Differentiation of embryonic stem cells into cardiomyocytes used to
investigate the cardioprotective effect of salvianolic acid B through BNIP3
involved pathway
Chih-Yang Huang 1,2,3, Shao-Yu Chen1, Ru-Huei Fu4,5, Yu-Chuen Huang3,6, Shih-Yin Chen3,6, Woei-Cherng Shyu4,5, Shinn-Zong Lin4,5,7,8, Shih-Ping Liu1,5,9
1Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan 2Department of Health and Nutrition Biotechnology, Asia University, Taichung, Taiwan 3Graduate Institute of Chinese Medical Science, College of Chinese Medicine, China Medical University, Taichung, Taiwan
4Graduate Institute of Immunology, China Medical University, Taichung, Taiwan 5Center for Neuropsychiatry, China Medical University Hospital, Taichung, Taiwan
6Genetics Center, Department of Medical Research, China Medical University Hospital, Taichung, Taiwan
7Department of Neurosurgery, China Medical University Beigan Hospital, Yunlin, Taiwan 8Department of Neurosurgery, Tainan Municipal An-Nan Hospital-China Medical University,
Tainan, Taiwan
9Department of Social Work, Asia University, Taichung, Taiwan
Address correspondence to Shih-Ping Liu, PhD, No. 2, Yuh-Der Road, Taichung, Taiwan
40447, Republic of China. Phone: 886-4-2205-2121 ext. 7828; Fax: 886-4-2205-2121 ext.
7810; spliu@mail.cmu.edu.tw.
ABSTRACT
Cardiovascular diseases are related to many risk factors, such as diabetes, high blood pressure, smoking, and obesity. Myocardial infarction (MI), a cardiovascular disease, is the most common cause of cardiomyocyte death. In MI, hypoxia induces cardiomyocyte apoptosis; in particular, diabetes combined with MI has a synergistic effect that exacerbates cardiomyocyte death. The hypoxia-inducible factor-1α (HIF1α) transcriptional factor and a BH-3 only protein, Bcl-2 adenovirus E1B 19 kDa interacting protein 3 (BNIP3), are known to play fundamental roles in both adaptive and cell-death processes in response to hypoxia. In addition, most cardioprotective studies used H9c2 cells that were not beating so H9c2 cells
may not be the best models for testing cardioprotective effects. Embryonic stem (ES) cells are pluripotent stem cells that are able to differentiate into several types of cells, including cardiomyocytes. In this study, we reveal a simple method to differentiate ES cells into cardiomyocytes by using poly - D - lysine coated plates combined with ITS and N2 containing
medium and characterized the ES cell-derived cardiomyocytes by cardiomyocyte marker
staining. The ES cell-derived cardiomyocytes were used to investigate the protective effect of salvianolic acid B (Sal-B) in high glucose combined with hypoxic conditions to mimic diabetes patients with ischemia. The results of MTT and TUNEL assays indicate that Sal-B suppresses the apoptotic effect of treatment with high glucose combined with hypoxia in ES cell-derived cardiomyocytes. In particular, Sal-B inhibited HIF1α, BNIP3, and cleavage-caspase 3 expression levels, thereby suppressing apoptosis. This is the first study to mention the correlation between BNIP3 and Sal-B for cardioprotective effects. In
conclusion, we suggest that Sal-B may be suitable for use as a future cardioprotective medicine.
INTRODUCTION
Several diseases, including stroke, myocardial infarction (MI), diabetes mellitus, and multiple organ failure, cause cell death by inducing hypoxia (14,20). In the heart, atherosclerosis causes severe ischemic heart disease (IHD) and MI, which lead to myocardial hypoxia (232). Cardiomyocytes undergoing apoptosis have previously been observed in the myocardium under a variety of pathological conditions such as dilated cardiomyopathy (DCM), ischemia/reperfusion injury, MI, and end-stage heart failure (28). Further evidence indicated that cardiomyocyte apoptosis contributes to MI progression in human (18) and animal (1,5,29) models. In previous studies, hypoxia was demonstrated to enhance apoptosis in primary cultures of cardiomyocytes (1,33).
