Sprague Dawley rats (male, 3 months old, 250–300 g, total number: 120) were obtained from The Experimental Animal Center of Zhejiang Chinese Medical University (Lot: SCXK; Shanghai 2007-0005). The animals were acclimated for 7 days in a controlled temperature (20–24°C) with 12-h light/dark cycle and free access to food and water before the experiments. All experiments were designed according to The National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by The Animal Care Committee of Zhejiang Chinese Medical University. Every effort was made to minimize the number and suffering of the animals in the present study.

Rats were anesthetized with sodium pentobarbital (50 mg/kg) containing heparin (300 IU) by intraperitoneal injection. The rats were sacrificed and hearts were immediately harvested and mounted on the Langendorff system for retrograde perfusion at a constant pressure of 75 mmHg with oxygenated (95% O₂ and 5% CO₂) Krebs-Henseleit (KH) buffer (118 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO₄, 1.25 mM CaCl₂, 1.2 mM KH₂PO₄, 25 mM NaHCO₃ and 11 mM glucose; pH 7.4) as previously described (25). Hemodynamic measurements for heart rate (HR), maximal rate of the increase of left ventricular pressure (dp/dt_max) and left ventricular developed pressure (LVDP) were assessed during the experiment. After 30 min for system equilibration, cardiac I/R injury was determined by the hearts experiencing 30 min ischemia (no flow) and 120 min reperfusion. The CRO treatment group was subjected to 20 min equilibration followed by 10 min CRO administration before I/R injury.

CRO (P0352; purity ≥95%) was purchased from Shanghai PureOne Biotechnology and dissolved in DMSO (100 mM) for storage, and then diluted in KH buffer before use. All the chemical reagents used in the present study were of analytical grade. Rats were divided into 5 groups (n=8; other rats were used to verify the success of the model and to test the surgical procedure): i) Sham, surgery without occlusion; ii) I/R, surgery with 30 min occlusion, followed by 120 min reperfusion; iii) I/R + CRO (10 µM); iv) I/R + CRO (20 µM); and v) I/R + CRO (40 µM).

The H9c2 cardiomyocyte cell line was obtained from the American Type Culture Collection (cat. no. CRL1446) and cultured in DMEM containing 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) in a humidified incubator (5% CO₂) at 37°C. Control siRNA (cat. no. sc-37007; scrambled sequence) and Nrf2 siRNA (cat. no. sc-37049) was purchased from Santa Cruz Biotechnology, Inc., and used according to the manufacturer's protocol. H9c2 cells were transiently transfected with Nrf2 siRNA (10 µM) using Lipofectamine^® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.) in opti-MEM (Gibco; Thermo Fisher Scientific, Inc.) for 6–12 h. Then, the medium was replaced with DMEM for the following 48 h.

The cardiac infarct size was determined by 2,3,5-triphenyltetrazolium chloride (TTC) staining, as previously described (26). The heart was frozen at −20°C for 15 min, then sliced into five 2 mm-thick transverse sections and immersed in 1% TTC solution in distilled deionized water for 15 min at 37°C and then fixed in 4% paraformaldehyde overnight in 4°C. The viable tissue was stained a deep red color and the infarcted zone was not stained. The infarcted size of each sliced heart section was measured and the percentage of the infarcted zone was calculated using ImageJ 1.48V (National Institutes of Health), image analyzing software.

SOD, CK, LDH, MDA and GSH-PX production from the coronary flow were measured using a commercial kit (Nanjing Jiancheng Bioengineering Institute) according to the manufacturer's protocol. In brief, after I/R injury, the coronary blood flow was collected for SOD, CK, LDH, MDA and GSH-PX determination. The activities of the control group were considered as 1.

The heart tissue was lysed and homogenized in 400 µl lysis buffer (RLT Buffer; Qiagen, Inc.). The total RNA of the tissue was isolated on spin columns with silica-based membranes (RNeasy Mini kit; Qiagen, Inc.) according to the manufacturer's protocol. Then, RNA was reverse transcribed (25°C for 10 min, following at 50°C for 15 min, terminate the reaction by heating at 85°C for 5 min) in a total volume of 20 µl using the RT High-Capacity RNA-to-cDNA kit (Applied Biosystems; Thermo Fisher Scientific, Inc.). Quantitative PCR was performed with the FastStart Universal SYBR-Green Master Rox (Roche Diagnostics) using the ViiA™ 7 real-time PCR system. The cycling protocol was as follows: 95°C for 2 min, followed by 40 cycles at 95°C for 10 sec and 60°C for 40 sec. All data were normalized to the housekeeping gene and the relative expression levels were calculated using the 2^−ΔΔCq method (27). Primer sequences for IL-1α, IL-1β, IL-6, IL-8 and TNF-α were used to quantify the mRNA levels, as well as β-actin used as an endogenous control. The primer sequences are listed in Table I.

