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. 2021 Apr 30:8:655619.
doi: 10.3389/fmolb.2021.655619. eCollection 2021.

Ischemic Postconditioning-Mediated DJ-1 Activation Mitigate Intestinal Mucosa Injury Induced by Myocardial Ischemia Reperfusion in Rats Through Keap1/Nrf2 Pathway

Rong Chen  1 , Wei Li  1 , Zhen Qiu  1 , Qin Zhou  1 , Yuan Zhang  1 , Wen-Yuan Li  1 , Ke Ding  1 , Qing-Tao Meng  1 , Zhong-Yuan Xia  1
Affiliations

Ischemic Postconditioning-Mediated DJ-1 Activation Mitigate Intestinal Mucosa Injury Induced by Myocardial Ischemia Reperfusion in Rats Through Keap1/Nrf2 Pathway

Rong Chen et al. Front Mol Biosci. .

Abstract

Intestinal mucosal barrier dysfunction induced by myocardial ischemia reperfusion (IR) injury often leads to adverse cardiovascular outcomes after myocardial infarction. Early detection and prevention of remote intestinal injury following myocardial IR may help to estimate and improve prognosis after acute myocardial infarction (AMI). This study investigated the protective effect of myocardial ischemic postconditioning (IPo) on intestinal barrier injury induced by myocardial IR and the underlying cellular signaling mechanisms with a focus on the DJ-1. Adult SD rats were subjected to unilateral myocardial IR with or without ischemic postconditioning. After 30 min of ischemia and 120 min of reperfusion, heart tissue, intestine, and blood were collected for subsequent examination. The outcome measures were (i) intestinal histopathology, (ii) intestinal barrier function and inflammatory responses, (iii) apoptosis and oxidative stress, and (iv) cellular signaling changes. IPo significantly attenuated intestinal injury induced by myocardial IR. Furthermore, IPo significantly increased DJ-1, nuclear Nrf2, NQO1, and HO-1 expression in the intestine and inhibited IR-induced apoptosis and oxidative stress. The protective effect of IPo was abolished by the knockdown of DJ-1. Conversely, the overexpression of DJ-1 provided a protective effect similar to that of IPo. Our data indicate that IPo protects the intestine against myocardial IR, which is likely mediated by the upregulation of DJ-1/Nrf2 pathway.

