Ischemia-reperfusion (I/R) plays an important role in myocardial damage, which has been widely recognized as a key procedure in the cardiovascular disease. A hypoxia/reoxygenation (H/R) model was established using H9c2 cardiomyocytes to investigate the possible positive effect of oxymatrine (OMT), an alkaloid originating from the traditional Chinese herb
Ischemic heart disease (IHD) has become a major public human issue, with a decreasing age of onset. Coronary heart disease (CHD) is a leading cause of death all over the world according to the World Health Organization (
Oxymatrine (OMT), an alkaloid that originates from the traditional Chinese herb
The H9c2 cardiomyocyte cell line was provided by the Tianjin Key Laboratory of Hepatopancreatic Fibrosis and Molecular Diagnosis and Treatment, and were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.) with 4,500 mg/l glucose containing 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin. The H9c2 cardiomyocytes were grown in an incubator with 100% humidity containing 95% air and 5% CO2 at 37˚C.
The H/R model was established according to previously published methods (
Cell groups were established and the concentrations and durations of treatment chosen with reference to previously published methods (
Cell viability was analyzed using the 3-(4,5-dimethylthiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The H9c2 cardiomyocytes in the various groups were grown to a density of 1x104 cells/well on 96-well plates, 20 µl MTT (5 mg/ml) was then added to each well, and the cells were incubated at 37˚C with 5% CO2 for 4 h. The medium was removed, and 100 µl dimethylsulfoxide was added to the H9c2 cells in each well to dissolve the formazan crystals. Finally, the absorbance was read at 490 nm using a microplate reader (5200Multi; Tanon Science and Technology Co., Ltd.).
Cell morphology was observed with hematoxylin and eosin (H&E) staining. The cell supernatant of each group was discarded after 5 min of centrifugation at 132 x g at 4˚C. The H9c2 cardiomyocytes were sequentially washed with phosphate-buffered saline (PBS) and deionized water two or three times, incubated with hematoxylin for 5 min at room temperature, and then placed in eosin solution for 2 min at room temperature. Finally, the H9c2 cardiomyocytes were dried under ventilated conditions, and the cell morphology was observed using an optical microscope.
The severity of H9c2 cardiomyocyte injuries was evaluated by detecting the release of LDH into the cell supernatant. This was performed using an LDH kit (A020-2-2; Nanjing Jiancheng Bioengineering Institute), with measurement of the absorbance at 450 nm according to the manufacturer's instructions.
The MDA levels, SOD activity and CAT activity of the cells were determined after the various treatments. H9c2 cardiomyocytes from the different groups were collected, washed three times with cold PBS, and then cell lysis buffer (RABLYSIS1; Sigma-Aldrich; Merck KGaA)was added for cell lysis. Following centrifugation at 206 x g for 5 min at 4˚C the supernatant was collected for detection. MDA levels, SOD activity and CAT activity were measured with corresponding kits (cat. nos. A003-3-1, A001-3-2 and A007-1-1; Nanjing Jiancheng Bioengineering Institute) at absorbances of 530, 450 and 405 nm, respectively, according to the manufacturer's instructions.
The percentage of apoptotic cells in each group was analyzed using an Annexin V-FITC/PI apoptosis kit (Beijing 4A Biotech Co., Ltd.) for flow cytometry according to the manufacturer's instructions. Following treatment, the H9c2 cardiomyocytes from the different groups were collected and washed twice with cold PBS. Then, 5 µl Annexin V/FITC was added to the cells, which were then incubated for 5 min in the dark at room temperature for the labeling of early apoptotic cells. This was followed by incubation with 10 µl PI (20 µg/ml) for 10 min in the dark at room temperature to label late apoptotic cells. The analysis was performed using a flow cytometer (BeamCyte-1026; Changzhou Beamdiag Biotech Co., Ltd.), and quantitative processing was performed using FlowJo 10.6.2 software (FlowJo LLC).
