Intonation of Nrf2 and Hif1-α pathway by curcumin prophylaxis: A potential strategy to augment survival signaling under hypoxia.

BACKGROUND: Pulmonary surfactant oxidation leads to alveolar collapse- a condition often noticed in high altitude pulmonary edema (HAPE). The present study was aimed to determine the effect of curcumin prophylaxis in augmenting the phase II antioxidant enzymes and surfactant proteins expression in enabling the pulmonary surfactant homeostasis under hypoxia. METHODS: A549 cells were exposed to 3% hypoxia for different time durations (1 h, 3 h, 6 h, 12 h and 24 h). The Cells were pretreated (1 h) with 10 µM curcumin and exposed to hypoxia. The in-vivo results were extrapolated into in-vivo system using male Sprague Dawley rats, exposed to a stimulated altitude of 7620 m for 6 h. The rats were supplemented with curcumin (50 mg/kgBW) 1 h prior to hypoxia exposure. RESULTS: Results showed that, the expression of surfactant proteins (SPs) A and B decreased from 3 h of hypoxic exposure, whereas expression of SP-C and SP-D proteins were increased within 1 h of hypoxic exposure over control cells. Hypoxic exposure resulted into significant increase in protein and lipid peroxidation (p < 0.001), reduced levels of antioxidants (GSH, GPx and SOD) (p < 0.001) along with significant down regulation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and Heme oxygenase-1 (HO-1) in A549 cells over control. However, the curcumin supplementation both in-vitro and in-vivo resulted into increased expressions of HO-1 and Nrf2 significantly (p < 0.001), which enabled the cells in balanced expression of SPs with reduced levels of oxidants. Further curcumin significantly enhanced the levels of antioxidant enzymes in BALF along with stabilized expression of hypoxia inducible factor 1(HIF-1α) followed by reduced expression of vascular endothelial growth factor (VEGF) in lungs of rats. The immunohistochemistry observations provided substantial evidence of enhanced surfactant protein expressions in lungs of curcumin administered hypoxia exposed rats. CONCLUSION: These results indicate that curcumin augment survival signaling by reinforcing the induction of phase II antioxidant enzymes thereby enabling the pulmonary surfactant homeostasis under hypoxia.

Curcumin prophylaxis mitigates the incidence of hypobaric hypoxia-induced altered ion channels expression and impaired tight junction proteins integrity in rat brain.

BACKGROUND: The present study was proposed to elucidate the prophylactic role of curcumin in the prevention of hypoxia-induced cerebral edema (HACE). METHODS: Rats were exposed to simulated hypobaric hypoxia at 7620 m for 24 h at 25 ± 1 °C. Transvascular leakage, expression of transcriptional factors (nuclear factor-kappa B (NF-κB) and hypoxia inducible factor 1 alpha (Hif-1α) and also the genes regulated by these transcriptional factors, sodium potassium-adenosine triphosphatase (Na(+)/K(+)-ATPase) and endothelial sodium channel (ENaC) levels and brain tight junction (TJ) proteins like ZO-1, junctional adhesion molecule C (JAMC), claudin 4 and claudin 5 levels were determined in the brain of rats under hypoxia by Western blotting, electro mobility shift assay, ELISA, immunohistochemistry, and histopathology along with haematological parameters. Simultaneously, to rule out the fact that inflammation causes impaired Na(+)/K(+)-ATPase and ENaC functions and disturbing the TJ integrity leading to cerebral edema, the rats were pre-treated with curcumin (100 mg/kg body weight) 1 h prior to 24-h hypoxia. RESULTS: Curcumin administration to rats, under hypoxia showed a significant decrease (p < 0.001) in brain water content (3.53 ± 0.58 wet-to-dry-weight (W/D) ratio) and transvascular leakage (136.2 ± 13.24 relative fluorescence units per gram (r.f.u./g)) in the brain of rats compared to control (24-h hypoxia) (7.1 ± 1.0 W/D ratio and 262.42 ± 24.67 r.f.u./g, respectively). Curcumin prophylaxis significantly attenuated the upregulation of NF-κB (p < 0.001), thereby leading to concomitant downregulation of pro-inflammatory cytokine levels (↓IL-1, IL-2, IL-18 and TNF-α), cell adhesion molecules (↓P-selectin and E-selectin) and increased anti-inflammatory cytokine (↑IL-10). Curcumin stabilized the brain HIF-1α levels followed by maintaining VEGF levels along with upregulated Na(+)/K(+)-ATPase and ENaC levels (p < 0.001) under hypoxia. Curcumin restored the brain ZO-1, JAMC, claudin 4 and claudin 5 levels (p < 0.001) under hypoxia. Histopathological observations revealed the absence of edema and inflammation in the brain of rats supplemented with curcumin. CONCLUSIONS: These results indicate that curcumin is a potent drug in amelioration of HACE as it effectively attenuated inflammation as well as fluid influx by maintaining the tight junction proteins integrity with increased ion channels expression in the brain of rats under hypoxia.