Cell viability was determined by evaluating the absorbance of MTT. Cells were cultured in 96-well microplates at a density of 1×10⁴ cells/ml. After siRNA interference and I/R injury, cells were incubated with medium containing MTT (500 µg/ml) for 3–4 h in the dark. Then, the MTT solution was aspirated and DMSO (150 µl) was added for 15 min. The absorbance was detected at 570 nm on a Multi-Mode Detection Platform (SpectraMax Paradigm; Molecular Devices, LLC). Cell viability of the control group was considered as 100%.

Heart tissue was homogenized in ice-cold RIPA buffer (Cell Signaling Technology, Inc.) containing protease inhibitors and incubated on ice for 20 min, followed by centrifugation at 13,000 × g for 10 min at 4°C. The supernatant was collected and the concentration of protein was quantified using the Bio-Rad protein assay kit (Bio-Rad Laboratories, Inc.). Proteins (30 µg/lane) of different groups were loaded and separated by SDS-PAGE on 10% gels and transferred to a PVDF membrane (pore size: 0.45 µm; EMD Millipore). The membranes were blocked with 5% bovine serum albumin in TBS with 0.1% Tween 20 (TBST) for 1 h at room temperature and incubated with Nrf2 (1:1,000, Cell Signaling Technology, Inc.; cat no. 12721), HO-1 (1:1,000, Cell Signaling Technology, Inc.; cat. no. 86806), PERK (1:500, Cell Signaling Technology, Inc.; cat. no. 5683), phosphorylated (p)-PERK (1:500, Cell Signaling Technology, Inc.; cat. no. 3179), IRE1 (1:1,000, Cell Signaling Technology, Inc.; cat. no. 3294), X-box binding protein 1 (XBP1; 1:1,000, Cell Signaling Technology, Inc.; cat no. 40435) and ATF6 (1:1,000; Cell Signaling Technology, Inc.; cat no. 65880) primary antibodies overnight at 4°C. β-Actin (1:10,000; Cell Signaling Technology, Inc.; cat no. 3700) was used as the loading control. After the primary antibody incubation, the membrane was washed with TBST 3 times (5 min) and incubated with the anti-mouse and anti-rabbit IgG HRP secondary antibodies (1:10,000; Cell Signaling Technology, Inc.; cat. nos. 7076, 7074) for 1 h at room temperature. The intensity of the bands was detected using the ChemiDoc Touch Imaging System (Bio-Rad Laboratories, Inc.; series no. 732BR2237).

All data are presented as the mean ± standard error of the mean with GraphPad Prism 7.0 (GraphPad Software, Inc.). The Student's t-test was used to analyze the differences between 2 groups and one-way analysis of variance with multiple comparisons (using Tukey's test) was used to analyze the differences between 3 or more groups. P<0.05 was considered to indicate a statistically significant difference.

The present study established a Langendorff perfusion system, an ex vivo model of I/R, to evaluate the cardiac protective effect of CRO in rat hearts. CRO was dissolved in perfusion KH buffer and administered for 10 min before 30 min ischemia followed by 120 min reperfusion as shown in Fig. 1A. During the whole process of the experiment, cardiac functional parameters were recorded and analyzed. Long-term I/R injury decreased the HR, dp/dt_max and LVDP. CRO (40 µM) markedly enhanced these cardiac functional parameters in the period of reperfusion, suggesting that CRO exhibited cardioprotection in rats (Fig. 1B-D).

