Ischemic Postconditioning Protects Against Intestinal Ischemia/Reperfusion Injury via the HIF-1α/miR-21 Axis

Intestinal ischemia/reperfusion (I/R) can lead to tissue damage associated with inflammation and mucosal apoptosis. Ischemic postconditioning (IPostC), a series of repeated, brief, intermittent periods of ischemia and reperfusion, has beneficial effects against I/R-induced injury in the heart and intestine, although the underlying mechanisms for these effects remain unclear. We evaluated the involvement of microRNA-21 (miR-21) in the protective effects of IPostC in a rat model of I/R induced by superior mesenteric artery occlusion and reopening. IPostC decreased I/R injury and suppressed apoptosis in the intestinal tissues concomitant with the induction of hypoxia inducible factor 1 alpha (HIF-1α) and the upregulation of miR-21. In vitro experiments in the intestinal epithelial cell line IEC-6 showed that hypoxia induced miR-21 and this effect was abolished by silencing HIF1-α, confirming the induction of miR-21 by HIF1-α, HIF1-α or miR-21 inhibition exacerbated I/R induced apoptosis, and programmed cell death 4 (PDCD4) and Fas-L was involved in miR-21 mediated anti-apoptotic effects on intestinal epithelial cells. Knockdown of miR-21 or inhibition of HIF1-α abolished the IPostC-mediated attenuation of intestinal injury and apoptosis and the downregulation of PDCD4 and Fas-L. A potential mechanism underlying the protective effect of IPostC may therefore involve the induction of miR-21 by HIF1-α and the attenuation of apoptosis via the downregulation of PDCD4 and Fas-L.

modulation of the PTEN/Akt signaling pathway 6 . Additionally, IPostC-mediated regulation of miR-1 and miR-21 have been found to attenuate apoptosis in patients undergoing valvular heart surgery 12 .
Hypoxia inducible factor 1 (HIF-1) is a transcription factor that is expressed under conditions of hypoxia and activates genes related to angiogenesis, glycolysis, vascular tone regulation, and erythropoiesis 13 . HIF-1α upregulation occurs in certain organs after exposure to hypoxia and is an early response to myocardial ischemia in humans, leading to angiogenesis and improving myocardial cell survival 14 . The expression of HIF-1α was significantly increased in the ischemic intestinal mucosa 15 . We also found that the expression of HIF-1α increased further after Ischemic postconditioning. However, little is known as to how the role of HIF-1α and miR-21 in IPostC protects against intestinal ischemia/reperfusion injury.
In the present study, we examined the involvement of HIF-1α and miR-21 in the progression of intestinal I/R in a rat model and explored the molecular mechanisms underlying the protective effects of IPostC after intestinal I/R.

IPostC attenuated intestinal I/R injury and was associated with the upregulation of HIF-1α and miR-21.
To investigate the effect of IPostC in I/R injury, we compared intestinal mucosal injuries sustained in the I/R and I/R + IPostC groups. Figure 1A shows representative photomicrographs of HE-stained intestinal tissues from sham-operated rats and from those subjected to I/R and I/R + IPostC. I/R significantly increased intestinal mucosal injury scores compared with the sham operation (3.67 ± 0.37 vs 0.42 ± 0.08; P < 0.01), and IPostC decreased the injury score in rats after I/R versus I/R alone (1.86 ± 0.27; P < 0.01) (Fig. 1B). Evaluation of apoptosis by TUNEL staining demonstrated few TUNEL-positive (apoptotic) cells in the sham group, with an increase in TUNEL-positive cells in the I/R group and a decrease in the IPostC group (Fig. 1C). Quantification of apoptosis demonstrated an approximately 7.5-fold increase in the apoptotic index in the I/R group compared with the index in the sham group (31.3 ± 3.3% vs 4.3 ± 0.7%; P < 0.01), with a decrease in the IPostC group  Supplementary Fig. S1. Data are expressed as mean ± SD, n = 6. ** P < 0.01 compared with sham group; ## P < 0.01 compared with I/R group.

