Myricitrin was provided from the Institute of Medicinal Plant Development (Beijing, China) (Sun et al., 2013a). Cell culture products were purchased from Gibco BRL (Grand island, NY, United States). The fluorescent dye JC-1 was purchased from Sigma-Aldrich (St. Louis, MO, United States). The Annexin V/propidium iodide (PI) apoptosis detection kit was obtained from Invitrogen Corporation (Eugene, OR, United States). Caspase-3 fluorometric assay kits were acquired from BioVision (Milpitas, CA, United States). Primary antibodies against Bcl-2, Bax, Caspase-3, and β-actin were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, United States). Primary antibodies against Hsp90, Akt, and p-Akt were purchased from Abcam (Cambridge, United Kingdom). Horseradish peroxidase (HRP)-conjugated secondary antibodies were obtained from CW Biotech (Beijing, China).

The H9c2 cardiomyocyte line was obtained from the Chinese Academy of Sciences Cell Bank (Shanghai, China) and cultured as previously described (Wang et al., 2014). Briefly, H9c2 cells were cultured in high glucose DMEM supplemented with 10% (v/v) fetal bovine serum, 1% penicillin/streptomycin (v/v), and 2 mM L-glutamine. The cells were maintained at 37°C with 100% relative humidity in a CO₂ incubator containing 5% CO₂.

The H/R procedures were modified from a previous study (Sun et al., 2013b; Wang et al., 2014). The H9c2 cardiomyocytes were cultured in an anaerobic glove box (Coy Laboratory, United States) under hypoxia for 6 h. Subsequently, the cells were removed from the anaerobic glove box and the medium was replaced with high glucose medium and maintained for 12 h in an incubator to mimic reperfusion. For all experiments, the cells were plated at an appropriate density according to the experimental design and grown for 24 h to reach 70 to 80% confluence prior to experimentation.

The cultured H9c2 cardiomyocytes were randomly divided into different groups. In the control (Con) group, the H9c2 cardiomyocytes were incubated under normoxic conditions for equivalent durations with high glucose DMEM. The H/R group was conducted as described in the preceding section. In the myricitrin-treated group (H/R+Myr), the H9c2 cardiomyocytes were pre-treated with myricitrin for 12 h prior to H/R. The inhibitor-treated groups were processed the same as the H/R+Myr group, but the cells were pre-incubated with 2 μM 17-AAG for 1 h, then removed the medium and treated with myricitrin. The appropriate concentration of the selective HSP90 inhibitor, 17-AAG, was determined on the basis of data found in the literature (Wu et al., 2012) and our preliminary experiments (Supplementary Figure 1).

Cell viability was determined using a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium (MTT) assay as previously described (Sun et al., 2012). Briefly, H9c2 cells were plated onto 96-well plates at a density of 1 × 10⁴ cells/well. After designated treatment, 20 μL of MTT (5 mg/mL) was added to each well and incubated for 4 h. The medium was subsequently removed, and the formazan crystals were dissolved in dimethyl sulfoxide (DMSO). The absorbance was read at 570 nm using a microplate reader (TECAN Infinite M1000, Austria).

The supernatant and cells were collected after the different treatments to measure the lactate dehydrogenase (LDH), catalase (CAT), glutathione peroxidase (GSH-Px) and malondialdehyde (MDA) levels as well as superoxide dismutase (SOD) activity using the corresponding detection kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions.

The effect of myricitrin on the intracellular ROS levels was monitored using the total ROS detection kit according to the manufacturer’s instructions (Enzo Life Sciences, Inc., Farmingdale, NY, United States). Briefly, the cells were preconditioned with myricitrin (40 μM) for 12 h, and further exposed to 6 h of hypoxia followed by 12 h reoxygenation. After treatment, the cells were harvested, placed into 5-mL round-bottom polystyrene tubes, and washed with 1× wash buffer. Subsequently, the cells were centrifuged for 5 min at 400 × g at room temperature and the supernatant was discarded. The cells were re-suspended in 500 μL of ROS detection solution, stained in the dark at 37°C for 30 min, and analyzed using flow cytometry (Sun et al., 2011).

