The cell extraction protocol for cardiomyocyte primary culture was approved by the Institutional Animal Care and Use Committee of Wuhan University (Wuhan, China). A total of 100 Sprague-Dawley rats (1-3-day-old) were purchased from the Centre of Experimental Animals at Wuhan University (Wuhan, China). Primary cultures of neonatal rat cardiomyocytes were prepared from the ventricles of rats. Briefly, the hearts were harvested and finely minced with scissors for about 1 min and then dissociated with 0.125% (w/v) trypsin and 0.08% collagenase I for five times at 37°C. The collected enzyme solution is next centrifuged (170 × g, 4 min, 4°C), the supernatant discarded, and cells re-suspended in Dulbecco's modified Eagle's medium (DMEM, Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) containing 15% (v/v) fetal bovine serum (FBS), 1% penicillin (100 U/ml) and 1% streptomycin (100 µg/ml). To reduce fibroblast contamination, cells were incubated at 37°C and 5% CO₂ for 1 h, then non-adherent cells (cardiomyocytes) in DMEM were collected and seeded in six-well plates at a density of 1×10⁶ cells/ml. Cardiomyocytes were incubated for 4 days prior to conducting the experiment, then the cells were randomly assigned into six groups. The group descriptions are as follows: Control group (Control), cardiomyocytes were incubated in DMEM F12 with 15% FBS; Hypoxia and reoxygenation (H/R) group, cardiomyocytes were incubated in DMEM following 24 h of synchronization, subjected to hypoxia for 1 h at 37°C and 95% N₂ at 5% (v/v) CO₂ prior to incubation in DMEM F12 for 4 h; H/R+anti-HMGB1 group, cardiomyocytes were pretreated with HMGB1-neutralizing antibody (1:100 dilution; #BM3965; Wuhan Boster Bioengineering Co, Ltd., Wuhan, China) at 37°C for 24 h before incubation in DMEM, and then were subjected to H/R, as described above; H/R+anti-IL-17A group, cardiomyocytes were pretreated with IL-17A-neutralizing antibody (1:100 dilution; #A00421; Wuhan Boster Bioengineering Co, Ltd.) at 37°C for 24 h before incubation in DMEM, and then were subjected to H/R, as described above; H/R+rHMGB1 group, cardiomyocytes were pretreated with recombinant HMGB1 (rHMGB1; 200 ng/ml) at 24 h before incubation in DMEM, and then were subjected to H/R, as described above; and the H/R+rIL-17A group, cardiomyocytes were pretreated with recombinant IL-17A (rIL-17A; 100 ng/ml) at 24 h before incubation in DMEM, and then were subjected to H/R, as described above.

The extent of cell injury was assessed by the concentrations of lactate dehydrogenase (LDH) and creatine kinase (CK) in the culture medium. The protocols were followed according to the manufacturer's instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China).

Cell viability was determined using the Cell Counting Kit (CCK)-8 assay (Dojindo Molecular Technologies, Inc., Kumamoto, Japan), and the experimental procedure was based on the manufacturer's recommendations. Cardiomyocytes were seeded in 96-well plates at 1×10⁵ cells/well and incubated for 4 d prior to treatments as described above. The absorbance of each well at 490 nm was measured with a microplate reader (Bio-Rad Laboratories, Hercules, CA). The percent cell viability was calculated using the following formula: % cell viability=(mean absorbance in test wells)/(mean absorbance in control wells) ×100.

Apoptosis rate was assessed by flow cytometric analysis of propidium iodide (PI) and Annexin V double staining. The cardiomyocytes were harvested after treatment, then rinsed in PBS and suspended in 500 µl binding buffer, and finally incubated with 5 µl Annexin V and 5 µl propidium iodide (Nanjing KeyGen Biotech. Inc., Nanjing, China). The stained cells were analyzed by using a BD flow cytometer (FACSCalibur; BD Biosciences, Franklin Lakes, NJ, USA).