Diabetes is a disease that affects the manner in which our bodies digest food for energy. In diabetic patients, the pancreas does not produce enough insulin (Type I) and/or cells in the liver, muscles, and fat (Type II) (21). In addition insulin is not properly used. As a result, the amount of glucose in the blood increases while the cells are starved of energy, causing high morbidity in diabetes (13). Moreover, there are many complications of diabetes, such as retinopathy, nephropathy, peripheral neuropathy, stroke (3), autonomic neuropathy, and heart disease; in particular, coronary heart disease is recognized to be the cause of death in 80% of people with diabetes. Diabetes can change the makeup of blood vessels (34), and this can lead to cardiovascular disease.
Previous studies revealed the probable mechanism of hypoxia-induced apoptosis: hypoxia stabilizes hypoxia-inducible factor-1α (HIF-1α) protein and increases nuclear translocation of this transcription factor, which further induces downstream proteins including Bcl-2 adenovirus E1B 19 kDa-interacting protein 3 (BNIP3) (41). At a later stage, BNIP3 forms stable homodimerization complexes that are inserted into the mitochondrial membrane, resulting in mitochondrial damage that promotes mitochondrial-dependent apoptosis (12,15,20).
BNIP3 belongs to the Bcl-2 homology domain, which includes (BH3)-only members of the Bcl-2 family and its domain structure containing the BH3 domain, conserved domain (CD), transmembrane domain (TM), and conserved Cys residue involved in stabilization of the BNIP3 homodimers (6,8,38). The C-terminal TM domain is a major functional domain found in BNIP3 that forms homodimers or heterodimers with Bcl-2 and Bcl-XL (2,31). Furthermore, BNIP3 localizes to the outer mitochondrial membrane and promotes cell death through its TM domain (4,7). The insertion of BNIP3 results in permeability transition (PT) pore opening and loss of mitochondrial membrane potential (ΔΨm), leading to mitochondrial dysfunction and cell death including: apoptosis (11), necrosis (10,27), and autophagic cell death (9,19).
Embryonic stem (ES) cells are pluripotent stem cells derived from the inner cell mass of blastocysts (243). Additionally, under certain conditions, ES cells are capable of indefinite propagation (39). This allows ES cells to be employed as useful tools for both research and regenerative medicine, because limitless numbers can be produced, and we can
differentiate ES cells into several cell types such as cardiomyocytes. Therefore, we attempted to determine a simple method to differentiate ES cells into cardiomyocytes in this study by using poly-D-lysine coating, three different kinds of culture media, and characterization by immunofluorescence (IF) analysis.
In China, Radix Salvia miltiorrhiza (Danshen) has been widely used to treat cardiovascular diseases for hundreds of years (16). Salvianolic acids are the most abundant water-soluble compounds extracted from Danshen. Salvianolic acids, especially salvianolic acids A and B (Sal-A and Sal-B, respectively), have been found to have potent anti-oxidative capabilities (16). Sal-B has also been reported to protect cardiomyocytes (37). However, the correlation between Sal-B and BNIP3 was unclear regarding cardioprotective effects.
H9c2 cells were the most common cell line used for the cardioprotective experiments.
However, H9c2 cells were suggested to be dissimilar to primary cardiomyocytes because
H9c2 cells do not beat. For this reason, ES cell-derived cardiomyocytes were the most
suitable cells for studying cardioprotective effects. In this manuscript we reveal a simple
method to differentiate ES cells into cardiomyocytes by employing poly-D-lysine coated
plates and using three different types of media to test the differentiation efficiency.
In addition, to investigate the protective effect of Sal-B, we used high glucose combined with hypoxia treatment in ES-derived cardiomyocytes to mimic diabetes patients with ischemia and treated them with Sal-B. Previous studies reported that HIF-1α transcription factor and BNIP3 protein expression increase production of cardiomyocytes under hypoxia (12,30). Specifically, our aim was to reveal the regulatory mechanisms
among hypoxia-related proteins such as HIF-1α, BNIP3, and cleaved caspase 3 in ES-derived cardiomyocytes under excessive hypoxia and high glucose conditions.
