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. 2014 Mar;175(3):476-84.
doi: 10.1111/cei.12229.

The kiwi fruit peptide kissper displays anti-inflammatory and anti-oxidant effects in in-vitro and ex-vivo human intestinal models

C Ciacci  1 , I Russo, C Bucci, P Iovino, L Pellegrini, I Giangrieco, M Tamburrini, M A Ciardiello
Affiliations

The kiwi fruit peptide kissper displays anti-inflammatory and anti-oxidant effects in in-vitro and ex-vivo human intestinal models

C Ciacci et al. Clin Exp Immunol. 2014 Mar.

Abstract

Literature reports describe kiwi fruit as a food with significant effects on human health, including anti-oxidant and anti-inflammatory activity. Fresh fruit or raw kiwi fruit extracts have been used so far to investigate these effects, but the molecule(s) responsible for these health-promoting activities have not yet been identified. Kissper is a kiwi fruit peptide displaying pore-forming activity in synthetic lipid bilayers, the composition of which is similar to that found in intestinal cells. The objective of this study was to investigate the kissper influence on intestinal inflammation using cultured cells and ex-vivo tissues from healthy subjects and Crohn's disease (CD) patients. The anti-oxidant and anti-inflammatory properties of kissper were tested on Caco-2 cells and on the colonic mucosa from 23 patients with CD, by challenging with the lipopolysaccharide from Escherichia coli (EC-LPS) and monitoring the appropriate markers by Western blot and immunofluorescence. EC-LPS challenge determined an increase in the intracellular concentration of calcium and reactive oxygen species (ROS). The peptide kissper was highly effective in preventing the increase of LPS-induced ROS levels in both the Caco-2 cells and CD colonic mucosa. Moreover, it controls the calcium increase, p65-nuclear factor (NF)-kB induction and transglutaminase 2 (TG2) activation inflammatory response in Caco-2 cells and CD colonic mucosa. Kissper efficiently counteracts the oxidative stress and inflammatory response in valuable model systems consisting of intestinal cells and CD colonic mucosa. This study reports the first evidence supporting a possible correlation between some beneficial effects of kiwi fruit and a specific protein molecule rather than generic nutrients.

Keywords: inflammation; lipopolysaccharide; reactive oxygen species.

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Figures

Figure 1
Figure 1
Kissper controls calcium release and reactive oxygen species (ROS) production in intestinal epithelial cells. (a) Immunofluorescence of intracellular ROS [chloromethyl-2′7′-dichlorodihydrofluorescein diacetate acetyl ester (CM-H2DCFDA, top panel] or a Wallac 1420 multi-label counter (a, bottom line) DCF, 5-(and-6)CM-H2DCFDA). Values are means ± standard error (SE), n = 6. Asterisks indicate that means differ from samples cultured with medium. *P < 0·05. (b) Caco-2 cells were loaded with Fura-2AM. [Ca2+] imaging pseudocolour ratiometric images. Colours correspond to the scale of [Ca2+]i increase. Red, high [Ca2+]i contents. (b, bottom line) Quantification of results from three independent experiments is shown in the histogram. Values are means ± standard error (s.e.). Asterisks indicate that means differ from samples cultured with medium alone. *P < 0·05.
Figure 2
Figure 2
Effect of kissper in the control of mucosal inflammation. (a) Reactive oxygen species (ROS) levels in control (n = 12) and Crohn's disease (CD) colonic mucosa (n = 23) before challenge and after challenge with medium alone, medium with Escherichia coli-lipopolysaccharide (EC-LPS) or EC-LPS with kissper. Values are means ± standard deviation (s.d.). Asterisks indicate that means differ from control samples, *P < 0·05 versus control by analysis of variance (anova). (b) Number of epithelial cells with p65 nuclear localization per 100 epithelial cells in control (n = 12) and CD colonic mucosa (n = 23) before and after challenge with medium alone, medium with EC-LPS or EC-LPS and kissper following 24 h of incubation. Values are means ± s.d. *P < 0·05 versus control by anova. (c) Tumour necrosis factor (TNF)-α protein after challenge with medium alone, medium with EC-LPS or EC-LPS with kissper following 24 h of incubation. Values are means ± s.d. of three experiments. *P < 0·05 versus control by anova. (d) Immunofluorescence of cyclooxygenase 2 (COX-2) and intercellular adhesion molecule 1 (ICAM-1) and number of COX-2- and ICAM-1-positive lamina propria cells per mm2 of mucosa in CD colonic mucosa (n = 23 after challenge with medium alone, medium with EC-LPS or EC-LPS and kissper following 24 h of incubation. Scale bar, 10 μm. Values are means ± s.d. of three experiments. *P < 0·05 versus control by anova.
Figure 3
Figure 3
Kissper modulates transglutaminase 2 (TG2) expression in Crohn's disease (CD) colonic mucosa and Caco-2 cells. (a) Immunofluorescence analysis of TG2 protein (green) and activity (red) expression in CD colonic mucosa challenged for 6 h and other 24 h with Escherichia coli-lipopolysaccharide (EC-LPS) in the presence or absence of kissper. Incubation with kissper reduces the TG2 expression and activity in EC-LPS-challenged CD biopsies. (b) Immunoblot analysis of TG2 in Caco-2 cells after 24 h of challenge with EC-LPS. β-actin was used as loading control. (b, bottom line) Densitometric analysis of the band intensity. Values are means ± standard deviation (s.d.) of three experiments. (c) Tumour necrosis factor (TNF)-α protein after challenge with medium alone, medium with EC-LPS or EC-LPS with kissper following 24 h of incubation. Values are means ± s.d. of three experiments. *P < 0·05 versus control by analysis of variance (anova).
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