**4. Discussion**

The use of microalgae recently attracted considerable attention worldwide due to their considerable application potential in the biopharmaceutical and nutraceutical industries. They are renewable, sustainable, and inexpensive sources of bioactive molecules and food with surprising pharmacological and biological qualities. In particular, blue-green algae were demonstrated to have remarkable antioxidant and anti-inflammatory properties due to the presence of many bioactive substances [9–12]. Among the blue-green algae extracts, AphaMax® contains high concentrated amount of AFA-phycocyanins and AFA-phytochrome, which are the components with the greatest antioxidant, anti-inflammatory, anticancer, and cardiovascular properties.

The aim of this study was to test the effects of AphaMax®, a commercial extract of the blue-green Klamath microalgae (AFA), on colonic inflammatory condition induced in an experimental model of colitis, the DNBS rats.

The rationale for this comes by the awareness that the current IBD therapies have significant side effects and modest results for long-term managemen<sup>t</sup> and that recently, much evidence supports the key role of diet and nutritional factors in IBD. Our results indicate the AphaMax® supplementation is able to reduce colon injury induced by DNBS in rats, mainly due to its antiinflammatory and anti-oxidant effects.

As previously reported [18–21], intracolonic administration of DNBS causes an acute colitis in rats, characterized by marked reduction in body weight, diarrhoea with occasional blood in the stool, colonic shortening, an augmented colonic wall thickness, with a significant increase in the colonic weight/length ratio, reliable indicator of tissue oedema, and an extensive transmural, granulomatous inflammation of the distal colon.

Fourteen days of AphaMax ® treatment leaded to a reduction of the aforementioned parameters as the deterioration of body weight, diarrhoea, and the increased colon weight/length ratio. Also histological analysis of the colon confirmed the macroscopic observations, suggesting that AphaMax ® presents an antiulcerogenic e ffect, as confirmed by preservation of the colon architecture and by the attenuation of mucosal disruption, as well as of histopathological damage and oedema.

Few studies have been conducted on the possible protective e ffects of BGA against experimental colitis, but this is the first study about the e ffect of Klamath algae in such conditions. Oral administration of cyanobacterium Spirulina Platensis was reported to have protective e ffects in other models of experimental colitis showing both antioxidant (reduction in oxidative stress and augmented endogenous antioxidant mechanisms) and anti-inflammatory (reduction in inflammatory cytokine levels and neutrophil infiltration) e ffects [33]. Moreover, in ulcerative colitis patients, diet supplementation with the green algae Chlorella pyrenoidosa accelerated wound healing [34].

Furthermore, we can speculate about the mechanisms underlying the intestinal beneficial e ffects of the AFA extract. Among the possible mechanisms we can suppose a relation with the well-known positive properties, ascribed to the majority of the blue-green algae extracts, due to their content in compounds, as phycocyanins.

In particular, Klamath algae contains the pigment C-phycocyanin (C-PHY) bound to a structural component phycoerythrocyanin (PEC). PEC is a photosynthetic component identified only in a limited number of cyanobacterial species, likely responsible for the higher antioxidant and anti-inflammatory effects of Klamath algae compared to other cyanobacters [14,15]. C-phycocyanins can act as radical scavenger in oxidative stress-induced diseases and they have strong antioxidant and anti-inflammatory proprieties as demonstrated by various in vitro and in in vivo evidences [35,36].

Moreover, in our experimental model of colitis, AphaMax ® treatment induced an attenuation of the neutrophil infiltration, as shown by both histological observations and biochemical data showing a significant reduction in colonic myeloperoxidase activity, confirming the ability of this extract to modulate the altered immune response, likely ascribable to the C-phycocyanins action. This observation is in agreemen<sup>t</sup> with Gonzalez et al. [37] where C-phycocyanins extracted from the Blue-green algae Arthospira maxima, have been reported to reduce significantly myeloperoxidase activity, inflammatory cell infiltration and colonic damage in acetic acid-induced colitis in rats. We are aware that the model of colitis generation in Gonzales et al. [37] is di fferent from our model; however, it is possible to observe that in our study the highest doses of AphaMax ® (25% AFA-PC concentration) was more e ffective in the reduction of myeloperoxidase activity of Spirulina (80% PC concentration) indicating the major anti-inflammatory and protective power of AFA-phycocyanins.

Although in our study, AphaMax ®, at all doses tested, attenuated the colitis signs in the colonic tissue, we can sugges<sup>t</sup> that the higher dose tested, 100 mg/Kg, containing a concentration of AFA-phycocyanins and AFA-phytochrome, carotenoids and polyphenols able to significantly improve all the analyzed scores, could be considered the best to attenuate the severity of colitis.

