Olmesartan decreased levels of IL-1β and TNF-α, down-regulated MMP-2, MMP-9, COX-2, RANK/RANKL and up-regulated SOCs-1 in an intestinal mucositis model

Abstract

Methotrexate (MTX) is a pro-oxidant compound that depletes dihydrofolate pools and is widely used in the treatment of leukaemia and other malignancies. The efficacy of methotrexate is often limited by mucositis and intestinal injury, which are major causes of morbidity in children and adults. The aim of this study was to evaluate the effect of olmesartan (OLM), an angiotensin II receptor antagonist, on an Intestinal Mucositis Model (IMM) induced by MTX in Wistar rats. IMM was induced via intraperitoneal (i.p.) administration of MTX (7 mg/kg) for three consecutive days. The animals were pre-treated with oral OLM at 0.5, 1 or 5 mg/kg or with vehicle 30 min prior to exposure to MTX. Small intestinal homogenates were assayed for levels of the IL-1β, IL-10 and TNF-α cytokines, malondialdehyde and myeloperoxidase activity. Additionally, immunohistochemical analyses of MMP-2, MMP-9, COX-2, RANK/RANKL and SOCS-1 and confocal microscopy analysis of SOCS-1 expression were performed. Treatment with MTX + OLM (5 mg/kg) resulted in a reduction of mucosal inflammatory infiltration, ulcerations, vasodilatation and haemorrhagic areas (p<0.05) as well as reduced concentrations of MPO (p<0.001) and the pro-inflammatory cytokines IL-1β (p<0.001) and TNF-a (p<0.01), and increase anti-inflammatory cytocine IL-10 (p<0.05). Additionally, the combined treatment reduced expression of MMP-2, MMP-9, COX-2, RANK and RANKL(p<0.05) and increased cytoplasmic expression of SOCS-1 (p<0.05). Our findings confirm the involvement of OLM in reducing the inflammatory response through increased immunosuppressive signalling in an IMM. We also suggest that the beneficial effect of olmesartan treatment is specifically exerted during the damage through blocking inflammatory cytocines.

Figure 1. OLM attenuates MTX effects on MPO and MDA.

The MTX alone group had significantly greater MPO and MDA than the Negative control (***p<.001 and *p<0.5, respectively). All three doses of OLM (0.5 mg/kg, 1 mg/kg, 5 mg/kg) countered the MTX effect on MPO significantly (###p<.001 vs. MTX). Although all doses of OLM appeared to reduce MDA subjectively, only the highest dose (5 mg/kg) produced a significant difference versus the MTX alone group (#p<.05). Segments of duodenum, jejunum and ileum/Duplicate Experiments.

Figure 2. OLM opposes MTX influence on cytokines.

MTX elevated levels of IL-1β (***p<0.001) and TNF-α (**p<0.01), while reducing IL-10 levels(***p<0.001) than the Negative control. OLM had dose-dependent MTX-countering effects on IL-1β (0.5 mg/kg, 1 mg/kg ##p<0.01 and 5 mg/kg ### p<0.001) and TNF-α (1 mg/kg #p<0.05 and 5 mg/kg ## p<0.01), but only OLM 5 mg/kg a significant MTX-countering effect on IL-10 levels (#p<0.05). Segments of duodenum, jejunum and ileum/Duplicate Experiments.

Figure 3. MTX and OLM effects on leukocyte density.

MTX alone and MTX-OLM 5 mg/kg reduced leukocyte density (**p<.001 and *** p<0.001, respectively vs. Negative control), indeed, the and MTX-OLM 5 mg/kg resulted in an even stronger reduction effect than MTX alone (##p<.01 vs. MTX). OLM 5 mg/kg increased leukocyte density significantly (###p<.001 vs. MTX).

Figure 4. Histological examination of jejunum specimens.

For each group, 5 animals were used, and three H&E sections from each animal were analyzed. Representative samples from each OLM treatment group are shown with graphs summarizing the group's mean histopathological score . Images and data from the low, middle, and high OLM dose groups are shown in panels A–C; D–F; G–I respectively (micrograph key: a, villus height; b, reduction of villus; c, inflammatory infiltrate). Control group slides are shown in A, D, and G (upper left image in each group). MTX alone group slides are shown in panels B, E, and H(middle images). Note that the MTX alone rats' jejunum exhibited intestinal mucositis with loss of crypt architecture, severe villous epithelial atrophy, degeneration and shortening of the villus length, and polymorphonuclear leukocyte infiltration in the lamina propria. Damage in mucosa as intense inflammation and villous epithelial atrophy persisted in the MTX-OLM 0.5 mg/kg (C) and MTX-OLM 1 mg/kg (F) groups (# p>.05 vs. MTX). (I) Conversely, reduced inflammation, reepithelization with decreased vasodilatation, decreased cellular infiltration, and reduction of hemorrhagic areas, ulcerations, and abscesses were observed in the jejunum from animals treated with MTX-OLM 5 mg/kg for 3 d. Magnification 20×, scale bar  = 100 µm.

Figure 5. Effect of OLM treatment on intestinal damage in rats.

For each group, 5 animals were used, and three H&E sections from each animal were analyzed. Representative samples from each OLM treatment group are shown with graphs summarizing the group's mean histopathological score. Values are expressed as means ± SEM (Compared to negative control ***p<0.001, compared to MTX *p<0.05; compared to MTX #p>0.05).

Figure 6. IHC examination of jejunum specimens.

For each antigen, three immunolabeled sections were analyzed per animal (N = 5/group). Generally, jejunum from MTX rats had greater MMP-2 (A–C), MMP-9 (D–F) and COX-2 (G–I) immunoreactivity. Magnification 40×, scale bar  = 100 µm.

Figure 7. IHC examination of jejunum specimens.

For each antigen, three immunolabeled sections were analyzed per animal (N = 5/group). Generally, jejunum from MTX rats had greater RANK (A–C), and RANK-L (D–F) immunoreactivity and reduced SOCs (G–I) immunoreactivity. Magnification 40×, scale bar  = 100 µm.

Figure 8. Effect of OLM treatment on intestinal damage in rats.

For each group, 5 animals were used, and three immunohistochemistry sections from each animal were analyzed. Representative samples from MTX-OLM 5 mg/Kg treatment group are shown with graphs summarizing the group's mean score, showing immunoreactivity to MMP-2, MMP-9, COX-2, RANK, RANK-L, and SOCs . Note that these effects were reversed (i.e. normalized) in the MTX- OLM 5 mg/kg group's jejunum . *p<.05 vs MTX and ***p<.001 negative control vs. MTX, Kruskal-Wallis test followed by Dunn's test.

Figure 9. OLM reverses MTX-induced suppression of SOCs-1 protein expression.

Representative confocal photomicrographs of SOCs-1 immunoreactivity (green) in jejunum from each group. The sections are nuclear counterstained with DAPI (blue). (A) negative control rat jejunum had moderately diffuse SOCs-1 labeling in all mucosa layers, 20×. (B) SOCs-1 labeling was absent in the MTX intestinal mucositis group, 20×. (C, C.1) Strong cytoplasmic SOCs-1 labeling (red arrows) was seen in the group treated with MTX-OLM 5 mg/kg, 20× and scale bar 50 µm and, 63× and scale bar 20 µm, respectively. (D) Densitometric analysis confirmed a significant reduction in SOCs-1 immunoreactivity in the MTX group that was blocked in the MTX-OLM 5 mg/kg group (*p<.05, Kruskal-Wallis test followed by Dunn's test).