**3. Discussion**

The Caco-2 cell line is extensively used as a model of the intestinal epithelial barrier. In particular, monolayers of differentiated Caco-2 cells are used for the investigation of the function and integrity of the intestinal barrier. Therefore, this cell model is often used to study the modulatory role that natural compounds (including polyphenols) may have on intestinal function [17,18]. Here, for the first time, POE, a polyphenol-rich phytocomplex, has been shown to inhibit glucose uptake, improve intestinal barrier integrity, and protect cells from oxidative stress in Caco-2 cells.

The inhibition of glucose uptake induced by POE was observed to the same extent in both the presence and absence of sodium. Moreover, a significant decrease in the GLUT2 levels in POE-treated cells was observed in our experimental conditions. On the contrary, no effect of POE on the SGLT1 protein levels was observed. These results suggest that the effect of POE on glucose could be mainly mediated by a modulation of the expression of glucose transporter GLUT2, in agreement with other studies that reported a reduction in the expression of intestinal glucose transporters by polyphenols [11]. In rat enterocytes, the apical membrane levels of both transporters can alter rapidly in response to cell signaling events [21,22]. GLUT2 has been detected at both the apical membrane and basolateral membrane of Caco-2 cells [23]. Further studies are required to address the effect of POE on cell signaling events and the cellular distribution of GLUT2 and SGLT1.

Elevated levels of intestinal glucose transporters have been reported in diabetic and obese animal models and this contributed directly to their hyperglycemic status [24,25]. Therefore, compounds that regulate glucose transporter expression may be useful as potential anti-hyperglycemic agents. Till today, this effect has been observed only in animal models. Gokce et al. (2008) have reported that the oral administration of a *P. oceanica* extract for 15 days (50, 150, and 250 mg/kg b.wt.) resulted in a dose-dependent decrease in blood glucose in alloxan-induced diabetic rats [4]. Further studies are needed to investigate the effects of POE intake on post-prandial glycaemia in normal and hyperglycemic subjects.

A dysfunctional intestinal barrier is associated also with dysmetabolic diseases, including diabetes and obesity [19,26,27]. Therefore, in this study, transepithelial electrical resistance (TEER) [28] and the levels of TJ proteins, such as Zonula occludens (ZO-1), have been evaluated to investigate the potential role of POE on the integrity of cell monolayer using differentiated Caco-2 cell, as a model of an intestinal barrier. A significant increase in TEER across the cellular monolayer in POE-treated cells was observed with respect to

untreated control cells. Furthermore, POE demonstrated a positive effect on intestinal cells by increasing the levels of ZO-1. The protein ZO-1 is involved in the regulation of intestinal barrier integrity and plays a crucial role as a key molecule in cell-to-cell contact and in maintaining the structure of TJ and the epithelial barrier function [26]. TJ barrier integrity is essential for human health and metabolic homeostasis [26]. The effects of POE on ZO-1 are in agreement with previous studies, which demonstrated that polyphenols (such as catechins and phenolic acids) modulate intestinal barrier function and increase the expression of several TJ proteins, including ZO-1, in in vitro models [12,13]. Some in vivo studies have demonstrated that, in older subjects, a polyphenol-rich dietary pattern improves intestinal permeability, evaluated as the serum Zonulin levels [29]. However, further in vivo studies will be required to verify the protective effect of POE on the intestinal barrier integrity.

Intestinal cells are exposed to dietary pro-oxidants, AGEs, lipid peroxidation products, and are susceptible to oxidative damage [30–32]. In addition, previous studies have also shown that high-sugar diets cause increased oxidative stress and inflammation [31].

ROS-induced oxidative stress is widely considered as a possible upstream mechanism of high-glucose-induced cell damage [20]. These cellular alterations may cause a dysfunction of intestinal barrier and lead to the onset of the intestinal bowel diseases [30,31]. Previous studies have reported that POE has an antioxidant role against intracellular ROS formation in macrophages activated by LPS [6]. In our experimental conditions, POE shows a protective role against ROS-induced oxidative stress under chronic high-glucose conditions in Caco-2 cells. The antioxidant role of POE was also confirmed by an inhibitory action against the ROS formation induced, in the same cell model, by treatment with TBHP, a molecule commonly used to study cellular alterations resulting from oxidative stress [33].

Chronic exposure to high glucose evokes oxidative stress; the resulting high levels of intracellular ROS can promote AGEs formation. Indeed, previous studies have shown that highly reactive intermediates of the Maillard reaction, such as glycolaldehyde (GA) and glyoxal, are involved in cross-linking of proteins and are precursors of AGEs [34]. In this study, Caco-2 cells treated with POE showed a significant decrease in the GA protein levels in chronic high-glucose conditions. Since GA proteins are useful markers of oxidative stress [20], our findings further support the protective role of POE against oxidative stress induced by high glucose. Overall, these results are in agreement with our previous studies that described an in vitro role of POE against glucose-induced glycation of human serum albumin [5]. Moreover, other studies have demonstrated the ability of polyphenols to exert a protective effect against oxidative stress and formation of AGEs in Caco-2 cells [14]. In addition, previous studies have reported that some phenolic compounds exert a protective effect against oxidative stress, either by reducing ROS production during the glycation process or by trapping of dicarbonyl species [35].

In conclusion, the data reported in this study demonstrate that POE reduces glucose transport by lowering the GLUT2 levels and promotes intestinal barrier integrity by positively modulating the ZO-1 levels. Furthermore, POE has a protective antioxidant effect against high-glucose-induced damage, in terms of lower production of intracellular ROS and AGE-modified proteins.

Inhibition of glucose uptake in the small intestine may prevent post prandial hyperglycemia, which is one of the risk factors for diabetes and metabolic syndrome. Our findings suggest that POE may have a positive impact by preventing the intestinal cell dysfunction involved in the development of inflammation-related intestinal diseases associated with oxidative stress.

The in vitro effect of POE was observed at a concentration of polyphenols of 15 μg GAE/mL (88 μmol/L). It has been reported that the bioavailability of polyphenols is related to the structural properties of molecules. The total levels of polyphenols are present in plasma at <1 μmol/L concentrations, but they are present in the stomach and intestinal lumen at much higher concentrations after consumption of vegetables rich in polyphenols. Saura-Calixto et al. (2007) demonstrated that polyphenols could act as antioxidants in the

intestine because they are present at millimolar concentrations after consumption of fruits and vegetables [36].
