Deepika Saraswat

Exogenous Sphingosine-1-Phosphate Boosts Acclimatization in Rats Exposed to Acute Hypobaric Hypoxia: Assessment of Haematological and Metabolic Effects

Background The physiological challenges posed by hypobaric hypoxia warrant exploration of pharmacological entities to improve acclimatization to hypoxia. The present study investigates the preclinical efficacy of sphingosine-1-phosphate (S1P) to improve acclimatization to simulated hypobaric hypoxia. Experimental Approach Efficacy of intravenously administered S1P in improving haematological and metabolic acclimatization was evaluated in rats exposed to simulated acute hypobaric hypoxia (7620m for 6 hours) following S1P pre-treatment for three days. Major Findings Altitude exposure of the control rats caused systemic hypoxia, hypocapnia (plausible sign of hyperventilation) and respiratory alkalosis due to suboptimal renal compensation indicated by an overt alkaline pH of the mixed venous blood. This was associated with pronounced energy deficit in the hepatic tissue along with systemic oxidative stress and inflammation. S1P pre-treatment improved blood oxygen-carrying-capacity by increasing haemoglobin, haematocrit, and RBC count, probably as an outcome of hypoxia inducible factor-1α mediated erythropoiesis and renal S1P receptor 1 mediated haemoconcentation. The improved partial pressure of oxygen in the blood could further restore aerobic respiration and increase ATP content in the hepatic tissue of S1P treated animals. S1P could also protect the animals from hypoxia mediated oxidative stress and inflammation. Conclusion The study findings highlight S1P’s merits as a preconditioning agent for improving acclimatization to acute hypobaric hypoxia exposure. The results may have long term clinical application for improving physiological acclimatization of subjects venturing into high altitude for occupational or recreational purposes.

Novel vascular endothelial growth factor blocker improves cellular viability and reduces hypobaric hypoxia-induced vascular leakage and oedema in rat brain.

Vascular endothelial growth factor (VEGF) is an important cerebral angiogenic and permeability factor under hypoxia. There is a need to find effective molecules that may ameliorate hypoxia‐induced cerebral oedema. In silico identification of novel candidate molecules that block VEGF‐A site were identified and validated with a Ramachandran plot. The active site residues of VEGF‐A were detected by Pocketfinder, CASTp, and DogSiteScorer. Based on in silico data, three VEGF‐A blocker (VAB) candidate molecules (VAB1, VAB2, and VAB3) were checked for improvement in cellular viability and regulation of VEGF levels in N2a cells under hypoxia (0.5% O2). Additionally, the best candidate molecules efficacy was assessed in male Sprague‐Dawley rats for its ameliorative effect on cerebral oedema and vascular leakage under hypobaric hypoxia 7260 m. All experimental results were compared with the commercially available VEGF blocker sunitinib. Vascular endothelial growth factor‐A blocker 1 was found most effective in increasing cellular viability and maintaining normal VEGF levels under hypoxia (0.5% oxygen) in N2a cells. Vascular endothelial growth factor‐A blocker 1 effectively restored VEGF levels, decreased cerebral oedema, and reduced vascular leakage under hypobaric hypoxia when compared to sunitinib‐treated rats. Vascular endothelial growth factor‐A blocker 1 may be a promising candidate molecule for ameliorating hypobaric hypoxia‐induced vasogenic oedema by regulating VEGF levels.

Nanocurcumin–pyrroloquinoline formulation prevents hypertrophy–induced pathological damage by relieving mitochondrial stress in cardiomyocytes under hypoxic conditions

This study investigates the therapeutic effect of a nanocurcumin formulation (NCF) containing nanocurcumin (NC) and pyrroloquinoline quinone (PQQ) on ameliorating hypoxia-induced stress in hypertrophied primary human ventricular cardiomyocytes (HVCM) under hypoxic conditions, as validated in a Sprague-Dawley rat model of chronic hypobaric hypoxia (cHH)-induced right ventricular hypertrophy (RVH). Based on our previous findings, here, we analyzed the improvement in the protective efficacy of NCF against mitochondrial damage. The electron transport chain Complexes’ activities were analyzed as a chief operational center for mitochondrial homeostasis, along with key gene and protein markers for mitochondrial biogenesis, redox function, fatty acid oxidation, bio-energetic deficit and cell survival. NCF supplementation imparts cyto-protection from hypoxia-induced hypertrophy and damage in both in vitro and in vivo models while maintaining mitochondrial homeostasis better than NC and PQQ alone. This study proposes the use of NCF as a potential candidate molecule for imparting protection from high altitude-induced maladies in ascendants.