The apoptosis of the H9c2 cardiomyocytes was also assessed using a TUNEL kit (KA4159; Abnova) according to the manufacturer's instructions. The H9c2 cardiomyocytes were fixed with xylene for 10 min at room temperature and washed with PBS three times. The cells were then blocked with FBS in a humid atmosphere at 37˚C for 60 min and incubated with the antibody from the kit at 4˚C overnight. Afterwards, the slides were rinsed with PBS three times, the TUNEL reaction mixture was added and the slides were incubated for 1 h at 37˚C in the dark. The apoptotic cells were incubated in the mounting medium containing 0.05% DAPI for 10 min in the dark and then analyzed under a fluorescence microscope; green fluorescence was observed at 520 nm with a standard fluorescence filter and blue DAPI was observed at 460 nm. Image-Pro Plus 6.0 software (Media Cybernetics) was used for quantification.
RT-qPCR was used to detect the expression of B cell lymphoma/leukemia-2 (Bcl-2), Bax, caspase-3, PI3K, Akt, GSK3β, Nrf2 and HO-1 in each group. TRIzol® (Invitrogen; Thermo Fisher Scientific, Inc.) was used to extract total RNA, and UV spectrophotometry was used to measure the purity. Then, RNA was reverse transcribed into cDNAs using a HiFiScript cDNA Synthesis Kit (CoWin Biosciences) according to the manufacturer's instructions. The cDNA templates were analyzed by qPCR using the UltraSYBR Mixture (Low ROX) kit (CoWin Biosciences) under the following conditions: 40 cycles of 10 sec at 95˚C, 30 sec at 60˚C and 32 sec at 72˚C. The nucleotide sequences of the forward and reverse primers are shown in
H9c2 cardiomyocytes from the various groups were washed three times with PBS, and then lysed in complete RIPA buffer (R0020; Beijing Solarbio Science & Technology Co., Ltd.) at 4˚C for 20 min. The total protein concentrations were determined using a BCA kit (A045-4-2; Nanjing Jiancheng Bioengineering Institute). Equal amounts of protein from each group (30 µg) were loaded onto 10% polyacrylamide gels for electrophoresis and transferred to nitrocellulose membranes (EMD Millipore). Then, the membranes were blocked with Tris-buffered saline and 0.05%Tween 20 buffer containing 5% skimmed milk for 3 h at room temperature, followed by incubation with the following primary antibodies overnight at 4˚C: Bax (cat. no. 50599-2-Ig; 1:2,000;), Bcl-2 (cat. no. 60178-1-Ig; 1:2,000), pro caspase-3 (cat. no. 66470-2-Ig; 1:1,000), cleaved caspase-3 (cat. no. 66470-2-Ig; 1:1,000), PI3K (cat. no. 20584-1-AP; 1:1,000), Akt (cat. no. 10176-2-AP, 1:1000), GSK3β (cat. no. 22104-1-AP; 1:1,000), Nrf2 (cat. no. 16396-1-AP, 1:1,000), HO-1 (cat. no. 16396-1-AP, 1:1,000) and β-actin (cat. no. 4970S; 1:1,000), all from ProteinTech Group, Inc.; phosphorylated (p-)PI3K (cat. no. bs-3332R, 1:1,000; BIOSS); p-Akt (cat. no. 4060s; 1:2,000; Cell Signaling Technology, Inc.) and p-GSK3β (cat. no. 9327s; 1:1,000; Cell Signaling Technology, Inc.). The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (cat. no. 7074V; 1:5,000; Cell Signaling Technology, Inc.) for 1 h at room temperature. Signals were observed using ECL reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Band densities were detected using ImageJ 1.52a software (National Institutes of Health).
Results are presented as the mean ± SD (n=10). Multigroup comparisons of the means were performed using one-way ANOVA followed by Tukey's post hoc test for multiple comparisons. SPSS version 25.0 (IBM Corp.) statistical software was used to perform the analysis. P<0.05 was considered to indicate a statistically significant result. All experiments were repeated three times.
