Background: Exaggerated Toll-like receptor (TLR)-mediated immune and inflammatory responses play a role in inflammatory bowel diseases. This report deals with the ability of a mixture of oxysterols widely present in cholesterol-rich foods to induce in vitro intestinal inflammation through TLR up-regulation. The anti-inflammatory action of four cocoa bean shell (CBS) extracts with different polyphenol content, was tested. Methods: Differentiated intestinal CaCo-2 cells were treated with a dietary oxysterol mixture (Oxy-mix) (60 µM). The expression and activation of TLR2 and TLR4, as well as the production of their downstream signaling effectors IL-8, IFNβ and TNFα were analyzed in the presence or absence of TLR antibodies. Honduras CBS extracts were characterized for their polyphenol contents; their anti-inflammatory action was analyzed in CaCo-2 cells treated with Oxy-mix. Results: Oxysterol-dependent TLR-2 and TLR4 over-expression and activation together with cytokine induction were abolished by blocking TLRs with specific antibodies. Polyphenol-rich CBS extracts consisting of high quantities of (−)-epicatechin and tannins also prevented TLR induction. Conclusions: TLR2 and TLR4 mainly contribute to inducing oxysterol-dependent intestinal inflammation. The fractionation method of CBS allowed the recovery of fractions rich in (−)-epicatechin and tannins able to counteract oxysterol-induced inflammation, thus highlighting the beneficial biological potential of specific CBS extracts.

Background: Cocoa bean shell (CBS), a main byproduct of cocoa processing, represents a source of components such as polyphenols and methylxanthines, which have been associated with a reduced risk of several diseases. Therefore, CBS has potential application as a food ingredient. Intestinal mucosa is exposed to immune and inflammatory responses triggered by dietary agents, such as oxysterols, which derive from cholesterol oxidation and are pro-oxidant compounds able to affect intestinal function. We aimed at investigating the capability of the Forastero cultivar CBS, added or not added to ice cream, to protect against the intestinal barrier damage induced by a dietary oxysterol mixture. Methods: Composition and antioxidant capacity of in vitro digested CBS and CBS-enriched ice cream were analyzed by high-performance liquid chromatography and 1,1-diphenyl-2-picryl-hydrazyl radical-scavenging assay, respectively. CaCo-2 cells differentiated into enterocyte-like monolayer were incubated with 60 µM oxysterol mixture in the presence of CBS formulations. Results: The oxysterol mixture induced tight junction impairment, interleukin-8 and monocyte chemoattractant protein-1 cell release, and oxidative stress-related nuclear factor erythroid 2 p45-related factor 2 response Nrf2. Both CBSs protected cells from these adverse effects, probably thanks to their high phenolic content. CBS-enriched ice cream showed the highest antioxidant capacity. Theobromine, which is in high concentrations of CBS, was also tested. Although theobromine exerted no effect on Nrf2 expression, its anti-inflammatory cooperating activity in CBS effect cannot be excluded. Conclusions: Our findings suggest that CBS-enriched ice cream may be effective in the prevention of gut integrity damage associated with oxidative/inflammatory reactions.

Cancer cachexia, a multifactorial syndrome characterized by body weight loss, muscle, and adipose tissue wasting, affects patients with cancer. Over time, the definition of cachexia has been modified, including inflammation as one of the main causal factors. Evidence has suggested that a range of proinflammatory mediators may be involved in the regulation of intracellular signaling, resulting in enhanced resting energy expenditure, metabolic changes, and muscle atrophy, all of which are typical features of cachexia. Physiologically speaking, however, inflammation is a response aimed at facing potentially damaging events. Along this line, its induction in the cancer hosts could be an attempt to restore the physiological homeostasis. Interesting observations have shown that cytokines such as interleukins 4 and 6 could improve muscle wasting, supporting the view that the same mediator may exert pro- or anti-inflammatory activity depending on the immune cells involved as well as on the tissue metabolic demand. In conclusion, whether inflammation is crucial to the occurrence of cachexia or just one contributor among others, is still unclear. Indeed, while inflammation is a trigger of cachexia, the alterations of energy and protein metabolism and of the hormonal homeostasis occurring in cachexia likely act as inflammatory stimuli on their own. Whether the causative role prevails over the compensatory one likely depends on the tumor type and stage, patient lifestyle, the presence of comorbidities, and the response to anticancer treatments paving the way to a holistic, personalized approach to cancer cachexia.

Adrenocortical carcinoma (ACC) is a rare cancer with poor prognosis. Mitotane, the standard treatment for ACC, impairs adrenocortical steroid biosynthesis and cholesterol metabolism. In the H295R cell line, a standard ACC in vitro model, mitotane was previously reported to enhance the production of some oxysterols. To verify the possible mechanistic involvement of oxysterols in the anti-ACC effect of mitotane, a gas chromatography mass spectrometry (GC-MS) profiling of oxysterols and the main cholesterol precursors was carried out in H295R cells. Among the oxysterols detected in mitotane-treated cells, 27OHC was markedly produced, as well as lanosterol and lathosterol cholesterol precursors. In this cell model, mitotane was confirmed to affect mitochondrial transmembrane potential and induce apoptosis. Such cytotoxic effects were perfectly matched by H295R cell treatment with a single identical micromolar amount of 27OHC. The mitotane-dependent strong increase in 27OHC was confirmed in vivo, in the plasma of ACC patients under treatment with the drug. Moreover, lanosterol, lathosterol, desmosterol and, to a minor extent, 24-hydroxycholesterol and 25-hydroxycholesterol plasma levels were significantly increased in those patients. The cytotoxic effect of mitotane on ACC cells may be partly related to the increased intracellular level of 27OHC induced by the drug itself.