Maternal exposure to low levels of corticosterone during lactation protects adult rat progeny against TNBS-induced colitis: A study on GR-mediated anti-inflammatory effect and prokineticin system

Abstract

The early phase of life represents a critical period for the development of an organism. Interestingly, early life experiences are able to influence the development of the gastrointestinal tract and the reactivity to colonic inflammatory stress. We recently demonstrated that adult male rats exposed to low doses of corticosterone during lactation (CORT-nursed rats) are protected against experimental colitis induced by the intracolonic infusion of 2,4,6-trinitrobenzenesulfonic acid (TNBS). Based on these interesting results, we wanted to better investigate which cellular actors could be involved in the protection of CORT-nursed rats from TNBS-induced experimental colitis. Therefore, in the present work, we focused our attention on different factors implicated in GR-mediated anti-inflammatory effect. To address this issue, colonic tissues, collected from control and CORT-nursed healthy animals and from control and CORT-nursed colitic rats, were processed and the following inflammatory factors were evaluated: the expression of (i) glucocorticoid receptors (GR), (ii) glucocorticoid-induced leucine zipper (GILZ), (iii) phospho-p65NF-κB, (iv) the pro-inflammatory cytokines IL-1β and TNF-α, (v) the prokineticins PK2 and PK2L and (vi) their receptors PKR1 and PKR2. We found that adult CORT-nursed rats, in comparison to controls, showed increased expression of colonic GR and reduced expression of pro-inflammatory molecules (IL-1β, TNF-α, PK2 and PK2L) in response to inflammatory colitis. The observed changes were associated with an increase in GILZ colonic expression and with a reduction in phospo-p65NF-κB colonic expression.

Fig 1. Effects of TNBS-induced colitis on colonic GR expression.

Four days after TNBS instillation, increased colonic GR expression was observed in both control and CORT-nursed colitic animals (Fisher’s LSD post-hoc, **p<0.01, ***p<0.001 vs. own healthy animals). The CORT-nursed colitic group showed GR expression levels greater than those of the control colitic group (Fisher’s LSD post-hoc, *p<0.05). The results are expressed as the ratio of optical density (OD) of the GR to that of the β-actin band (control healthy n = 4, control colitic n = 5, CORT-nursed healthy n = 4, CORT-nursed colitic n = 4). A representative western blot image was reported.

Fig 2. Effects of TNBS-induced colitis on colonic GILZ expression.

Four days after TNBS instillation, an increase in colonic GILZ expression was observed in CORT-nursed colitic animals (Fisher’s LSD post-hoc, ***p<0.001 vs. CORT-nursed healthy). GILZ expression was significantly greater in CORT-nursed colitic rats with respect to control colitic rats (Fisher’s LSD post-hoc, ***p<0.001). Tissue sections were incubated with a primary antibody against GILZ (red signal). Nuclei were visualised by Hoechst 33258 (blue signal). Results are expressed as percentage (%) of positive labelled with respect to the total area (control healthy n = 4, control colitic n = 5, CORT-nursed healthy n = 4, CORT-nursed colitic n = 4). Representative immunofluorescence images are shown.

Fig 3. Effects of TNBS-induced colitis on colonic phospho-p65NF-κB expression.

Four days after TNBS instillation, an increase in colonic phospo-p65NF-κB expression was observed in colitic animals (***p<0.001 colitic vs. healthy group). A reduction in phospo-p65NF- κB was observed in CORT-nursed rats (**p<0.01 CORT-nursed vs. control group). Tissue sections were incubated with a primary antibody against phospho-p65NF-κB (red signal). Nuclei were visualised by Hoechst 33258 (blue signal). Results are expressed as percentage (%) of positive labelled with respect to the total area (control healthy n = 4, control colitic n = 5, CORT-nursed healthy n = 4, CORT-nursed colitic n = 4). Representative immunofluorescence images are shown.