Quercetin as a prophylactic measure against high altitude cerebral edema.

The present study was undertaken to elucidate the intervention of quercetin against high altitude cerebral edema (HACE) using male Sprague Dawley rats as an animal model. This study was also programmed to compare and correlate the effect of both quercetin (flavonoid) and dexamethasone (steroid) against HACE. Six groups of animals were designed for this experiment, (I) normoxia, (II) hypoxia (25,000 ft, 24 h), (III) normoxia+quercetin (50 mg/kg body wt), (IV) normoxia+dexamethasone (4 mg/kg body wt), (V) hypoxia+quercetin (50 mg/kg body wt), (VI) hypoxia+dexamethasone (4 mg/kg body wt). Quercetin at 50 mg/kg body wt, orally 1h prior to hypoxia exposure, was considered as the optimum dose, due to a significant reduction in the level of brain water content and cerebral transvascular leakage (P < 0.001), as compared to control (24 h hypoxia). Dexamethasone was administered at 4 mg/kg body wt, orally, 1h prior to hypoxia exposure. Both drugs (quercetin and dexamethasone) could efficiently reduce the hypoxia-induced hematological changes. Quercetin was observed to be a more potent antioxidative and anti-inflammatory agent. It blocks nuclear factor kappa-beta (NFκB) more significantly (P < 0.05) than the dexamethasone-administered hypoxia-exposed rats. Histopathological findings demonstrate the absence of an edema and inflammation in the brain sections of quercetin-administered hypoxia-exposed rats. The present study reveals quercetin to be a potent drug against HACE, as it efficiently attenuates inflammation as well as cerebral edema formation without any side effects of steroid therapy (dexamethasone).

Selenium protects the hypoxia induced apoptosis in neuroblastoma cells through upregulation of Bcl-2.

Selenium (Se) is an essential micronutrient as well as a toxic trace element in animal and human nutrition. The effects of Se in the immune system and some diseases are well documented. The objective of the present study was to examine the role of Se in reducing the hypoxia induced apoptosis in neuroblastoma cell line. Hypoxia showed an enhanced cytotoxicity, increased free radical production and apoptosis (p<0.001) which was measured in terms of DNA break down by comet assay. Hypoxia has decreased reduced Glutathione (GSH) content, Glutathione Reductase (GR), Glutathione peroxidase (GPx) and Superoxide Dismutase (SOD) activities as compared to control cells. During hypoxic condition the expression of cytochrome C, pro and active caspase-3 levels were enhanced significantly followed by nonsignificant upregulation of Bcl-2. But, the Se supplementation inhibited the cytotoxicity, free radical generation and stabilized the HIF-1alpha accumulation in cells under hypoxia. The GSH content, GR, GPx and SOD activities increased significantly in Se-treated hypoxic cells, as compared to control. Further there was an appreciable inhibition of apoptosis by upregulation of Bcl-2 proteins, in the presence of Se under hypoxia. Selenium supplementation to cells significantly inhibited the hypoxia induced DNA fragmentation and restored the antioxidant status back to control levels. This study suggests that Se supplementation prevented the cells from hypoxia induced apoptosis by triggering upregulation of Bcl-2 protein and reducing the oxidative stress.

