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In a Western or Westernized diet, the abundant cholesterol is invariably associated with the presence of biochemically reactive oxysterols, the amount of which mainly depends upon the autoxidation degree of cholesterol itself, during food harvesting, production and storage. Oxysterols, in the average amount and composition detected in a high-cholesterol diet, display remarkable pro-inflammatory and cytotoxic effects on the gut epithelium. Moreover, in a low micromolar range, they may change the physiological level and membrane localization of tight junctions of the intestinal epithelial barrier, which then become leaky and permeable to microbiota. This combination of toxic effects possibly exerted by dietary oxysterols likely contributes to the impairment of the microbiota–gut–brain axis, through both direct and indirect mechanisms hereby reviewed. Importantly, dietary oxysterols are absorbed like cholesterol and circulate in the bloodstream, mainly within LDLs, rendering these micelles more oxidized and dangerous. Last but not the least, dietary oxysterols may deeply interfere with correct gut–brain signalling because of the redox pathways they are hyper-regulating and sustaining. In conclusion, protective dietary measures should be adopted, including restricted consumption of cholesterol-rich food and reduction of cholesterol autoxidation in food production and storage, for instance by supplementation of food with flavonoids and/or other bioactive substances with strong anti-oxysterol properties.
Department of Translational Medicine, University of East Piedmont, Novara, Italy
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Graphical abstract
Redox-induction of expression and synthesis of ATP binding cassette transporters in CaCo-2 cells by externally added 27-hydroxycholesterol.
27OHC: 27-hydroxycholesterol; NOX: NADPH oxidase; ROS: reactive oxygen species; ABCA1: ATP binding cassette A1; ABCG8: ATP binding cassette G8; DPI: diphenylene iodonium.
Abstract
Objective
We tested the effect of 27-hydroxycholesterol (27OHC) on the expression and synthesis of two membrane transporters involved in sterols extrusion from the intestinal epithelium into the gut lumen: ATP-binding cassette A1 (ABCA1) and G8 (ABCG8). Special attention was given to ABCG8, a key player in the intestinal cell discharge of plant sterols.
Methods
Differentiated CaCo-2 intestinal cells were supplemented with 27OHC, and added to the cell incubation medium at a final concentration of 1 or 5 µM. These 27OHC externally added amounts were proven to reach intracellular oxysterol levels within the range of those normally recovered in the human peripheral blood.
Results
An up-regulation of the ABCA1 and ABCG8 mRNAs was observed in the CaCo-2 cells supplemented with 27OHC. Moreover, both 1 µM and 5 µM 27OHC induced a net, and steady, statistically significant, increase of both ABCA1 and ABCG8 protein levels. Of interest, the cellular pre-treatment with diphenylene iodonium, a selective inhibitor of NADPH oxidase, i.e. a major intracellular source of reactive oxygen species, fully inhibited the 27OHC enhancement of both ABCA1 and ABCG8 protein synthesis.
Conclusion
This in vitro study shows for the first time that the addition of 27OHC to intestinal epithelial cells up-regulates ABCG8, the transporter discharging plant sterols into the gut lumen, besides confirming to induce ABCA1 as well. Importantly, the 27OHC-dependent up-regulation of the two transporters appears to involve a redox mechanism rather than the canonical liver-X-receptors-dependent pathway.
Significance statement
The 27OHC introduced with the diet might modulate the plant sterol extrusion in the gut, in parallel with that of cholesterol.
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Background
In both Western and Westernized diets, together with a relatively high amount of cholesterol, variable amountsof its oxidized metabolites, oxysterols, are consistently consumed. These oxysterols, mostly of non-enzymatic origin, are produced during sterol autoxidation in foodstuff manufacturing and storage.
Objective
This study aimed to analyze the potential enterotoxic effects of all main oxysterols of non-enzymatic origin so far identified in a variety of foods.
Experimental plan
Differentiated human intestinal cell monolayers (CaCo-2) were incubated up to 48 h in the presence or absence of 0.5, 1 or 5 µM with one out of seven non-enzymatic oxysterols, prior to the verification of minimal irreversible cell damage within the chosen concentration range.
Results
All tested oxysterols were proven to exert damaging effects on cell monolayers in vitro. The inflammatory interleukin-8 and monocyte chemotactic protein-1 were mostly upregulated by 7-ketocholesterol and 7β-hydroxycholesterol, respectively, then to a lower extent by 5α,6α-epoxycholesterol, 7α-hydroxycholesterol and 5β,6β-epoxycholesterol. 7-Ketocholesterol and 7β-hydroxycholesterol also appeared to be most effective in impairing claudin-1, occludin and E-cadherin proteins, followed by 25-hydroxycholesterol and triol.
Conclusions
The oxysterols consistently derived by food autoxidation were tested; they potentially impaired the integrity of the intestinal epithelial barrier and triggered an inflammatory response within 0.5–5 µM concentrations, easily reachable in a single Western meal.
Significance Statement
This comprehensive analysis focused on the potential impairment of the intestinal barrier by the main dietary non-enzymatic oxysterols, should guide further nutrition research aiming at defining a threshold amount of these cholesterol derivatives in order not to derange the physiological gut–brain axis.
