The mitochondrially targeted antioxidant MitoQ protects the intestinal barrier by ameliorating mitochondrial DNA damage via the Nrf2/ARE signaling pathway

Abstract

Disruption of the mucosal barrier following intestinal ischemia reperfusion (I/R) is life threatening in clinical practice. Mitochondrial dysfunction and oxidative stress significantly contribute to the early phase of I/R injury and amplify the inflammatory response. MitoQ is a mitochondrially targeted antioxidant that exerts protective effects following I/R injury. In the present study, we aimed to determine whether and how MitoQ protects intestinal epithelial cells (IECs) from I/R injury. In both in vivo and in vitro studies, we found that MitoQ pretreatment downregulated I/R-induced oxidative stress and stabilized the intestinal barrier, as evidenced by MitoQ-treated I/R mice exhibiting attenuated intestinal hyperpermeability, inflammatory response, epithelial apoptosis, and tight junction damage compared to controls. Mechanistically, I/R elevated mitochondrial 8-hydroxyguanine content, reduced mitochondrial DNA (mtDNA) copy number and mRNA transcription levels, and induced mitochondrial disruption in IECs. However, MitoQ pretreatment dramatically inhibited these deleterious effects. mtDNA depletion alone was sufficient to induce apoptosis and mitochondrial dysfunction of IECs. Mitochondrial transcription factor A (TFAM), a key activator of mitochondrial transcription, was significantly reduced during I/R injury, a phenomenon that was prevented by MitoQ treatment. Furthermore, we observed that thee protective properties of MitoQ were affected by upregulation of cellular antioxidant genes, including HO-1, NQO-1, and γ-GCLC. Transfection with Nrf2 siRNA in IECs exposed to hypoxia/reperfusion conditions partially blocked the effects of MitoQ on mtDNA damage and mitochondrial oxidative stress. In conclusion, our data suggest that MitoQ exerts protective effect on I/R-induced intestinal barrier dysfunction.

Fig. 1. MitoQ protects against intestinal I/R injury in mice.

a Chemical structure of MitoQ. b Representative images of intestinal histology (H&E staining, original magnification ×200) and Chiu’s score of the intestine following intestinal I/R. c Levels of serum I-FABP, ALT, creatinine, and LDH. d The Kaplan–Meier survival curves compared by the log-rank test. e Cell viability in H/R-treated IEC-6 cells analyzed with CCK-8 assay. Data are expressed as the mean ± SD. ^*P < 0.05 vs sham or control group; ^#P < 0.05 vs I/R or H/R group

Fig. 2. Effects of MitoQ on intestinal permeability and bacterial translocation.

a Serum FD-40 was measured to evaluate in vivo permeability. b, c In vitro permeability of the mouse intestine measured by Ussing chamber analyses. MLN, CLN, blood, and PF were collected and cultured at 37 °C for 24 h. Culture results of bacterial growth were considered positive when more than 10² colonies/g of tissue were observed. Data are expressed as the mean ± SD. ^*P < 0.05 vs sham or control group; ^#P < 0.05 vs I/R or H/R group

Fig. 3. MitoQ restored the impaired intestinal barrier dysfunction in I/R-injured mice.

Localization of ZO-1 (a), claudin-1 (b), occludin (c), and DAPI (DNA) within intestinal tissue sections assessed by immunofluorescence at 6 h after intestinal I/R. TJ proteins (green) DAPI stain (blue), and merged TJ proteins and DAPI images are presented. d The protein levels of occludin, claudin-1, and ZO-1 in intestinal mucosa were also measured by western blot at 6 h after I/R. n = 6 mice per group

Fig. 4. MitoQ attenuates intestinal I/R injury induced enterocyte apoptosis in vivo.

a1 Representative images and a2 the apoptotic index of in situ TUNEL assay of intestinal epithelial cell apoptosis of mice. b Enterocyte apoptosis assessed by western blot of cleaved caspase-3 and cytochrome C. ^*P < 0.05 vs sham group; ^#P < 0.05 vs I/R. Values are expressed as the mean ± SD, n = 6

