Dexmedetomidine ameliorates endotoxin-induced acute lung injury in vivo and in vitro by preserving mitochondrial dynamic equilibrium through the HIF-1a/HO-1 signaling pathway

Abstract

Increasing lines of evidence identified that dexmedetomidine (DEX) exerted protective effects against sepsis-stimulated acute lung injury via anti-inflammation, anti-oxidation and anti-apoptosis. However, the mechanisms remain unclear. Herein, we investigated whether DEX afforded lung protection by regulating the process of mitochondrial dynamics through the HIF-1a/HO-1 pathway in vivo and in vitro. Using C57BL/6J mice exposed to lipopolysaccharide, it was initially observed that preemptive administration of DEX (50μg/kg) alleviated lung pathologic injury, reduced oxidative stress indices (OSI), improved mitochondrial dysfunction, upregulated the expression of HIF-1α and HO-1, accompanied by shifting the dynamic course of mitochondria into fusion. Moreover, HO-1-knockout mice or HO-1 siRNA transfected NR8383 cells were pretreated with HIF-1α stabilizer DMOG and DEX to validate the effect of HIF-1a/HO-1 pathway on DEX-mediated mitochondrial dynamics in a model of endotoxin-induced lung injury. We found that pretreatment with DEX and DMOG distinctly relieved lung injury, decreased the levels of mitochondrial ROS and mtDNA, reduced OSI, increased nuclear accumulation of HIF-1a and HO-1 protein in wild type mice but not HO-1 KO mice. Similar observations were recapitulated in NC siRNA transfected NR8383 cells after LPS stimulation but not HO-1 siRNA transfected cells. Concertedly, DEX reversed the impaired mitochondrial morphology in LPS stimulated-wild type mice or NC siRNA transfected NR8383 cells, upregulated the expression of mitochondrial fusion protein, while downregulated the expression of fission protein in HIF-1a/HO-1 dependent pathway. Altogether, our data both in vivo and in vitro certified that DEX treatment ameliorated endotoxin-induced acute lung injury by preserving the dynamic equilibrium of mitochondrial fusion/fission through the regulation of HIF-1a/HO-1 signaling pathway.

Keywords: Acute lung injury; Dexmedetomidine; Endotoxin; Heme oxygenase-1; Hypoxia-inducible factor 1; Mitochondrial dynamics.

Fig. 1

Pretreatment with DEX protected the lung against LPS-driven oxidative injury in mice. A, Photomicrographs of histopathologic changes of lung sections stained with hematoxylin and eosin at 12h after LPS challenge (original magnification, ×200). Scale bar: 100μm. B, Semiquantitative analysis of lung tissues by lung injury scores. Values are expressed as medians (range) using the Mann-Whitney U test (n = 6 mice/group). C-E, Serum levels of total oxidant status (TOS), total antioxidant status (TAS) and oxidative stress index (OSI) calculated as TOS/TAS ratio. Data were represented as mean ± SD in bar graph C, D and E. Analysis of variance (ANOVA), Bonferroni multiple comparison test, n = 6 per group. Significant differences were indicated with an asterisk: *P < 0.05, **P < 0.01, ***P < 0.001. DEX, dexmedetomidine; LPS, lipopolysaccharide; N·S., not significant; TOS, total oxidant status; TAS, total antioxidant status; OSI, oxidative stress index.

Fig. 2

Pretreatment with DEX mitigated LPS-induced mitochondrial oxidative damage in the lung tissue of mice. A, Mitochondrial ROS production detected spectro-fluorometrically. using DCFH-DA as a fluorescent dye. B, Mitochondrial DNA (mtDNA) content using real time PCR. C, The morphological alterations of mitochondria by transmission lectron microscopy (original magnification, ×5000). Scale bar: 2μm. Red arrows denoted deformed mitochondria manifested as isolated, small, rounded mitochondria with loss of clearly defined cristae. White arrows relatively marked normal shape of mitochondria in the DEX alone group. Data were. representative of mean ± SD in bar graph A, B (n = 6), *P < 0.05, **P < 0.01, ***P < 0.001. one-way ANOVA corrected with Bonferroni coefficient. DEX, dexmedetomidine; ROS, reactive oxygen species; LPS, lipopolysaccharide; DCFH-DA, 2’,7’-dichlorofluorescein diacetate. . (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig.3

