Protective Effects of Dexmedetomidine on the Vascular Endothelial Barrier Function by Inhibiting Mitochondrial Fission via ER/Mitochondria Contact

Abstract

The damage of vascular endothelial barrier function induced by sepsis is critical in causing multiple organ dysfunctions. Previous studies showed that dexmedetomidine (Dex) played a vital role in protecting organ functions. However, whether Dex participates in protecting vascular leakage of sepsis and the associated underlying mechanism remains unknown yet. We used cecal ligation and puncture induced septic rats and lipopolysaccharide stimulated vascular endothelial cells (VECs) to establish models in vivo and in vitro, then the protective effects of Dex on the vascular endothelial barrier function of sepsis were observed, meanwhile, related mechanisms on regulating mitochondrial fission were further studied. The results showed that Dex could significantly reduce the permeability of pulmonary veins and mesenteric vessels, increase the expression of intercellular junction proteins, enhance the transendothelial electrical resistance and decrease the transmittance of VECs, accordingly protected organ functions and prolonged survival time in septic rats. Besides, the mitochondria of VECs were excessive division after sepsis, while Dex could significantly inhibit the mitochondrial fission and protect mitochondrial function by restoring mitochondrial morphology of VECs. Furthermore, the results showed that ER-MITO contact sites of VECs were notably increased after sepsis. Nevertheless, Dex reduced ER-MITO contact sites by regulating the polymerization of actin via α₂ receptors. The results also found that Dex could induce the phosphorylation of the dynamin-related protein 1 through down-regulating extracellular signal-regulated kinase1/2, thus playing a role in the regulation of mitochondrial division. In conclusion, Dex has a protective effect on the vascular endothelial barrier function of septic rats. The mechanism is mainly related to the regulation of Drp1 phosphorylation of VECs, inhibition of mitochondrial division by ER-MITO contacts, and protection of mitochondrial function.

Keywords: Drp1; ER-MITO contact; dexmedetomidine; sepsis; vascular endothelial barrier function.

FIGURE 1

Protective effect of dexmedetomidine on vascular permeability of septic rats. (A) Vascular permeability of the lung, measured by the leakage of Evans Blue, n = 8. (B,C) Vascular permeability of the lung, measured by the mean optical density of intravenously injected FITC-BSA in vivo (Bar, 50 μm), n = 8. (D,E) The FITC-BSA leakage of mesentery microvessels in rats, dynamically measured by intravital microscopy in vivo (Bar, 100 μm), n = 8. (F) Western blot analysis of ZO-1, Occludin and VE-cadherin in the superior mesenteric vein of rats treated with CLP, n = 3. (G) Representative transmission electron microscope microphotographs of tight junctions of pulmonary venules (Bar, 200 nm), n = 8. EC, endothelial cell; RBC, red blood cell; TJ, tight junction; L, lumen; Sham, sham group; Sep, sepsis group; CT, conventional treatment group; Dex, dexmedetomidine group. ^∗∗P < 0.01, as compared with sham group; ^##P < 0.01, as compared with CT group; ^#P < 0.05, as compared with CT group.

FIGURE 2

The influence of dexmedetomidine on the permeability of vascular endothelial cells after sepsis. (A) Effects of dexmedetomidine on the TER (transendothelial electrical resistance) of VEC monolayers after sepsis, n = 3. (B) Effects of dexmedetomidine on the infiltration rate of FITC-BSA in monolayer VECs after sepsis, n = 3. (C) Measurement of the expression of ZO-1(green) after sepsis in VECs by immunofluorescence (Bar, 25 μm), n = 3. (D–G) Western blot analysis of ZO-1, Occludin and VE-cadherin in VECs after sepsis, n = 3. Normal: nomal group; LPS: lps group; Dex: dexmedetomidine group. ^∗∗P < 0.01, as compared with normal group; ^##P < 0.01, as compared with lps group; ^#P < 0.05, as compared with lps group.

