Clean-grade Sprague Dawley rats (male; age, 8–10 weeks; weight, 200–220 g; n=40) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Animals were housed at 21–25°C and 50–60% humidity, under a 12-h light/dark cycle, with free access to food and water. Sprague-Dawley rats were randomly divided into four groups (n=10): Sham operation group, MI model group, MI + crocin (Amyjet Scientific, Inc.) low-dose group (100 mg/kg) and MI + crocin high-dose group (200 mg/kg). Rats in the MI + crocin low-dose group and MI + crocin high-dose group were intraperitoneally injected with crocin once per day (volume 0.2 ml/100 g) for 14 days. Rats in the sham operation and MI groups were intraperitoneally injected with an equal volume of normal saline for 14 days. The rats were anesthetized 30 min after the last administration by intraperitoneal injection of 2% sodium pentobarbital (40 mg/kg). Following endotracheal intubation, an artificial respirator was connected (tidal volume, 1.5–2 ml/100 g; frequency, 48–54 times/min). The right common carotid artery was carefully separated and the left ventricle was intubated. An incision was made between ribs 3 and 4 on the left side of the chest of rats. The pericardium was excised to completely expose the heart. With the exception of the sham operation group, the left anterior descending coronary artery was ligated. After ligation for 30 min, the ligature was loosened for reperfusion for 120 min. Left ventricular end-diastolic pressure, left ventricular systolic pressure and cardiac function were all evaluated following ischemia and reperfusion in each group. At the end of the experiment, rats were euthanized via carbon dioxide; rats were placed within a box and 100% CO₂ was used at a displacement rate of 30% vol/min. After 10 min, the rats were observed to no longer be breathing and had discolored eyes. Minimum CO₂ flow was maintained for a further 2 min following respiratory arrest. Animal death was then determined. All animal experiments were approved by the Ethics Committee of Zhongda Hospital Southeast University (approval no. 190501017; Nanjing, China).

The mouse leukemia RAW264.7 cell line was purchased from the American Type Culture Collection and seeded in RPMI 1640 media (Gibco; Thermo Fisher Scientific, Inc.) containing 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.). The cells were cultured in an incubator at 37°C with 5% CO₂. After 24 h, the culture medium was changed to serum-free medium overnight. An inflammatory response model was established using recombinant mouse high mobility group box 1 (rmHMGB1; 1 µg/ml; cat. no. ab255799; Abcam) and recombinant mouse heat shock protein 60 (rmHSP60; 1 mol/ml; cat. no. ab92364; Abcam) for 24 h at 37°C. Crocin (low dose, 25 µg/ml; high dose, 50 µg/ml) was used to pretreat RAW264.7 cells 1 h before establishment of the inflammatory response model. The cells in the control group were not treated with crocin, rmHMGB1 or rmHSP60.

RAW264.7 cells (2×10⁵ cells/well) were transfected with 50 nM small interfering RNA (si)-negative control (NC; 5′-UUCUCCGAACGUGUCACGU-3′) and 50 nM si-KBTBD7 (5′-GGAUUAAUAUAGGCACCAU-3′), synthesized by Shanghai GenePharma Co., Ltd. Lipofectamine^® 2000 (Thermo Fisher Scientific, Inc.) reagent was used for transfection according to the manufacturer's protocol. The transfection was performed at 37°C for 24 h.

Cardiac tissue apoptosis was detected using the TUNEL assay (cat. no.; 40307ES50; Shanghai Yeasen Biotechnology Co., Ltd.) according to the manufacturer's protocol. Briefly, RAW264.7 cells were fixed with 4% paraformaldehyde at 4°C for 25 min and permeabilized using 0.2% Triton X-100 at room temperature for 5 min. After washing with PBS, the cells were stained with 50 µl TdT buffer at 37°C for 1 h. The nuclei were stained with 2 µg/ml DAPI at room temperature for 5 min in the dark. Images were captured using a confocal microscope and 10 randomly-selected high-magnification fields were observed. The apoptotic rate was calculated as follows: Apoptotic nuclei/total nuclei ×100.