Pretreatment with DEX reinforced mitochondrial fusion while restrained mitochondrial fission in the context of septic lung injury in mice. A and B, Representative western blots of mitochondrial fusion (Mfn1, Mfn2, OPA1) and fission (Drp1 and Fis1) markers shown on right and quantitation shown on left. Analyses of band intensity on the western blotting images were presented as their relative ratios compared with β-actin. C, Expressions of the mRNA levels of mitochondrial fusion/fission markers detected by real-time PCR. Values were presented as mean ± SD of six separate experiments using one-way ANOVA and the Bonferroni test for multiple comparisons. *Significant difference from sham group (P< 0.05). ^#Significant difference from LPS-exposed mice (P < 0.05). DEX, dexmedetomidine; LPS, lipopolysaccharide; Mfn1, mitofusin 1; Mfn2, mitofusin 2; OPA1, optic atrophy 1; Drp1, dynamin-related protein 1; Fis1, fission 1.

Fig. 4

HIF-1a/HO-1 pathway mediated the regulation of DEX on mitochondrial dynamics following LPS challenge. A, Immunofluorescence assays of HO-1 and HIF-1a protein by fluorescence microscope (original magnification, ×400). Green stood for FITC-HO-1 stained sections, red stood for TRITC-HIF-1a stained sections, while blue stood for images of DAPI- stained nuclei. HO-1 was found to colocalize with HIF-1a in the nucleus of the lung tissue section. Scale bar: 10μm. B, Quantification and example bands from western blotting to assess the expressions of HO-1 and HIF-1a protein in the lung tissue of mice. β-actin served as a standard for protein loading. C, The expressions of mRNA of HO-1 and HIF-1a detected by real-time PCR in in the lung tissue of mice. Values were expressed as mean ± SD of six individual samples using one-way ANOVA corrected with Bonferroni coefficient for multiple comparisons. *Significant difference from sham group (P< 0.05). ^#Significant difference from LPS-treated mice (P < 0.05). DEX, dexmedetomidine; LPS, lipopolysaccharide; HO-1, heme oxygenase-1; HIF-1α, hypoxia inducible factor-1α. . (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

HO-1 knockout counteracted the effects of DEX-mediated lung protection in mice subjected to LPS. A, The histopathologic changes of lung sections stained with hematoxylin and eosin following 50mg/kg DMOG intraperitoneally exposed 0.5h before DEX pretreatment for 0.5h prior to LPS challenge (original magnification, ×200). Scale bar: 50μm. B, Semiquantitative analysis of lung tissues by lung injury scores. Values are expressed as medians (range) using the Mann–Whitney U test (n = 6 mice/group). C-E, Serum levels of TOS, TAS and OSI. F, The morphological alterations of mitochondria by transmission electron microscopy (original magnification, ×7800). Scale bar: 500nm. The right panels (original magnification, ×19000) were enlarged transmission electron microscopy images of mitochondria in the left panels. Scale bar: 200nm. Red arrows marked as isolated, small, rounded mitochondria with loss of clearly defined crista. G, Mitochondrial ROS generation. H, Mitochondrial DNA contents. Data were represented as mean ± SD in bar graph C, D, E, G, H, and analyzed by paired T-test (n = 6 per group). Significant differences were indicated with an asterisk: *P < 0.05, **P < 0.01, ***P < 0.001. DEX, dexmedetomidine; LPS, lipopolysaccharide; HO-1, heme oxygenase-1; DMOG, dimethyloxalylglycine; TOS, total oxidant status; TAS, total antioxidant status; OSI, oxidative stress index; ROS, reactive oxygen species; mtDNA, Mitochondrial DNA. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6

HO-1 knockout reversed the effects of DEX-afforded preservation of mitochondrial dynamics in mice septic lung injury. A-B, Quantification and example bands from Western blot analysis to determine the expression levels of Mfn1, Mfn2, OPA1, Drp1, Fis1 protein required for mitochondrial fusion/fission, as well as HO-1 and HIF-1a protein in the lung tissue of mice subjected to DEX and DMOG pretreatment. Band density was quantified relatively to β-actin. C, Representative mRNA levels of Mfn1, Mfn2, OPA1, Drp1, Fis1, HO-1 and HIF-1a expression assessed by real-time PCR in the lung tissue of DEX and DMOG-pretreated mice. Values representative of mean ± SD obtained from six independent experiments using paired T-test. *P < 0.05 versus DEX + DMOG + LPS-treated wild type mice. DEX, dexmedetomidine; LPS, lipopolysaccharide; HO-1, heme oxygenase-1; DMOG, dimethyloxalylglycine; HIF-1α, hypoxia inducible factor-1α; Mfn1, mitofusin 1; Mfn2, mitofusin 2; OPA1, optic atrophy 1; Drp1, dynamin-related protein 1; Fis1, fission 1.

