Exogenous sphingosine-1-phosphate boosts acclimatization in rats exposed to acute hypobaric hypoxia: assessment of haematological and metabolic effects.

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.Efficacy of intravenously administered S1P in improving haematological and metabolic acclimatization was evaluated in rats exposed to simulated acute hypobaric hypoxia (7620 m for 6 hours) following S1P pre-treatment for three days.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.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

Effect of S1P on oxidative stress markers and arginase activity in acute hypobaric hypoxia exposed rats.

<p>Values are means ± SD (<i>n</i> = <b>6</b>).</p><p>*<i>p</i><0.05 compared with the normoxic control,</p><p>**<i>p</i>≤0.01 compared with the normoxic control,</p><p>***<i>p</i>≤0.001 compared with the normoxic control,</p>†<p><i>p</i><0.05 compared with the hypoxic control,</p>††<p><i>p</i>≤0.01 compared with the hypoxic control,</p>†††<p><i>p</i>≤0.001 compared with the hypoxic control.</p

Effect of S1P treatment on circulatory pro- and anti-inflammatory markers.

<p>Markers of inflammation – IFN-γ, IL-6, TNF-α, MCP-1, TGF-β, C-Reactive protein (C-RP) and anti-inflammatory cytokine IL-10 were quantified post-exposure in plasma of experimental animals using sandwich ELISA, each animal represented in triplicate. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098025#pone-0098025-g004" target="_blank">Figure 4 a</a>) describes TGF-β, TNF-α, MCP-1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098025#pone-0098025-g004" target="_blank">Figure 4 b</a>) describes IL-6, IFN-γ, C-RP, IL-10 levels in the systemic circulation. Values are representative of mean ± SD (n = 6). Statistical significance was calculated using ANOVA/<i>post hoc</i> Bonferroni. NC: Normoxia control, HC: Hypoxia control, 1: 1 µg S1P/kg b.w., 10: 10 µg S1P/kg b.w., 100: 100 µg S1P/kg b.w., *: p<0.05 compared with the normoxic control, **: p≤0.01 compared with the normoxic control, ***: p≤0.001 compared with the normoxic control,†: p<0.05 compared with the hypoxic control, ††: p≤ 0.01 compared with the hypoxic control, †††: p≤ 0.001 compared with the hypoxic control.</p

Effect of S1P treatment on bioenergetics status in hepatic tissue.

<p>Homogenate of hepatic tissue was analyzed for ATP content, tissue glucose, glycogen reserve, indicator of anaerobic metabolism (Lactate Dehydrogenase), glycolysis (Hexokinase) and citric acid cycle (Succinate dehydrogenase and Citrate Synthase). Each assay was carried out for each experimental animal thrice. Data is represented as mean percentage fold change against normoxic control. Values are representative of mean ± SD (n = 6). Statistical significance was calculated using ANOVA/<i>post hoc</i> Bonferroni. NC: Normoxia control, HC: Hypoxia control, 1: 1 µg S1P/kg b.w., 10: 10 µg S1P/kg b.w., 100: 100 µg S1P/kg b.w., *: p<0.05 compared with the normoxic control, **: p≤0.01 compared with the normoxic control, ***: p≤0.001 compared with the normoxic control, †: p<0.05 compared with the hypoxic control, ††: p≤0.01 compared with the hypoxic control, †††: p≤0.001 compared with the hypoxic control.</p

Effect of S1P treatment on HIF-1α accumulation and downstream gene expression.

<p>a) Renal HIF-1α accumulation and Epo accumulation in plasma. HIF-1α accumulation in the renal tissue homogenate and build-up of erythropoietin in plasma was quantified. b) Hepatic HIF-1α accumulation. c) Effect S1P pre-treatment on circulatory VEGF. Vascular endothelial growth factor (VEGF) was quantified in plasma of experimental animals. These estimations were carried out using sandwich ELISA, and were carried out in triplicates for each experimental animal. Values are representative of mean ± SD (n = 6). Statistical significance was calculated using ANOVA/<i>post hoc</i> Bonferroni. NC: Normoxia control, HC: Hypoxia control, 1: 1 µg S1P/kg b.w., 10: 10 µg S1P/kg b.w., 100: 100 µg S1P/kg b.w., *: p<0.05 compared with the normoxic control, **: p≤0.01 compared with the normoxic control, ***: p≤0.001 compared with the normoxic control, †: p<0.05 compared with the hypoxic control, ††: p≤0.01 compared with the hypoxic control, †††: p≤0.001 compared with the hypoxic control.</p

Effect of S1P treatment on S1P₁ expression in renal tissue.