Total RNA was extracted from rat myocardium and RAW264.7 cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). An ultraviolet spectrophotometer was used to determine the concentration and purity of the RNA. Total RNA was reverse transcribed into cDNA using the following components: High-Fidelity Script reverse transcriptase, dNTPs, primers, RNA, RT buffer, dithiothreitol and RNase-free water (Invitrogen; Thermo Fisher Scientific, Inc.). RT was performed at 37°C for 15 min followed by 85°C for 5 sec. qPCR was subsequently performed using the UltraSYBR Mixture (CoWin Biosciences) according to the manufacturer's protocol. The following thermocycling conditions were used for qPCR: Pre-denaturation at 95°C for 10 min; 35 cycles of denaturation at 95°C for 15 sec, and annealing and elongation at 60°C for 1 min; followed by final extension at 95°C for 15 sec, 60°C for 1 min, 95°C for 15 sec and 60°C for 15 sec. The following primer pairs were used for qPCR: KBTBD7 forward (F), 5′-AGCTCAGTTGTATCGGTAGCA-3′ and reverse (R), 5′-CCGGAATAAGGGTCGTAACAGA-3′; interleukin (IL)-1β F, 5′-GGTGTGTGACGTTCCCATTAGAC-3′ and R, 5′-CATGGAGAATATCACTTGTTGGTTGA-3′; IL-6 F, 5′-TAGTCCTTCCTACCCCAATTTCC-3′ and R, 5′-TTGGTCCTTAGCCACTCCTTC-3′; tumor necrosis factor α (TNFα) F, 5′-AAGCCTGTAGCCCACGTCGTA-3′ and R, 5′-GGCACCACTAGTTGGTTGTCTTTG-3′; Bax F, 5′-AGTGTCTCAGGCGAATTGGC-3′ and R, 5′-CACGGAAGAAGACCTCTCGG-3′; Bcl-2 F, 5′-ACTGAGTACCTGAACCGGCATC-3′ and R, 5′-GGAGAAATCAAACAGAGGTCGC-3′; and GAPDH F, 5′-AGGTCGGTGTGAACGGATTTG-3′ and R, 5′-TGTAGACCATGTAGTTGAGGTCA-3′. mRNA expression levels were quantified using the 2^−∆∆Cq method (32) and normalized to the internal reference gene GAPDH.

After 30 min of ischemia and 120 min of reperfusion, 100 mg myocardial tissue was immediately extracted from the ischemic area in the left ventricle of the rats. Tissue was subsequently washed with 1X PBS before tissue lysis solution was added. The tissue was then incubated with RIPA buffer (Beyotime Institute of Biotechnology) on ice for 30 min. Total protein content was determined by bicinchoninic acid (BCA) assay (Thermo Fisher Scientific, Inc.). In addition, RAW264.7 cells were collected, cells were cleared on ice, and total protein was extracted using RIPA buffer and was quantified using a BCA assay. Total protein (20 µg) was separated by SDS-PAGE on 12% gels. After blocking with 5% skim-milk at room temperature for 1 h, the PVDF membranes were incubated with primary antibodies against HMGB1 (1:1,000; cat. no. ab227526; Abcam), HSP60 (1:1,000; cat no. ab190828; Abcam), p38 (1:1,000: cat. no. 8690; Cell Signaling Technology, Inc.), phosphorylated (p)-p38 (1:1,000; cat. no. 4511; Cell Signaling Technology, Inc.), JNK (1:1,000; cat. no. 9252; Cell Signaling Technology, Inc.), p-JNK (1:1,000; cat. no. 9251; Cell Signaling Technology, Inc.), p65 (1:1,000; cat. no. 8242; Cell Signaling Technology, Inc.), p-p65 (1:1,000; cat. no. 3033; Cell Signaling Technology, Inc.) and GAPDH (1:1,000; cat. no. 5174; Cell Signaling Technology, Inc.) overnight at 4°C. Horseradish peroxidase-labeled secondary antibodies (1:3,000; cat. no. 7074; Cell Signaling Technology, Inc.) were then added to incubate with the membranes at room temperature for 2 h. Protein bands were visualized using ECL (Shanghai Yeasen Biotechnology Co., Ltd.). The ratio of gray values was used for semi-quantitative analysis with GAPDH as the loading control using ImageJ software (version 1.8.0; National Institutes of Health).

The RAW 264.7 cell supernatant solution was collected. ELISA was performed according to the manufacturers' instructions. The levels of cytokines in each treatment group were determined using the standard curve and optical density. The following ELISA kits were used: IL-1β ELISA Kit [cat. no. EK201B/3; Hangzhou Multi Sciences (Lianke) Biotech, Co., Ltd.], TNFα ELISA Kit [cat. no. EK282/4; Hangzhou Multi Sciences (Lianke) Biotech, Co., Ltd.], IL-6 ELISA Kit [cat. no. EK206/3; Hangzhou Multi Sciences (Lianke) Biotech, Co., Ltd.], HSP60 ELISA Kit (cat. no. EM1136; FineTest), and HMGB1 ELISA Kit (cat. no. EM0382; FineTest).

Data were analyzed using SPSS 19.0 software (IBM Corporation) from three independent experiments. Data are presented as the mean ± standard deviation. Unpaired Student's t-test was used for comparisons between two groups. One-way ANOVA and Tukey's post-hoc test was used for comparisons between multiple groups (33–35). P<0.05 was considered to indicate a statistically significant difference.

