Image_1_Astaxanthin supplementation counters exercise-induced decreases in immune-related plasma proteins.TIFF

ObjectivesAstaxanthin is a dark red keto-carotenoid found in aquatic animals such as salmon and shrimp, and algae (Haematococcus pluvialis). Astaxanthin has a unique molecular structure that may facilitate anti-oxidative, immunomodulatory, and anti-inflammatory effects during physiological stress. The primary objective of this study was to examine the efficacy of 4-week ingestion of astaxanthin in moderating exercise-induced inflammation and immune dysfunction using a multi-omics approach.MethodsThis study employed a randomized, double blind, placebo controlled, crossover design with two 4-week supplementation periods and a 2-week washout period. Study participants were randomized to astaxanthin and placebo trials, with supplements ingested daily for 4 weeks prior to running 2.25 h at close to 70%VO2max (including 30 min of 10% downhill running). After the washout period, participants repeated all procedures using the counterbalanced supplement. The astaxanthin capsule contained 8 mg of algae astaxanthin. Six blood samples were collected before and after supplementation (overnight fasted state), immediately post-exercise, and at 1.5, 3, and 24 h-post-exercise. Plasma aliquots were assayed using untargeted proteomics, and targeted oxylipin and cytokine panels.ResultsThe 2.25 h running bout induced significant muscle soreness, muscle damage, and inflammation. Astaxanthin supplementation had no effect on exercise-induced muscle soreness, muscle damage, and increases in six plasma cytokines and 42 oxylipins. Notably, astaxanthin supplementation countered exercise-induced decreases in 82 plasma proteins (during 24 h recovery). Biological process analysis revealed that most of these proteins were involved in immune-related functions such as defense responses, complement activation, and humoral immune system responses. Twenty plasma immunoglobulins were identified that differed significantly between the astaxanthin and placebo trials. Plasma levels of IgM decreased significantly post-exercise but recovered after the 24 h post-exercise recovery period in the astaxanthin but not the placebo trial.DiscussionThese data support that 4-week astaxanthin versus placebo supplementation did not counter exercise-induced increases in plasma cytokines and oxylipins but was linked to normalization of post-exercise plasma levels of numerous immune-related proteins including immunoglobulins within 24 h. Short-term astaxanthin supplementation (8 mg/day during a 4-week period) provided immune support for runners engaging in a vigorous 2.25 h running bout and uniquely countered decreases in plasma immunoglobulin levels.</p

Data_Sheet_2_Astaxanthin supplementation counters exercise-induced decreases in immune-related plasma proteins.xlsx

ObjectivesAstaxanthin is a dark red keto-carotenoid found in aquatic animals such as salmon and shrimp, and algae (Haematococcus pluvialis). Astaxanthin has a unique molecular structure that may facilitate anti-oxidative, immunomodulatory, and anti-inflammatory effects during physiological stress. The primary objective of this study was to examine the efficacy of 4-week ingestion of astaxanthin in moderating exercise-induced inflammation and immune dysfunction using a multi-omics approach.MethodsThis study employed a randomized, double blind, placebo controlled, crossover design with two 4-week supplementation periods and a 2-week washout period. Study participants were randomized to astaxanthin and placebo trials, with supplements ingested daily for 4 weeks prior to running 2.25 h at close to 70%VO2max (including 30 min of 10% downhill running). After the washout period, participants repeated all procedures using the counterbalanced supplement. The astaxanthin capsule contained 8 mg of algae astaxanthin. Six blood samples were collected before and after supplementation (overnight fasted state), immediately post-exercise, and at 1.5, 3, and 24 h-post-exercise. Plasma aliquots were assayed using untargeted proteomics, and targeted oxylipin and cytokine panels.ResultsThe 2.25 h running bout induced significant muscle soreness, muscle damage, and inflammation. Astaxanthin supplementation had no effect on exercise-induced muscle soreness, muscle damage, and increases in six plasma cytokines and 42 oxylipins. Notably, astaxanthin supplementation countered exercise-induced decreases in 82 plasma proteins (during 24 h recovery). Biological process analysis revealed that most of these proteins were involved in immune-related functions such as defense responses, complement activation, and humoral immune system responses. Twenty plasma immunoglobulins were identified that differed significantly between the astaxanthin and placebo trials. Plasma levels of IgM decreased significantly post-exercise but recovered after the 24 h post-exercise recovery period in the astaxanthin but not the placebo trial.DiscussionThese data support that 4-week astaxanthin versus placebo supplementation did not counter exercise-induced increases in plasma cytokines and oxylipins but was linked to normalization of post-exercise plasma levels of numerous immune-related proteins including immunoglobulins within 24 h. Short-term astaxanthin supplementation (8 mg/day during a 4-week period) provided immune support for runners engaging in a vigorous 2.25 h running bout and uniquely countered decreases in plasma immunoglobulin levels.</p