<p>Representative immune-blot of S1P1. Densitometric analysis of blot normalized against the loading control (α-tubulin). Values are representative of mean ± SD (n = 6). Statistical significance was calculated using ANOVA/<i>post hoc</i> Bonferroni. NC: Normoxia control, HC: Hypoxia control, 1: 1 µg S1P/kg b.w., 10: 10 µg S1P/kg b.w., 100: 100 µg S1P/kg b.w., *: p<0.05 compared with the normoxic control, **: p≤0.01 compared with the normoxic control, ***: p≤0.001 compared with the normoxic control, †: p<0.05 compared with the hypoxic control, ††: p≤0.01 compared with the hypoxic control, †††: p≤0.001 compared with the hypoxic control.</p

Effect of S1P on oxygen-carrying-capacity in acute hypobaric hypoxia exposed rats.

<p>Values are means ± SD (<i>n</i> = <b>6</b>).</p><p>*<i>p</i><0.05 compared with the normoxic control,</p><p>**<i>p</i>≤0.01 compared with the normoxic control,</p><p>***<i>p</i>≤0.001 compared with the normoxic control,</p>†<p><i>p</i><0.05 compared with the hypoxic control,</p>††<p><i>p</i>≤0.01 compared with the hypoxic control,</p>†††<p><i>p</i>≤0.001 compared with the hypoxic control.</p

Hypothesis for underlying basis of the observed protection conferred by S1P preconditioning.

<p>Exposure to hypobaric hypoxia evokes pathological (red boxes in circle) as well as adaptive (green boxes in circle) responses in the body, as an outcome of compromised systemic oxygen bioavailability. The strength of adaptive responses in unacclimatized individuals is insufficient to confer protection, arising the need for pharmacological mitigation. The study reports sphingosine-1-phosphate mediated preconditioning (black text outside the circle) to potentially confer protection against pathological milieu as well as boost the adaptive responses. Sphingosine-1-phosphate mediated boost in haemoglobin, haematocrit, RBC count, serum iron, TIBC, haemo-concentration and oxygen bioavailability culminates into successful acclimatization.</p

Redox control mechanisms in AtCyp19-3 and TaCypA-1 (A)

<p>Potential metal binding site (Site1) in TaCypA-1 comprising of Cys-122, His-99 and Cys-126 amino acids (yellow) as predicted by TEMSP. Cys-122-Cys-126 pair (3.905 <i>Å</i>) in TaCypA-1 which may be involved in Redox 2-Cys mechanism similar to SmCypA, is also shown. <b>(B)</b> Second potential metal binding site (Site 2) consisting of Cys-40, His-54 and Cys-168 (red). Disulfide bond between Cys-40 and Cys-168 (5.489 <i>Å</i>), and hydrogen bond network linking side chains of Glu-83(red) with divergent loop residues Lys-48 and Ser-49 (magenta) in TaCypA-1 are implicated in allosteric control similar to Redox 2-Cys Mechanism in CsCypA. <b>(C)</b> Cartoon showing His-54 and disulfide bridge forming pair Cys-40-Cys-168 (5.4 <i>Å</i>) in AtCyp19-3. This triad (yellow) may form a metal binding site in AtCyp19-3. <b>(D)</b> Hydrogen bond connecting side chain carboxyl group of Glu-83(red) with main chain amide group of Lys-48 (green) from the divergent loop in AtCyp19-3. Formation of Cys-40-Cys-168 disulfide bond disrupts this interaction and closes the active site. Distances have been shown as black dashed lines while hydrogen bonds as black solid lines.</p

Comparative analysis of biochemical parameters of various cyclophilins.

<p>Comparative analysis of biochemical parameters of various cyclophilins.</p