Quercetin prophylaxis protects the kidneys by modulating the renin-angiotensin-aldosterone axis under acute hypobaric hypoxic stress.

The study presented here aims at assessing the effects of hypobaric hypoxia on RAAS pathway and its components along with mitigation of anomalies with quercetin prophylaxis. One hour prior to hypobaric hypoxia exposure, male SD rats were orally supplemented with quercetin (50 mg/kg BW) and acetazolamide (50 mg/kg BW) and exposed them to 25,000 ft. (7,620 m) in a simulated environmental chamber for 12 h at 25 ± 2 °C. Different biochemical parameters like renin activity, aldosterone, angiotensin I, ACE 2 were determined in plasma. As a conventional response to low oxygen conditions, oxidative stress parameters (ROS and MDA) were elevated along with suppressed antioxidant system (GPx and catalase) in plasma of rats. Quercetin prophylaxis significantly down regulated the hypoxia induced oxidative stress by reducing plasma ROS & MDA levels with efficient enhancement of antioxidants (GPx and Catalase). Further, hypoxia mediated regulation of renin and ACE 2 proves the outstanding efficacy of quercetin in repudiating altercations in RAAS cascade due to hypobaric hypoxia. Furthermore, differential protein expression of HIF-1α, NFκB, IL-18 and endothelin-1 analyzed by western blotting approves the biochemical outcomes and showed that quercetin significantly aids in the reduction of inflammation under hypoxia. Studies conducted with Surface Plasmon Resonance demonstrated a binding among quercetin and ACE 2 that indicates that this flavonoid might regulate RAAS pathway via ACE 2. Henceforth, the study promotes the prophylaxis of quercetin for the better adaptability under hypobaric hypoxic conditions via modulating the RAAS pathway.

Hypoxia-mediated alterations in pulmonary surfactant protein expressions: Beneficial effects of quercetin prophylaxis.

We have compared the prophylactic efficacies of quercetin and salbutamol in preventing pulmonary surfactants oxidation under hypoxia. Male SD rats supplemented orally with quercetin (50 mg/Kg BW) and salbutamol (2 mg/Kg BW) were exposed to hypobaric hypoxia (7,620 m for 6 h). Hypoxia-mediated elevation in oxidative stress, inflammation, and extravasations of LDH & albumin content in BALF of rats were assessed. Western blotting and mRNA studies determined the differential expressions of Nrf-2, HO-1, and associated surfactant proteins (SP-A, SP-B, SP-C, & SP-D) in rat lungs. Later, the lung configuration under hypoxia was assessed histopathologically. Quercetin and salbutamol pretreatment considerably restored the expressions of Nrf-2, HO-1, and surfactant proteins to normal by attenuating the increase in oxidative stress, inflammation, and extravasations of plasma proteins in the animals under hypoxia. The histopathology has also evidenced the protective effect of quercetin in retaining normal lung architecture under hypoxia over salbutamol. The present study indicates the effectiveness of quercetin prophylaxis in preventing pulmonary surfactants oxidation under hypoxia over salbutamol.

Quercetin: a savior of alveolar barrier integrity under hypoxic microenvironment.

High altitude pulmonary edema (HAPE) is generally characterized by the loss of alveolar epithelial barrier integrity. The current study was undertaken to assess the noninvasive approaches of HAPE diagnosis and to evaluate the prophylactic potential of quercetin in preventing alveolar junction impairments. Male SD rats fed with quercetin 1 h prior to hypoxia (7,620 m, for 6 h) were selected. PET/CT imaging was performed to visualize the lung uptake of 18F-FDG in animals under hypoxia. Further, oxidant status, catalase activity, hematological & blood gas parameters were evaluated. Moreover, tight junction (TJ) proteins (ZO-1, JAM-C, Claudin-4, and occludin) expression analysis was accomplished using immune-blotting. The structural differences in lung epithelia were noted by TEM imaging. Quercetin prophylaxis has significantly reduced the FDG uptake in rat lungs under hypoxia. It has also dramatically alleviated the protein oxidation followed by an elevation in catalase activity in the lungs under hypoxia. The TJ protein expression in the lungs has also been restored to normal upon quercetin pre-treatment. Concomitantly, the quercetin preconditioning has elicited the stable blood gas and hematological parameters under hypoxia. The observations from TEM imaging have also implicated the normal lung epithelial structures in the quercetin pretreated animals under hypoxia. Quercetin prophylaxis has significantly restored alveolar epithelium integrity by abating oxidative stress in the lungs under hypoxia.Abbreviations: CT- Computed Tomography18F-FDG- Fluorodeoxyglucose (18FHAPE- High Altitude Pulmonary EdemaHb- HemoglobinHCT- HematocritHCO3- BicarbonateJAM- Junctional Adhesion MoleculeKBq- Killo BecquerelPaO2- Partial pressure of arterial oxygenPaCO2- Partial pressure of arterial carbon di-oxidePET- Positron Emission TomographyRBC- Red Blood CorpusclesSD- Sprague DawleyTJ- Tight JunctionsTEM- Transmission Electron MicroscopyWBC- White Blood CorpusclesZO- Zona Occludin.