Keywords: DJ-1; intestinal barrier injury; ischemic postconditioning; myocardial ischemia reperfusion; nuclear factor (erythroid-derived 2)-like 2.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
The schematic illustration of animal grouping and the experimental protocols. Animals were randomly treated with a mock treatment (control), AAV9-control, AAV9-DJ-1(+), or AAV9-DJ-1(–) before the establishment of the IR model. The AAV9-control group was subjected to only the sham operation as described in the figure, and then, the animals in the remaining groups, including the animals treated with a mock treatment, AAV9-DJ-1(+), or AAV9-DJ-1(–) were randomly allocated into three subgroups: (1) sham (S), (2) IR, and (3) IPo. A total of 60 rats in 10 groups were used in the experiments. The groupings and treatments (n = 6/group) are shown in the figure.
FIGURE 2
FIGURE 2
Ischemic postconditioning alleviated myocardial IR-induced myocardial and intestinal injury. (A) Infarct area relative to the area at risk (IA/AAR × 100%). (B) Serum CK-MB level. (C) Intestinal water W/D ratios. (D) Histopathologic changes of the small-intestinal mucosa under light microscopy imaging (H&E staining, Scale bar = 300 μm). Intestinal mucosa injury was graded by Chiu’s score. Serum concentrations of D-LA (E) and I-FABP (F). (G) Expression of occludin. The levels of inflammatory cytokines (IL-1β, IL-10, and TNF-α) in the serum (H) and intestine (I). β-actin were detected as loading control. S, Sham; IR, ischemia reperfusion; IPo, ischemic postconditioning. Data are mean ± SD or Box-Whisker’s plot (n = 6); *P < 0.05.
FIGURE 3
FIGURE 3
Ischemic postconditioning ameliorates myocardial IR-induced apoptosis and suppresses oxidative stress. (A) The TUNEL staining for cell apoptosis rates. (B) Expression of apoptosis-related proteins Bax, Bcl-2, and caspase-3. (C) SOD. (D) MDA. (E) GSH/GSSG ratio. β-actin was detected as loading control, and β-action gel is the same as in Figure 2. S, Sham; IR, ischemia reperfusion; IPo, ischemic postconditioning. Data are mean ± SD (n = 6); *P < 0.05.
FIGURE 4
FIGURE 4
Ischemic postconditioning activates DJ-1, induces Nrf2 nuclear translocation, and upregulates HO-1 and NQO1 expression. (A) Expression of DJ-1, Keap-1, Cytosolic Nrf2. (B) Expression of Nrf2 in the nucleus and (C) of its downstream targets HO-1 and NQO1. Lamin B1 and β-actin were detected as loading controls, respectively, and β-action gel is the same as in Figure 2. S, Sham; IR, ischemia reperfusion; IPo, ischemic postconditioning. Data are presented as the mean ± SD (n = 6). *P < 0.05.
FIGURE 5
FIGURE 5
Overexpression of DJ-1 promoted IPo-induced protection against myocardial IR-induced intestinal injury. (A,B) Expression of DJ-1 level. (C) Infarct area relative to the area at risk (IA/AAR × 100%). (D) serum CK-MB level. (E) Intestinal water W/D ratios. (F) Histopathologic changes of the small-intestinal mucosa under light microscopy imaging (H&E staining, Scale bar = 300 μm). Intestinal mucosa injury was graded by Chiu’s score. Serum concentrations of D-LA (G) and I-FABP (H). (I) Expression of occludin. The levels of inflammatory cytokines (IL-1β, IL-10, and TNF-α) in the serum (J) and intestine (K). β-actin was detected as loading control. S, Sham; IR, ischemia reperfusion; IPo, ischemic postconditioning. Data are mean ± SD or Box-Whisker’s plot (n = 6); *P < 0.05.
FIGURE 6
FIGURE 6
Overexpression of DJ-1 promoted IPo-induced protection via ameliorated myocardial IR-induced apoptosis and suppressed oxidative stress by regulating Nrf2 pathway. (A) The TUNEL staining for cell apoptosis rates. (B) Expression of apoptosis-related proteins Bax, Bcl-2, and caspase-3. (C) SOD. (D) MDA. (E) GSH/GSSG ratio. (F) Expression of Keap-1 and Nrf2 in the cytosolic and nucleus, (G) Expression of HO-1 and NQO1. Lamin B1 and β-actin were detected as loading controls, respectively, and β-actin gel is the same as in Figure 5. S, Sham; IR, ischemia reperfusion; IPo, ischemic postconditioning. Data are presented as the mean ± SD (n = 6). *P < 0.05.
FIGURE 7
FIGURE 7
Knockdown of DJ-1 abolished IPo induced protection against myocardial IR-induced intestinal injury. (A,B) Expression of DJ-1 level. (C) Infarct area relative to the area at risk (IA/AAR × 100%). (D) serum CK-MB level. (E) Intestinal water W/D ratios. (F) Histopathologic changes of the small-intestinal mucosa under light microscopy imaging (H&E staining, Scale bar = 300 μm). Intestinal mucosa injury was graded by Chiu’s score. Serum concentrations of D-LA (G) and I-FABP (H). (I) Expression of occludin. The levels of inflammatory cytokines (IL-1β, IL-10, and TNF-α) in the serum (J) and intestine (K). β-actin was detected as loading control. S, Sham; IR, ischemia reperfusion; IPo, ischemic postconditioning. Data are mean ± SD or Box-Whisker’s plot (n = 6); *P < 0.05.
FIGURE 8
FIGURE 8
Knockdown of DJ-1 abrogated IPo-induced protection via ameliorated myocardial IR-induced apoptosis and suppressed oxidative stress by regulating Nrf2 pathway. (A) The TUNEL staining for cell apoptosis rates. (B) Expression of apoptosis-related proteins Bax, Bcl-2 and Caspase-3. (C) SOD. (D) MDA. (E) GSH/GSSG ratio. (F) Expression of Keap-1, Nrf2 in the cytosolic and nucleus, (G) Expression of HO-1 and NQO1. Lamin B1, β-action were detected as loading controls, respectively, and β-action gel is the same as in Figure 7. S, Sham; IR, ischemia reperfusion; IPo, ischemic postconditioning. Data are presented as the mean ± SD (n = 6). *P < 0.05.
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