Fig 4. Effects of TNBS-induced colitis on colonic IL-1β and TNF-α mRNA expression.

(A) An increase in colonic IL-1β mRNA expression was observed in control colitic rats (Fisher’s LSD post-hoc, ***p<0.001 vs. control healthy rats). A reduction in IL-1β mRNA expression was observed in CORT-nursed colitic rats (Fisher’s LSD post-hoc, ***p<0.001 vs. control colitic rats). (B) An increase in colonic TNF-α mRNA expression was observed in control colitic rats (Fisher’s LSD post-hoc, ***p<0.001 vs. control healthy rats). A reduction in TNF-α mRNA expression was observed in CORT-nursed colitic rats (Fisher’s LSD post-hoc, ***p<0.001 vs. control healthy rats). The colonic relative mRNA expression levels are expressed in relation to β-actin and presented as fold increases relative to control rats. (Control healthy n = 4, control colitic n = 5, CORT-nursed healthy n = 4, CORT-nursed colitic n = 4).

Fig 5. Effects of TNBS-induced colitis on colonic PK2 mRNA and protein expression.

(A) Four days after TNBS instillation, an increase in colonic PK2 mRNA expression was found in control colitic rats (Fisher’s LSD post-hoc, ***p<0.001 vs. control healthy rats). A reduction in PK2 mRNA levels was observed in CORT-nursed colitic animals (Fisher’s LSD post-hoc, ***p<0.001 vs. control colitic rats). The colonic relative mRNA expression levels are expressed in relation to β-actin and presented as fold increase relative to control rats. (B) Western blot analysis showed an increase in PK2 in colitic rats (**p<0.01colitic vs. healthy group). A reduction in PK2 was observed in CORT-nursed rats (*p<0.05 CORT-nursed vs. control group). Results are expressed as the ratio of the optical density (OD) of the PK2 and the β-actin band. (control healthy n = 4, control colitic n = 5, CORT-nursed healthy n = 4, CORT-nursed colitic n = 4). A representative western blot image is shown.

Fig 6. Effects of TNBS-induced colitis on colonic PK2L mRNA expression.

Four days after TNBS instillation, an increase in colonic PK2L mRNA expression was revealed in control colitic rats (Fisher’s LSD post-hoc ***p<0.001 vs. control healthy rats). PK2L mRNA levels in CORT-nursed colitic animals were significantly lower (Fisher’s LSD post-hoc ***p<0.001 vs. control colitic). The colonic relative mRNA expression levels are expressed in relation to β-actin and presented as fold increase relative to control rats. (control healthy n = 4, control colitic n = 5, CORT-nursed healthy n = 4, CORT-nursed colitic n = 4).

Fig 7. Effects of TNBS-induced colitis on colonic PKR1 mRNA and protein expression.

(A) Four days after TNBS instillation, no significant differences were observed in colonic PKR1 mRNA expression between groups. The colonic relative mRNA expression levels are expressed in relation to β-actin and presented as fold increase relative to control rats. (B) Analysis of PKR1 protein expression showed no differences between experimental groups. Results are expressed as the ratio of the optical density (OD) of the PKR1 and the β-actin band. (control healthy n = 4, control colitic n = 5, CORT-nursed healthy n = 4, CORT-nursed colitic n = 4). A representative western blot image is shown.

Fig 8. Effects of TNBS-induced colitis on colonic PKR2 mRNA and protein expression.

(A) Four days after TNBS instillation, an increase in colonic PKR2 mRNA expression was observed in colitic rats (***p<0.001colitic vs. healthy group). The colonic relative mRNA expression levels are expressed in relation to β-actin and presented as fold increase relative to control rats. (B) Western blot analysis showed no differences between experimental groups. Results are expressed as the ratio of the optical density (OD) of the PKR2 and the β-actin band. (control healthy n = 4, control colitic n = 5, CORT-nursed healthy n = 4, CORT-nursed colitic n = 4). A representative western blot image is shown.