A rat model of MIRI was constructed to study the pharmacological effects of crocin, the chemical structure of which is shown in Fig. 1A. To assess the inflammatory response and the degree of myocardial injury, mRNA expression levels of IL-1β, IL-6 and TNFα in myocardial tissue were determined. The mRNA expression levels of IL-1β, IL-6 and TNFα were significantly increased in the MI group compared with those in the sham group. However, the mRNA expression levels of IL-1β, IL-6 and TNFα were significantly decreased following crocin treatment compared with in the MI group (Fig. 1B-D). Furthermore, the TUNEL assay was used to detect apoptosis. Apoptosis was observed following myocardial I/R in the MI group, this was significant compared with the sham group; however, the apoptotic rate in the low and high crocin treatment groups was significantly reduced compared with that in the MI group (Fig. 1E). The mRNA expression levels of the apoptosis-related protein Bax and antiapoptotic protein Bcl-2 were also detected. The mRNA expression levels of Bax in the myocardial tissue of the MI group were significantly increased compared with those in the sham group; however, Bax expression was significantly reduced following treatment with high and low doses of crocin compared with in the MI group (Fig. 1F). The mRNA expression levels of Bcl-2 were significantly downregulated in the MI group compared with those in the sham group, and were significantly upregulated following high and low levels of crocin treatment compared with in the MI group (Fig. 1G). These results therefore suggested that crocin could inhibit myocardial apoptosis caused by MIRI.

Changes in the levels of HMGB1 and HSP60 in myocardial tissue following MI were determined. HMGB1 and HSP60 levels, measured using ELISA, were significantly increased in the MI group compared with those in the sham group (Fig. 2A and B). Western blotting results also demonstrated that the protein expression levels of HMGB1 and HSP60 were significantly increased in the MI group compared with those in the sham group (Fig. 2C). Subsequently, rmHMGB1 and rmHSP60 were used to stimulate an inflammatory response in RAW264. 7 cells and the effect of crocin on these macrophages was observed. The results demonstrated that the levels of IL-1β, IL-6 and TNFα following rmHMGB1 treatment were significantly downregulated by the addition of crocin, in a dose-dependent manner, compared with the control (no crocin treatment) (Fig. 2D). These results were further reflected upon RAW 264. 7 cells treatment with rmHSP60 (Fig. 2E). These results indicated that crocin may inhibit the inflammatory response triggered by HMGB1 and HSP60.

Western blotting was used to detect the expression levels of proteins involved in the MAPK and NF-κB signaling pathways following treatment with rmHMGB1, rmHSP60 and crocin. The results demonstrated that the protein expression levels of p-p38, p-JNK and p-p65 were significantly increased following treatment with rmHMGB1 and rmHSP60 compared with those in the control group. However, the protein expression levels of p-p38, p-JNK and p-p65 were significantly reduced following high-dose crocin treatment compared with cells that received no crocin (Fig. 3A and B). Therefore, the results indicated that rmHMGB1 and rmHSP60 macrophage stimulation may activate the p38 and NF-κB signaling pathways. The results also suggested that crocin may suppress the production of inflammatory cytokines through inhibition of the MAPK and NF-κB signaling pathways.

KBTBD7 plays a crucial role in MI, especially in the response to inflammation and regulation of myocardial function (6). Therefore, the present study investigated whether crocin could function through regulating KBTBD7. qPCR detection demonstrated that KBTBD7 was significantly upregulated following MI compared with in the sham group. Furthermore, high-dose crocin treatment significantly downregulated KBTBD7 mRNA expression levels compared with those in the MI group (Fig. 4A). In RAW264.7 cells, rmHMGB1 induction significantly upregulated KBTBD7 mRNA expression levels compared with in the control, whereas crocin inhibited KBTBD7 mRNA expression levels in rmHMGB1-treated cells (Fig. 4B). Similar results were also determined in the rmHSP60-induced RAW264.7 cells (Fig. 4C). These results therefore suggested that KBTBD7 is a target of crocin.

The effect of KBTBD7 on DAMP-induced inflammatory cytokines in RAW264.7 cells was further observed. The transfection efficiency of si-KBTBD7 was first analyzed. The results demonstrated that si-KBTBD7 could significantly reduce mRNA expression levels of KBTBD7 compared with si-NC (Fig. 5A). Furthermore, the results demonstrated that KBTBD7 knockdown significantly decreased the levels of the rmHMGB1- and rmHSP60-induced inflammatory cytokines compared with si-NC (Fig. 5B and C).

The molecular mechanism by which KBTBD7 affects the inflammatory response was investigated. Western blotting was used to detect the expression levels of proteins associated with the MAPK and NF-κB pathways following rmHMGB1, rmHSP60 and si-KBTBD7 treatments. The results demonstrated that the protein expression levels of p-p38, p-JNK and p-p65 were significantly increased following rmHMGB1 and rmHSP60 treatment compared with those in the control group. However, KBTBD7 knockdown significantly reduced the protein expression levels of p-p38, p-JNK and p-p65 compared with the rmHMGB1 and rmHSP60 only treatments (Fig. 6A and B). These results indicated that rmHMGB1 and rmHSP60 stimulation activated the p38 and NF-κB signaling pathways, whereas KBTBD7 knockdown potentially inhibited the production of inflammatory cytokines.