Dr Shipeng Dai, Department of Cardiology, Cangzhou Central Hospital, 16 Xinhua West Road, Yunhe, Cangzhou, Hebei 061000, P.R. China
The present study investigated whether the protective effect and mechanism of astragaloside IV (AS-IV) on heart failure (HF) involves small ubiquitin-like modifier (SUMO)-specific protease 1 (Senp1). Mouse HF was established by aortic constriction, inducing pressure overload. The model was confirmed by echocardiography 6 weeks after surgery. Mice were randomly divided into control, HF, HF+AS-IV, and AS-IV groups. Ventricular function was examined by echocardiography. Morphological changes of myocardial tissues were examined by H&E staining. The protein levels of the apoptosis-related proteins, cleaved caspase-3, caspase-3, Bcl2, Bax, and SUMO-Senp1 were determined by Western blotting. H2O2 in isolated mitochondria and cells was determined by Amplex Red. A reactive oxygen species (ROS) detection kit determined ROS levels in isolated mitochondria and HL-1 cells. JC-1 reagent measured mitochondrial membrane potential (ΔΨm). Apoptosis of HL-1 cells was examined by terminal deoxynucleotidyl transferase dUTP nick end labeling. Compared with the control group, the heart weight and heart mass/body weight ratio increased in the HF group (P<0.05). Furthermore, the ejection fraction and left ventricular shortening fraction decreased (P<0.05), while the left ventricular end-diastolic diameter (LVID;d) and end-systolic diameter (LVID;s) increased (P<0.05). Finally, mitochondrial ROS and H2O2 increased (P<0.05), while the ΔΨm decreased (P<0.05). However, AS-IV improved the cardiac function of HF mice, decreased the level of ROS and H2O2 in the myocardium, suppressed the decrease in ΔΨm, and decreased the apoptosis of myocardial cells (P<0.05). AS-IV also decreased the Senp1-overexpression. Furthermore, in HL-1 cells, Senp1-overexpression significantly inhibited the protective effects of AS-IV. AS-IV decreased oxidative stress in cardiomyocytes, decreased mitochondrial damage, inhibited ventricular remodeling, and ultimately improved cardiac function by inhibiting HF-induced Senp1-overexpression. This mechanism provides a novel theoretical basis and clinical treatment for HF.
Chronic heart failure (CHF) is a worldwide public health problem that severely threatens human health. Its main manifestation is abnormal cardiac structure, leading to impaired cardiac filling or ejection function (
Traditional Chinese Medicine, including astragalus, ginseng, ginsenoside and pepperweed seed, has a long history in the treatment of HF, and has the advantages of multiple targets, low cost and few side effects (
Ubiquitin is a small molecule protein that exists in the majority of eukaryotic cells. The main function of protein ubiquitination is to mark the proteins for degradation, followed by removal by proteolytic enzyme hydrolysis. More than 80% of cellular proteins are degraded by the ubiquitin proteasome system (
In the present study, a mouse HF model was established by aortic coarctation, and the protective effects of AS-IV on cardiac function and oxidative stress of myocardial cells were examined
Male C57BL/6J mice (8 weeks old) were purchased from the Institute of Experimental Animals, Chinese Academy of Medical Sciences. The mouse atrial muscle HL-1 cell line was purchased from ATCC. A lentiviral plasmid containing HA-
Animal experiments were performed according to the regulations and guidelines approved by the Animal Ethics Committee of The Fifth Central Hospital of Tianjin (approval. no. TJWZX2019018). Animal studies adhered to the Guide for the Care and Use of Laboratory Animals (8th edition, 2011, the Institute for Laboratory Animal Research of the National Research Council in the USA). A total of 24 male C57BL/6J mice (8-week-old; mean weight, 20 g) were purchased from the Animal Center of Nanjing University. Animals were housed in the Experimental Animal Center of The Fifth Central Hospital of Tianjin, and maintained under a controlled temperature (22-24˚C), stable humidity (40-60%) and a 12 h-light/dark cycle with
HL-1 cardiomyocytes from mouse heart tissue were purchased from ATCC. The cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum, 100 U penicillin/streptomycin and 2 mM glutamine at 37˚C, 5% CO2, 95% air and 95% humidity. Following vector or HA-
The mitochondria and cytoplasm were separated by a Tissue Mitochondrial Separation kit (Beyotime Institute of Biotechnology) using differential centrifugation, according to the manufacturer's protocols. Briefly, heart tissue blocks of the same weight and from the same region were washed with precooled PBS solution, cut into small pieces and transferred to a precooled glass homogenizer. Mitochondrial isolation solution containing protease inhibitors was added and the tissue was homogenized in an ice bath ~10 times. The supernatant was extracted following centrifugation at 4˚C for 5 min at 600 x g. Subsequently, the supernatant was discarded following centrifugation at 4˚C for 10 min at 11,000 x g. The remaining precipitate comprised the isolated mitochondria.