H9c2 cardiomyocytes were treated with different concentrations of OMT for 12 h under normoxic conditions to explore the effects of OMT on these cells. As evidenced by the MTT assay, OMT did not exert marked cytotoxic effects or reduce the viability of H9c2 cardiomyocytes pretreated with 10, 30 or 50 µM OMT under normoxic conditions (
As shown in the images of H&E staining, the H9c2 cardiomyocytes in the control group (
The activities of SOD and CAT and the quantity of MDA in each group were detected using the corresponding kits, to investigate whether the protective effect of OMT on H9c2 cardiomyocytes exposed to H/R was associated with the suppression of oxidative stress. Compared with the control group, the activities of the antioxidants SOD and CAT were decreased and the content of the lipid peroxide marker MDA was increased in H9c2 cardiomyocytes in the model group, indicating that H/R injury increased the oxidative stress response. No differences in results were detected between the model group and the model + LY294002 group, indicating that LY294002 had no effect on the cells. Compared with the model group, the H9c2 cardiomyocytes pretreated with 10, 30 and 50 µM OMT exhibited significantly increased SOD and CAT activities and significantly decreased MDA content, suggesting that the protective effect of OMT was associated with the suppression of oxidative stress. The SOD and CAT activities and MDA content in the cells treated with LY294002 prior to OMT pretreatment were comparable with those in the model group. These results indicate that the OMT pretreatment protected H9c2 cardiomyocytes from H/R injury by preserving their antioxidant capacity, which may be associated with the PI3K/Akt signaling pathway (P<0.05;
TUNEL staining (
The PI3K/Akt/GSK3β and Nrf2/HO-1 signaling pathways were analyzed using western blotting and RT-qPCR to examine the molecular mechanism of OMT in H9c2 cardiomyocytes with H/R injury (
The Nrf2/HO-1 pathway is a crucial component of the antioxidant defenses against H/R injury. Western blots (
IHD is a serious threat to human health worldwide. Acute myocardial infarction (AMI) is one of the main diseases that constitute IHD. Patients with AMI often have a history of coronary atherosclerotic heart disease (CAD), and the basic pathological change in patients with CAD is atherosclerosis (
OMT is an alkaloid that has been widely used clinically and possesses various biological activities. OMT pretreatment has been shown to have a protective effect on cardiomyocytes exposed to I/R injury, but the protective mechanism has not been fully elucidated (
Under normal physiological conditions, the serum concentration of LDH is low, and LDH in cells is released only after cell membrane damage (
Oxidative stress is an imbalance between oxidant levels and antioxidant activity in the body. It is often accompanied by the infiltration of a large number of inflammatory cells and increased lipid oxidation and decomposition. Oxidative stress is considered one of the pathological processes that promotes apoptosis in I/R injury (
In-depth study of myocardial I/R injury has demonstrated that the Nrf2/HO-1 pathway, a downstream signaling pathway of the PI3K/Akt pathway (
The PI3K/Akt/GSK3β signaling pathway is an important pathway involved in the intracellular transduction of signals from transmembrane receptors that serve key roles in cell survival (
Apoptosis is a type of programed death characterized by morphological changes, such as cell shrinkage, nucleolysis and DNA fragmentation (
In summary, the present study provides new insights into the protective effects of OMT against myocardial I/R injury. The reperfusion injury salvage kinase signaling pathway, Nrf2/HO-1 signaling pathway and mitochondrial apoptosis pathway were used as entry points to clarify that the PI3K/Akt signaling pathway is involved in the protective effect of OMT on H9c2 cardiomyocytes subjected to H/R injury. The mechanism is hypothesized to be as follows: When H/R injury occurs in cardiomyocytes, upstream signaling activates PI3K via the stimulation of membrane receptors to transduce a signal in the cell. PI3K then transmits the extracellular signal to the downstream kinase Akt and activates it. Akt phosphorylates the downstream protein GSK3β to inactivate it, and finally apoptosis is inhibited via regulation of mitochondrial permeability. Concurrently, Nrf2, an important transcription factor downstream of the PI3K/Akt/GSK3β signaling pathway, is also activated by Akt, functions as an antioxidant and inhibits cell apoptosis by increasing the expression of the anti-apoptotic protein Bcl-2. When the H9c2 cardiomyocytes were exposed to H/R injury after OMT pretreatment, OMT significantly increased the expression of proteins involved in the Akt/GSK3β/Nrf2/HO-1 signaling pathway, while the PI3K inhibitor LY294002 blocked this biological effect. The occurrence of this phenomenon strongly suggests that the PI3K/Akt signaling pathway is involved in the protective effects of OMT. The OMT pretreatment protected H9c2 cardiomyocytes from H/R-induced cell damage, oxidative stress and cell apoptosis via a common upstream PI3K/Akt pathway. Based on these findings, OMT might be a potential candidate treatment for myocardial I/R injury.
