World Congress of Gastroenterology

WCOG 2019


 
DEFLAMIN, A NON-TOXIC OLIGOMER FROM LUPINUS ALBUS SEEDS, RESISTS DIGESTION AND INHIBITS COLITIS BY TARGETING MMP-9 ACTIVITY IN VITRO AND IN VIVO
JOãO BOAVIDA FERREIRA 1 Joana Mota 2 Ana Lima 2 Rosa Direito 3 João Rocha 3 Maria Eduardo Figueira 3 Anabela Raymundo 2 Ricardo Boavida Ferreira 2 António Guimarães 1 António Moreira 1

1- INSTITUTO PORTUGUêS DE ONCOLOGIA DE LISBOA FRANCISCO GENTIL
2- INSTITUTO SUPERIOR DE AGRONOMIA
3- FACULDADE DE FARMÁCIA DA UNIVERSIDADE DE LISBOA
 
Background/Aims:

Matrix metalloproteinases (MMPs), particularly MMP-9, are important mediators of inflammation in inflammatory bowel disease (IBD). Whilst MMP-9 inhibitors (MMPIs) have been demonstrated to effectively reduce inflammation in IBD, their efficacy has been hampered by low specificity, high toxicity and overall severe secondary effects. Deflamin, an oligomer isolated from lupin seeds, is a novel type of MMPI that is edible and proteinaceous in nature, and survives boiling and digestion. The aim of this work was to understand if deflamin can be used as an anti-inflammatory agent in diet, using in vitro and in vivo models of colitis.

Materials and Methods:

Deflamin was isolated from lupin seeds using a clean extraction method (food-compatible). MMPI activities as well as anti-proliferative and anti-invasion activities were determined in colon adenocarcinoma (HT-29) cells. Anti-inflammatory activities were tested using in vivo models of experimental TNBS-induced colitis in mice with oral and intraperitoneal administrations of 15 and 10 mg.kg-1, respectively. Histological expression of COX-2, iNOS and MMP-9 was analyzed in colons. Food products (cookies) containing deflamin were produced and assessed for their digestibility and efficacy in in vivo models of AA-induced colitis. The expression of inflammatory and oxidative stress-related biomarkers such as lipid peroxidation, SOD and GPx was also evaluated.

Results:

In vitro studies showed that deflamin did not affect cell growth or metabolism in HT-29 cells (suggesting low cell toxicity) but successfully impaired cell invasion while reducing MMP-9 activity in a dose-dependent manner (Figure 1). With the in vivo model of experimental colitis, a reduction of the extent of visible injury (ulcer formation) (Figure 2) was seen, along with a reduction of general histological features of colon inflammation, particularly in the oral administrations (Figure 3). A significant decrease in the expression of COX-2 and iNOS and in the activity of MMP-9 was also observed in the colon tissue. Deflamin-containing cookies also reduced colitis injuries, whilst significantly reducing oxidative stress markers, namely SOD activity and lipid peroxidation.

 

 

Figure 1. HT-29 cell invasion after exposure to different concentrations of deflamin, as determined by the wound healing assay. Cells were grown until reaching 80% confluence and the monolayer was scratched with a pipette tip (0h). Cells were then exposed to 100, 50, 10 and 5 μg.ml-1 deflamin and cell migration was recorded after 48 h. Results show that deflamin inhibits the gelatinolytic activity of HT-29 cells and reveals a dose-response effect, which can be useful for future studies.

 

Figure 2. Effect of deflamin administration on the macroscopic observation of colon. (A) Sham group (n=6); (B) EtOH group (n=6); (C) TNBS group (n=8); (D) TNBS+deflamin p.o. group (15 mg.kg-1; n=9). There was a clear attenuation of colon injury in animals treated with deflamin when compared to the TNBS-induced colitis. 

 

Figure 3Effect of deflamin administration on the colon tissue expression of inflammation markers COX-2 and iNOS. (A) COX-2 expression: (1) Sham group, (2) TNBS group, (3) TNBS+deflamin p.o. group, (4) TNBS+deflamin i.p. group; (B) iNOS expression: (1) Sham group, (2) TNBS group, (3) TNBS+deflamin p.o. group (15 mg.kg-1, n=9), (4) TNBS+deflamin i.p. group (10 mg.kg-1, n=10). Results show that TNBS treatment induced a marked increase in COX2 and iNOS expression along the remaining crypts, indicated by brown color when compared with control samples. Administration of deflamin led to a reduced staining for COX-2 and iNOS, indicating that it impaired the expression of COX-2 and iNOS in the injured intestinal tissue.

Conclusion:

Our work validates deflamin as a novel type of MMPI that survives cooking and digestion and is effective when administered orally, strongly suggesting a therapeutic potential use in gut inflammation. 

Keywords:

deflamin, inflammatory bowel disease, matrix metalloproteinase-9, colitis.