Efficacy of Quercetin as a potent sensitizer of ß2-AR in combating the impairment of fluid clearance in lungs of rats under hypoxia.

BACKGROUND: Hypoxia reportedly increases free radical generation in the body, causing oxidative stress and inhibiting ß2-AR signaling. The present study correlates the prophylactic potential of quercetin and salbutamol in ameliorating fluid clearing capacity of lungs by re-sensitizing ß2-AR signaling under hypoxia. METHODS: Male SD rats supplemented orally with quercetin (50 mg/Kg BW), and salbutamol (2 mg/Kg BW) were exposed to hypobaric hypoxia at 7620 m for 6 h. Western blotting and ELISA quantitated NFĸB and related genes and GPCR pathway proteins. The binding affinities of drugs with receptor were determined by SPR spectroscopy and further confirmed insilico. RESULTS: Quercetin and salbutamol pre-treatment significantly up-regulated the expressions of ß2-AR, GPR-1, GPR-10, GCSα, cAMP content, and down-regulated GRK-2, ß-arrestin, ROS, NFκB (p < 0.001), thus, enhancing alveolar fluid clearance (AFC). SPR and insilico findings revealed a higher binding affinity of ß2-AR with quercetin over salbutamol. CONCLUSION: Results indicated quercetin to be a better prophylactic that augmented AFC in rats exposed to hypoxia by attenuating inflammation and stimulating ß2-AR.

Prophylactic efficacy of Quercetin in ameliorating the hypoxia induced vascular leakage in lungs of rats.

The objective of the study was to find out the prophylactic efficacy of Quercetin in ameliorating the hypoxia induced vascular leakage in lungs of rats. Male SD rats received different doses of quercetin @ 25mg, 50mg, 100mg and 200mg/Kg BW, 1h prior to hypobaric hypoxia exposure (7,620m, for 6h). Quercetin 50 mg/kg BW supplemented orally 1h prior to hypoxia exposure was considered to be the optimum dose, due to significant reduction (p<0.001) in lung water content and lung transvascular leakage compared to control (hypoxia, 6h). Further, biochemical analysis (ROS, MDA, GSH, GPx, LDH, and albumin) and differential expressions of proteins (IKK-α/ß, NFĸB, Nrf-2,TNF-α, ICAM-1, VCAM, P-selectin, Hif-1α, VEGF, TNF-α, TGF-ß, INF-γ and IL-4) were determined by western blotting and ELISA. Changes in lung parenchyma were assessed by histopathology. Quercetin (50 mg/kg BW) prophylaxis under hypoxia showed significant reduction in oxidative stress (ROS and MDA), concomitant increase in antioxidants (GSH, GPx and SOD) followed by decreased LDH and albumin extravasation in BAL fluid over hypoxia. Quercetin prophylaxis significantly down regulated hypoxia induced increase in IKKα/ß and NFĸB expressions leading to reduction in the levels of pro-inflammatory cytokines (TNF-α and INF-γ) followed by up regulation of anti-inflammatory cytokines (IL-4 and INF-γ) in lungs. Further, hypoxia mediated increase in HIF-1α was stabilized and VEGF levels in lungs were significantly down regulated by quercetin supplementation, leading to reduction in vascular leakage in lungs of rats under hypoxia. However, Quercetin has also enacted as Nrf-2 activator which significantly boosted up the synthesis of GSH under hypoxic condition compared to hypoxia. Histopathological observations further confirmed that quercetin preconditioning has an inhibitory effect on progression of oxidative stress and inflammation via attenuation of NFκB and stabilization HIF-1α in lungs of rats under hypoxia.These studies indicated that quercetin prophylaxis abrogates the possibility of hypobaric hypoxia induced pulmonary edema in rats.