Knockdown of miR-21 abolished the effects of IPostC on alleviating I/R injury and suppressing apoptosis by upregulating PDCD4 and Fas-L.
To further explore the biological involvement of miR-21 in IPostC-mediated intestinal protection, miR-21 expression was blocked through the delivery of antagomir-21 or scramble antagomir into the intestines of rats 24 hours before IPostC. After 3 hours of reperfusion, miR-21 was upregulated in the intestinal mucosa in IPostC-pretreated rats relative to the I/R group (relative 4.2 ± 0.39 vs 3.4 ± 0.31; P < 0.01), and this effect was abolished by miR-21 knockdown (1.59 ± 0.14 vs 4.2 ± 0.39; P < 0.01), as determined by qRT-PCR (Fig. 4A). Determination of the injury score in HE-stained sections showed that IPostC significantly attenuated I/R-induced intestinal injury at 3 hours of reperfusion (2.6 ± 0.20 vs 3.7 ± 0.31; P < 0.01), and anti-miR-21 abolished these protective effects (3.8 ± 0.35 vs 2.6 ± 0.20; P < 0.01) ( Fig. 4B and C). Assessment of apoptosis by TUNEL staining showed a similar pattern in which IPostC attenuated apoptosis (19.0 ± 2.1% vs 39.4 ± 3.5%; P < 0.01), whereas knockdown of miR-21 suppressed the protective effects of IPostC, restoring the apoptotic index to that of tissues exposed to I/R (41.0 ± 0.39% vs 19.0 ± 2.1%; P < 0.01) ( Fig. 4D and E). Western blot analysis showed that PDCD4 and Fas-L were downregulated by IPostC, whereas their levels were restored by miR-21 knockdown (Fig. 4F). Taken together, these results indicated that the protective effects of IPostC on I/R-induced intestinal injury may be mediated by the modulation of apoptosis by miR-21 and its target PDCD4.

HIF-1α inhibition downregulated miR-21 and abolished the protective effects of IPostC mediated by the inhibition of apoptosis.
The involvement of HIF-1α was assessed by treating rats exposed to IPostC with the HIF-1α inhibitor 2-ME, which significantly decreased HIF-1α levels in the nucleus compared with the vehicle group ( Fig. 5A; P < 0.01). Knockdown of miR-21 had no effect on HIF-1α expression. Inhibition of HIF-1α resulted in a significant downregulation of miR-21 (0.39 ± 0.05 vs 1.0 ± 0.11; P < 0.01) concomitant with a significant increase in the intestinal injury score (3.3 ± 0.32 vs 1.3 ± 0.19; P < 0.01) in rats exposed to IPostC, and this effect was not enhanced by treatment with anti-miR-21 ( Fig. 5B-D), indicating that the protective effects of IPostC may be mediated by a regulatory loop involving HIF-1α and miR-21 in response to hypoxia. To further examine the role of apoptosis in I/R-induced intestinal injury and the protective effects of IPostC, we examined TUNEL-stained sections and assessed the expression of PDCD4 and Fas-L. HIF-1α inhibition increased the apoptosis index (35.9 ± 3.1% vs 14.9 ± 2.1%; P < 0.01) and upregulated PDCD4 and Fas-L in tissues exposed to IPostC, whereas knockdown of miR-21 under these conditions had no effect on apoptosis or PDCD4 and Fas-L expression ( Fig. 6A-C). Taken together, these data indicated that IPostC inhibits apoptosis via the HIF-1α-mediated modulation of miR-21 and its downstream targets.

Discussion
The protective effects of IPostC were originally described in the heart of an in vivo dog model, where it was shown to reduce neutrophil accumulation and improve endothelial function 17 . Subsequent studies suggested possible mechanisms of IPostC-mediated cardioprotection, such as the attenuation of reactive oxygen species (ROS) production, activation of prosurvival kinase pathways such as ERK1/2 and phosphoinositol 3-kinase/Akt, and mitochondrial modulation [18][19][20] . In the intestine, I/R injury can lead to tissue damage associated with the generation of oxygen free radicals, neutrophil infiltration, and mucosal apoptosis 21 . However, the mechanisms underlying the protective effects of IPostC against intestinal I/R remain unclear. Here, we used a rat model of intestinal I/R to study the involvement of miR-21 in IPostC and explored the underlying mechanisms.
A recent prospective clinical study of patients undergoing valve replacement with or without IPostC analyzed apoptosis-related miRNAs and identified miR-21 as a differentially regulated miRNA, suggesting its involvement in the mechanism of IPostC 12 . In a rat model of myocardial infarction, miR-21 was shown to be downregulated in infarcted areas, and its overexpression inhibited apoptosis through the modulation of PDCD4 22 . miR-21 is downregulated in cardiomyocytes in response to hypoxia, and its overexpression decreases infarct size, exerting an antiapoptotic effect by suppressing the expression of Fas-L 16 . The protective effect of ischemic preconditioning in the heart was shown to be mediated by the upregulation of miR-21 and the downregulation of its target PDCD4 10 . However, studies have also shown that overexpression of miR-21 for longtime leads to fibrosis in the kidney 23 .
In the present study, we showed that IPostC upregulated miR-21 concomitant with a reduction in I/R injury in a rat model of intestinal I/R. Furthermore, the upregulation of miR-21 by IPostC was associated with the activation of HIF-1α, the suppression of apoptosis, and the downregulation of PDCD4 and Fas-L.
We examined the levels of HIF-1α during I/R and IPostC to clarify the roles of hypoxia, HIF-1α, miR-21, and apoptosis in IPostC. Our results showed that HIF-1α was upregulated in intestinal tissues exposed to I/R, whereas IPostC reduced the injury score and apoptosis concomitant with a further upregulation of HIF-1α and miR-21. The upregulation of HIF-1α and miR-21 by hypoxia was abolished by siRNA-mediated silencing of HIF-1α, confirming the modulation of miR-21 expression by HIF-1α. This modulation of miR-21 by hypoxia-induced HIF-1α was shown previously in human pancreatic cancer cells, where the regulation of miR-21 by HIF-1α decreased apoptosis and promoted cell survival 24 , similar to the findings of the present study.
The cardioprotective effects of the HIF-1α/miR-21 axis were reported by Liu et al. 25 , who showed that hypoxia induced miR-21 and HIF-1α, and knockdown of HIF-1α or miR-21 induced cardiomyocyte apoptosis. Similar to the hypoxic tumor microenvironment, myocardial ischemia is characterized by hypoxia, and the consequent activation of HIF-1α plays a critical role in triggering cellular mechanisms of protection against the consequences of oxygen deprivation in the myocardium. HIF-1α is a heterodimeric transcription factor, the levels of which are regulated by cellular oxygen concentration, and its role in the maintenance of oxygen homeostasis has been