We used 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) (Sigma-Aldrich, St. Louis, MO, United States) to analyze changes in the mitochondrial transmembrane potential as previously described (Sun et al., 2012). The cells were pre-incubated with 40 μM myricitrin for 12 followed by H/R procedure. After treatment, the cells were incubated with 2 μM JC-1 for 30 min in the dark and washed twice with phosphate-buffered saline (PBS). The cells labeled with JC-1 were analyzed by BD FACSCalibur flow cytometry using 488 nm excitation and green (525 nm) or orange-red (575 nm) emission wavelengths with CellQuest software. The JC-1 positive rate was expressed as the percentage of the red/green fluorescence intensity relative to that in the control group.

The percentages of early apoptosis and necrosis were measured using an Annexin V-FITC/PI apoptosis kit for flow cytometry according to the manufacturer’s instructions (Invitrogen). After treatment, the cells were harvested and washed twice with cold PBS and subsequently incubated with 5 μL of FITC-Annexin V and 1 μL of the PI working solution (100 μg/mL) in 100 μL of cell suspension for 15 min in the dark at room temperature. Cellular fluorescence was measured by flow cytometry analysis (FACS Calibur^TM, BD Biosciences, San Jose, CA, United States).

Caspase-9 activity (Sun et al., 2011) and caspase-3 activation (Sun et al., 2013a) were measured using the corresponding Fluorometric Assay Kits (BioVision, CA, United States) according to the manufacturer’s instructions. To assess caspase-9 activity, the cells were resuspended in lysis buffer and kept on ice for 10 min. Then, 50 ml of 2× reaction buffer containing 10 mM dithiothreitol was added to each sample with 5 ml of 1 mM substrate LEHD-AFC for 1.5 h at 37°C. The samples were determined using a microplate reader (TECAN Infinite M1000, Austria) at the excitation wavelength of 400 nm and the emission wavelength of 505 nm. For analysis of caspase-3 activation, the cells were harvested and incubated with 1 μL substrate FITC-DEVD-FMK for 1 h at 37°C, and then centrifuged at 3000 rpm for 5 min; the supernate was removed. After washing twice with cold PBS, the cells were re-suspended in 100 μL of wash buffer and then transfer the cell suspension to each well in the black microtiter plate. The fluorescence intensity was detected using microplate reader with excitation wavelength of 485 nm and emission wavelength of 535 nm.

After treatment, H9c2 cells were harvested, washed with PBS, and lysed with cell lysis buffer containing 1% phenylmethylsulfonyl fluoride. Lysate preparation and Western blot analysis were performed as previously described (Sun et al., 2012). The immunoblots were developed using an electrochemiluminescence (ECL) kit.

The molecular targets of myricitrin were predicted using Discovery Studio (DS) 4.5 (BIOVIA Software, Inc., San Diego, CA, United States). Molecular structure of myricitrin was submitted to Monte Carlo based conformational analysis (Discovery Studio’s FAST algorithm) allowing maximal 255 conformers with less than 20 kcal/mol above the energy minimum. PharmaDB is the only pharmacophore database implemented in DS 4.5. In the profiling with PharmaDB, all pharmacophore models with the shape of the binding pocket were selected for virtual screening using the default settings of the Ligand Profiler module of DS 4.5. The RIGID mode was used as the molecular mapping algorithm. The screening was conducted using the default settings and with a minimal inter-feature distance of 0.5 Å. After inputting all parameters, the job was run, and a result sheet was returned indicating which models were hits and how well myricitrin mapped into each model (fit value), respectively. A higher fit value represents a better fit (Rollinger et al., 2009; Yi et al., 2016).

The results are expressed as the means ± standard deviation. Comparisons were performed using Student’s t-test or one-way ANOVA followed by Tukey’s multiple comparison test using Prism 5.00 software. Statistical significance was set at p < 0.05. All data are the result of at least three independent experiments.

The protective effect of myricitrin against H/R-induced cell death was detected using an MTT assay. As shown in Figure 1A and Supplementary Figure 2, myricitrin pretreatment exhibited a strong protective effect after pretreatment for 12 h. The survival rate increased from 65.48 ± 6.42% (with H/R treatment alone) to 78.92 ± 5.41%, 83.56 ± 5.89%, and 87.48 ± 7.68% after treatment with 10, 20, and 40 μM myricitrin, respectively. LDH, which is secreted from cells after plasma membrane disruption, can be used as an indicator of cell injury. As shown in Figure 1B, myricitrin pretreatment decreased the LDH levels in the culture medium in a concentration-dependent manner (p < 0.05 or p < 0.01). This effect is consistent with its protective effects on cell viability, as assessed using an MTT assay.