Co-expression of S. Typhi GroEL and IL-22 gene augments immune responses against Salmonella infection

Recombinant DNA vaccines represent a novel method for generating in situ expression of vaccine antigens. Intramuscular injections of naked DNA are able to elicit potent humoral and cellular immune responses but still numerous factors limit the immunogenicity of DNA vaccines. Co‐expression of cytokines with antigen encoding genes in DNA vectors can improve the immune responses and modify Th1/Th2 balance. In this study, the immunomodulatory effect of Interleukin 22 (IL‐22) as an adjuvant was studied by DNA vaccination with S. Typhi Heat shock protein 60 (HSP60/GroEL) in mice. Further, DNA construct of IL‐22 gene fused with GroEL was developed and immunization studies were carried out in mice. DNA vaccination with GroEL alone stimulated humoral and cell‐mediated immune responses. Co‐immunization (IL‐22+GroEL) further resulted in increase in T‐cell proliferative responses, antibody titres (IgG, IgG1, IgG2a) and secretion of IFNγ (Th1), IL‐1β and Th2 (IL‐4, IL‐6) cytokines. Co‐expression (IL‐22‐GroEL DNA) also promoted antibody titres and cytokine levels were significantly higher as compared to co‐immunized group. A reduction in bacterial load in spleen, liver and intestine was seen in all the immunized groups as compared to control, with least organ burden in fusion DNA construct group (co‐expression). Improved protective efficacy (90%) against lethal challenge by Salmonella was observed with IL‐22‐GroEL co‐expressing DNA vector as compared with plasmid encoding GroEL only (50–60%) or co‐immunization group (75–80%). This study thus shows that co‐expression of IL‐22 and GroEL genes enhances the immune responses and protective efficacy, circumventing the need of any adjuvant.

The MAPK-activator protein-1 signaling regulates changes in lung tissue of rat exposed to hypobaric hypoxia

This study reports the role of MAPKs (JNK, ERK, and p38), and activator protein‐1 (AP‐1) transcription factor in the hypobaric hypoxia induced change in lung tissue. Healthy male Sprague–Dawley rats were exposed to hypobaric hypoxia for 6, 12, 24, 48, 72, and 120 hr. Hypoxia resulted in significant increase in reactive oxygen species (ROS), vascular endothelial growth factor (VEGF) and decreased nitric oxide (NO), these act as signaling molecules for activation of MAPK and also contribute in development of vascular leakage (an indicator of pulmonary edema) as confirmed by histological studies. Our results confirmed JNK activation as an immediate early response (peaked at 6–48 hr), activation of ERKs (peaked at 24–72 hr) and p38 (peaked at 72–120 hr) as a secondary response to hypoxia. The MAPK pathway up regulated its downstream targets phospho c‐Jun (peaked at 6–120 hr), JunB (peaked at 24–120 hr) however, decreased c‐Fos, and JunD levels. DNA binding activity also confirmed activation of AP‐1 transcription factor in lung tissue under hypobaric hypoxia. Further, we analyzed the proliferative and inflammatory genes regulated by different subunits of AP‐1 to explore its role in vascular leakage. Increased expression of cyclin D1 (peaked at 12–72 hr) and p16 level (peaked at 48–120 hr) were correlated to the activation of c‐jun, c‐Fos and JunB. Administration of NFκB inhibitor caffeic acid phenethyl ester (CAPE) and SP600125 (JNK inhibitor) had no effect on increased levels of Interferon‐γ (IFN‐γ), Interleukin‐1 (IL‐1), and Tumor Necrosis Factor‐α (TNF‐α) thereby confirming the involvement of AP‐1 as well as NFκB in inflammation. Expression of c‐jun, c‐Fos were correlated with activation of proliferative genes and JunB, Fra‐1 with pro‐inflammatory cytokines. In conclusion immediate response to hypobaric hypoxia induced c‐Jun:c‐Fos subunits of AP‐1; responsible for proliferation that might cause inhomogeneous vasoconstriction leading to vascular leakage and inflammation at increased duration of hypobaric hypoxia exposure.