Not applicable.
All data generated or used during the study are included in this published article.
ZZ, YL, WZ and MZ designed the experiments, conducted the experiments and wrote the manuscript. ZZ, FC and ZW performed RT-qPCR, MTT and H&E staining assays. XQ, RC and CL performed flow cytometry and TUNEL assays. ZZ, CL, ZW, RC and WZ analyzed the datasets and supervised the project. All authors reviewed the data and provided feedback on the manuscript. ZZ and MZ confirm the authenticity of all the raw data. All authors read and approved the final manuscript.
Not applicable.
Not applicable.
The authors declare that they have no competing interests.
OMT increases the viability of H9c2 cardiomyocytes exposed to H/R. (A) MTT assay was performed to detect the effect of OMT on the viability of H9c2 cardiomyocytes cultured under normal conditions and after treatment with various concentrations of OMT. (B) H9c2 cardiomyocytes were pretreated with OMT at different experimental concentrations in the presence or absence of LY294002 prior to H/R injury, and cell viability was measured using the MTT assay. (C) Cytotoxicity was also measured by determining the LDH content. *P<0.05 compared with the control group; #P<0.05 compared with the model group. OMT, oxymatrine; H/R, hypoxia/reoxygenation; LDH, lactate dehydrogenase; OD, optical density at 490 nm.
Representative images of H9c2 cardiomyocytes stained with hematoxylin and eosin. (A) The cells in the control group exhibited a long spindle-like morphology with a full cytoplasm and complete structure. (B) Cells in the model group exposed to H/R exhibited a marked loss of basic structure. After pretreatment with (C) 10, (D) 30 and (E) 50 µM OMT, the morphology of the cells gradually recovered in a concentration-dependent manner. (F) In the cells treated with LY294002 and exposed to H/R, no differences were observed compared the model. (G) LY294002 inhibited the protective effect of OMT on the cells. Magnification, x100. OMT, oxymatrine; H/R, hypoxia/reoxygenation.
OMT suppresses oxidative stress in H9c2 cardiomyocytes exposed to hypoxia/reoxygenation. Markers of oxidative stress, namely (A) MDA content, (B) SOD activity and (C) CAT activity were detected using kits. After pretreatments with 10, 30 and 50 µM OMT, the SOD and CAT activities were increased and the MDA content was decreased in a concentration-dependent manner compared with those the model group. However, LY294002 inhibited the changes induced by OMT. *P<0.05 compared with the control group; #P<0.05 compared with the model group. OMT, oxymatrine; MDA, malondialdehyde; SOD, superoxide dismutase; CAT, catalase.