MATERIALS AND METHODS
Mouse embryonic fibroblast and ES cell cultures
Mouse embryonic fibroblast (MEF) isolation was performed as previously described (265). Cells were isolated from 13.5-d-old C57BL/6 mice embryos that were retrieved by Cesarean section of a pregnant mouse from Taiwan National Laboratory Animal Center (Taipei, Taiwan). Internal organs, legs, and heads were removed, and the remaining parts of the embryo were digested with trypsin (Gibco-BRL, Grand Island, NY, USA). Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% heat-inactivated fetal bovine serum (FBS), penicillin (100 U/mL), streptomycin (100 μg/mL), nonessential amino acids (0.1 mM), and L-glutamine (2 mM) in a humidified incubator (37°C) with 5% CO2 (all reagents were from Gibco-BRL). Mouse ES cells (from Taiwan Bioresource Collection and Research Center, Hsinchu, Taiwan) were cultured in DMEM with 15% FBS (Hyclone, Logan, UT, USA), nonessential amino acids (0.1 mM), L-glutamine (2 mM), β-mercaptoethanol (0.2 mM; Gibco-BRL), and Leukemia inhibitory factor (LIF) (4 ng/mL; Millipore, Billerica, MA, USA) in a humidified incubator (37°C) with 5% CO2. All experimental protocols were approved by the Institutional Animal Care and Use Committee of China Medical University.
Poly-D-lysine coating and three culture media for differentiation experiment
that is soluble in water; therefore, poly-D-lysine was recommended as a cell culture substrate when using 0.1 mg/mL solution to coat culture dishes or plates. In addition, we used three types of media to test differentiation efficiency: 1. LIF-out medium, comprised of DMEM with 15% FBS (Hyclone), nonessential amino acids (0.1 mM, Gibco-BRL), L-glutamine (2 mM, Gibco-BRL), β-mercaptoethanol (0.2 mM; Gibco-BRL); 2. ITS medium, comprised of DMEM/F12 (Hyclone), L-glutamine (2 mM; Gibco-BRL), ITS (1%, Invitrogen, Carlsbad, CA, USA), fibronectin (5 μg/mL, R&B); and 3. N2 medium, comprised of DMEM/F12 (Hyclone), L-glutamine (2 mM; Gibco-BRL), N2 (1%, Gibco-BRL), and basic fibroblast growth factor (bFGF) (20 ng/mL, Invitrogen, Carlsbad, CA, USA).
Six strategies to differentiate ES cells into cardiomyocytes
In the differentiation experiment, ES cells were harvested by trypsinization and transferred to ultra-low attached culture dishes in LIF-out medium. After 3 d, aggregated cells were separated to six groups: 1. poly-D-lysine-coated culture dishes and LIF-out medium for 6 d; 2. poly-D-lysine-coated culture dishes and ITS medium for 6 d; 3. poly-D-lysine-coated and ITS medium for 2 d and then N2 medium for 4 d; 4. non-poly-D-lysine-coated culture dishes and LIF-out medium for 6 d; 5. non-poly-D-lysine-coated culture dishes and ITS medium for 6 d; and 6. non-poly-D-lysine-coated culture dishes and ITS medium for 2 d and then N2 medium for 4 d. Cell types were characterized in the six groups of cells by IF analysis.
IF analysis
troptonin (Millipore) were used to characterize the ES cell-derived cardiomyocytes. Briefly,
cells cultured in plates were fixed with 4% paraformaldehyde (Sigma-Aldrich) in phosphate buffered saline (PBS) (Gibco-BRL) for 15 min and permeabilized with 0.1% Triton X-100 (Tedia, Fairfield, OH, USA) in 0.1% sodium citrate for 2 min. The cells were rinsed with 1× PBS and blocked for 60 min with blocking buffer. The cells were then incubated with the indicated dilution of the antibody (1:100) for 24 h at 4°C, washed with 1× PBS, incubated with a secondary fluorescein isothiocyanate (FITC)-conjugated antibody (Invitrogen) (1:500 dilution) for 1 h at 37°C, rinsed, and mounted. The cellular nuclei were stained with 4 ,6-′ diamidino-2-phenylindole (DAPI, Sigma-Aldrich) to observe the markers.
