Protective effect of dexmedetomidine in cecal ligation perforation-induced acute lung injury through HMGB1/RAGE pathway regulation and pyroptosis activation

Abstract

Dexmedetomidine (DEX) has been reported to attenuate cecal ligation perforation (CLP)-stimulated acute lung injury (ALI) by downregulating HMGB1 and RAGE. This study aimed to further investigate the specific mechanisms of RAGE and its potential-related mechanisms of DEX on ALI models in vitro and in vivo. The in vitro and in vivo ALI models were established by lipopolysaccharide treatment in MLE-12 cells and CLP in mice, respectively. The effect of DEX on pathological alteration was investigated by HE staining. Thereafter, the myeloperoxidase (MPO) activity and inflammatory cytokine levels were respectively detected to assess the lung injury of mice using commercial kits. The expression levels of HMGB1, RAGE, NF-κB, and pyroptosis-related molecules were detected by RT-qPCR and Western blot. HE staining showed that lung injury, increased inflammatory cell infiltration, and lung permeability was found in the ALI mice, and DEX treatment significantly attenuated lung tissue damage induced by CLP. The MPO activity and inflammatory cytokines (TNF-α, IL-1β, and NLRP3) levels were also significantly reduced after DEX treatment compared with those in the ALI mice. Moreover, DEX activated the HMGB1/RAGE/NF-κB pathway and upregulated the pyroptosis-related proteins. However, the protective DEX effect was impaired by RAGE overexpression in ALI mice and MLE-12 cells. Additionally, DEX treatment significantly suppressed HMGB1 translocation from the nucleus region to the cytoplasm, and this effect was reversed by RAGE overexpression. These findings suggested that DEX may be a useful ALI treatment, and the protective effects on ALI mice may be through the inhibition of HMGB1/RAGE/NF-κB pathway and cell pyroptosis.

Keywords: Dexmedetomidine; HMGB1; RAGE; NF-κB; acute lung injury; pyroptosis.

Figure 1.

DEX protected against lung injury in CLP-induced mice model. (a) Pathological changes of the lung samples in CLP-induced mice. Representative HE staining of lung tissue slices from sham, CLP, and different DEX doses groups. DEX-L low dose of DEX (2.5 μg/kg), DEX-M medium dose of DEX (5 μg/kg), and DEX-H high dose of DEX (10 μg/kg). Scale bars 50 µm. (b) Pathological score of the lung sample in each group. (c) The W/D weight ratio of the lung in each group. (d) MPO activity of the lung samples in mice. (e) Inflammatory cytokines level of TNF-α, IL-1β, and NLRP3 in BALF sample. The mice were i.p. injected with 2.5, 5, and 10 μg/kg of DEX 30 min after CLP procedure. Three animals in each group were sacrificed to collect lung and BALF after 24 h. N = 6. Data were expressed as mean ± SD. *P < 0.05, vs. sham; ^#P < 0.05, vs. CLP mice

Figure 2.

The viral infection efficiency after RAGE overexpression mice construction. (a) The relative mRNA expression of RAGE after infection using RT-qPCR. (b) The relative protein RAGE expression after infection by Western blot. (c) The eGFP-oeRAGE expression in the lung tissues of the RAGE overexpression mice. N = 6. Data were expressed as mean ± SD. *P < 0.05 vs. sham + vector; ^#P < 0.05 vs. CLP + vector

Figure 3.

Protective effect of DEX on the pathological manifestation of lung tissue could be impaired by injection of RAGE lentiviral. (a) Representative HE staining of lung tissue slices from sham, CLP, CLP + DEX (10 μg/kg), and CLP +DEX + RAGE overexpression (oeRAGE) group. Scale bars = 50 µm. RAGE-overexpressed mice model was constructed by i.v injection of RAGE-overexpressed lentivirus supernatants. (b) Pathological score of lung samples in each group. (c) The W/D weight ratio of the lung in each group. (d) MPO activity of the lung sample in mice. (e) Inflammatory cytokine release of TNF-α, IL-1β, and NLRP3 in BALF sample of mice. RAGE-overexpressed mice model was constructed by i.v injection of RAGE-overexpressed lentivirus supernatants. The mice were treated with DEX (10 μg/kg) 30 min after the CLP procedure. Three animals in each group were sacrificed to collect BALF samples after 24 h. = 6. Data were expressed as mean ± SD. *P < 0.05 vs. sham; ^#P < 0.05 vs. CLP mice; ^$P < 0.05, vs. CLP + DEX

Figure 4.

RT-qPCR (a) and Western blot (b) analyses of HMGB1/RAGE pathway and pyroptosis-related proteins in mice. The expression of mRNAs and proteins was normalized with a housekeeping gene GAPDH. ˆ = 6. Data were expressed as mean ± SD. *P < 0.05, vs. sham; ^# P < 0.05, vs. CLP mice; ^$P < 0.05, vs. CLP + DEX

Figure 5.

Inflammatory cytokine release in LPS-exposed MLE-12 cell (a) and immunofluorescence analysis of HMGB1 location in LPS-stimulated MLE-12 cells (b). After 24 h of LPS (1 μg/mL) exposure, the MLE-12 cells were treated with DEX (10 μM) or RAGE-overexpressed lentivirus for 18 h. Subsequently, cell supernatant was collected, and inflammatory cytokines were determined by ELISA. N = 6. Data were expressed as mean ± SD. *P < 0.05, vs. LPS; ^#P < 0.05, vs. LPS + DEX

Figure 6.

RT-qPCR (a) and Western blot (b) analyses of HMGB1/RAGE pathway and pyroptosis-related proteins in LPS-treated MLE-12 cell. N = 6. Data were expressed as mean ± SD. ^#P < 0.05, vs. LPS; ^$P < 0.05, vs. LPS + DEX