The promising future of microalgae: new pharmacological applications for human health

Microalgae are the oldest group of autotrophic microorganisms and are largely used for a wide range of biotechnological applications especially as dietary supplements for animal and human nutrition thanks to the presence of numerous bioactive compounds (High-Value Molecules) such as carotenoids (e.g., astaxanthin, fucoxanthin, β-carotene), omega-3 (ω-3) fatty acids, polyphenols and vitamins. Among microalgae, the most largely used is Arthrospira, commercially identified as Spirulina platensis, a cyanobacterium globally known for its high content of proteins (up to 70%). The marine microalgae Tisochrysis lutea is not available for human consumption but already known for its valuable nutritional profile especially for the high content of Fucoxanthin (FX). Chronic inflammation is the principal cause of diseases as osteoarthritis, rheumatoid arthritis, inflammatory bowel diseases (Crohn’s disease), metabolic syndrome-associated disorders, retinal neovascularization and cancers, however, the events that lead to localized chronic inflammation are relatively unknown. Metabolic Syndrome (MetS) is a complex pathophysiologic state characterized by the imbalance between calorie intake and energy storage and utilization, highly associated with hypertension, central obesity, insulin resistance, and atherogenic dyslipidemia, leading to an increased risk for developing type 2 diabetes and cardiovascular disease (CVD). The aim of this PhD project was to investigate the pharmacological bases of the positive effects of A. platensis F&M-C256 and T. lutea F&M-M36 for human health by testing concentrations/doses of translational interest and investigating the molecular mechanisms underlying certain findings. I focused on the two most representative health problems worldwide: inflammation and metabolic disorders. The anti-inflammatory effects of Spirulina and Tisochrysis lutea are actually ascribed to the presence of phycocyanin and fucoxanthin, respectively; both compounds are in fact active in many experimental contexts but, often at concentrations with no human applicability. The positive effects of spirulina on metabolic alterations are also known and highlighted by some clinical trials. However hardly anyone has tried to explain the mechanisms associated with these effects. The effect on inflammation was characterized by using murine macrophages RAW 264.7 stimulated with LPS (1μg/mL), while the effect on metabolic disorders was evaluated on rats fed a diet rich in fat (30%), with low fibers. In the first sets of experiments I compared the effects of a methanolic extract of T. lutea F&M-M36 and of pure FX, tested at the same concentration found in the extract, to evaluate the relevance of this pigment on the anti-inflammatory activity of this microalga. T. lutea F&M-M36 methanolic extract and FX, at equivalent concentrations, exert both, anti-inflammatory activities by regulating several pro-inflammatory mediators. In detail, while the methanolic extract of T. lutea F&M-M36 (1-100 μg/mL) was the most effective in counteracting the activation induced by LPS of the COX-2/PGE2 axis, the analysis of other inflammatory markers, such as IL-6, IL-10, Arg1 and HO-1, demonstrated that, in this case, the anti-inflammatory effect was mainly due to the FX content. Following the same concept, in the second sets of experiment I compared the effect of an aqueous extract of A. platensis F&M-C256 and its main water- 7 soluble bioactive compounds Phycocyanin (PC). The analysis on PGE2 production revealed that PC alone (56 and 560 μg/mL) exert anti-inflammatory effects whereas the aqueous extract was ineffective, probably due to a less PC bioavailability. Moreover, I conducted also a set of experiments on methanolic extracts from three different Arthrospira strains with the aim to correlate the antinflammatory activity to the different metabolic profile. The results indicated that A. platensis F&M-C260 (5-25 μg/mL) and A. platensis F&M-C256 (25 μg/mL) were able to counteract the inflammatory status induced by LPS acting on NF-κB/iNOS/COX-2 axis, while A. maxima F&M-C258 (25 μg/mL) was the most effective on the NLRP3/IL-1β signaling pathway. These results suggest that the anti-inflammatory properties of A. platensis and T. lutea are mediated by the multiple actions of a phytocomplex with higher activity than single components due to additive and/or synergistic effects. In the second part of my research project, I focused on the possible use of both microalgae as complementary strategy for the controls of the various metabolic syndrome components. At first, I evaluated the safety profile of T. lutea testing the effect of a microalgae biomass-rich diet (20% w/w) and using A. platensis F&M-C256 as negative reference. The results of this sub-acute toxicity study did not highlight any negative effect produced by T. lutea F&M-M36 consumption, neither by A. platensis F&M-C256, as expected. Interestingly, both microalgae-rich diets showed remarkable effects on plasmatic levels of total cholesterol, HDL cholesterol and triglycerides. Thus, these effects were further investigated in an appropriate model represented by male Sprague-Dawley rats fed with a high fat diet supplemented with 5% of A. platensis F&M-C256 (HFA) for three months and compared to those observed in rats fed a High Fat Diet (HF) or a normocaloric diet (NF). Despite no differences among the experimental groups on body weight and no significant effects on the main adipose tissues, A. platensis F&M-C256 rich diet was able to significantly reduce liver weight, plasma levels of triglycerides and total cholesterol, associated with a significantly increased fecal lipid excretion compared with the HF group. Interestingly, rats fed with A. platensis F&M-C256 showed a significant reduction of blood pressure parameters compared to the other groups. The molecular mechanism responsible of these effects is the metabolic shift of white adipose tissue (WAT) toward a brown-like through the activation of PRDM-16 and UCP-1 gene expression in abdominal WAT. The metabolic activation of the WAT stimulated the thermogenesis mechanisms due to ANGPTL3 that increased the lipolysis rate, resulting in a release of energy in the form of heat. In the same experimental model, I also studied the effect of T. lutea F&M-M36 (HFT), by comparing its effect with the NF and HF diets and with a Fenofibrate 100 mg/Kg (w/w) treatment (HFF). As expected, Fenofibrate significantly decreased the plasma level of triglycerides and blood glucose. In the same way, T. lutea F&M-M36-rich diet induced an, almost, comparable effect. Moreover, T. lutea F&M-M36-rich diet significantly decreased the renal adipose weight. Interestingly, rats fed with T. lutea F&M-M36 showed a significant reduction in diastolic blood pressure and on Mean Arterial Pressure. The analysis of the possible molecular mechanisms revealed a shift from white to brown adipocytes thanks to the activation of the β-adrenergic receptors (βARs) signaling. Western blot and microarray analysis on visceral adipose tissue 8 demonstrated that T. lutea F&M-M36 feeding increased the protein expression of β3ADr (upstream receptor) and UCP-1 (downstream target). On the contrary, Fenofibrate did not exert any effect on this process. However, both treatments significantly induced the protein expression of GLP1r. As expected, Fenofibrate increased liver weight and the steatosis score. Overall, these data demonstrated for the first time, that the hypolipidemic effect of A. platensis is linked to its action on ANGPTL3/Lpl axis and the lipolytic process, and that T. lutea F&M-M36 induced the browning of white fat through the activation of β3ADr/UCP-1 pathway, affecting also GLP-1 signaling, without the negative effects of Fenofibrate on liver physiology. In conclusion, these results demonstrated the positive effects of microalgae against inflammation and metabolic disorders and their mechanisms, suggesting that the marine microalga T. lutea deserves to be explored for human consumption.