H9C2 cardiomyocytes were purchased from The Cell Bank of Type Culture Collection of the Chinese Academy of Sciences. Cells were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 100 g/ml streptomycin, 100 g/ml penicillin and 10% FBS (Gibco; Thermo Fisher Scientific, Inc.), and maintained in a humidified atmosphere of 5% CO₂ at 37˚C. H9C2 cells were treated with 1.25, 2.5 or 5 µM butorphanol (1mg/mL, Butorphanol (Jiangsu Hengrui Medicine Co. Ltd.) for 24 h at 37˚C for further analysis. Cells were divided into the following four groups: i) H9C2 group, in which cells were cultured in basic culture medium; ii) LPS group, in which cells were treated with 10 µg/ml LPS from Escherichia coli O111:B4 (Sigma-Aldrich; Merck KGaA) for 24 h; iii) LPS + 1.25, 2.5 or 5 µM butorphanol group, in which cells were pretreated with 1.25, 2.5 or 5 µM butorphanol for 2 h prior to treatment with 10 g/ml LPS for 24 h (a preliminary experiment was conducted, which determined that 2 h was the appropriate pre-conditioning time of butorphanol); and iv) LPS + 5 µM butorphanol + KOR antagonist nor-binaltorphimine (Nor-BNI; Abcam) group, in which cells were pretreated with 10 µmol/l Nor-BNI (20) for 30 min prior to the treatment described for the LPS + 5 µM butorphanol group. After the experimental treatment, the cells were used for further analysis.

H9C2 cells were digested with Trypsin (cat. no. R001100; Gibco; Thermo Fisher Scientific, Inc.) and seeded into 96-well plates at a density of 3x10⁵ cells/well. Following treatment with 10 µg/ml LPS and 1.25, 2.5 or 5 µM butorphanol for 24 h, the cells were incubated with 10 µl CCK-8 reagent (MedChemExpress) for 4 h at 37˚C. The absorbance of each well was measured at a wavelength of 450 nm using a microplate reader. The cell viability was calculated using the formula: Cell viability (%) = [A_drug - A_blank/A_0drug - A_blank] x 100. A_drug, absorbance of wells containing cells, CCK8 solution and drug solution; A_blank, absorbance of wells containing medium and CCK-8 solution, without cells; A_0drug, absorbance of wells containing cells and CCK-8 solution, but no drug solution.

After H9C2 cells (1x10⁶ cells) were seeded into 6-well plates and treated as aforementioned, they were harvested and centrifuged at 3,000 x g for 5 min. The levels of tumor necrosis factor (TNF)-α (cat. no. ab181421), IL-1β (cat. no. ab214025) and IL-6 (cat. no. ab178013) in the H9C2 cell supernatant were detected using the respective commercial ELISA kits (Abcam) according to the manufacturer's protocols.

Total protein was extracted from H9C2 cells using RIPA lysis buffer (Beyotime Institute of Biotechnology). Protein concentration was quantified using a BCA assay kit (cat. no. ab102536; Abcam) according to the manufacturer's protocol. A total of 50 µg protein was loaded into each lane, separated by SDS-PAGE on 12% gels and subsequently transferred onto PVDF membranes (EMD Millipore), which were blocked with 5% skimmed milk at room temperature for 1 h. The membranes were then incubated with the following primary antibodies: Anti-phosphorylated (p)-P65 (cat. no. ab183559; 1:1,000; Abcam), anti-P65 (cat. no. ab32536; 1:1,000; Abcam), anti-cyclooxygenase 2 (cat. no. ab179800; 1:1,000; Abcam), anti-inducible nitric oxide synthase (cat. no. ab178945; 1:1,000; Abcam), anti-Bax (cat. no. ab32503; 1:1,000; Abcam), anti-cleaved caspase 3 (cat. no. ab32042; 1:1,000; Abcam), anti-cleaved poly(ADP)-ribose polymerase (PARP1; cat. no. ab32064; 1:1,000; Abcam), anti-Bcl-2 (cat. no. ab32124; 1:1,000; Abcam) and anti-GAPDH (cat. no. ab9485; 1:1,000; Abcam). Following incubation with the primary antibody at 4˚C overnight, the membranes were incubated with the corresponding HRP-conjugated secondary antibody: i) rabbit anti-mouse secondary (cat. no. SA5-10317; 1:10,000; Invitrogen; Thermo Fisher Scientific, Inc.) and ii) goat anti-rabbit (cat. no. A16104SAMPLE; 1:10,000; Invitrogen; Thermo Fisher Scientific, Inc.) at room temperature for 2 h. Protein bands were visualized using enhanced chemiluminescence reagent (Pierce; Thermo Fisher Scientific, Inc.) and analyzed using ImageJ software version 1.46r (National Institutes of Health). Protein expression levels were normalized to GAPDH expression levels.

