| dc.description.abstract | Metabolic disease is a combination of conditions that manifest together, elevating the risk for cardiovascular disease, type 2 diabetes and stroke. Inflammation is a major contributor in the pathophysiology of metabolic syndrome. Toll-like receptor (TLR) yields one of the main targets for anti-inflammatory pharmacotherapy. As macrophages and associated cytokines are strongly implicated in the inflammatory component of obesity, and metabolic disease, we aimed to investigate the effect of supplementation with AquaminTM a mineral extract from red marine algae ? rich in calcium (34%), in modulating inflammatory responses in vitro.
Using ELISA, and RT-PCR, it was demonstrated that Aquamin significantly reduces the protein and RNA expression of signature inflammatory cytokines such as TNF-? and IL-6 in dendritic cells, human monocytes, as well as murine and human macrophages that were exposed to LPS which is a TLR4 ligand.
To empower our insight concerning the anti-inflammatory effect of Aquamin, the protein expression of iNOS was measured by western blotting. The results revealed notable reduction of LPS-induced iNOS expression in murine macrophages by Aquamin.
Furthermore, significant reduction of TNF-? and IL-6 of TLR3 driven macrophages upon Aquamin treatment was reported by ELISA by exposing the cells to dsRNA, while no significant alteration was observed in TLR9-stimulated macrophages. That finding led to the conceivable assumption that Aquamin exerts its anti-inflammatory effect by exhibiting a TRIF-dependent immunomodulary role. Murine macrophages were exposed to LPS and transcriptional levels of MyD88 and TICAM1 adaptor molecules were measure by RT-PCR. Interestingly, our results revealed that gene expression of TICAM1 is significantly reduced by Aquamin, while no effect was seen on MyD88. These results further corroborate the hypothesis that Aquamin blocks TLR in a TRIF-dependent manner. IFN regulatory factor 3 (IRF3) is a transcription factor downstream of TLR3 as well as TRIF (TICAM1)-dependent TLR4 signaling. Activation of the receptors results to nuclear translocation of IRF3 and subsequent production of IFN-?. Using immunohistochemistry, the effect of Aquamin in the activation of IRF3 was assessed. The results demonstrate significant reduction of Poly(I:C)-induced nuclear translocation of IRF3 as well as reduction of the IFN-? gene expression which was measured by RT-PCR.
Analysis of the macrophage transcriptome under LPS alone and NLRP3 exposure in the presence and absence of Aquamin and simvastatin can shed light on the mechanisms associated with Aquamin treatment. Here, the results of an in vitro study of murine BMDMs transcriptome response during LPS and LPS with cholesterol crystals (CC) stimulation using RNA-seq are described. Primary macrophages were extracted from murine bone marrow and were treated with Aquamin and simvastatin prior to exposure to TLR4 and NLRP3 stimulants. Initially ELISA was carried out to determine the TNF-? and IL-1? protein concentration. Our results highlighted that Aquamin has a more potent inhibitory effect than simvastatin against both cytokines in vitro. Treatment with Aquamin led to a significantly higher number of differentially expressed genes (DEGs) compared to simvastatin, both when compared to untreated control and in comparison, with LPS or LPS- and CC-induced samples. Functional analyses of these DEGs, including pathway enrichment, revealed potential functional roles for genes that have not been previously described. Verification of some relative genes was performed by RT-PCR.
Interestingly, this study evaluated the effect of Aquamin in a clinically relevant model of diet-induced obesity and inflammation. The mineral extract was formulated into a high fat diet and fed to C57BL/6 mice over 10 weeks, where diet induced obesity markers are assessed. A decreasing pattern in actual and percentage weight gain in animals fed the mineral extract supplemented diet was observed compared to control high fat fed animals. No change in food and water intake between the animal groups is observed. Mineral supplemented animals also responded better to insulin and glucose tolerance tests.
Collectively, the data generated in this thesis suggests that Aquamin?s anti-inflammatory effects under TLR4 and TLR3 stimulation might be a result of TRIF dependent signaling mechanism. In summary the natural marine mineral extract may be beneficial in ameliorating diet induced obesity and modulates the associated inflammation in a TRIF-dependent manner. | en |
