Stimulation of Brain AMP-Activated Protein Kinase Attenuates Inflammation and Acute Lung Injury in Sepsis

Abstract

Sepsis and septic shock are enormous public health problems with astronomical financial repercussions on health systems worldwide. The central nervous system (CNS) is closely intertwined in the septic process but the underlying mechanism is still obscure. AMP-activated protein kinase (AMPK) is a ubiquitous energy sensor enzyme and plays a key role in regulation of energy homeostasis and cell survival. In this study, we hypothesized that activation of AMPK in the brain would attenuate inflammatory responses in sepsis, particularly in the lungs. Adult C57BL/6 male mice were treated with 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR, 20 ng), an AMPK activator, or vehicle (normal saline) by intracerebroventricular (ICV) injection, followed by cecal ligation and puncture (CLP) at 30 min post-ICV. The septic mice treated with AICAR exhibited elevated phosphorylation of AMPKα in the brain along with reduced serum levels of aspartate aminotransferase, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6), compared with the vehicle. Similarly, the expressions of TNF-α, IL-1β, keratinocyte-derived chemokine and macrophage inflammatory protein-2 as well as myeloperoxidase activity in the lungs of AICAR-treated mice were significantly reduced. Moreover, histological findings in the lungs showed improvement of morphologic features and reduction of apoptosis with AICAR treatment. We further found that the beneficial effects of AICAR on septic mice were diminished in AMPKα2 deficient mice, showing that AMPK mediates these effects. In conclusion, our findings reveal a new functional role of activating AMPK in the CNS to attenuate inflammatory responses and acute lung injury in sepsis.

Figure 1

Phosphorylation of AMPK in the brain. The hypothalamus and surrounding tissue of sham, vehicle- and AICAR-treated mice were harvested at 20 h after CLP. The tissue was lysed and subjected to Western blotting against phosphorylated (p-AMPKα) and total AMPK (t-AMPKα). Histogram shows mean densitometric analysis of bands after normalizing with t-AMPK and β-actin. Expression in the sham group is designated as 1 for comparison. Data are expressed as mean ± SE (n = 6–8 per group). *P < 0.05 versus sham and ^#P < 0.05 versus vehicle.

Figure 2

Alterations in serum levels of organ injury marker and proinflammatory cytokines. Blood of sham, vehicle- and AICAR-treated mice were harvested at 20 h after CLP for measuring (A) AST, (B) TNF-α, (C) IL-1β, and (D) IL-6. Data are expressed as mean ± SE (n = 6–8 per group). *P < 0.05 versus sham and ^#P < 0.05 versus vehicle.

Figure 3

Morphological changes in the lungs. The lung tissues of sham, vehicle- and AICAR-treated mice were harvested at 20 h after CLP and subjected to histological analysis with hematoxylin–eosin staining. Representative images of lung histology from n = 6–8 per group. Original magnification 100× (top panels); 200× (bottom panels).

Figure 4

Apoptosis in the lungs after CLP. The lung tissues of sham, vehicle- and AICAR-treated mice were harvested at 20 h after CLP and subjected to TUNEL assay. (A) Representative images of TUNEL staining (green fluorescent) and nuclear counterstaining (red fluorescent) of lung sections. Original magnification 100×. Scale bar, 50 μm. (B) The number of apoptotic cells in the lung quantified from TUNEL staining. Data are expressed as mean ± SE (n = 6–8 per group). *P < 0.05 versus sham and ^#P < 0.05 versus vehicle.

Figure 5

Alterations in expression of proinflammatory cytokines in the lungs. The lung tissues of sham, vehicle- and AICAR-treated mice were harvested at 20 h after CLP for measuring the mRNA levels of (A) TNF-α and (C) IL-1β by real-time RT-PCR as well as protein levels of (B) TNF-α and (D) IL-1β by ELISA. The mRNA expression in the sham group is designated as 1 for comparison. Data are expressed as mean ± SE (n = 6–8 per group). *P < 0.05 versus sham and ^#P < 0.05 versus vehicle.

Figure 6

Alterations in expression of chemokines and myeloperoxidase activity in the lungs. The lung tissues of sham, vehicle- and AICAR-treated mice were harvested at 20 h after CLP for measuring the mRNA levels of (A) KC and (B) MIP-2 by real-time RT-PCR. (C) Lung myeloperoxidase (MPO) activity was determined spectrophotometrically. The mRNA expression in the sham group is designated as 1 for comparison. Data are expressed as mean ± SE (n = 6–8 per group). *P < 0.05 versus sham and ^#P < 0.05 versus vehicle.

Figure 7

Alterations in serum levels of organ injury marker and proinflammatory cytokines in AMPKα2 KO mice. Blood of sham, vehicle- and AICAR-treated AMPKα2 KO mice were harvested at 20 h after CLP for measuring (A) AST, (B) TNF-α and (C) IL-1β. Data are expressed as mean ± SE (n = 6–8 per group). P = nonsignificant (NS).

Figure 8

Alterations in expression of proinflammatory cytokines and chemokines in the lungs of AMPKα2 KO mice. The lung tissues of sham, vehicle- and AICAR-treated AMPKα2 KO mice were harvested at 20 h after CLP for measuring (A) TNF-α, (B) IL-1β, (C) KC and (D) MIP-2 by real time RT-PCR. The expression in the sham group is designated as 1 for comparison. Data are expressed as mean ± SE (n = 6–8 per group). P = nonsignificant (NS).