Isolated mitochondria or HL-1 cells were incubated with JC-1 (10 µg/ml) for 20 min at 37˚C. Mitochondria were examined by a fluorescence reader and HL-1 cells were examined by a laser scanning confocal microscope (Olympus FV 1200; Olympus Corporation; magnification x60). The excitation and emission wavelengths of the green fluorescence of JC-1 monomer were 488 and 525 nm, respectively, and of the red fluorescence of JC-1 aggregate were 543 and 590 nm, respectively. The change in fluorescence intensity of each experimental group is expressed as polymer/monomer. The temperature was maintained at 37˚C.
Isolated myocardial mitochondria or HL-1 cells were incubated with DCFH-DA (10 µM) for 20 min at 37˚C. Mitochondria were examined by a fluorescence reader and HL-1 cells were examined by a laser scanning confocal microscope (Olympus FV 1200; Olympus Corporation; magnification x100). The excitation and emission wavelengths of DCFH-DA were 488 and 525 nm, respectively. The fluorescence intensity of DCFH-DA is expressed as a percentage of the control group.
The Amplex Red
Total protein was extracted from cells or tissues using RIPA buffer (Beijing Solarbio Science & Technology Co., Ltd.) supplemented with 1 mM PMSF and 20 mM N-ethylmaleimide. The protein supernatant was collected following centrifugation at 10,000 x g at 4˚C for 15 min. Protein concentration was measured using a BCA protein assay (Beijing Solarbio Science & Technology Co., Ltd.). Equal amounts of protein lysates (40 µg per lane; 2 µg/µl) were separated on SDS-polyacrylamide gel using 10% gels and were transferred onto PVDF membranes. The membranes were then blocked with 5% skimmed milk for 1 h at room temperature. Next, the membranes were incubated overnight with the aforementioned primary antibodies at 4˚C overnight, and then with secondary antibodies for 1 h at room temperature. Finally, the protein bands were visualized by enhanced chemiluminescence (EMD Millipore). Densitometric semi-quantification analysis of the Western blot bands was performed using image analysis software (ImageJv1.48; National Institutes of Health). Protein band intensity was normalized to β-actin and expressed as a percentage of the naive control.
To detect apoptosis, a TUNEL kit (Roche Diagnostics GmbH) was used. Following treatment, HL-1 cells were fixed in 4% paraformaldehyde for 40 min at 25˚C, blocked with 3% H2O2 for 10 min at 25˚C, and permeated with 0.1% Triton X-100 for 3 min. Apoptotic cells were labeled with the TUNEL reaction mixture and nuclei were stained with DAPI (1 µg/ml) for 3 min at 25˚C. Cells were washed twice with PBS for 5 min at room temperature and mounted with ProLong Gold Antifade reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Images were randomly obtained using a fluorescence microscope (Olympus ix73; Olympus Corporation; dp73 camera; magnification x10).
Eight weeks after the operation, mice were anesthetized with pentobarbital sodium (30 mg/kg) and placed on a warm pad. Vevo 770® (Visualsonics Inc.) and a 716 probe were used to dynamically evaluate the cardiac function of mice by echocardiography. Left ventricular end-systolic diameter (LVESD) and left ventricular end-diastolic diameter (LVEED) were obtained by M-type tracing. Ejection fraction (EF) and shortening fraction (FS) were automatically obtained by high-resolution echocardiography (ECG).
Myocardial tissue was fixed with 4% paraformaldehyde for 24 h at 4˚C, dehydrated, paraffin embedded and sectioned (4 µm thick). Pathological sections were stained with hematoxylin for 5 min at 4˚C and eosin for 3 min at 4˚C (Nanjing SenBeiJia Biological Technology Co., Ltd.). H&E staining was observed under a light microscope (Olympus Medical Systems; magnification, x20). The hypertrophy of cardiomyocytes was quantified by counting the cross-sectional area of cardiomyocytes using Image-Pro Plus 6.0 (Media Cybernetics, Inc.).
Total RNA was extracted from the HL-1 cells using a TRIzol® RNA extraction kit (Tiangen Biotech Co., Ltd.). The RNA extracted from HL-1 cells was used to obtain the full-length cDNA of mouse
Cell viability was examined using a Cell Counting Kit-8 (CCK-8; Beijing Solarbio Science & Technology Co., Ltd.) assay. Hl-1 cells were cultured in 96-well plates (2x103 cells/well) and treated as described in ‘cell culture’. Subsequently, 10 µl CCK-8 solution was added to each well and incubated at 37˚C for 2 h. A microplate reader (VersaMax™ Microplate Reader; Molecular Devices, LLC) was used to analyze the absorbance at 450 nm. To increase the reliability of measures, each experiment was performed three times.