Examination of protein expression by western blotting
ES-derived cardiomyocytes were plated onto 6-well plates, treated with high glucose, incubated at 37°C for 12 h, and then placed into a hypoxic environment for another 24 h. To isolate total proteins, cells were washed with cold PBS and resuspended in lysis buffer (50 mM Tris, pH 7.5, 0.5 M NaCl, 1.0 mM EDTA, pH 7.5, 10% glycerol, 1 mM BME, 1% IGEPAL-630, and a proteinase inhibitor cocktail; Roche Molecular Biochemicals, Mannheim, Germany). After incubation for 30 min on ice, the supernatant was collected by centrifugation at 12000 × g for 15 min at 4°C. The protein concentration was determined using the Bradford method (Bio-rad, Hercules, CA, USA). Samples containing equal amounts of protein (50 μg) were loaded and analyzed by western blot analysis. Briefly, proteins were separated by 10% SDS-PAGE and transferred onto PVDF membrane (Millipore). Membranes
were blocked with blockingbuffer (5% non-fat dry milk, 20 mM Tris-HCl, pH 7.6, 150 mM NaCl, and 0.1% Tween 20) (Sigma-Aldrich) for at least 1 h at room temperature. Membranes were incubated with primary antibodies (HIF-1α, and β-actin (1:100 dilutions): Santa Cruz Biotechnology, Santa Cruz, CA, USA; BNIP3 and cleaved-caspase-3 (1:100 dilutions): Cell Signaling, Beverly, MA, USA), in the above solution on an orbit shaker at 4°C overnight. Following primary antibody incubation, membranes were incubated with horseradish peroxidase-linked secondary antibodies (anti-rabbit, anti-mouse, or anti-goat IgG) (1:500 dilutions) (GeneTex, San Antonio, TX, USA). A western blotting substrate kit (Thermoscientific, Waltham, MA, USA) was used to stain the membranes, and signals were obtained using a UVP BioSpectrum Imaging System (Upland, CA, USA).
Sal-B treatment and MTT assay
Sal-B from Salvia miltiorrhiza extract (mol weight 718.61) was purchased from ChromaDex, Inc. (Irvine, CA, USA). Sal-B was dissolved in pure water to a concentration of 1 μM and stored at −20°C until use as a stock solution. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assays (Sigma-Aldrich) were performed using 0.0125, 0.025, 0.05, and 0.1 nM concentrations. The same concentrations were used for determining gene expression profiles and maintaining stem cell self-renewal.
RNA extract and Real-Time PCR
TRIzol (Invitrogen) was used to extract total RNA from MEF and ES cells. RNA concentrations were determined by spectrophotometry. Complementary DNA was produced from mRNA
(5 μg) using a SuperScript III Reverse Transcriptase Kit (Invitrogen). Real-time PCR was performed as previously described (2226) to determine the expression levels of BNIP3 (primer sequence: forward-CCTTCCATCTCTGTTACTGTCTCATC; reverse-TCAGACGCCTTCCAATGTAGATC) and GAPDH (primer sequence: forward-TGGTATCGTGGAAGGACTCA; reverse-AGTGGGTGTCGCTGTTGAAG).
TUNEL assay
The TUNEL (TdT-mediated digoxigenin-dUTP nick-end labeling) method was carried out with a commercially available In Situ Apoptosis Detection Kit (Roche Molecular Biochemicals). To explore the Sal-BTMP effect on hypoxia-induced cardiomyocytes. Apoptosis staining was performed according to the manufacturer’s protocol. TUNEL-positive cells were identified with a fluorescent microscope (Zeiss, Oberkochen, Germany) using an excitation wavelength in the 450–500 nm range and a detection wavelength in the 515–565 nm range (green). The percentage of apoptotic cells was calculated by dividing TUNEL-positive cells by the total number of cells visualized in the same field. Three digitized images with similar total cell numbers were selected from each cover slip, counted, averaged, and considered one independent experiment. Three independent experiments were then averaged and statistically analyzed.
Gene overexpression through transient transfection
Cells with 50% confluence were placed into fresh culture medium containing serum 2 h before transient transfection. HIF-1α and BNIP3 genes containing plasmids (gift from Prof
Chih-Yang Huang) were then transfected into the cells for 24 h using the Fugene HD (Roche Molecular Biochemicals) following the manufacturer’s protocol.
Statistical Analysis
Results are presented as mean ± standard error (SE). One-way analysis and Tukey’s test were used to compare the means among different treatments. Statistical analyses were performed using SPSS software package (version 18.0) and p < 0.05 were considered significant.
RESULTS
Induction of ES cells into cardiomyocytes
In this study, we attempted to determine a simple method to differentiate ES cells into cardiomyocytes for future clinical application. The three steps differentiation strategy was used (Figure 1A). ES cells were passaged for embryoid body formation and then cultured on poly-D-lysine coating culture plates. We discovered that poly-D-lysine differentiates ES cells into cardiomyocytes. This finding was accidental because we initially aimed to obtain neural cells from ES cells. However, we observed some of the cells were “beating” and the morphology was similar to cardiomyocytes (Figure 1B).
