Oleuropein Decreases Cyclooxygenase-2 and Interleukin-17 Expression and Attenuates Inflammatory Damage in Colonic Samples from Ulcerative Colitis Patients

Tiziana Larussa, Manuela Oliverio, Evelina Suraci, Marta Greco, Roberta Placida, Serena Gervasi, Raffaella Marasco, Maria Imeneo, Donatella Paolino, Luigi Tucci, Elio Gulletta, Massimo Fresta, Antonio Procopio, Francesco Luzza, Tiziana Larussa, Manuela Oliverio, Evelina Suraci, Marta Greco, Roberta Placida, Serena Gervasi, Raffaella Marasco, Maria Imeneo, Donatella Paolino, Luigi Tucci, Elio Gulletta, Massimo Fresta, Antonio Procopio, Francesco Luzza

Abstract

Oleuropein (OLE) is the major phenolic secoiridoid of olive tree leaves, and its antioxidant and anti-inflammatory activities have been demonstrated in in vitro and in vivo animal models. The aim of this study was to investigate the activity of OLE in the colonic mucosa from patients with ulcerative colitis (UC). Biopsies obtained during colonoscopy from 14 patients with active UC were immediately placed in an organ culture chamber and challenged with lipopolysaccharide from Escherichia coli (EC-LPS) at 1 μg/mL in the presence or absence of 3 mM OLE. The expression of cyclooxygenase (COX)-2 and interleukin (IL)-17 was assessed in total protein extracts from treated colonic biopsies by Western blotting. Levels of IL-17 were also measured in culture supernatant by ELISA. A microscopic evaluation of the cultured biopsies was performed by conventional histology and immunohistochemistry. The expression of COX-2 and IL-17 were significantly lower in samples treated with OLE + EC-LPS compared with those treated with EC-LPS alone (0.80 ± 0.15 arbitrary units (a.u.) vs. 1.06 ± 0.19 a.u., p = 0.003, and 0.71 ± 0.08 a.u. vs. 1.26 ± 0.42 a.u., p = 0.03, respectively) as were the levels of IL-17 in culture supernatants of OLE + EC-LPS treated colonic samples (21.16 ± 8.64 pg/mL vs. 40.67 ± 9.24 pg/mL, p = 0.01). Histologically, OLE-treated colonic samples showed an amelioration of inflammatory damage with reduced infiltration of CD3, CD4, and CD20 cells, while CD68 numbers increased. The anti-inflammatory activity of OLE was demonstrated in colonic biopsies from UC patients. These new data support a potential role of OLE in the treatment of UC.

Keywords: cyclooxygenase-2; interleukin-17; oleuropein; olive oil; ulcerative colitis.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Oleuropein (OLE) decreases cyclooxygenase (COX)-2 expression in colonic biopsy culture. Levels of COX-2 observed by Western blotting of total protein extracts from colonic biopsiesof patientswith ulcerative colitis (n = 14) treated with medium (untreated samples) or lipopolysaccharide from Escherichia coli (EC-LPS) at 1 µg/mL, in the absence or presence of 3 mM OLE for 20 h in an organ culture chamber. β-actin was used as loading control. Values are expressed as mean values ± SD of arbitrary units (a.u.). The immunoblot panel is one representative experiment.
Figure 2
Figure 2
Oleuropein (OLE) decreases interleukin (IL)-17 expression in colonic biopsy culture. Levels of IL-17 observed by Western blotting of total protein extract from colonic biopsies of patients with ulcerative colitis (n = 14) treated for 20 h in an organ culture chamber with medium (untreated samples) or lipopolysaccharide from Escherichia coli (EC-LPS) at 1 µg/mL, in the absence or presence of 3 mM OLE. β-actin was used as loading control. Values are expressed as mean values ± SD of arbitrary units (a.u.). The immunoblot panel is one representative experiment.
Figure 3
Figure 3
Oleuropein (OLE) decreases interleukin (IL)-17 levels in supernatant of colonic biopsy culture. Levels of IL-17 by ELISA observed in supernatant from colonic biopsies of patients with ulcerative colitis (n = 14) treated for 20 h in an organ culture chamber with medium (untreated samples) or lipopolysaccharide from Escherichia coli (EC-LPS) at 1 µg/mL, in the absence or presence of 3 mM OLE. Values are given in pg/mL as scattered plots with mean values.
Figure 4
Figure 4
Anti-inflammatory effects of oleuropein (OLE) in colonic biopsies from patients with ulcerative colitis. Representative images of tissue sections of OLE-treated and untreated samples from the same patient. (A) Dense inflammatory infiltrate of leukocytes in the lamina propria, with epithelial damage, necrosis of the surface cells, and loss of mucin secretion (score +++; H & E stain, 400× magnification) in a biopsy specimen treated with lipopolysaccharide from Escherichia coli (EC-LPS) alone; (B) Decreased infiltration of leukocytes, mainly mononuclear cells, preservation of mucin secretion and presence of goblet cells in the superficial portion of the glands (score +; H & E stain, 400× magnification) in a biopsy specimen treated with OLE + EC-LPS; (C) A greater number of intra-epithelial and sub-mucosal T lymphocytes infiltration (score +++; immunohistochemical stain for CD3, 400× magnification) in a biopsy specimen treated with EC-LPS alone; (D) Decreased T lymphocytosis in the submucosa and lamina propria, and preservation of mucin secretion (score +; immunohistochemical stain for CD3, 400× magnification) in a biopsy specimen treated with OLE + EC-LPS; (E) Enhanced infiltration of intraepithelial and sub-mucosal T helper lymphocytes in a biopsy specimen treated with EC-LPS alone (score +++; immunohistochemical stain for CD4, 40× magnification); (F) Decreased infiltration of intraepithelial and sub-mucosal T helper lymphocytes in a biopsy specimen treated with OLE + EC-LPS (score +; immunohistochemical stain for CD4, 40× magnification); (G) A greater number of B lymphocytes are observed as the dominant cell types in the aggregates of biopsies treated with EC-LPS alone (score ++; immunohistochemical stain for CD20, 200× magnification); (H) The aggregates are significantly reduced in size and are confined in the submucosa as inflammatory residuals in biopsy specimens treated with OLE + EC-LPS (score +; immunohistochemical stain for CD20, 200× magnification); (I) Histiocytic infiltration consistent with the degree of the inflammation in the submucosa of the biopsy specimens treated with EC-LPS (score +; immunohistochemical stain for CD68, 400× magnification); (J) Increased histiocytic infiltration along with restoration of mucin secretion in biopsy specimens treated with OLE + EC-LPS (score ++; immunohistochemical stain for CD68, 400× magnification).

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