Combinatorial therapy of exercise-preconditioning and nanocurcumin formulation supplementation improves cardiac adaptation under hypobaric hypoxia

Abstract Background: Chronic hypobaric hypoxia (cHH) mediated cardiac insufficiencies are associated with pathological damage. Sustained redox stress and work load are major causative agents of cardiac insufficiencies under cHH. Despite the advancements made in pharmacological (anti-oxidants, vasodilators) and non-pharmacological therapeutics (acclimatization strategies and schedules), only partial success has been achieved in improving cardiac acclimatization to cHH. This necessitates the need for potent combinatorial therapies to improve cardiac acclimatization at high altitudes. We hypothesize that a combinatorial therapy comprising preconditioning to mild aerobic treadmill exercise and supplementation with nanocurcumin formulation (NCF) consisting of nanocurcumin (NC) and pyrroloquinoline quinone (PQQ) might improve cardiac adaptation at high altitudes. Methods: Adult Sprague-Dawley rats pre-conditioned to treadmill exercise and supplemented with NCF were exposed to cHH (7620 m altitude corresponding to pO2~8% at 28±2°C, relative humidity 55%±1%) for 3 weeks. The rat hearts were analyzed for changes in markers of oxidative stress (free radical leakage, lipid peroxidation, manganese-superoxide dismutase [MnSOD] activity), cardiac injury (circulating cardiac troponin I [TnI] and T [cTnT], myocardial creatine kinase [CK-MB]), metabolic damage (lactate dehydrogenase [LDH] and acetyl-coenzyme A levels, lactate and pyruvate levels) and bio-energetic insufficiency (ATP, p-AMPKα). Results: Significant modulations (p≤0.05) in cardiac redox status, metabolic damage, cardiac injury and bio-energetics were observed in rats receiving both NCF supplementation and treadmill exercise-preconditioning compared with rats receiving only one of the treatments. Conclusions: The combinatorial therapeutic strategy showed a tremendous improvement in cardiac acclimatization to cHH compared to either exercise-preconditioning or NCF supplementation alone which was evident from the effective modulation in redox, metabolic, contractile and bio-energetic homeostasis.

Lingzhi or Reishi Medicinal Mushroom, Ganoderma lucidum (Agaricomycetes), as a Cardioprotectant in an Oxygen-Deficient Environment

Imbalanced oxygen availability is detrimental to normal cell function. Oxygen-sensitive cells such as cardiomyoblasts experience severe irreversible pathophysiological damage under conditions of reduced oxygen availability, such as hypoxia. A number of natural therapeutic agents have been explored for their potential cytoprotective effects, of which medicinal mushrooms are an important source. Ganoderma lucidum, commonly known as lingzhi, is one such mushroom that has been elaborately studied for its potential pharmacological properties. In this study, aqueous and alcoholic extracts of a natural Himalayan variety of G. lucidum were evaluated for their efficiency as remedial agents in treating hypoxic injury to H9c2 cardiomyoblasts. The alcoholic extract of G. lucidum effectively restored cellular viability at a concentration of 600 μg/mL and aided in maintaining cellular redox balance under hypoxia. Substantial reduction in caspase-3 and -7 activation was observed with fluorescent-activated cell sorting. Alcoholic extract of G. lucidum minimized oxidative stress as indicated by measuring reactive oxygen species, lipid peroxidation, and reduced glutathione-to-oxidized glutathione ratio, and also by determining changes in hypoxia-inducible factor 1α and associated genes. To ascertain these positive outcomes of administration of G. lucidum extracts, certain phytoconstituents (nucleobases and flavonoids) were identified using high-performance thin-layer chromatography; antioxidant potential was also evaluated. Results indicated that both extracts contained notable quantities of nucleobases and flavonoids. The extracts also effected high free radical scavenging activities.