Fig. 5. MitoQ attenuates mtDNA damage caused by intestinal I/R injury, both in vivo and in vitro.

a Representative images of COX IV (green), 8-OHdG immunostaining (red), and DAPI (blue) in intestinal sections. The yellow in the merged images of green and red fluorescence indicates mitochondrial 8-OHdG-positive cells. b, c mtDNA copy number (b) and mtDNA transcript levels (c) in intestinal tissues analyzed by quantitative real-time PCR. mtDNA levels were normalized to the internal control GAPDH. Mitochondrial genes ND1 and COX3 were chosen to indicate mtDNA transcription. d Representative images of MitoTracker red (red) and 8-OHdG immunostaining (green) in IEC-6 cells. ^*P < 0.05 vs control; ^#P < 0.05 vs I/R. Data are expressed as the mean ± SD. mtDNA mitochondrial DNA, COX IV cytochrome c oxidase subunit IV, 8-OHdG 8-hydroxyguanine, ND1 NADH dehydrogenase subunits 1, COX3 cytochrome c oxidase subunit 3

Fig. 6. MitoQ alleviates oxidative stress and mitochondrial dysfunction in response to I/R injury.

a MitoQ effects on MDA, SOD, GSH, and GSH-Px activities in intestinal tissue after I/R (n = 6). b Effects of MitoQ (0.1, 0.5, 1.0 μM) on cellular ROS levels in IEC-6 cells. c Effects of MitoQ (0.1, 0.5, 1.0 μM) on mitochondrial ROS levels in IEC-6 cells. d Effects of MitoQ (0.1, 0.5, 1.0 μM) on membrane potential (△Ψm) in IEC-6 cells. e ATP concentration quantified using a commercially available kit and expressed as a percentage of the sham group. f Effects of MitoQ on ATP content in H/R-treated cells. Data are expressed as the mean ± SD (n = 5). ^*P < 0.05 vs sham or control group; ^#P < 0.05 vs I/R or H/R group

Fig. 7

Depletion of mtDNA by EtBr-induced mitochondrial dysfunction and IEC-6 cell damage. a mtDNA copy number and b mtDNA transcript levels in IEC-6 cells quantified by real-time PCR after treatment with vehicle (control) or EtBr at the indicated times. Mitochondrial ROS (c),△Ψm (d), ATP content (e), and cell viability (f) quantification. ^*P < 0.05 vs control. EtBr ethidium bromide

Fig. 8

Effects of MitoQ on activation of the Nrf2 antioxidant pathway. a Western blot analysis of total Nrf2, HO-1, NQO-1, and γ-GCLC in MitoQ pretreatment mouse intestine 6 h after reperfusion and MitoQ-treated IEC-6 cells subjected to H/R injury. b Immunofluorescence analysis of the effect of MitoQ preconditioning on expression of Nrf2 after intestinal I/R injury. c Immunofluorescence staining showing changes in Nrf2 fluorescence. MitoQ increases nuclear translocation of Nrf2 (endogenous) in H/R-treated IEC-6 cells. ^*P < 0.05 vs sham or control group; ^#P < 0.05 vs I/R or H/R group

Fig. 9. Protective effect of MitoQ on mtDNA damage, ROS production and apoptosis via Nrf2.

IEC-6 cells were transfected with a specific siRNA against Nrf2 or a non-silencing control. a Confocal microscopic images showing that MitoQ ameliorates mtDNA oxidative damage in IEC-6 cells subjected to H/R treatment. This effect was partially blocked by Nrf2 siRNA. mtDNA copy number in IEC-6 cells analyzed by quantitative real-time PCR (b). c, d, e) Bar graphs represent intracellular ROS, mitochondrial ROS, and apoptosis levels, as demonstrated using DCFH-DA, MitoSOX, and TUNEL assays. ^*P < 0.05 vs control group; ^#P < 0.05 vs H/R group; ^$P < 0.05 vs H/R+MitoQ group