Dexmedetomidine attenuates subarachnoid hemorrhage-induced acute lung injury through regulating autophagy and TLR/NFκB signaling pathway

Abstract

Background: Acute lung injury (ALI) is the most serious complication of subarachnoid hemorrhage (SAH). We investigated role of autophagy and inflammatory signaling pathways in lung damage and therapeutic effects of dexmedetomidine (DEX).

Methods: Fifty male Wistar rats were randomly divided into five groups: sham, SAH, SAH+ DEX5, SAH+DEX25, and SAH+DEX50. SAH was induced using endovascular perforation technique. All rats received mechanical ventilation for 60 minutes. At 2 and 24 h of SAH induction, SAH+DEX groups were treated with 5, 25, and 50 µg/kg of DEX, respectively. Histological ALI score and pulmonary edema were assessed after 48 h. Lung expression of LC3B, ATG3, p62, TLR4, TLR9, and NFκB was assessed using western blotting and quantitative PCR. Blood levels of IL-6, IL-1β, IFN-γ, and TNFα were also assessed.

Results: SAH induced ALI and pulmonary edema, which were attenuated in SAH+DEX5 (P < 0.001 for both) and SAH+DEX25 groups (P = 0.001 and P < 0.001 for ALI and edema, respectively). Lung expressions of LC3B and ATG3 were upregulated in SAH group, which was attenuated in SAH+DEX5 and SAH+DEX25 groups. Lung expressions of TLR4, TLR9, and NFκB were increased in SAH group, which was attenuated in SAH+DEX5 group. Blood IL-6 level was increased in SAH group and attenuated in SAH+DEX5 and SAH+DEX25 groups. Blood IFN-γ level was lower in SAH group than in sham group, and it was increased in SAH+DEX25 group.

Conclusions: Low-dose DEX treatment after SAH may protect against ALI by disrupting pathological brain-lung crosstalk and alleviating autophagy flux and TLR-dependent inflammatory pathways.

Keywords: Acute lung injury; Autophagy; Dexmedetomidine; Inflammation; Subarachnoid hemorrhage; Toll-like receptors..

Fig. 1.

Effects of DEX on severity of lung injury and pulmonary edema. (A) Representative histology images of lung tissue assessed by hematoxylin–eosin staining at 48 h after SAH induction. (B) A quantification of severity of lung injury assessed by modified ALI score that ranges from 0 to 4. (C) A quantification of pulmonary edema assessed by ratio of lung wet/dry weight. n = 7–10 per group and three technical replicates per each rat. Values are presented as means and SD. ^*P < 0.05 vs. sham group, ^†P < 0.05 vs. SAH group. ALI: acute lung injury, SAH: subarachnoid hemorrhage, DEX5, 25, 50: dexmedetomidine 5, 25, 50 µg/kg administered at 2 and 24 h after surgery.

Fig. 2.

Effects of DEX on expression of autophagy-regulatory factors in lung tissue. (A) Representative western blots and a quantification of protein levels of LC3B, ATG3, and p62 normalized to GAPDH. n = 7–10 per group and three technical replicates per each tissue sample. (B) Representative LC3B immunohistochemistry images. Black arrows indicate LC3B-positive cells (brown color). Values are presented as means and SD. ^*P < 0.05 vs. sham group, ^†P < 0.05 vs. SAH group. SAH: subarachnoid hemorrhage, DEX5, 25: dexmedetomidine 5, 25 µg/kg administered at 2 and 24 h after surgery.

Fig. 3.

Effects of DEX on expression of TLRs and NFκB in lung tissue. (A) Representative western blots and a quantification of protein levels of TLR4, and TLR9 normalized to GAPDH and phosphorylated NFκB normalized to NFκB. (B) A quantification of mRNA levels of TLR4 and TLR9 normalized to GAPDH assessed by quantitative PCR. n = 7–10 per group and three technical replicates per each tissue sample. Values are presented as means and SD. ^*P < 0.05 vs. sham group, ^†P < 0.05 vs. SAH group. SAH: subarachnoid hemorrhage, DEX5, 25: dexmedetomidine 5, 25 µg/kg administered at 2 and 24 h after surgery.

Fig. 4.

Effects of DEX on circulating levels of inflammatory cytokines. (A-D) A quantification of blood serum levels of IL-1β, IL-6, IFN-γ, and TNF-α. n = 7–10 per group and three technical replicates per each tissue sample. Values are presented as means and SD. ^*P < 0.05 vs. sham group, ^†P < 0.05 vs. SAH group. SAH: subarachnoid hemorrhage, DEX5, 25: dexmedetomidine 5, 25 µg/kg administered at 2 and 24 h after surgery.