All HL-1 cell culture dishes and mice were randomly assigned to different experimental groups. Data are presented as the mean ± standard deviation. Statistical analysis was performed using GraphPad Prism 6.0 software (GraphPad Software, Inc.). The differences between the experimental and control groups were tested using unpaired t-test. For comparing differences among more than two experimental groups, the means were compared using one-way analysis of variance (ANOVA), followed by Tukey's test, or two-way ANOVA, followed by Bonferroni's test. Normal distribution was assessed with the Shapiro-Wilk test. P<0.05 was considered to indicate a statistically significant difference.
Compared with the control group, the heart weight and the heart mass/body weight ratio were increased in the HF group (P<0.05); however, AS-IV reversed this increase induced by pressure overload-driven heart failure (P<0.05;
H&E staining demonstrated that, compared with the control group, myocardial cells in the HF group exhibited hypertrophy, disordered arrangement, increased intercellular stroma and inflammatory infiltration. However, AS-IV reversed the morphological changes of the myocardial tissue induced by TAC (
Western blotting demonstrated that, compared with the control group, the cleaved-caspase-3/caspase-3 ratio was increased, but the Bcl2/Bax ratio was decreased in the HF group, indicating that apoptosis of cardiomyocytes in the HF group was increased. This effect was abolished by AS-IV (
To further investigate the effect of Senp1-overexpression on oxidative stress in HL-1 cells, the present study examined the effect of different concentrations of AS-IV on the survival of HL-1 cells subjected to ISO, which induces myocardial injury. The CCK-8 results demonstrated that HL-1 cells treated with ISO had significantly decreased cell viability. AS-IV at 50 and 100 mmol/l significantly improved the cell viability, but the protective effect of 25 and 200 mmol/l was not notable (
Mitochondria are associated with apoptosis, and the decline in Δψm is an important indicator of early apoptosis. Confocal detection of JC-1 demonstrated that, compared with the HF group, AS-IV inhibited the decrease in Δψm, while Senp1-overexpression in HL-1 cells abrogated the effect of AS-IV (
CHF is the end-stage of numerous cardiovascular diseases, which severely affect the life span and quality of life of patients and is a major public health problem. According to epidemiological data, there are more than 5.8 million patients with CHF in the United States and 23 million worldwide (
The effect of astragalus in the treatment of heart failure has been verified in a variety of animal models. Its protective effects involve improving myocardial contraction, protecting myocardial cells, regulating the neuroendocrine system and inhibiting left ventricular remodeling (
Basic and clinical studies have demonstrated that ROS (superoxide, hydrogen peroxide, hydroxyl radical) produced by HF myocardium are increased. Excessive ROS accumulation will not only cause non-specific oxidative damage to DNA, protein and lipids, but also regulate redox-related signaling cascades, leading to further myocardial damage (
The present study demonstrated that AS-IV decreased oxidative stress and mitochondrial damage, inhibited ventricular remodeling and improved cardiac function by decreasing Senp1-upregulation in HF mice. The discovery of this mechanism provides a novel theoretical basis and clinical reference for the treatment of HF. However, the present study only investigated the role of Senp1 in AS-IV-treated cardiomyocytes; therefore, the specific target of Senp1 and the free radical types regulated by Senp1 remain to be identified in order to provide a more comprehensive theoretical basis for the protective effect of AS-IV against cardiovascular diseases.
Not applicable.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
JL and XL designed the experiments. YL, XB and NX performed the experiments, and collected and analyzed the data. SD and JY drafted the manuscript. JY analyzed the data. SD interpreted data and wrote the manuscript. All authors agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy and integrity of any part of the work are appropriately investigated and resolved. XL and SD confirm the authenticity of all the raw data. All authors read and approved the final manuscript.
The present study was performed in accordance with the Guidelines of the Ethics Committee of Tianjin Medical University Cancer Institute and Hospital (Tianjin, China). Animal experiments were approved by the Animal Ethics Committee of Tianjin Fifth Central Hospital (Tianjin, China).
Not applicable.
The authors declare that they have no competing interests.
AS-IV treatment improved the contraction function in transverse aortic constriction-induced HF. (A) Representative M-mode echocardiography images. (B) Summarized data of HW/BW, ejection fraction, fractional shortening; LVIDs, LVIDd, (n=6). Data are presented as the mean ± standard deviation. One-way analysis of variance, followed by Tukey's test. *P<0.05 vs. control; #P<0.05 vs. HF. AS-IV, astragaloside; HF, heart failure; HW/BW, heart mass/body weight; EF, ejection fraction; FS, fractional shortening; LVIDs, left ventricular internal systolic diameter; LVIDd, left ventricular internal diastolic diameter; DMSO, dimethyl sulfoxide.