Comparison of six differentiation strategies for inducing ES cells into cardiomyocytes We used six differentiation strategies to evaluate the differentiate efficiency of ES cells into cardiomyocytes, which are described in the Materials and Methods. As shown in Figure 2,
the beating percentage after culture using the six differentiation strategies demonstrates that poly-D-lysine induced the ES cells into cardiomyocytes compared with non-poly-D-lysine coated groups (Figure 2A). In addition, ITS medium and N2 medium also induced ES cells to differentiate into cardiomyocytes. The beating percentage of the cells in the best performing group, poly-D-lysine-coated and ITS medium for 2 d and then N2 medium for 4 d (group 3), was about 80%.
In the IF analysis, we used Desmin, Actinin and Troptonin as the cardiomyocyte markers to characterize the ES cell-derived cardiomyocytes. The results showed that the cells positively expressed Desmin, Actinin and Troptonin in three poly-D-lysine coating groups (Figure 2B). The western blot data also showed that Desmin expression levels in the poly-D-lysine coated groups were higher than non-poly-D-lysine coated groups (Figure 2C). These results indicate that we successfully differentiated the ES cells into cardiomyocytes using a simple method. The ES cell-derived cardiomyocytes were then used for the Sal-B cardioprotection experiment.
Sal-B inhibited the apoptosis of cardiomyocytes treated with high glucose plus hypoxia After we successfully differentiated the ES cells into cardiomyocytes, we used these cells for the Sal-B cardioprotection experiment. In the cell viability test, the cell survival rate of ES cell-derived cardiomyocytes treated with high glucose and/or hypoxia significantly decreased by MTT assay. However, after Sal-B treatment, the cell survival rate of ES cell-derived cardiomyocytes treated with high glucose combined with hypoxia significantly increased compared with the non-Sal-B treatment groups, especially at a concentration of
0.1 nM (Figure 3A). In the TUNEL assay, we found that the cell apoptosis rate of ES cell-derived cardiomyocytes treated with high glucose combined with hypoxia significantly increased, but was inhibited by 0.1 nM Sal-B treatment (Figure 3B). These data indicate that Sal-B inhibited high glucose combined with hypoxia-induced cardiomyocyte apoptosis.
Sal-B inhibited cell apoptosis by down-regulating HIF1α, BNIP3, and cleaved-caspase 3 In order to investigate the mechanism by which Sal-B inhibits cardiomyocyte apoptosis, we detected BNIP3 expression levels by real-time PCR. As shown in Figure 4A, the BNIP3 expression levels increased with high glucose and/or hypoxia treatment, while addition of 0.0125, 0.025, 0.05, and 0.1 nM Sal-B inhibited BNIP3 expression. The western blot data demonstrate that Sal-B inhibited HIF1α, BNIP3, and cleaved-caspase 3 levels and has a dose-response effect (Figure 4B). These data indicate that Sal-B inhibits cardiomyocyte apoptosis by down-regulating HIF1α, BNIP3, and cleaved-caspase 3 signaling pathways.
HIF1α and BNIP3 overexpression inhibits the effect of Sal-B
In order to confirm the mechanism by which Sal-B inhibits expression, we overexpressed
HIF1α and BNIP3 in ES cell-derived cardiomyocytes using plasmids containing HIF1α or
BNIP3. As previously stated, Sal-B inhibited HIF1α, BNIP3 and cleaved-caspase 3 levels. However, after HIF1α or BNIP3 were overexpressed, these effects disappeared (Figure 4C). These data confirm that Sal-B inhibits cell apoptosis by down-regulating HIF1α, BNIP3, and cleaved-caspase 3 signaling pathways (Figure 5).
DISCUSSION
Pluripotent stem cells, including ES cells and induced pluripotent stem (iPS) cells (254,32), have the ability to differentiate into several types of cells, including cardiomyocytes. In heart disease experiments, the H9c2 cell line is commonly used (40). However, H9c2 cells may not be the best cell model for cardiovascular disease research. For this reason, we attempted to obtain cardiomyocytes from ES cells that were more similar to real cardiomyocytes. In a previous study, TGF-β1 was reported to induce cardiomyocyte differentiation (36), but there was no mention of whether poly-D-lysine could induce cardiomyocyte differentiation. In this paper, we successfully established a simple method to differentiate ES cells into cardiomyocytes (Figure 1). In particular, ES cell-derived cardiomyocytes are very important for future heart disease research because these cells are more similar to real cardiomyocytes than the currently used cells.