FIGURE 3

The effects of dexmedetomidine on mitochondrial fission and mitochondrial functions after sepsis. (A) TEM (transmission electronic microscopy) images to observe mitochondrial morphology of pulmonary venules in septic rats (Bar, 200 nm), n = 8. (B) Confocal images to observe mitochondrial morphology of VECs after sepsis (Bar, 25 μm), n = 50. (C,D) Time-lapse images of mitochondrial morphologic alternation of VECs per 15 s after sepsis by confocal immunostaining (Bar, 20 μm), n = 3. (E) Effects of dexmedetomidine on the ATP of VECs after sepsis, n = 3. (F–H) Representative confocal images of ROS(Bar, 100 μm) and △Ψm (Bar, 25 μm) after sepsis in VECs, n = 3. (I) Effects of dexmedetomidine on the respiratory control ratio in septic rats, n = 3. Sham: sham group; Sep: sepsis group; CT: conventional treatment group; Normal: nomal group; LPS: lps group; Dex: dexmedetomidine group. ^∗∗P < 0.01, as compared with normal group; ^##P < 0.01, as compared with lps group.

FIGURE 4

The effects of dexmedetomidine on F-actin and ER-MITO contact of vascular endothelial cells after sepsis. (A) Representative fluorescent images of ER-MITO contact transformed with ERtracker and Mitotracker in VECs after sepsis (Bar, 5 μm), n = 3. The sites of mitochondrial fission (yellow arrows) correspond to black arrows of contact sites on the line scan. (B) Confocal images to observe the effect of dexmedetomidine on the polymerization of actin (Bar, 25 μm), n = 3. (C) Confocal images to observe mitochondria and F-actin of VECs after sepsis(Bar, 20 μm), n = 3. (D) The statistical analysis of mitochondrial morphology of VECs in different groups. Quantitation was performed in triplicate and scored into three categories: long, middle, and short mitochondria, with 50 cells scored per group. (E) The intersections of F-actin and mitochondria of VECs in different groups were calculated by Image J software. N, nomal group; LPS, lps group; Dex, dexmedetomidine group; Ati, Atipamezole group. ^∗∗P < 0.01, as compared with normal group; ^##P < 0.01, as compared with lps group; ^@@P < 0.01, as compared with dex group.

FIGURE 5

Dexmedetomidine regulates ERK1/2-mediated mitochondrial fission. (A) Effects of dexmedetomidine on the phosphorylation of Drp1 after sepsis in VECs detected by Western Blotting, n = 3. (B) Effects of dexmedetomidine on the mitochondrial translocation of Drp1 after sepsis in VECs, n = 3. (C,D) The co-location of Drp1 and mitochondria of VECs in each group (Bar, 25 μm), n = 3. (E) The length of mitochondria of VECs in each group. (F) Drp1 protein phosphorylation network (PPN) predicted by iGPS 1.0 software. (G) Effects of dexmedetomidine on the phosphorylation of ERK1/2 after sepsis in VECs, n = 3. (H) Effects of dexmedetomidine and ERK1/2 inhibitor on the phosphorylation of ERK1/2 after sepsis in VECs, n = 3. (I) Mitochondrial morphology in PD98059-treated VECs after sepsis (Bar, 15 μm), n = 3. (J,K) Effects of PD98059 on the TER and FITC-BSA infiltration rate of VEC monolayers after sepsis, n = 3. Normal, normal group; LPS, lps group; Dex, dexmedetomidine group. PD98059: ERK1/2 inhibitor group. ^∗∗P< 0.01, as compared with normal group; ^##P < 0.01, as compared with lps group.

FIGURE 6

The influence of dexmedetomidine on the survival rate, survival time and organ function of septic rats. (A–F) Statistical histogram of pH, PaO₂, PaCO₂, ALT, CK-MB, and Crea levels in each group, n = 8. (G) Survival rate and survival time, n = 16. (H) Effects of dexmedetomidine on the phosphorylation 616 site of Drp1 and ERK1/2 in septic rats detected by Western Blotting, n = 3. Sham, sham group; Sep, sepsis group; CT, conventional treatment group; Dex. dexmedetomidine group. ^∗∗P< 0.01, as compared with sham group; ^##P < 0.01, as compared with CT group; ^#P < 0.05, as compared with CT group.

FIGURE 7

Schematic diagram of dexmedetomidine regulating mitochondrial fission pathways after sepsis. Dexmedetomidine could inhibite actin polymerization of VECs, leading to the decrease of ER-MITO contact sites. Meanwhile, it also down-regulated the phosphorylation of ERK1/2 and Drp1 ser616, leading to the decrease of the mitochondrial translocation of Drp1 after sepsis in VECs.