The Antioxidant MitoQ Protects Against CSE-Induced Endothelial Barrier Injury and Inflammation by Inhibiting ROS and Autophagy in Human Umbilical Vein Endothelial Cells

Abstract

Chronic obstructive pulmonary disease (COPD) is a common disease characterized by persistent airflow limitation. Pulmonary vascular endothelial barrier injury and inflammation are increasingly considered to be important pathophysiological processes in cigarette smoke extract (CSE)-induced COPD, but the mechanism remains unclear. To identify the cellular mechanism of endothelial barrier injury and inflammation in CSE-treated human umbilical vein endothelial cells (HUVECs), we investigated the effect of the mitochondrion-targeting antioxidant mitoquinone (MitoQ) on endothelial barrier injury and inflammation. We demonstrated that MitoQ restored endothelial barrier integrity by preventing VE-cadherin disassembly and actin cytoskeleton remodeling, as well as decreased inflammation by the NF-κB and NLRP3 inflammasome pathways in endothelial cells. In addition, MitoQ also maintained mitochondrial function by reducing the production of ROS and excess autophagy. Inhibition of autophagy by 3-MA protected against cytotoxicity that was induced by CSE in HUVECs. Overall, our study indicated that mitochondrial damage is a key promoter in the induction of endothelial barrier dysfunction and inflammation by CSE. The protective effect of MitoQ is related to the inhibition of ROS and excess autophagy in CSE-induced HUVEC injury.

Keywords: Autophagy; Chronic obstructive pulmonary disease; Cigarette smoke extract; Endothelial barrier; Inflammation; ROS.

Figure 1

The effect of CSE on the expression of VE-cadherin and the actin cytoskeleton in HUVECs. (A) The expression levels of the VE-cadherin and (B) F-actin in cell lysates from HUVECs were detected by Western blotting. Expression of VE-cadherin (C) and F-actin (D) in HUVECs after CSE treatment was analyzed by immunofluorescent staining. Scale bar is 10μm. Green indicates cells labeled with F-actin and VE-cadherin; blue, DAPI. All data were compared with control group and presented as mean ±SEM (n=5 in each group), *P < 0.05, **P < 0.01.

Figure 2

CSE induced NF-κB and NLRP3 inflammasome activation in HUVECs. (A) Expression of protein IκBα and p-IκBα in cell lysates from HUVECs were detected by Western blotting. GAPDH served as the standards. Expression of protein nuclear p65 was detected by cytoplasmic separation Kit. Histone was used as loading control. Quantification analysis of protein p-IκBα (B) and nuclear p65 (C) levels. (D) Nuclear translocation of p65 (red, 400×) was detected by immunofluorescent staining. Expression of protein NLRP3 (E), Caspase-1 (F) and IL-1β (G) was detected by Western blotting. All data were compared with control group and presented as mean ±SEM (n=5 in each group), *P < 0.05, **P < 0.01.

Figure 3

CSE caused mitochondrial dysfunction in HUVECs. (A) HUVECs were incubated with CSE at different concentration for 24h. The cell viability was measured by CCK8 kit. (B) Reactive oxygen species levels were analyzed by DCFH-DA kit. (C) After treatment the cells were subjected JC-1 staining (400×) for mitochondrial membrane potential assessment. Red staining indicates polarized mitochondria in JC-1 staining. Green staining indicates depolarized mitochondria in JC-1 staining. (D) Immunofluorescence staining for caspase-3 (red) and nuclei (blue) (400×). (E) Expression of protein TOMM20 in HUVECs was shown by Western blotting. GAPDH served as the standards. (F) Representative mitochondrial morphology (1000×) in HUVECs incubated for 24h with CSE. Blue: DAPI; green: TOMM20 (mitochondria). All data were compared with control group and presented as mean ±SEM (n=5 in each group), *P < 0.05, **P < 0.01.

Figure 4

The effect of MitoQ on endothelial barrier dysfunction induced by CSE. (A) Western blotting was used to detect expression of protein VE-cadherin and F-actin. Quantification analysis of protein VE-cadherin (B) and F-actin (C) levels. Expression of protein VE-cadherin (green) (D) and F-actin (green) (E) were detected by immunofluorescence. The HUVECs were grown to about 90% confluence. Scale bar is 10μm. Data are expressed as mean ± SEM (n = 5). *P < 0.05, **P < 0.01.

Figure 5

The effect of MitoQ in CSE-induced NF-κB and NLRP3 inflammasome activation in HUVECs. (A) Expression of protein IκBα, p-IκBα and nuclear p65 was detected by Western blotting. GAPDH and histone as the standards. Quantification analysis of p-IκBα (B) and nuclear p65(C) levels. (D) Immunofluorescence staining of p65 (red) using confocal microscopy (400×). Expression of protein NLRP3 (E), Caspase-1 (F) and IL-1β (G) was detected by Western blotting. Data are expressed as mean ± SEM (n = 5). *P < 0.05, **P < 0.01.

Figure 6

MitoQ ameliorated mitochondrial damage induced by CSE in HUVECs. HUVECs were stimulated by 5% CSE and (or) 100nM MitoQ for 24h. (A) Cell viability was measured by CCK8 kit. (B) Reactive oxygen species levels were analyzed by DCFH-DA kit. (C) Immunofluorescence staining (400×) for caspase-3 (red) and nuclei (blue). (D) Mitochondrial membrane potential in HUVECs was analyzed by JC-1 staining (400×). (E) Expression of protein TOMM20 was detected by Western blotting. (F) Expression of TOMM20 was detected by immunofluorescence staining (1000×). Data are expressed as mean ± SEM (n = 5). *P < 0.05, **P < 0.01.

Figure 7

MitoQ inhibited CSE-induced autophagy in HUVECs. (A) Expression of LC3 and Beclin-1 were detected by Western blotting and quantification analysis. (B) Expression of protein P62 was detected by Western blotting. (C) Cell viability in HUVECs treated with CSE and (or) 3-MA was measured by CCK8 kit. (D) Expression of LC3 was detected by Western blotting and quantification analysis. Data are expressed as mean ± SEM (n = 5). *P < 0.05, **P < 0.01.