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 Freund's 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.

Effect of adjuvants on immune response and protective immunity elicited by recombinant Hsp60 (GroEL) of Salmonella typhi against S. typhi infection.

Heat shock proteins (Hsps) have been reported to be dominant antigens for the host immune response to various pathogens and thus, have great potential for use in vaccination. In the present study, we evaluated the immunogenicity and protective efficacy of GroEL of Salmonella enterica serovar Typhi against lethal infection by S. typhi Ty2 in mice with or without adjuvants. Anti GroEL-IgG titers were significantly higher in mice immunized with either GroEL-alone or in combination with alum/Complete Freund's adjuvant (CFA) as compared to the control. Analysis of antibody isotypes suggested predominance of Th2 type immune response in GroEL + alum immunized animals as revealed by higher IgG1/IgG2a ratio. Whereas, immunization of animals with GroEL + CFA or GroEL-alone shifted the immune response toward Th1 phenotype. Mice immunized with GroEL with or without adjuvants, showed a significant increase in lymphocyte proliferation and cytokine levels. The animals immunized with GroEL + CFA or GroEL-alone showed higher IFN-gamma and IL-2 levels than alum group, indicating Th1 response whereas IL-4 levels (Th2 response) were found to be highest in alum group as compared to other two immunized groups. Immunization of mice with GroEL-alone, GroEL + alum, and GroEL + CFA provided 70, 50 and 80% protection, respectively, against lethal challenge by S. typhi in mice. The differences in the percentage protection among various groups were attributed to the differences in the immune responses generated by respective immunizations. The present study shows that GroEL forms an ideal candidate molecule to develop a recombinant protein based vaccine against human typhoid.

Cloning, expression and characterization of heat shock protein 60 (groEL) of Salmonella enterica serovar Typhi and its role in protective immunity against lethal Salmonella infection in mice.

Heat shock proteins (Hsps) represent dominant antigens in numerous microbial infections, suggesting a potential use of pathogen-derived Hsps for vaccination. The present study evaluates the immunogenicity and protective efficacy of groEL (Hsp60) of Salmonella enterica serovar Typhi against lethal challenge by S. Typhi Ty2 and Salmonella enterica serovar Typhimurium in mice. The groEL gene was cloned and expressed in Escherichia coli BL21 and purified by affinity chromatography. Immunization of mice with groEL resulted in a significant increase in antibody titers. Antibody isotyping revealed that groEL immunization induces both IgG1 and IgG2a antibodies. There was a significant increase in lymphocyte proliferation, interleukin-4 and interferon-gamma levels in cells isolated from immunized mice as compared to control. Immunization of mice with recombinant groEL protein with or without adjuvant conferred 70-90% protection against lethal infections either by S. Typhi Ty2 or S. Typhimurium. Passive immunization with anti-groEL sera also protected 50% mice against lethal infection.

Studies on immunogenicity and protective efficacy of DnaJ of Salmonella Typhi against lethal infection by Salmonella Typhimurium in mice.

In the present study DnaJ (HSP40) of Salmonella enterica serovar Typhi has been evaluated for its immunogenicity and efficacy in protecting mice against lethal challenge by S. enterica serovar Typhimurium infection. DnaJ was amplified by PCR of the genomic DNA of S. Typhi and subsequently cloned in pQE-30 expression vector. The protein was induced by IPTG and purified using Ni-NTA chromatography under denaturing conditions. After refolding in vitro the immune response was evaluated by injecting 40 microg DnaJ protein/mouse i.p. on 0th, 7th and 28th day. The results showed a significant increase in antibody titre and lymphocyte proliferation in animals immunised with DnaJ as compared to control. Further there was an appreciable increase in IL-2, IL-4, IFN-gamma production in lymphocytes isolated from immunised mice as compared to control. In this limited study, immunisation of mice with DnaJ was found to provide 70% protection against lethal challenge by S. Typhimurium indicating the possible use of DnaJ as vaccine candidate against typhoid.