Fig. 7

HO-1 knockdown offset the effects of DEX-mitigated LPS-induced oxidative injury in NR8383 alveolar macrophages. A, The viability of NR8383 cells preincubated with different concentrations of DEX prior to LPS conducted by CCK8 assay. Data were analyzed using the Mann-Whitney U test followed by Bonferroni correction for six individual experiments. *Significant difference from sham group (P<0.05). #Significant difference from LPS-incubated cells (P<0.05). B-D, The levels of TOS, TAS and OSI to reflect the oxidative status of LPS-primed NR8383 cells pretreated with 100μmol/L DMOG 1h before 10μmol/L DEX for 2h. E, Mitochondrial ROS production in cells. F, mtDNA contents in cells. Data in B, C, D, E and F were presented as mean ± SD using paired T-test (n = 6 replicates for each group). Significant differences were indicated with an asterisk: *P < 0.05, **P < 0.01, ***P < 0.001. DEX, dexmedetomidine; LPS, lipopolysaccharide; HO-1, heme oxygenase-1; DMOG, dimethyloxalylglycine; TOS, total oxidant status; TAS, total antioxidant status; OSI, oxidative stress index; ROS, reactive oxygen species; mtDNA, Mitochondrial DNA.

Fig. 8

HO-1 knockdown abrogated the effects of DEX-regulated mitochondrial dynamics in in NR8383 alveolar macrophages exposed to LPS. A, Expression of HO-1 and HIF-1a in LPS-cultured NR8383 cells preincubated with DMOG and DEX detected by immunofluorescence assay. Green stood for HO-1 or HIF-1a-FITC stained sections, and nuclear was counter stained with DAPI. The overlay color of blue staining in nucleus, along with green staining both in cytoplasm or nucleus was considered to be positive. The right panels were enlarged images of the orange boxed areas in the panels of merge. Six representative fields of each slice were randomly selected from one of three individual experiments. B–C, Fluorescence intensity of HO-1 and HIF-1a quantified by Image J version1.49r software. The proportion of positive cells were calculated as the number of positive cells relative to the number of DAPI-positive cells. D-E, Representative western blots of mitochondrial fusion/fission markers including Mfn1, Mfn2, OPA1, Drp1 and Fis1 as well as HO-1 and HIF-1a protein shown on right and quantitation shown on left. β-actin was served as a loading control. F, Representative mRNA levels of Mfn1, Mfn2, OPA1, Drp1, Fis1, HO-1 and HIF-1a expression assessed by real-time PCR in LPS-primed NR8383 cells pretreated with DMOG and DEX. Values were expressed as mean ± SD in bar graph B, C, E, D and F. Paired T-test was used to detect statistically significant changes, n = 6/per group. Significant differences from DEX + DMOG + LPS-treated NC siRNA-transfected NR8383 cells were shown by (*) (**) and (***) which respectively indicated the P<0.05, P<0.01 and P<0.001 levels. DEX, dexmedetomidine; LPS, lipopolysaccharide; HO-1, heme oxygenase-1; DMOG, dimethyloxalylglycine; HIF-1α, hypoxia inducible factor-1α; Mfn1, mitofusin 1; Mfn2, mitofusin 2; OPA1, optic atrophy 1; Drp1, dynamin-related protein 1; Fis1, fission 1. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 9

Graphical illustration of proposed DEX-mediated preservation of mitochondrial fusion and fission involved in regulating HIF-1a/HO-1 pathway. When LPS was administrated to C57BL/6J mice or NR8383 alveolar macrophages, DEX activated HIF-1a/HO-1 pathway, led to HIF-1a translocated into nuclear, heterodimerization with HIF-1β, which binds to hypoxia-response elements (HREs) to induce target gene of HO-1 expression. Meanwhile, pretreatment with DEX upregulated the expressions of mitochondrial fusion proteins Mfn1, Mfn2, OPA1, yet downregulated the expressions of mitochondrial fusion proteins Drp1, Fis1, accompanied with improved mitochondrial morphology and dysfunction in HIF-1a/HO-1 dependent pathway. Of note, DEX as a specific agonist of α₂-adrenoceptor exerted efficacy of decreased sympathetic tone while increased vagal tone, thus, further studies were needed to validate the upstream mechanism of HIF-1a/HO-1pathway that DEX aimed at, and the relationship between HIF-1a/HO-1pathway and sympathetic or vagal tone in this area of research.