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. 2018 Mar 23;16(1):78.
doi: 10.1186/s12967-018-1455-1.

Dexmedetomidine attenuates lung apoptosis induced by renal ischemia-reperfusion injury through α2AR/PI3K/Akt pathway

Juanjuan Li  1 , Qian Chen  1 , Xinhai He  1 , Azeem Alam  2 , Jiaolin Ning  1 , Bin Yi  1 , Kaizhi Lu  1 , Jianteng Gu  3
Affiliations

Dexmedetomidine attenuates lung apoptosis induced by renal ischemia-reperfusion injury through α2AR/PI3K/Akt pathway

Juanjuan Li et al. J Transl Med. .

Abstract

Background: Acute lung injury caused by renal ischemia-reperfusion is one of the leading causes of acute kidney injury-related death. Dexmedetomidine, an α2-adrenergic agonist sedative, has been found to have protective effects against acute kidney injury and remote lung injury. We sought to determine whether dexmedetomidine can exert its anti-apoptotic effects in acute lung injury after acute kidney injury, in addition to its common anti-inflammatory effects, and to determine the underlying mechanisms.

Methods: In vivo, acute kidney injury was induced by 60 min of kidney ischemia (bilateral occlusion of renal pedicles) followed by 24 h of reperfusion. Mice received dexmedetomidine (25 µg/kg, i.p.) in the absence or presence of α2-adrenergic antagonist atipamezole (250 µg/kg, i.p.) before IR. Histological assessment of the lung was conducted by HE staining and arterial blood gases were measured. Lung apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay. The expression of caspase 3 and p-Akt in lung tissue was detected by western blot. In vitro, C57BL/6J mice pulmonary microvascular endothelial cells were treated with serum from mice obtained following sham or IR. Dexmedetomidine was given before serum stimulation in cells, alone or with atipamezole or LY294002. Cell viability was assessed by CCK 8 assay. Cell apoptosis was examined by Hoechst staining and Annexin V-FITC/PI staining flow cytometry analysis. Mitochondrial membrane potential was measured by flow cytometry. The expression of p-Akt, caspase 3, Bcl-2 and Bax was measured by western blot.

Results: In vivo, dexmedetomidine remarkably mitigated pathohistological changes and apoptosis and significantly increased p-Akt expression in the lung. In addition, dexmedetomidine also slightly improved oxygenation in mice after IR, which can be abolished by atipamezole. In vitro, dexmedetomidine significantly inhibited IR serum-induced loss of viability and apoptosis in PMVECs. Dexmedetomidine increased p-Akt in a time- and dose-dependent manner, and down-regulated the expression of caspase 3 and Bax and up-regulated the Bcl-2 expression in PMVECs. The changes of MMP were also improved by dexmedetomidine. Whilst these effects were abolished by Atipamezole or LY294002.

Conclusion: Our results demonstrated that dexmedetomidine attenuates lung apoptosis induced by IR, at least in part, via α2AR/PI3K/Akt pathway.

Keywords: Acute lung injury; Akt pathway; Apoptosis; Renal ischemia–reperfusion; α2-Adrenoceptor.

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Figures

Fig. 1
Fig. 1
Dexmedetomidine preserved lung architecture in renal IR—induced lung injury. C57B/6J mice were pre-treated with dexmedetomidine (Dex) alone or in combination with α2-adrenoceptor antagonist atipamezole (Atip) followed by clamping of the bilateral renal pedicle for 60 min and reperfusion for 24 h. Sham animals were used as control. Representative photomicrographs of pulmonary histology (a) and lung injury scores (b) were evaluated under different conditions. Bar represents a length of 500 μm on histology. Data are shown as mean ± SD. n = 6 per group, **p < 0.01 versus sham group, ##p < 0.01 versus IR group
Fig. 2
Fig. 2
Dexmedetomidine inhibited lung apoptosis induced by IR in mice. C57B/6J mice were pre-treated with dexmedetomidine (Dex) alone or in combination with α2-adrenoceptor antagonist atipamezole (Atip) followed by clamping of the bilateral renal pedicle for 60 min and reperfusion for 24 h. a Apoptosis in the lungs was assessed by a TUNEL assay. b Quantitative analysis of apoptotic cells in kidneys. c Representative western blotting results and quantitative analysis of cleaved caspase-3 in lung tissues. The scale bars in all images represent 50 μm. All data are expressed as the mean ± SD (n = 6). **p < 0.01 versus sham group, #p < 0.05 versus IR group. HPF means high-power field
Fig. 3
Fig. 3
Dexmedetomidine inhibited mice IR serum-induced loss of viability in PMVECs. a Cell viability of PMVECs treated with different concentrations (10, 15, 20, 25%) of sham or IR serum. b Cell viability of PMVECs treated with different concentrations (0.1, 1, 10 μM) of Dexmedetomidine. c Cells were treated with 0.1–10 μM DEX 1 h before receiving 15% IR serum stimulation. The 15% sham serum stimulated group were used as control. **p < 0.01 versus Control group, ##p < 0.01 versus IR group
Fig. 4
Fig. 4
Dexmedetomidine inhibits IR serum-Induced apoptosis of PMVECs. Cells were treated with 0.1–10 μM DEX 30 min before receiving 15% IR serum stimulation. a Hoechst 33258 staining in PMVECs. b Cell apoptosis measured by flow cytometry. c The percentage of apoptotic cells. The 15% sham serum stimulated group was used as control. Bar represents a length of 50 μm. Data are shown as mean ± SD. **p < 0.01 versus sham group, ##p < 0.01 versus IR group
Fig. 5
Fig. 5
Effects of dexmedetomidine on the expression of p-Akt in vivo and vitro. a Representative western blotting results and quantitative analysis of p-Akt in lung tissues. b Effects of dexmedetomidine (0.1 μM) on the expression of p-Akt in PMVECs in different time (5, 10, 20, 40 min). c Effects of different concentrations (0.1, 1, 10 μM) of dexmedetomidine on the expression of p-Akt in PMVECs. **p < 0.01 versus control group, #p < 0.05 versus IR group, ##p < 0.01 versus IR group
Fig. 6
Fig. 6
The α2AR/PI3K/Akt signaling pathway may be involved in the PMVECs protection of Dex. PMVECs were pretreated with Atipamezole or the PI3K inhibitor LY-294002 30 min before 0.1 μM dexmedetomidine treatment for 20 min, followed by 15% IR serum stimuli for 24 h. The 15% sham serum stimulated group was used as the control. a The cleaved caspase in PMVECs was detected by western blot. b Cells were stained with JC-1 and mitochondrial membrane potential was determined by flow cytometry. c The expression of Bcl-2 and Bax in PMVECs. **p < 0.01 versus control group, #p < 0.05 versus IR group, ##p < 0.01 versus IR group
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