Data_Sheet_1_Astaxanthin supplementation counters exercise-induced decreases in immune-related plasma proteins.xlsx

ObjectivesAstaxanthin is a dark red keto-carotenoid found in aquatic animals such as salmon and shrimp, and algae (Haematococcus pluvialis). Astaxanthin has a unique molecular structure that may facilitate anti-oxidative, immunomodulatory, and anti-inflammatory effects during physiological stress. The primary objective of this study was to examine the efficacy of 4-week ingestion of astaxanthin in moderating exercise-induced inflammation and immune dysfunction using a multi-omics approach.MethodsThis study employed a randomized, double blind, placebo controlled, crossover design with two 4-week supplementation periods and a 2-week washout period. Study participants were randomized to astaxanthin and placebo trials, with supplements ingested daily for 4 weeks prior to running 2.25 h at close to 70%VO2max (including 30 min of 10% downhill running). After the washout period, participants repeated all procedures using the counterbalanced supplement. The astaxanthin capsule contained 8 mg of algae astaxanthin. Six blood samples were collected before and after supplementation (overnight fasted state), immediately post-exercise, and at 1.5, 3, and 24 h-post-exercise. Plasma aliquots were assayed using untargeted proteomics, and targeted oxylipin and cytokine panels.ResultsThe 2.25 h running bout induced significant muscle soreness, muscle damage, and inflammation. Astaxanthin supplementation had no effect on exercise-induced muscle soreness, muscle damage, and increases in six plasma cytokines and 42 oxylipins. Notably, astaxanthin supplementation countered exercise-induced decreases in 82 plasma proteins (during 24 h recovery). Biological process analysis revealed that most of these proteins were involved in immune-related functions such as defense responses, complement activation, and humoral immune system responses. Twenty plasma immunoglobulins were identified that differed significantly between the astaxanthin and placebo trials. Plasma levels of IgM decreased significantly post-exercise but recovered after the 24 h post-exercise recovery period in the astaxanthin but not the placebo trial.DiscussionThese data support that 4-week astaxanthin versus placebo supplementation did not counter exercise-induced increases in plasma cytokines and oxylipins but was linked to normalization of post-exercise plasma levels of numerous immune-related proteins including immunoglobulins within 24 h. Short-term astaxanthin supplementation (8 mg/day during a 4-week period) provided immune support for runners engaging in a vigorous 2.25 h running bout and uniquely countered decreases in plasma immunoglobulin levels.</p

Exposure to CS increased HNE (A) and ACR protein adducts (B) in HaCaT cells measured by Western blot and this is confirmed also by ICC staining for HNE and ACR (C) (see arrows).

