Isoproterenol hydrochloride was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Rosuvastatin was kindly provided by AstraZeneca (Shanghai, China). The aspartate transaminase (AST), alanine transaminase (ALT), creatine kinase (CK-MB), and lactate dehydrogenase (LDH) kits were procured from Nanjing Jiancheng Bioengineering Institute (Jiangsu, China). Superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) activities as well as reduced glutathione (GSH) and malondialdehyde (MDA) levels in heart tissues were measured by commercially available kits (Beyotime Institute of Biotechnology, Haimen, China). Commercially ELISA kits for IL-1β and IL-18 were obtained from R&D Systems (Minneapolis, MN, USA). Antibodies of NLRP3 and ASC were obtained from Cell Signaling Technology (Danvers, MA, USA), antibodies of caspase-1 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). All other chemicals and reagents used in this study were of analytical grade.

Forty-five male Wistar-Albino rats (180–200 g) were purchased from the Experimental Animal Center of Fudan University (Shanghai, China). The animals were maintained under standard laboratory conditions at 25±2°C and 50±15% humidity with an alternate 12 h cycle of light and dark. They were acclimatized to the conditions of the animal house for 1 week before the experiment and allowed free access to standard laboratory diet and water ad libitum. All animal procedures were done in accordance with the guidelines for the care and use of laboratory animals approved by the Ethics Committee for Animal Experimentation of Fudan University.

Rats were randomly divided into two groups, NC group (n=9) and ISO group (n=36). A rat model of myocardial ischemia was induced by subcutaneous injection of ISO hydrochloride for 2 consecutive days, while NC group were injected with normal saline for 2 consecutive days. After one week, ISO group were randomly divided into four subgroups: Model group: rats received 1 ml/kg/day 1% Tween-80 suspension in distilled water by oral gavage for 8 consecutive weeks, RSV5, RSV10 and RSV15 group: rats received rosuvastatin (5, 10 or 15 mg/kg, respectively) in distilled water by oral gavage for 8 consecutive weeks. The dose of ISO and RSV was selected based upon previous studies (24,28,29). After the end of the animal experiment, rats were anesthetized and sacrificed, blood samples were collected and centrifuged to obtain serum for the biochemical assays.

After blood collection, the heart tissues were rapidly removed, then the cardiac apex was immediately fixed in 4% paraformaldehyde, processed in ethanol and embedded in paraffin wax. The cardiac apex were stained with hematoxylin and eosin (H&E) and examined under a light microscope (Olympus, Tokyo, Japan).

Enzyme immunoassay of IL-1β and IL-18 in heart homogenate was performed by using ELISA kits according to manufacturer's instructions (R&D Systems). The color intensity was read at 450 nm with a microplate reader (Tecan Ltd., Mannedorf, Switzerland) and the cytokines levels were expressed as pg/mg of tissue.

The serum was used to assay AST, ALT, CK-MB and LDH activities. The activities of AST, ALT, CK-MB and LDH were assayed using commercial kits purchased from Jiancheng Bioengineering Institute (Nanjing, China) according to the manufacturer's instructions.

After experimental treatment, the homogenates of heart tissues were centrifuged at 16,000 rpm for 10 min. The supernatant was used to assay MDA levels, and SOD, CAT, GPX activities, as well as GSH concentrations according to the manufacturer's instructions, on a microplate reader at 560 and 532 nm. The commercially available assay kits were purchased from Jiancheng Bioengineering Institute.

The mRNA expression levels of NLRP3, ASC and caspase-1 were analyzed via qRT-PCR; total RNA sample from rat heart tissues was extracted and purified using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions. Total RNA was then reverse transcribed into cDNA with an M-MLV reverse transcriptase kits according to the manufacturer's protocol. Following the reverse transcription, qPCR was performed to quantify the RNA levels of NLRP3, ASC and caspase-1 using SYBR-Green Supermix (Bio-Rad, Hercules, CA, USA) and the data were analyzed using the 2^−ΔΔCt method. GAPDH was used as a housekeeping gene for mRNA analysis. The primer sequences are listed in Table I.

Total protein was loaded per well, resolution on a 10% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE), and then transferred onto a polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA). The membrane was then blocked with 5% skim milk and subsequently incubated with primary antibodies for NLRP3 (1:1,000), ASC (1:1,000) and caspase-1 antibodies (1:500) overnight at 4°C. Membranes were subsequently incubated with appropriate HRP-conjugated secondary antibody at room temperature for 1 h. Immunoreactive bands were visualized via enhanced chemiluminescence (Millipore) and quantified via densitometry using ImageJ (National Institutes of Health).

All data were expressed as mean ± SD, and analyzed using one-way ANOVA followed by a post-hoc test to determine the statistical difference between groups. Statistical analysis was performed using the GraphPad Prism 5 (GraphPad Software, Inc., San Diego, CA, USA). A value of P<0.05 was considered statistically significant.

To investigate whether rosuvastatin could ameliorate ISO-induced myocardial injury, we analyzed the myocardial injury marker activities in heart tissue of all groups. As illustrated in Fig. 1, two subcutaneous injections of ISO significantly induces cardiac dysfunction as evidenced by greatly increase serum activities of AST, ALT, CK-MB and LDH in comparison to normal control group (P<0.05). However, by administration of rosuvastatin, serum AST, ALT, CK-BA and LDH activities were obviously relieved in this animal model (P<0.05).

Fig. 2 shows that ISO induced a significant infarction area in comparison to the normal control group (P<0.05). Compared with the model group, myocardial infarction area of rosuvastatin treated groups were remarkably diminished (P<0.05) (Fig. 2). Furthermore, results of histopathological examination also confirmed the protective effect of rosuvastatin in ISO-induced myocardial injury. As illustrated in Fig. 3, NC group showed a normal histoarchitecture, while obvious necrosis of myofibers with cell infiltration, as well as extravasations of red blood cells were observed in the heart tissue of ISO rats (Fig. 3). Importantly, rosuvastatin significantly ameliorated these changes in ISO rats.

Then, we investigated the effect of rosuvastatin on ISO-induced lipid peroxidation and oxidative stress in rats. As shown in Fig. 4, marked increase of MDA, a lipid peroxidation byproduct, was observed in the heart of ISO group. Moreover, rats administered ISO showed decreased activities of antioxidants such as SOD, CAT and GPX, downregulated non-enzymatic antioxidant (GSH) concentrations, as compared to normal control rats. However, rosuvastatin significantly reduced cardiac MDA levels, increased SOD, CAT and GPX activities and GSH concentrations in ISO-treated rats (P<0.05). These results suggest that ISO injection causes oxidative stress in heart of rats, which is suppressed by the treatment of rosuvastatin.

The production of pro-inflammatory cytokines such as IL-1β and IL-18 in heart tissues are shown in Fig. 5. Compared to normal control group, injection of ISO significantly increased the secretion levels of IL-1β and IL-18 in the heart (P<0.01). Treatment with rosuvastatin reduced ISO-induced elevation of cardiac IL-1β and IL-18 secretion in this animal model.

In order to evaluate whether rosuvastatin alleviated ISO-induced myocardial injury via NLRP3 inflammasome, the mRNA and protein expression levels of NLRP3, ASC and caspase-1 in the heart of all experimental groups were detected. ISO injection obviously induced the activation of NLRP3, characterized by significantly increased cardiac mRNA (Fig. 6) and protein (Fig. 7) expression levels of NLRP3, ASC and caspase-1 (P<0.05). Rosuvastatin markedly decreased NLRP3, ASC and caspase-1 at both mRNA and protein levels.