After I/R injury, the heart was harvested for evaluation of the infarct size and the perfused buffer from the coronary flow was collected for determination of cardiac enzyme activities. A significant increase in the infarcted area was observed in the I/R group, ~20% compared with the sham group (P<0.001). Dose-dependent decreases in infarct size were identified in the CRO pretreatment group, particularly in the CRO 40 group in which the infarct size was <10% (Fig. 2A and B). Then, the activities of two enzymes, CK and LDH, were analyzed, which represented cardiac toxicity in the perfused buffer from the coronary flow. Both CK and LDH displayed downregulation in I/R hearts pretreated with CRO, suggesting that CRO could reduce cardiac toxicity from I/R injury (Fig. 2C and D). The antioxidant effect of CRO was further evaluated by determining the activities of SOD, MDA and GSH-PX. The changes of these three enzymes were significant in the I/R group and CRO regulated their activities in a dose-dependent manner (P<0.001; Fig. 2E-G). Collectively, these observations demonstrated that CRO exhibited a protective effect to alleviate cardiac I/R injury.

As the inflammatory response accelerated tissue injury in the reperfusion period after the lethal ischemic damage, the levels of pro-inflammatory cytokines were detected. Cardiac mRNA levels of IL-1α, IL-1β, IL-6, IL-18 and TNF-α significantly increased in the I/R group (P<0.001). CRO significantly reduced the mRNA expression of these pro-inflammatory cytokines in a dose-dependent manner (P<0.001; Fig. 3A). Nrf2 is sensitive to oxidative stress and activates the transcription of HO-1 and NAD(P)H: Quinine oxidoreductase 1 (NQO1) in I/R injury (15). Nrf2-mediated signaling plays a key role in the innate immune/inflammatory pathway and regulates pro-inflammatory biomarkers, including IL-1 and TNF-α (28). Due to the importance of Nrf2/HO-1 signaling in the inflammatory response induced by I/R, their protein expression was evaluated. I/R injury significantly reduced the protein expression of Nrf2, HO-1 and NQO1, and CRO upregulated their expression in a dose dependent fashion (P<0.001; Fig. 3B). These results suggested that CRO could attenuate cardiac I/R injury via Nrf2/HO-1-mediated anti-inflammation signaling.

The UPR is composed of three principle branches; IRE1, PERK and ATF6. The IRE1 branch is the central branch of the UPR; its activation cleaves the mRNA of XBP-1 and activates XBP-1 to translocate to the nucleus for transcription of genes involved in ER stress (16,17). The UPR has been demonstrated to become activated when myocardial reperfusion occurs. Drugs with cardioprotective effects are demonstrated to attenuate the toxic UPR (29). Due to the important role of the UPR in I/R injury, immunoblot analysis was performed to evaluate the expression of proteins in the UPR system. The results showed that expression of p-PERK, IRE1, ATF6 and XBP-1 significantly increased in cells induced by I/R, suggesting I/R injury activated ER-stress signaling (P<0.001; Fig. 4). Pretreatment with CRO significantly decreased the expression levels of these proteins, displaying a beneficial effect by downregulating ER-stress signaling (P<0.05; Fig. 4).

Nrf2 transcription activated antioxidant and anti-inflammatory gene expression against cardiac I/R injury. Therefore, it was further evaluated whether CRO protected the heart against I/R injury in a Nrf2-dependent manner. An in vitro system was first applied using Nrf2 siRNA to downregulate Nrf2 expression in H9c2 cardiomyocytes. The results showed that both the protein and mRNA expression reduced >1/2 after 54–60 h siRNA treatment (Fig. 5A and B). Subsequently, it was investigated whether downregulation of UPR protein expression by CRO pretreatment was dependent on Nrf2 activation. In the group with control siRNA (non-target) treatment, CRO pretreatment significantly reduced the expression of IRE1, ATF6 and XBP1. However, in the group with Nrf2 siRNA, the expression of IRE1, ATF6 and XBP1 did not reduce even with CRO pretreatment, suggesting that CRO attenuated the I/R-induced UPR in a Nrf2-dependent manner (Fig. 5C). The mRNA levels of pro-inflammatory cytokines were also measured and the results clearly showed that deficiency of Nrf2 suppressed the anti-inflammatory ability of CRO (Fig. 5D). From these results, it was determined whether reduction of Nrf2 expression inhibited the cardioprotective effect of CRO. An MTT assay was performed to evaluate cell survival and the results showed that CRO significantly increased cell viability in H9c2 cells induced by I/R. In the group with Nrf2 siRNA, CRO could not enhance cell survival, suggesting that the cardioprotective effect of CRO required Nrf2 activation (Fig. 5E).