Oxidant stress has long been associated with heart injury in response to I/R (Kalogeris et al., 2012). Membrane lipid oxidation is one of the primary events in oxidative damage, which can be assessed according to the degradation product MDA (Rodrigo et al., 2013). As shown in Figure 2A, the H/R group showed a significant increase in intracellular MDA levels, whereas a significant reduction in the levels of MDA was observed in the myricitrin pretreatment group compared with the H/R group. To confirm the antioxidative effects of myricitrin, the activities of the endogenous antioxidative enzymes SOD, CAT, and GSH-Px were detected. Myricitrin pretreatment significantly enhanced the SOD, CAT, and GSH-Px activities compared to the H/R group (Figures 2B–D, p < 0.05 or p < 0.01).

Based on flow cytometry staining, we examined the effect of myricitrin treatment on the intracellular ROS levels. As shown in Figures 2E,F, H/R significantly increased the ROS levels by nearly threefold compared with the control, as demonstrated by a shift in fluorescence from left to right. However, myricitrin pretreatment (40 μM) partially blocked this effect, suggesting that myricitrin pretreatment decreased the ROS levels.

Oxidative stress-induced apoptosis is associated with a loss of mitochondrial membrane potential (ΔΨm), an early marker of apoptosis (Sun et al., 2011). Mitochondrial depolarization was indicated as a decrease in the red/green fluorescence intensity ratio of JC-1 staining. The H/R group exhibited an increase in green fluorescence intensity, indicating ѱm dissipation (Figures 3A,B), while pretreatment with myricitrin attenuated ΔΨm depolarization, as indicated by the increased red fluorescence (p < 0.05). We further confirmed the anti-apoptotic effect of myricitrin through quantitative analysis of FITC-Annexin V/PI staining using flow cytometry. The percentage of apoptotic cells in the H/R group significantly increased from 1. 23 ± 0.56% to 8.56 ± 0.97% compared with the control group (Figures 3C,D). By contrast, pretreatment with myricitrin dramatically decreased the ratio of apoptotic cells to 3.89 ± 0.62%.

Caspase activity was also used to assess the level of apoptosis (Zhuang et al., 2000). As shown in Figures 4A,B, the caspase-9 activity and active caspase-3 levels markedly increased after H/R treatment. Myricitrin pretreatment significantly inhibited these effects (p < 0.05). To understand the mechanism of myricitrin treatment on the regulation of cell apoptosis, we measured the effects of myricitrin on the expression of apoptosis-related protein expression using Western blot analysis. As shown in Figures 4C,D, the H/R group showed a decrease in expression of the anti-apoptotic protein Bcl-2 and an increase in expression of the pro-apoptotic protein Bax. Pretreatment with myricitrin increased the Bcl-2/Bax expression ratio compared with the H/R group (1. 28 ± 0.31 vs. 0.46 ± 0.12, respectively).

The molecular targets of myricitrin were predicted using Discovery Studio software (Figure 5). The ranking according to fit score in descending order and the top 10 disease-related targets are shown in Table 1. Among these targets, we focused on heat shock protein 90 (Hsp90), a critical target in the protection of the myocardium from I/R injury.

Accumulating evidence has shown that upregulation of Hsp90 expression protects cardiomyocytes from I/R-induced damage (Wu et al., 2012). Thus, we detected the expression levels of Hsp90 in myricitrin–treated H9c2 cardiomyocytes. As shown in Figure 6A, when H9c2 cells were treated with 40 μM myricitrin for 0–12 h, a significant increase in the expression level of Hsp90 was observed. Next, we detected the effects of myricitrin on the Hsp90 expression levels in H/R-treated cardiomyocytes. Figure 6B shows that myricitrin treatment significantly inhibited the downregulation of the H/R-induced Hsp90 expression.

To determine whether such elevated expression of Hsp90 is responsible for the cell protective effect of myricitrin, a selective inhibitor of Hsp90, 17-AAG, was utilized. As shown in Figure 7, the Hsp90 inhibitor 17-AAG reversed the protection against H/R injury by decreased cell viability (Figure 7A), phosphorylation levels of the prosurvival protein Akt (Figure 7B), and mitochondrial membrane potential (Figures 7C,D). In addition, in the presence of 17-AAG, the decreased mitochondrial ROS levels induced by myricitrin were also abolished (Figures 7E,F). These results suggest that Hsp90 is involved in the protective effect of myricitrin against H/R injury in H9c2 cells.