Protective effects of fenofibrate against acute lung injury induced by intestinal ischemia/reperfusion in mice

Abstract

This experiment was conducted to evaluate whether pretreatment with fenofibrate could mitigate acute lung injury (ALI) in a mice model of intestinal ischemia/reperfusion (I/R). Male C57BL/6 mice were randomly assigned into three groups (n = 6): sham, intestinal I/R + vehicle, and intestinal I/R + fenofibrate. Intestinal I/R was achieved by clamping the superior mesenteric artery. Fenofibrate (100 mg/kg) or equal volume of vehicle was injected intraperitoneally 60 minutes before the ischemia. At the end of experiment, measurement of pathohistological score, inflammatory mediators and other markers were performed. In addition, a 24-hour survival experiment was conducted in intestinal I/R mice treated with fenofibrate or vehicle. The chief results were as anticipated. Pathohistological evaluation indicated that fenofibrate ameliorated the local intestine damage and distant lung injury. Pretreatment with fenofibrate significantly decreased inflammatory factors in both the intestine and the lung. Consistently, renal creatine levels and hepatic ALT levels were significantly decreased in the fenofibrate group. Moreover, serum systemic inflammatory response indicators were significantly alleviated in the fenofibrate group. In addition, fenofibrate administration significantly improved the survival rate. Collectively, our data indicated that pretreatment with fenofibrate prior to ischemia attenuated intestinal I/R injury and ALI.

Figure 1. Alteration in gut morphology after I/R.

(A) Representative images are shown here for the gross morphological alterations of the intestine at the end of the reperfusion. Black arrowheads indicate normal intestine in the sham group. White arrows point to mild I/R injury in the fenofibrate group. Black arrows show severely injured intestine in the vehicle-treated group. (B) Histological findings indicate that intestine I/R caused widespread mucosal destruction, loss of villi and infiltration of inflammatory cells, while fenofibrate administration showed protective effects. Red arrows depict mildly injured villi tips, a protective result of fenofibrate. Asterisks indicate severe injury with denuding of villi tips. Original magnification: X500. Scale bars represent 100 μm (C) Pathological score of the intestinal injury in the sham, fenofibrate- and vehicle-treated groups after I/R. Data are expressed as mean ±SD (n = 6/group) and compared by Kruskal-Wallis test and Mann-Whitney U test (*P < 0.05 vs. vehicle and ^#P < 0.05 vs. sham).

Figure 2

Inflammatory cytokine expression of TNF-α, IL-6, IL-1β, HMGB-1 (A–D) and apoptotic variables of NF-κB p65 (E) and Iκ Bα (F) in the small intestinal tissue of mice of the sham, fenofibrate and vehicle groups (n = 6/group). Cytokines were measured by ELISA and the values were normalized to total cellular protein. Bar graphs shows the mean ±SD. *P < 0.05 vs. vehicle and ^#P < 0.05 vs. sham by one-way ANOVA and LSD test.

Figure 3. Histological evaluations of the lung after intestinal I/R.

(A) Representative photomicrography of the lung section. Asterisks indicate the alveolar exudation. Arrowheads depict the edema. Black arrows show the hemorrhagic area. Red arrows represent intra-alveolar hemorrhage/debris. Yellow arrows show cellular hyperplasia. (HE staining; original magnification X200; Scale bars represent 100 μm); (B) Lung tissue injury in mice submitted to sham, vehicle and fenofibrate-treated groups. The data represent the mean ± SD (n = 6/group). *P < 0.05 vs. vehicle and ^#P < 0.05 vs. sham by Kruskal-Wallis test and Mann-Whitney U test.

Figure 4. Alterations of lung injury variables after intestinal I/R.

(A) NO, (B) iNOS, (C) NF-κB p65, (D) Iκ Bα were measured by ELISA in the sham, fenofibrate and vehicle groups; (E) Caspase-3 expressions of the lung tissues were assessed by the western blot: results representative of six to ten separate experiments are shown and β-actin is used as internal control. Data are expressed as mean ± SD (n = 6/group) and compared by one-way ANOVA and LSD test (*P < 0.05 vs. vehicle and ^#P < 0.05 vs. sham).

Figure 5. Fenofibrate decreased the distant organ injury after intestinal I/R.

(A) Renal creatine levels were measured by ELISA. (B)Liver tissues were collected and the ALT expressions were evaluated by western blot. Data are expressed as mean ± SD and compared by one-way ANOVA and LSD test (*P < 0.05 vs. vehicle and ^#P < 0.05 vs. sham).

Figure 6. Survival analysis of the fenofibrate and vehicle treated mice after intestinal I/R.

Mice subjected to a single dose of fenofibrate (i.p. 100 mg/kg) or vehicle (DMSO) one hour before the ischemia were observed for 24 hours. Each point in the figure represents the mean survival rate at each time point. The survival rate is compared by Kaplan-Meier method and the log-rank test (n = 15/group, *P < 0.05 vs. vehicle).