaccharide/Concanavalin A) and enhanced DNA fragmentation. Geriforte relieved the inhibitory effect of tert-BHP on lymphocyte proliferation and decreased DNA fragmentation appreciably. The results indicate that Geriforte possesses cytoprotective and immunomodulatory properties which could be due to its anti-oxidant activity.

Improvement in high altitude hypoxic stress tolerance in albino rats on oral glutamate supplementation

Hypoxia results in body weight loss due to anorexia and hypophagia and reduced human performance. The present study was undertaken to evaluate the role of oral glutamate administration with food and water in improvement in hypoxic tolerance, if any. The placebo controlled study revealed that supplementation of optimal dose of glutamate (Glu) promoted hypoxic tolerance in male Sprague Dawley Albino rats fed one dose daily for 5 days and exposed to continuous intermittent hypoxia of 6 hrs per day at 10,668m in an animal exposure chamber. Effect of varying dose of Glu on body weight (bw),food intake and water uptake were studied which revealed the optimal dose of Glu to be 27mgKg bw. Oral feeding of Glu resulted in improved tolerance of combined stress of cold and hypoxia as measured by fall in rectal temperature from 37’C to 23’C and faster rewarming. It enhanced 5.8 times hypoxic tolerance 36% bw gain and 24% food intake in hypoxia possibly by acting as a conditionally essential amino acid under hypoxia.