H&E staining showing the protective effect of AS-IV on transverse aortic constriction-induced HF. (A) Myocardial cells showed hypertrophy, disordered arrangement, increased intercellular stroma and inflammatory infiltration in the HF group, which were reversed by AS-IV. (B) Summarized data of H&E staining (n=6). Data are presented as the mean ± standard deviation. One-way analysis of variance, followed by Tukey's test. *P<0.05 vs. control; #P<0.05 vs. HF. AS-IV, astragaloside; HF, heart failure; DMSO, dimethyl sulfoxide.
Effects of AS-IV on oxidative stress and mitochondrial membrane potential in transverse aortic constriction-induced heart failure. (A) ROS levels were tested by DCFH-DA. (B) H2O2 was tested by an Amplex Red kit (n=6). (C) The mitochondrial membrane potential was tested by JC-1 (n=6). Data are presented as the mean ± standard deviation. One-way analysis of variance, followed by Tukey's test. *P<0.05 vs. control; #P<0.05 vs. HF. AS-IV, astragaloside; HF, heart failure; H2O2, hydrogen peroxide; DMSO, dimethyl sulfoxide.
AS-IV reversed the transverse aortic constriction-induced expression of apoptosis-related proteins and Senp1. (A) Apoptosis-related proteins and Senp1 expression was examined by Western blotting. (B) Quantification of the results in A (n=6). Data are presented as the mean ± standard deviation. One-way analysis of variance, followed by Tukey's test. *P<0.05 vs. control; #P<0.05 vs. HF. AS-IV, astragaloside; Senp1, small ubiquitin-like modifier-specific protease 1; HF, heart failure; DMSO, dimethyl sulfoxide.
Effect of Senp1-overexpression on H2O2 in HL-1 cells. (A) The effects of different concentrations of AS-IV (25, 50, 100 or 200 µmol/l) on the survival of HL-1 cells subjected to ISO. One-way ANOVA, followed by Tukey's test. (B) The expression of Senp1 protein was examined by Western blotting following HA-
Effect of Senp1-overexpression on ROS generation in HL-1 cells. (A) Compared with the control (dimethyl sulfoxide, 0.1%), DCF fluorescence was increased in 20 µmol/l ISO-induced HL-1 cells. AS-IV (50 µmol/l, n=6) prevented the ISO-induced increase in DCF fluorescence, which was inhibited by Senp1-overexpression. (B) Summarized data of DCF fluorescence (n=7). Data are presented as the mean ± standard deviation. Two-way analysis of variance, followed by Bonferroni's test. *P<0.05 vs. control; #P<0.05 vs. HF; &P<0.05 vs. HF+AS-IV. Senp1, small ubiquitin-like modifier-specific protease 1; ROS, reactive oxygen species; ISO, isoprenaline; AS-IV, astragaloside.
Effect of Senp1-overexpression on mitochondrial membrane potential in HL-1 cells. (A) Compared with the control (dimethyl sulfoxide, 0.1%), the JC-1 ratio (aggregate/monomer) was decreased in 20 µmol/l ISO-induced HL-1 cells. AS-IV (50 µmol/l, n=7) prevented the ISO-induced decrease in the JC-1 ratio, which was inhibited by Senp1-overexpression (n=8). (B) Summarized data of the JC-1 ratio. Data are presented as the mean ± standard deviation. Two-way analysis of variance followed by Bonferroni's test. *P<0.05 vs. control; #P<0.05 vs. HF; &P<0.05 vs. HF+AS-IV. Senp1, small ubiquitin-like modifier-specific protease 1; ISO, isoprenaline; AS-IV, astragaloside.
(A) TUNEL staining was used to examine cell apoptosis in HL-1 cells. Compared with the control (dimethyl sulfoxide, 0.1%), cell apoptosis was increased in 20 µmol/l ISO-induced HL-1 cells. AS-IV (50 µmol/l, n=10) prevented the ISO-induced increase in cell apoptosis, which was inhibited by Senp1-overexpression. (B) Summarized data of the cell apoptotic rate (n=7). Two-way analysis of variance followed by Bonferroni's test. Data are presented as the mean ± standard deviation. *P<0.05 vs. control; #P<0.05 vs. HF; &P<0.05 vs. HF+AS-IV. ISO, isoprenaline; AS-IV, astragaloside; Senp1, small ubiquitin-like modifier-specific protease 1.