The cultured primary cardiomyocytes were extracted to harbor protein solution in radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Nantong, China) after indicated experimental protocols. A BCA Protein Assay Kit (Wuhan Boster Biological Technology Co., Ltd.) was used to detect concentration of the proteins. Extracts containing 20 µg of protein were added into each well and subjected to 10% SDS-PAGE, which were then transferred onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). Subsequently, the membranes were blocked by 5% BSA for 60 min at 37°C, paralleling the incubation of primary antibodies overnight at 4°C with anti-HMGB1 (1:1,000 dilution; #SAB2701809; Sigma-Aldrich; Merck KGaA), anti-IL-17A (1:800 dilution; #13838; Cell Signaling Technology, Inc., Danvers, MA, USA), anti- microtubule-associated proteins 1A/1B light chain 3B (LC3) (1:600 dilution; #SAB1306269; Sigma-Aldrich; Merck KGaA), anti-Beclin-1 (1:800 dilution; #3738; Cell Signaling Technology, Inc.), anti-Bcl-2 (1:800 dilution; #15071; Cell Signaling Technology, Inc.) and anti-Bcl-2-associated X protein (Bax) (1:600 dilution; #2774; Cell Signaling Technology, Inc.). After being washed in Tris buffered saline with Tween 20 for three times (10 min), the membranes were exposed to horseradish peroxidase-conjugated secondary antibody (mouse anti-rat IgG antibody; 1:1,000 dilution; #bs-0293M; Beijing Bioss Biological Technology Co., Ltd, Beijing, China) at 37°C for 2 h. The enhanced chemiluminescence reagent (Pierce; Thermo Fisher Scientific, Inc., Waltham, MA, USA) was used to visualize protein blots. The expression of protein was normalized to β-actin (mouse anti-β-actin antibody; 1:500 dilution; #bsm-33036M; Beijing Bioss Biological Technology Co., Ltd) expression. Image Lab software 3.0 (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was adopted to measure the grey value of each band.

Statistical analysis was conducted with SPSS 18.0 software package (SPSS Inc., Chicago, IL, USA). All data are expressed as the mean ± standard deviation. One-way analysis of variance or the Welch test was used for comparisons among groups, and the Student-Newman-Keuls test or Dunnett's T3 test was used for post-hoc multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.

As demonstrated in Fig. 1A, cardiomyocytes subjected to hypoxia and reoxygenation were able to cause the significant release of LDH and CK, when compared with the control group (P<0.05). Furthermore, rHMGB1 or rIL-17A increased the levels of LDH and CK, while HMGB1 or IL-17A antibody were able to suppress their release (all P<0.05 vs. H/R group).

The authors assessed cell viability with CCK-8 assays in each groups. The cell viability was markedly inhibited following H/R treatment, when compared with the control group (P<0.05). Interestingly, pretreatment with HMGB1 or IL-17A antibody significantly improved cell viability, while rHMGB1 or rIL-17A could inhibit the cell viability (all P<0.05 vs. H/R group). These findings indicated that HMGB1 or IL-17A could inhibit the cell viability in cardiomyocyte subjected to H/R (Fig. 1B).

Annexin-V FITC/PI staining was used to quantify the pro-apoptotic effects of HMGB1 and IL-17A. The apoptosis rate in H/R group was significantly increased (P<0.05 vs. Control). However, pretreatment with HMGB1 or IL-17A antibody could significantly attenuate the elevated H/R-induced cardiomyocyte apoptosis, while rHMGB1 or rIL-17A could aggravate the cardiomyocyte apoptosis (all P<0.05 vs. H/R group) (Fig. 2A). In addition, Bcl-2 (anti-apoptotic protein) and Bax (pro-apoptotic protein) expression were also measured by western blotting (Fig. 2B). A significant decrease in Bcl-2 expression and a increase in Bax expression were observed in H/R group, when compared with the control group (P<0.05). Meanwhile, HMGB1 or IL-17A antibody could increase the expression of Bcl-2 and decrease the expression of Bax (both P<0.05 vs. H/R group). In contrast, treatment with rHMGB1 or rIL-17A could attenuate the increased expression of Bcl-2 and decreased the expression of Bax (both P<0.05 vs. H/R group). These results strongly demonstrated that HMGB1 or IL-17A could aggravate H/R-induced cardiomyocyte apoptosis.

It is well-understood that autophagy is regulated by autophagy-related proteins, such as, LC3-II/LC3-I and Beclin-1 (10). The ratio of LC3-II to LC3-I and the Beclin-1 expression were significantly increased in H/R group, when compared with the control group (P<0.05). The HMGB1 or IL-17A antibody could markedly decreased the ratio of LC3-II to LC3-I and the Beclin-1 expression when compared to H/R group, while those were even higher in H/R+rHMGB1 group or H/R+rIL-17A group (all P<0.05). It can be concluded that the autophagy was upregulated in cardiomyocytes exposed to H/R, and enhanced by HMGB1 or IL-17A prior to H/R (Fig. 3).

According to the western blotting, both HMGB1 and IL-17A expression in H/R group were markedly increased compared to those in control groups (P<0.05). Compared with the H/R group, IL-17A expression was significantly decreased in H/R+anti-HMGB1 group, while it was increased in H/R+rHMGB1 group (both P<0.05). Meanwhile, IL-17A antibody or rIL-17A had no significant effect on HMGB1 expression when compared to H/R groups (both P>0.05) (Fig. 4).