Moreover, the observation that AphaMax ® extract, even at the highest dose tested, did not a ffect sham rats, might indicate that AphaMax ® extract exerts its anti-inflammatory and antioxidant e ffects, only in the course of inflammation.

Interestingly, AphaMax ® treatment is su fficient to attenuate activation of NF-κB p65. NF-κB, is a redox-sensitive transcription factor, key regulator of inflammation, innate immunity, and tissue integrity. NF-κB phosphorylation and its nuclear translocation correlate with the severity of intestinal inflammation [38,39] due to the regulation of gene expression of molecules playing a pivotal role in inflammation as molecules of adhesion, chemokines, and cytokines [40]. We can suppose that the attenuation of DNBS colitis features, observed in our study, could be the consequence of the inhibition of early steps of inflammation. A reduced activation of NF-κB would lead to the infiltrating cells to decrease the amounts of inflammatory mediators and subsequently to preserve mucosal integrity. Once more, such an effect could be also positively related to the high concentration of C-phycocyanins present in the extract, which include C-phycocyanins + PEC (phycoerythrocyanins), and C-phycocyanins by themselves have been reported to be able to suppress activation of NF-κB in RAW 264.7 macrophages stimulated with LPS [41].

The DNBS model is also commonly associated with the T-helper (Th)1 response, and with an overproduction of proinflammatory cytokines, as IL-6 and IL 1β. Many of the IBD treatments act to regulate the levels of proinflammatory cytokines [42]. These immunoregulatory cytokines are involved in the initiation of the inflammatory response in colitis, amplifying the inflammatory reaction by triggering a cascade of immune cells, impairing intestinal permeability, and causing severe colonic infiltration [43] In addition, IL-1β contributes to the induction of the epithelial cell necrosis. Our results indicated that AphaMax® treatment caused a dose-dependent reduction of both IL-6 and IL- 1β cytokine levels in colitis rats, thus ameliorating the deregulated immune response typical of experimental colitis. Once more, such an effect could be likely due to the high concentration of AFA C-phycocyanins.

Moreover, AphaMax® significantly attenuated expression of the enzymes COX-2 and iNOS which were up-regulated in colitis rats. COX-2 is a key enzyme in the pathogenesis of IBD involved in the biosynthesis of prostaglandin [44], and target of many drugs used in the treatment of human IBD, including aminosalicylates [45,46]. This inhibition is not unexpected since C-phycocyanins are reported to be selective COX-2 inhibitors [31,47]. The expression of iNOS at sites of inflammation, acting in synergy with COX-2 to produce excessive inflammatory mediators has been reported. Their products may be detrimental to the colon integrity and contribute to the intestinal hypomotility and subsequent bacterial overgrowth [48], a typical features of the inflammatory reaction [49]. Accordingly, AphaMax® treatment reduced nitrite levels in colitis rats. Considering that expression of both COX-2 and iNOS is regulated by NF-κB [50,51] the observed inhibition of NF-κB and its downstream effectors, COX-2 and iNOS, can be crucial for the protective role played by AphaMax® treatment in DNBS colitis rats.

Lastly, in colorectal biopsies of human IBD [52,53] and in different experimental models of colitis, including the DNBS model, a mucosal production of reactive oxygen mediators was described, which would contribute to tissue damage during chronic intestinal inflammation [54].

The major effects of oxidative stress reported are local intestinal cell damage and activation of several signaling pathways, initiating inflammation [54].

In our preparations, AphaMax® treatment was able to modulate the redox status by scavenging ROS and to reduce the severity of the colitis, effect that is likely ascribable to AFA-phycocyanins, which possess antioxidant activity 75 to 200 times greater than that other more common phycocyanins [14]. Phycocyanins were reported to be able to scavenge hydroxyl and alcoxyl radicals in an acetic acid-induced colitis model [36]. Thereafter, since free radical generation could contribute to the initial infiltration of neutrophils in the colonic mucosa, we can speculate that ability of our extract in preserving the colonic mucosa from oxidative insult could participate to decrease the neutrophil infiltration DNBS-induced. However, whether the antioxidative effect precedes the anti-inflammatory one or not is still uncertain and has ye<sup>t</sup> to be explored.

We focused our attention on the AphaMax® effects on the local production of ROS and oxidant insult in colonic tissue; however, since a plasmatic increase of advanced oxidation protein products formed during oxidative stress in IBD patients was also reported [55], further studies investigating possible AphaMax® systemic effects are warranted.