<p>CS increased carbonyl groups expression (D) in HaCaT cells. Cells were exposed to CS for 50 min and cells were harvested at different time points (0–12 hrs). Western blot shown in the top is representative of five experiments. Quantification of the SR-B1 bands is shown as ratio of SRB1/β-actin (bottom panel). Data are expressed as arbitrary units (averages of five different experiments, *p<0.05; **p<0.01). β-actin was used as loading control.</p

Exposure to CS decreased SR-B1 protein levels in HaCaT cells. Cells were exposed to CS for 50 min and cells were harvested at different time points (0–24 hrs).

<p>The Western blot shown in the top is representative of five experiments. Quantification of the SR-B1 bands is shown in the bottom panel. Data are expressed in arbitrary units (averages of five different experiments, *p<0.05). β-actin was used as loading control. Immunogold for SR-B1 confirm the decreased protein levels after CS exposure (B). IHC for SR-B1 is shown in the C panel (arrows).</p

Cigarette Smoke exposure induces changes in SR-B1 levels and localization in human keratinocytes.

<p>Immunocytochemistry of HaCaT cells showing localization of SR-B1 (green) before and after CS exposure for different time points. Images are merged and are representative of at least 100 cells viewed in each experiments (n = 5). Nuclei (blue) were stained with DAPI. A) Cells overview at different time points 40×; B) Representative Western blot of proteins extracted from the membranes of cells exposed to Cigarette Smoke at different time points. The signals of SR-B1 protein levels were determined by densitometric analysis of the scanned images (bottom panel). Data are expressed in arbitrary units and are averages of the values for five different experiments. (*p<0.05).</p

Possible mechanism involved in the degradation of SR-B1.

<p>Among the components present in CS there are acrolein and H₂O₂ that beside to react with the membrane lipids (1) are able to cross the cell membrane (2), once H₂O₂ is inside the cells, there will be the formation of OH. (Fenton reaction) (3) that will react with the cytosolic membrane lipids and the formation of lipid peroxidation products such as ACR and HNE (4). ACR and HNE can from SR-B1 adducts (5 and 6) and HNE can also activate NOX by inducing the translocation of the cytoplasmic submit to the membrane (7). Activation of NOX lead to the increased production of O₂⁻ that can be dismutated (SOD) in H₂O₂ (8) that via Fenton reaction will further increase the level of peroxidation (9). The formation of HNE-SR-B1 adducts is recognized by the ubiquitination apparatus of the cell (10) that will ubiquitinate the protein that subsequent will be dregraded by the proteosome (11).</p

The decreased levels of SR-B1 after CS exposure was reversed by catalase (CAT) (left panel) or Diphenyleneiodonium Chloride (DPI) (right panel).

<p>Cells pretreated with CAT or DPI were exposed to CS for 50 min and harvested at different time points (0–24 hrs). Western blot shown is a representative of five independent experiments. Quantification of the SR-B1 bands is expressed under the blot as ratio of SR-B1/β-actin (arbitrary units).</p

CS exposure increased H2O2 levels and mitochondrial superoxide production.

<p>Cells were exposed to CS for 15, 30 or 50 min. (A) Concentration of H₂O₂ in the media with (close bars) or without cells (open bars). Data are presented as average of triplicate measurements from each sample and expressed as arbitrary units. (B) Mitochondrial ROS production was evaluated by Mitosox fluorescence. Cells were loaded with Mitosox before and after CS exposure and subjected to live cell imaging.</p

CS induces the increase of HNE/SRB1 adducts.

<p>Immunocytochemistry of HaCaT cells showing localization of HNE-adducts (left column, green color), SR-B1 (central column, red color) and HNE/SR-B1 adducts (right column, yellow color) before, and several time points after, CS exposure (A). Images are merged in the right panel and the yellow color indicates overlap of the staining. These data were confirmed by immunoprecipitation for SR-B1 (B). HaCaT cells were exposed to CS and cell lysates were immunoprecipitated using anti SR-B1. Immunoprecipitated proteins were separated by SDS-PAGE, and then transferred to a nitrocellulose membrane and immunoblotted with anti-HNE. Western blot shown is representative of five independent experiments.</p