**5.4 Other in vivo studies with animal models**

The IL-10 deficient mouse is a classic knockout model that develops spontaneous colitis under pathogen-free conditions. Some authors have explored the influence of GSPE in this model, supplementing colitic animals with 0.1–1 g 100 g<sup>1</sup> of dry feed weight for 12–16 days [86, 87]. These studies evidenced a reduction of multiple inflammatory markers in the jejunum and colon, such as TNF-α, IL-1β, IL-6, and IFN-γ gene expressions, as well as MPO activity. This anti-inflammatory effect was associated with the inhibition of the NF-κB signaling. Interestingly, GSPE supplementation also increased the density of goblet cells in the jejunum of treated animals, suggesting that there is an alternative mechanism by which inflammation is attenuated.

Cardoso et al. [13] recently tested both dietary (75 mg kg<sup>1</sup> ) and pharmacological doses of GSPE (375 mg kg<sup>1</sup> ) in a rat model of mild intestinal dysfunction induced by intraperitoneal injection of LPS. GSPE was administered daily by oral gavage for 15 days prior to LPS-induced intestinal dysfunction. LPS enhanced intestinal permeability and induced both oxidative stress and inflammation. GSPEtreated animals reduced OVA permeation to the circulation, MPO activity and COX-2 in the small intestine tissues, and reactive oxygen species (ROS) levels in the colon. Furthermore, a gene expression analysis with a low-density microarray technique revealed that unlike the dietary dose of GSPE, the pharmacological dose had a striking effect on the LPS-gene expression profile, showing a strong modulation of multiple genes associated with chemokines and ILs, including upregulation of the anti-inflammatory cytokine IL-13.

#### **5.5 Human ex vivo studies**

Although the use of animal models is the predominate approximation at preclinical stages for testing novel therapies in intestinal permeability, there is a strong trend in the industry towards replacing animal experiments with human cellculture based models [92, 93]. Nevertheless, advantages related to the usefulness of in vitro models for screening of bioactives and exploring action mechanisms, are offset by limitations regarding the mimicking of the in vivo situation and translation to the human [103]. Thus, some human ex vivo models have been proposed to test immunomodulatory properties of drug candidates in intestinal explants from IBD patients [104, 105]. Intestinal function can also be studied with UCh-based protocols. The UCh system consists of two halves with an opening between them, where mucosal tissue is adapted, thus isolating the apical and basolateral sides of the tissue. This technique has been applied for studying drug absorption [106] and secretion of enterohormones [107] in human endoscopic biopsies. An advantage of UCh models over explant-based models is that UCh models make it possible to measure the electrophysiological parameters, including TEER [106]. All these setups permit analyzing the cytokine profiling of intestinal explants or biopsies retaining their in situ conditioning in a polarized fashion [105, 108]. We have employed the UCh to determine TEER and cytokine release (TNF-α) in intestinal tissues from cafeteria diet-induced obese rats treated with GSPE [8, 15]. It could also be useful for testing the effect of bioactives on dysfunctional human intestine.

A feature of ex vivo models is that screening of drug effects does not compromise the patients by exposing them to unknown outcomes.
