Methylene Blue Attenuates Lung Injury Induced by Hindlimb Ischemia Reperfusion in Rats

Abstract

Objective: This study was aimed to investigate the protective effect of methylene blue against lung injury induced by reperfusion of ischemic hindlimb in a rat model.

Methods: Twenty-four healthy adult male Sprague-Dawley rats were equally randomized into three groups: sham (SM) group, ischemia reperfusion (IR) group, and methylene blue (MB) group. Rats in both IR and MB groups were subjected to 4 h of ischemia by clamping the left femoral artery and then followed by 4 h of reperfusion. Treatment with 1% methylene blue (50 mg/kg) was administrated intraperitoneally at 10 min prior to reperfusion in the MB group. After 4 h of reperfusion, malondialdehyde (MDA) level, myeloperoxidase (MPO), and superoxide dismutase (SOD) activities in lung tissue were detected; inflammatory cytokines, including IL-1β and IL-6, were measured in bronchoalveolar lavage fluid (BALF); correspondingly, the morphological changes and water content in both gastrocnemius muscle and lung samples were evaluated.

Results: Hindlimb IR caused remarkable morphological abnormalities and edema in both muscle and lung tissues. SOD activity was decreased, both the MPO activity and MDA level in lung tissue, as well as IL-1β and IL-6 levels in BALF, were increased in the IR group (p < 0.05). Compared with the IR group, SOD activity was increased, whereas MPO activity and MDA level in lung tissue and IL-1β and IL-6 levels in BALF were decreased in the MB group (p < 0.05). Also, the histological damage and edema in both lung and muscle tissues were significantly attenuated by the treatment of methylene blue.

Conclusion: Methylene blue attenuates lung injury induced by hindlimb IR in rats, at least in part, by inhibiting oxidative stress.

Figure 1

Histological changes in muscle tissue. (a) SM group; (b) IR group; (c) MB group; (d) histological damage score in muscle tissue. ^ap < 0.05 versus SM group; ^bp < 0.05 versus IR group.

Figure 2

Histological changes in lung tissue. (a) SM group; (b) IR group; (c) MB group; (d) lung injury score; (e) neutrophil number. ^ap < 0.05 versus SM group; ^bp < 0.05 versus IR group.

Figure 3

Water content, XO activity, and inflammatory cytokines. (a) Water contents in muscle and lung tissues; (b) uric acid levels in muscle and serum; (c) TNF-α level in serum and muscle; (d) IL-1β and IL-6 levels in BALF. ^ap < 0.05 versus SM group; ^bp < 0.05 versus IR group.

Figure 4

MPO and SOD activities and MDA level in lung tissue. (a) MPO activity in lung tissue; (b) SOD activity in lung tissue; (c) MDA level in lung tissue. ^ap < 0.05 versus SM group; ^bp < 0.05 versus IR group.