Immunogenicity and protective efficacy of DnaJ (hsp40) of Streptococcus pneumoniae against lethal infection in mice.

The present study was carried out to evaluate the immunogenicity and protective efficacy of DnaJ (hsp40) of Streptococcus pneumoniae, by cloning the full-length DnaJ of S. pneumoniae and expressing in heterologous host E. coli BL-21 (DE3). PCR amplified DnaJ was ligated in pQE-30 expression vector and subsequently transformed in E. coli DH5alpha strain. Cloning of DnaJ was confirmed by double digestion and PCR, followed by DNA sequencing. The His-tag containing recombinant protein was purified by Ni-NTA affinity chromatography. To determine the immunogenicity of DnaJ, the mice (10 mice/group) were immunized by injecting 40 microg DnaJ protein/mouse i.p. There was a significant increase in IgG titres (2 x 10(5)) in mice immunized with DnaJ protein. Isotyping studies revealed that antibodies produced are predominantly IgG2a type indicating the predominance of Th1 response. A significant increase in lymphocyte proliferation was observed in mice immunized with DnaJ protein as compared to the control mice. Further, there was a significant increase in IL-2 and gamma-IFN levels in culture supernatants of splenocytes isolated from immunized mice. To determine the efficacy of DnaJ vaccination in eliciting protection, the mice were challenged with 1 x 10(5)cells of S. pneumoniae A66 type 3 capsular strain intra-nasally after 7 days of last immunization. All the control mice died within 2 days of post-infection, while 70% of animals immunized with DnaJ survived the lethal challenge by S. pneumoniae. The study reveals that immunization of mice with DnaJ elicits protective immunity against S. pneumoniae infection.

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 body's 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.

Cyto-protective and immunomodulating properties of Amla (Emblica officinalis) on lymphocytes: an in-vitro study.

The fruits extracts of Emblica officinalis (Amla) has been reported to have strong anti-oxidant properties. There is a paucity of studies on the immunomodulatory properties of fruit extracts of Amla in immuno-compromised states, with the emphasis on lymphocytes. Therefore, the aim of the study was to determine the anti-oxidant and immunomodulatory properties of Amla using chromium (VI) as an immunosuppressive agent. Chromium (Cr) treatment results in enhanced cytotoxicity, free radical production, lipid peroxidation and decreased glutathione peroxidase (GPx) activity and diminished glutathione (GSH) levels. There was a significant inhibition of both lipopolysaccharide and concanavalin-A-stimulated lymphocyte proliferation. Chromium also inhibited Con A stimulated interleukin-2 and gamma-interferon production significantly. Further, there was enhanced apoptosis and DNA fragmentation in the presence of Cr. Amla significantly inhibited Cr-induced free radical production and restored the anti-oxidant status back to control level. Amla also inhibited apoptosis and DNA fragmentation induced by Cr. Interestingly, Amla relieved the immunosuppressive effects of Cr on lymphocyte proliferation and even restored the IL-2 and gamma-IFN production considerably.

Antioxidant effect of beta-carotene on hypoxia induced oxidative stress in male albino rats.

Hypoxia is known to induce oxidative stress in organisms leading to tissue injury. In the present study beta-carotene (BC) given at 10 mg/kg body weight (BW) in reducing the oxidative stress induced by hypoxia was evaluated on male albino rats. Hypoxia exposure caused an increase in malondialdehyde (MDA) levels in plasma and tissues, a concurrent decrease in blood glutathione (GSH), glutathione peroxidase (GPx), plasma protein and plasma BC content. Hemoglobin concentration, Red blood corpuscles (RBC) and White blood corpuscles (WBC) count were also increased under hypoxia. BC supplementation reversed the trend, inducing a significant decrease (P<0.05) in MDA and subsequent increase in plasma and tissue GSH levels in animals exposed to hypoxia. Blood GPx and plasma protein also increased significantly in BC supplemented animals. BC supplementation did not alter the changes in Hb concentration, RBC and WBC count. BC has potent antioxidant activities in reducing the oxidative stress induced by hypobaric hypoxia.