OMT inhibits apoptosis in H9c2 cardiomyocytes exposed to hypoxia/reoxygenation. (A) The apoptosis of H9c2 cardiomyocytes in different groups was determined using the TUNEL assay. TUNEL-positive cells are green, and nuclei are stained blue with DAPI (magnification, x100). (B) Flow cytometric analysis of the apoptosis of H9c2 cardiomyocytes in the (a) control, (b) model, (c) 10 µM OMT, (d) 30 µM OMT and (e) 50 µM OMT pretreatment, (f) model with LY294002 and (g) OMT + LY294002 pretreatment groups. (C) Apoptosis rates determined using (a) TUNEL assay and (b) flow cytometry. (D) The levels of apoptosis-associated proteins, namely Bax, Bcl-2, pro caspase-3 and cleaved caspase-3, were detected using western blot analysis. (E) The mRNA expression levels of the apoptosis-associated proteins (a) Bcl-2, (b) Bax and (c) caspase-3 were measured using RT-qPCR. *P<0.05 compared with the control group; #P<0.05 compared with the model group. OMT, oxymatrine; Bcl-2, B cell lymphoma/leukemia-2.
OMT protects H9c2 cardiomyocytes exposed to H/R by activating the Akt/GSK3β/Nrf2/HO-1 pathway. (A) After H/R injury and treatment with different concentrations of OMT, the levels of proteins involved in the PI3K/Akt/GSK3β pathway were detected using western blotting. (B) The expression of (a) PI3K, (b) Akt and (c) GSK3β mRNAs measured using RT-qPCR. (C) In addition, the levels of Nrf2 and HO-1 proteins, which are downstream targets of the PI3K/Akt/GSK3β pathway, were detected using western blotting and (D) the expression of (a) Nrf2 and (b) HO-1 mRNAs were measured using RT-qPCR. *P<0.05 compared with the control group; #P<0.05 compared with the model group. OMT, oxymatrine; H/R, hypoxia/reoxygenation; GSK3β, glycogen synthase kinase-3β; Nrf2, nuclear factor erythroid-2-related factor 2; HO-1, heme oxygenase-1; PI3K, phosphatidylinositol 3-kinase; RT-qPCR, reverse transcription-quantitative PCR.
Sequences of the primer pairs used for quantitative PCR.
Primer | Sequence |
---|---|
Bcl-2 | F: 5'-ATAACCGGGAGATCGTGATGA-3' |
R: 5'-CTCTCAGGCTGGAAGGAGAAG-3' | |
Bax | F: 5'-CCACCAGCTCTGAACAGATCA-3' |
R: 5'-GCTCCATGTTGTTGTCCAGT-3' | |
Caspase-3 | F: 5'-GAGCAGAGTCAAAGGCTGGT-3' |
R: 5'-TGTCGTCATGTCCACCACT-3' | |
Nrf2 | F: 5'-TCCTCTGCTGCCATTAGTCA-3' |
R: 5'-GTGCCTTCAGTGTGCTTCT-3' | |
HO-1 | F: 5'-TCTGGAATGGAAGGAGATGC-3' |
R: 5'-AGTTCTGGGGCTCTGTTGC-3' | |
PI3K | F: 5'-GACTCCAAGATGAAGAAGATGTG-3' |
R: 5'-GAGCATTCGCAGGTCCAAGCC-3' | |
Akt | F: 5'-CGAGGCCCAACACCTTCATC-3' |
R: 5'-CCGGAAGTCCATCGTCTCCT-3' | |
GSK3β | F: 5'-CCAGGTGGAGGACCATTTGC-3' |
R: 5'-ACTCTACACCAGCAGCAGCC-3' | |
β-actin | F: 5'-TCAGGTCATCACTATCGGCAAT-3' |
R: 5'-AAAGAAAGGGTGTAAAACGCA-3' |
Bcl-2, B cell lymphoma/leukemia-2; PI3K, phosphatidylinositol 3-kinase; GSK3β, glycogen synthase kinase-3β; Nrf2, nuclear factor erythroid-2-related factor 2; HO-1, heme oxygenase-1; F, forward; R, reverse.