H9C2 cells were harvested, washed with PBS three times and fixed with 4% paraformaldehyde at room temperature for 20 min. The TUNEL assay was then conducted using an In Situ Cell Death Detection Kit, Fluorescein (Roche Diagnostics) according to the manufacturer's protocol. The nuclei were counterstained with DAPI (0.1 µg/ml) at room temperature in the dark for 5 min. TUNEL-positive cells were visualized using a fluorescence microscope (Olympus Corporation) and the percentage of apoptotic cells was calculated in five randomly selected fields of view. The apoptotic rate was analyzed using ImageJ 1.52r software and calculated as follows: Apoptosis rate (%) = (Green fluorescence intensity/blue fluorescence intensity) x 100.

Total RNA was extracted from H9C2 cells treated with LPS using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). RNA concentration was measured using a spectrophotometer. Total RNA was reverse-transcribed into cDNA using a Reverse Transcription kit (Beijing TransGen Biotech Co., Ltd.) according to the manufacturer's protocol. qPCR was performed using TransScript II Green Two-Step qRT-PCR SuperMix (cat. no. AQ301-01; TransGen Biotech Co., Ltd.) on a QuantStudio 6 Flex Real-Time PCR system (Thermo Fisher Scientific, Inc.). The thermocycling conditions were as follows: Initial denaturation at 94˚C for 30 sec; 40 cycles at 94˚C for 5 sec, annealing at 55˚C for 15 sec and extension at 72˚C for 10 sec. The relative mRNA expression levels of KOR were quantified using the 2^-∆∆Cq method (21) and normalized to GAPDH. The primers used were as follows: KOR forward, 5'-GATGTG GATGTCATTGAATGCTCCTTGCAG-3' and reverse, 5'-GC TGTGCTGTGGGAGGTGCTGCCTAGAGCC-3'. GAPDH forward, 5'-AGGCCGGTGCTGAGTATGTC-3' and reverse, 5'-TGCCTGCTTCACCACCTTCT-3'.

Experiments were repeated at least three times and data are expressed as the mean ± SD. Statistical analysis was performed using GraphPad Prism software, version 7.0 (GraphPad Software, Inc.). Statistical differences among different groups were performed using one-way ANOVA followed by Tukey's post hoc test for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.

To determine the effects of butorphanol on normal cardiomyocytes, the viability of H9C2 cells was detected through CCK-8 assay after butorphanol treatment at different concentrations. The results of the CCK-8 assay revealed that H9C2 cell viability was not altered, which indicated that butorphanol did not affect the viability of normal cardiomyocytes (Fig. 1A). H9C2 cells were subsequently induced with LPS to establish an in vitro sepsis cell model. As shown in Fig. 1B, LPS significantly reduced the viability of H9C2 cells, whereas butorphanol treatment restored the reduced cell viability induced by LPS in a dose-dependent manner. Inflammation is an important process involved in sepsis-induced cardiac injury (22); thus, the levels of TNF-α, IL-1β and IL-6 were detected. As shown in Fig. 1C and D, LPS markedly elevated the levels of these inflammatory factors and upregulated the expression levels of inflammation-related proteins containing p-P65, iNOS and Cox2, whereas treatment with increasing doses of butorphanol gradually downregulated the levels of all inflammatory markers.

Apoptosis is an important process in the occurrence of SIMD (23); therefore, the effect of butorphanol on the apoptosis of LPS-induced H9C2 cells was investigated. As shown in Fig. 2, the apoptosis of H9C2 cells was significantly elevated by LPS; however, butorphanol treatment reduced the apoptotic rate of H9C2 cells. Concurrently, the expression levels of proapoptotic proteins (Bax, cleaved caspase 3 and cleaved PARP) were upregulated, whereas the expression levels of anti-apoptotic protein Bcl-2 were downregulated following LPS stimulation. These trends were all reversed following treatment of cells with butorphanol (Fig. 3A). These results suggested that butorphanol may suppress the apoptosis of LPS-induced H9C2 cells.

Butorphanol is known to act as a partial agonist of KOR in the G-protein activation signaling pathway (24). The results of western blot analysis revealed that LPS stimulation downregulated the expression levels of KOR in H9C2 cells, whereas the expression levels of KOR were upregulated following butorphanol treatment (Fig. 3B). Of note, treatment with 5 µM butorphanol upregulated the expression levels of KOR to the greatest extent. Additionally, 1.25 and 2.5 µm butorphanol significantly increased the expression level of KOR compared with LPS group. These results suggested that butorphanol may upregulate the expression of KOR in LPS-induced H9C2 cells.

It was subsequently hypothesized that butorphanol may exert its anti-inflammatory and anti-apoptotic effects by activating KOR. The LPS-induced increases in secretory levels of inflammatory factors and expression levels of inflammation-related proteins were reduced following butorphanol treatment, whereas further addition of Nor-BNI, an inhibitor of KOR, reversed this trend (Fig. 4A and B). Western blot analysis also revealed that butorphanol treatment downregulated the expression levels of proapoptotic proteins; Bax, cleaved caspase 3 and cleaved PARP; however, these trends were subsequently reversed by the addition of Nor-BNI (Fig. 4C). There was no significant difference in the expression levels of the anti-apoptotic protein Bcl-2 between the butorphanol and Nor-BNI groups. Taken together, these findings indicated that butorphanol may suppress the release of inflammatory factors and the apoptosis of LPS-induced H9C2 cells by upregulating KOR expression.