Selected identification data obtained from shotgun analysis of cytoplasmic and secretome fractions of HUVECs.

<p>Accession, Uniprot Accession number; Gene, gene name, Peptide IDs, number of distinct peptide identification; C, cytoplasmic fraction; S, secretome fraction. A semi-quantitative abundance measure is provided via emPAI values. Con, untreated cells; IL1b, stimulated with Interleukin 1-beta; m rac, stimulated with Interleukin 1-beta and treated with <i>M rac</i> extract. While inflammatory stimulation increased almost all emPAI values of the listed inflammatory mediators, treatment with <i>M rac</i> extract of stimulated cells resulted in the down-regulation of most emPAI values.</p

Two-dimensional representation of an LC-MS analysis of the <i>M rac</i> extract.

<p>The analytes were separated using HPLC and detected with a Q Exactive orbitrap in the positive ion mode. Each spot annotates a distinct molecular feature, which can be counted dependent on the applied sensitivity threshold. Several hundred different constituents can easily be distinguished. The x-axis shows the retention time between 10 and 26 min and the y-axis shows the mass range of <i>m/z</i> 150–750.</p

2D-PAGE of cytoplasmic proteins isolated from peripheral blood mononuclear cells (PBMCs).

<p>Cells were metabolically labelled while being inflammatory stimulated and treated with <i>M rac</i> extract (1:10’000 dilution), proteins were detected by autoradiography. A) untreated control. B) inflammatory stimulation with LPS and PHA strongly induced inflammatory mediators such as MX1, IFIT1, WARS and GBP5. C) treatment of PBMCs with <i>M rac</i> extract hardly induced any proteome alterations. D) treatment of stimulated PBMCs with <i>M rac</i> extract attenuated the induction of IFIT1, while other inflammation markers remained largely unchanged. Proteins were identified by proteolytic digestion of each spot and subsequent LC-MS/MS analysis.</p

Targeted proteomic analysis of pro-inflammatory cytokines secreted by inflammatory stimulated and treated HUVECs.

<p>Experiments were performed using two biological with two technical replicates. Inflammatory stimulation (IL-1β) up-regulated the levels of all cytokines in comparison to untreated controls (con). Treatment with dexamethasone (dex) down-regulated all cytokines as expected for a strong anti-inflammatory drug. Remarkably, also treatment with <i>M rac</i> extract was capable of significantly down-regulating these cytokines (M rac compared to IL-1β: *, p<0.01; **, p<0.001). “Area” refers to the area under the curve of the chromatographic peak in the nLC-MRM experiment of the quantifier transition of each peptide.</p

Regulation of selected metabolites assessed by the AbsoluteIDQ p180 metabolomics kit illustrating the contrasting inflammation modulating effects of dexamethasone (dex) and M rac.

<p>Again, HUVECs were stimulated with IL-1βeta (IL-1β) and additionally treated with dexamethasone or M rac extract (M rac). Error bars are derived from two technical and two biological replicates. (p-values: *, p<0.05 and **, p<0.005.)</p

2D-PAGE of cytoplasmic proteins isolated from human umbilical vein endothelial cells (HUVECs).

<p>Cells were metabolically labelled while being inflammatory stimulated and treated with <i>M rac</i> extract (1:10’000 dilution), proteins were detected by autoradiography. A) untreated control. B) inflammatory stimulation with interleukin 1-beta strongly induced inflammatory mediators such as MX1, IFIT1, WARS and GBP2 as well as the stress-related protein STIP1. C) treatment of HUVECs with <i>M rac</i> had little effect on the synthesis of cytoplasmic proteins. D) treatment of stimulated HUVECs with <i>M rac</i> extract attenuated the induction of IFIT1, GBP2 and STIP1. Proteins were identified by proteolytic digestion of each spot and subsequent LC-MS/MS analysis.</p

Proteomic and Metabolomic Analyses Reveal Contrasting Anti-Inflammatory Effects of an Extract of Mucor Racemosus Secondary Metabolites Compared to Dexamethasone

<div><p>Classical drug assays are often confined to single molecules and targeting single pathways. However, it is also desirable to investigate the effects of complex mixtures on complex systems such as living cells including the natural multitude of signalling pathways. Evidence based on herbal medicine has motivated us to investigate potential beneficial health effects of <i>Mucor racemosus</i> (M rac) extracts. Secondary metabolites of M rac were collected using a good-manufacturing process (GMP) approved production line and a validated manufacturing process, in order to obtain a stable product termed SyCircue (National Drug Code USA: 10424–102). Toxicological studies confirmed that this product does not contain mycotoxins and is non-genotoxic. Potential effects on inflammatory processes were investigated by treating stimulated cells with M rac extracts and the effects were compared to the standard anti-inflammatory drug dexamethasone on the levels of the proteome and metabolome. Using 2D-PAGE, slight anti-inflammatory effects were observed in primary white blood mononuclear cells, which were more pronounced in primary human umbilical vein endothelial cells (HUVECs). Proteome profiling based on nLC-MS/MS analysis of tryptic digests revealed inhibitory effects of M rac extracts on pro-inflammatory cytoplasmic mediators and secreted cytokines and chemokines in these endothelial cells. This finding was confirmed using targeted proteomics, here treatment of stimulated cells with M rac extracts down-regulated the secretion of IL-6, IL-8, CXCL5 and GROA significantly. Finally, the modulating effects of M rac on HUVECs were also confirmed on the level of the metabolome. Several metabolites displayed significant concentration changes upon treatment of inflammatory activated HUVECs with the M rac extract, including spermine and lysophosphatidylcholine acyl C18:0 and sphingomyelin C26:1, while the bulk of measured metabolites remained unaffected. Interestingly, the effects of M rac treatment on lipids were orthogonal to the effect of dexamethasone underlining differences in the overall mode of action.</p></div