. (A) Overexpression of PDCD4 by Ad-PDCD4 (30 MOI) in intestinal epithelial cells. (B) Ad-PDCD4 (30 MOI) increased intestinal epithelial cell apoptosis induced by H/R as determined by TUNEL staining. (C) Modulation of PDCD4 expression in intestinal epithelial cells by anti-miR-21 (30 nM) and pre-miR-21 (30 nM). (D) Overexpression of Fas-L by Ad-Fas-L (30 MOI) in intestinal epithelial cells. (E) Ad-Fas-L (30 MOI) increased intestinal epithelial cell apoptosis induced by H/R as determined by TUNEL staining. (F) Modulation of Fas-L expression in intestinal epithelial cells by anti-miR-21 (30 nM) and pre-miR-21 (30 nM).
Data are expressed as mean ± SD. n = 3. ** P < 0.01 compared with Ad-GFP control in A, B, D and E. * P < 0.05 and ** P < 0.01 compared with normal control in (C and F). demonstrated previously 13,26 . The results of the present study showing the upregulation of HIF-1α during IPostC suggest its involvement in the mechanism of IPostC. Zhao et al. 27 showed that IPostC upregulated HIF-1α and hypothesized that repetitive periods of reperfusion and ischemia may result in temporary hypoxia and thus the induction of HIF-1α. Our results were consistent with these findings and suggest a potential mechanism underlying the protective effects of IPostC mediated by the induction of miR-21by HIF-1α, leading to the downregulation of PDCD4 and the inhibition of apoptosis. Future studies should be aimed at exploring the involvement of survival pathways such as PI3K/Akt and ERK1/2 in the effects of IPostC mediated by miR-21 in the intestine, as suggested in previous studies of the heart.  Supplementary Fig. S1. Data are expressed as mean ± SD, n = 6. ** P < 0.01 compared with I/R group; ## P < 0.01 compared with I/R + IPostC group. Apoptosis is an important mechanism of cell death in the intestine induced by I/R 28 . IPostC has previously been shown to attenuate I/R-induced intestinal injury by inhibiting apoptosis in the intestinal mucosa in a rat model 21 . These effects of IPostC on alleviating intestinal injury and mucosal cell apoptosis have been shown to be mediated by the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway, which plays an important role in I/R-induced intestinal injury 29 . In the present study, we showed that IPostC suppressed apoptosis and I/R injury by upregulating miR-21 and downregulating PDCD4 and Fas-L. miR-21 was shown to protect rat hearts against I/R injury by decreasing apoptosis via the modulation of PDCD4 10 . However, Gao et al. 12 failed to show the involvement of PDCD4 in IPostC-induced cardioprotection, indicating that IPostC attenuated apoptosis by regulating miR-21 and the downstream effectors BCL2 and BAX without affecting PDCD4 levels. Additional studies specific to I/R injury in the intestine are necessary to clarify the mechanisms underlying the effects of IPostC.
In normoxic condition, HIF-1α was hydroxylated by prolyl hydroxylase domain (PHD) proteins, and subsequently degraded via the recruitment of an ubiquitin-protein ligase von Hippel-Lindau (VHL) 30 . Whereas in hypoxic condition, the hydroxylation of HIF-1α by PHD was decreased, resulting in the HIF-1α activation 31 . As a transcription factor, HIF-1α could activate the expression of numerous miRNAs under hypoxia 32 , via the modulation of their transcription factors 33 , include miR-21. Liu, et al. 25 showed that HIF-1α regulate the expression of miR-21 in response to hypoxia, via the binding sites 1 on miR-21 promoter in cardiomyocytes.
miR-21 and HIF-1α was found to be an anti-apoptotic miRNA and factor. Upregulation of HIF-1α and miR-21 in hypoxic condition should be a protective mechanism against hypoxia, however it could not resist the damage caused by hypoxia. Intermittent hypoxia could further up-regulate the expression of HIF-1α and miR-21, making it more likely to counteract the damage caused by hypoxia. But this short time up-regulation is different from that caused by the overexpression vector transfection.

