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 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

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