Preconditioning with ethyl 3,4-dihydroxy benzoate augments aerobic respiration in rat skeletal muscle

Muscle respiratory capacity decides the amount of exertion one’s skeletal muscle can undergo, and endurance exercise is believed to increase it. There are also certain preconditioning methods by which muscle respiratory and exercise performance can be enhanced. In this study, preconditioning with ethyl 3,4-dihydroxybenzoate (EDHB), a prolyl hydroxylase domain enzyme inhibitor, has been investigated to determine its effect on aerobic metabolism and bioenergetics in skeletal muscle, thus facilitating boost in physical performance in a rat model. We observed that EDHB supplementation increases aerobic metabolism via upregulation of HIF-mediated GLUT1 and GLUT4, thus enhancing glucose uptake in muscles. There was also a twofold rise in the activity of enzymes of tricarboxylic acid (TCA) cycle and glycolysis, ie, hexokinase and phosphofructokinase. There was an increase in citrate synthase and succinate dehydrogenase activity, resulting in the rise in the levels of ATP due to enhanced Krebs cycle activity as substantiated by enhanced acetyl-CoA levels in EDHB-treated rats as compared to control group. Increased lactate dehydrogenase activity, reduced expression of monocarboxylate transporter 1, and increase in monocarboxylate transporter 4 suggest transport of lactate from muscle to blood. There was a concomitant decrease in plasma lactate, which might be due to enhanced transport of lactate from blood to the liver. This was further supported by the rise in liver pyruvate levels and liver glycogen levels in EDHB-supplemented rats as compared to control rats. These results suggest that EDHB supplementation leads to improved physical performance due to the escalation of aerobic respiration quotient, ie, enhanced muscle respiratory capacity.

Rapid Acclimatization Strategies for High-Altitude Induction

High altitude (HA) is defined as elevation above 9,000 ft. At this altitude, most people develop acute mountain sickness (AMS). If untreated, this may lead to high-altitude pulmonary oedema (HAPE) or high-altitude cerebral oedema (HACE), both of which are potentially life-threatening. In emergencies/warlike conditions, rapid deployment of military personnel to high altitude frequently occurs without giving the adequate degree of altitude acclimatization, resulting in acute mountain sickness (AMS). Acclimatization to high altitude is the best strategy to prevent AMS, and this can be achieved by hypoxia preconditioning by the use of interventions like hypoxia mimetics. Efficacy of hypoxia mimetics, viz. cobalt chloride (CoCl2), ethyl 3, 4-dihydroxybenzoate (EDHB), sphingosine-1-phosphate (S1P) and other pharmacological agent nanocurmin in facilitating acclimatization to high altitude in animal model, has been discussed. An alternative approach to induce acclimatization and reduce incidence of AMS is the use of intermittent hypoxic exposure (IHE). This study was conducted to evaluate the effect of IHE exposure at sea level on incidence of AMS during acute ascent to 3,500 m altitude in Indian military personnel. The army volunteers were divided into two groups, viz. control and experimental. Experimental group of subjects were exposed to intermittent normobaric hypoxia consisting of 12 % FIO2 (altitude – air equivalent 4,350 m) for 4 h per day for 4 consecutive days. After giving IHT, the subjects were inducted to 3,500 m altitude (Leh) by air and different physiological parameters like AMS score (LLS), pulse arterial oxygen saturation (SaO2) and ventilatory parameters (V E, VO2, V T/Ti) were recorded daily. IHE-treated group showed a significant reduction in AMS at HA in comparison to control. IHE may be considered as an alternative approach to induce the altitude acclimatization at low altitude-based soldiers before their deployment to high-altitude operations in emergency-like conditions.