In heart disease research, chronic hypoxia in the presence of high glucose leads to progressive acidosis of cardiomyocytes in culture. A previous study demonstrated that HIF-1α is an important transcription factor in the ischemic embryonic rat heart and is increased following high glucose treatment (17). Another study indicated that hypoxia/acidosis genes such as BNIP3, which is downstream of HIF-1α in hypoxia conditions, are important (41). In this study, we were interested in the correlation between Sal-B and cardioprotective effects, specifically in the HIF1α and BNIP3 signaling pathway. Sal-B is a pure compound that is isolated from Danshen, a Chinese herbal medicine that has been widely used to treat cardiovascular diseases for hundreds of years (16). Sal-B has been found to have potent
anti-oxidative capabilities (16); moreover, it has been reported that Sal-B has a protective effect on cardiomyocytes (37), but not under conditions of high glucose combined with hypoxia. In this study, we demonstrated that Sal-B increased the cell survival rate in cardiomyocytes that were treated with high glucose combined with hypoxia (Figure 3A). This effect occurred through the anti-apoptosis pathway (Figure 5).
A previous study also indicated that Sal-B inhibited apoptosis in cardiomyocytes (35,37) by the PI3K/Akt pathway. In this study, we focused on the HIF1α/BNIP3 signaling pathway. Our data indicated that Sal-B inhibited apoptosis by down-regulating HIF1α, BNIP3, and cleaved-caspase 3 (Figure 4). This is the first study to determine that Sal-B downregulates
BNIP3, which affects apoptosis. We thus confirmed the effect of Sal-B in animal models
suffering from MI.
In summary, we demonstrated the cardioprotective effect of salvianolic acid B through BNIP3 involved pathway; moreover, Sal-B can inhibit the effect of high glucose combined with hypoxia-induced cell apoptosis via down-regulation of HIF1α, BNIP3, and cleaved-caspase 3 expression. Consequently, Sal-B could potentially be a powerful medicine for cardiovascular disease in the future.
ACKNOWLEDGMENTS: This study is supported in part by Taiwan Ministry of Health and Welfare Clinical Trial and Research Center of Excellence (MOHW104-TDU-B-212-113002) and China Medical University (DMR-103-055). The authors declare no conflicts of interest.
FIGURE CAPTIONS
FIG. 1. Induction of ES cells into cardiomyocytes. A. The three-step strategy to differentiate ES cells into cardiomyocytes. B. The morphology of the ES cells, embryoid body, and cardiomyocytes.
FIG. 2. Comparison of six differentiation strategies for inducing ES cells into cardiomyocytes. A. The beating percentages of the cells using six differentiation strategies. B. The cardiomyocyte markers Desmin, Actinin and Troptonin were used to stain the cells of the six differentiation strategies. * P < 0.01 versus the poly-D-lysine coating combined with LIF-out group. C. The Desmin protein expression levels of the six differentiation strategies
by western blot analysis.
FIG. 3. ES cell-derived cardiomyocyte viability under high glucose combined with hypoxia and Sal-B treatment. A. Cardiomyocytes viability decreased under high-glucose combined with hypoxia but increased with 0.1 nM Sal-B treatment. * P < 0.05 and ** P <0.01 versus the non-treatment control group. # P<0.05 and ## P<0.01 vers us the hypoxia combined with high glucose group. B. Cardiomyocyte apoptosis rate by TUNEL assay. Cardiomyocyte apoptosis rate increased when suffering from high glucose and hypoxia conditions while inhibited by Sal-B treatment. * P < 0.05 and ** P <0.01 versus the control group. # P<0.01 versus the hypoxia combined with high glucose group.
glucose combined with hypoxia. A. Sal-B treatment decreased the BNIP3 RNA expression level of cardiomyocytes under high glucose combined with hypoxia conditions, as determined by real-time PCR. * P<0.05 versus the hypoxia combined with high glucose group. B. The protein levels of HIF-1a, BNIP3, and cleavage-caspase 3 in the cardiomyocytes under high glucose and hypoxia conditions, as determined by western blot. C. Overexpression of HIF1α and BNIP3 inhibited the Sal-B protective effect in cardiomyocytes under high glucose and hypoxia conditions, as determined by western blot.
Figure 5. Model for the Sal-B involved in cardioprotective effect.
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