Conclusion
IPostC attenuates intestinal I/R injury by suppressing apoptosis via a regulatory axis involving the induction of miR-21 by HIF-1α, leading to the downregulation of the apoptotic mediators PDCD4 and Fas-L. Further research is necessary to confirm these findings and to clarify the role of other survival mediators and pathways in the protective effect of IPostC against intestinal injury.

Methods
Animals and surgical procedure. The study was approved by the Animal Care Committee of TongJi University, and all animal procedures were performed in compliance with National Institutes of Health guidelines. Sixty adult pathogen-free male Sprague-Dawley rats weighing 220 to 250 g were housed in individual cages in a temperature-controlled room with 12-hour light-dark cycles and free access to water ad libitum.
Rats were anesthetized with 20% urethane (6 mL/kg) intraperitoneally, tracheostomized, and mechanically ventilated with room air. A midline laparotomy was performed to access the small intestine. Intestinal ischemia was established by occluding the superior mesenteric artery (SMA) and confirmed by immediately blanching the small intestine and cecum. A return to original color indicated restoration of blood flow to the gut after declamping of the SMA. Normothermia (36 °C-38 °C) was maintained with heating pads during the procedure.
Cell culture and conditions. Intestinal epithelial cells (IEC-6) were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) and cultured in Dulbecco's Modified Eagle's Medium supplemented with 4.5 g/L D-glucose, 10% v/v fetal bovine serum, and 1% penicillin/streptomycin (Gibco; Invitrogen Ltd., Shanghai, China) at 37 °C in a 5% CO 2 and 21% O 2 incubator. Hypoxia was induced by culturing cells in a modular incubator equipped with an O 2 sensor, which enabled the mixing of N 2 and air to achieve hypoxic conditions (10% or 2% O 2 and 5% CO 2 balanced with N 2 ). For reoxygenation, cells were transferred to normoxic conditions.

Scoring of intestinal mucosal injury.
Sections of the small intestine were stained with hematoxylin-eosin (HE) and examined with light microscopy. Damage to the intestinal mucosa was evaluated in a blinded manner by two independent pathologists. The degree of injury was scored according to the method described by Chiu et al. 34 with this method, the score is based on changes in the villi and glands of the intestinal mucosa. In brief, mucosal damage was graded as follows: grade 0: normal mucosal villi; grade 1: development of subepithelial gruenhagen space at the apex of the villi and capillary congestion; grade 2: extension of the subepithelial space with moderate lifting of the epithelial layer from the lamina propria; grade 3: extensive epithelial lifting with few denuded tips; grade 4: denuded villi with exposure of lamina propria and dilated capillaries and increased cellularity of the lamina propria; and grade 5: digestion and disintegration of the lamina propria, hemorrhage, and ulceration.
Intestinal mucosal epithelial apoptosis evaluation. Apoptosis was quantified using an in situ apoptosis detection kit (Roche, Switzerland) as described previously 27 . Briefly, transmural intestinal tissue blocks were fixed using 4% paraformaldehyde in PBS, embedded in paraffin, and subjected to TUNEL staining according to the manufacturer's protocol. The apoptosis index was determined by measuring the percentage of apoptotic nuclei from the total nuclei in each slide.
Western blotting. For western blot detection of the HIF-1α protein, tissue blocks (50 mg) were lysed and homogenized. The protein concentration in the supernatant was determined using the BCA method. Protein samples (20 μg) were separated through 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. Membranes were blocked with 3% bovine serum albumin for 2 hours at room temperature and then incubated with primary antibody at 4 °C overnight. The membranes were incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (1:1,000 polyclonal) at room temperature for 1 hour. The primary antibodies used were anti-mouse monoclonal HIF-1α (1:200, Santa Cruz Biotechnology; Santa Cruz, CA, USA), anti-mouse monoclonal β-actin (1:1,000, Santa Cruz Biotechnology), and anti-mouse monoclonal LAMIN-B (1:100, Santa Cruz Biotechnology). Bands were detected using electrochemiluminescence. Taqman real-time PCR. Total RNA was isolated from tissues using the Trizol reagent (Invitrogen; Carlsbad, CA, USA), and miRNA was quantified by real-time PCR with Taqman chemistry (Applied Biosystems; Carlsbad, CA, USA) as described previously 35 . As an internal control, U6 was used for template normalization for miR-21 expression.