2021 年 44 巻 3 号 p. 396-403
Ferulic acid (FA) has potential therapeutic effects in multiple diseases including cardiovascular diseases. However, the effect and molecular basis of FA in heart failure (HF) has not been thoroughly elucidated. Herein, we investigated the roles and mechanisms of FA in HF in isoproterenol (ISO)-induced HF rat model. Results found that FA ameliorated cardiac dysfunction, alleviated oxidative stress, reduced cell/myocardium injury-related enzyme plasma level, inhibited cardiocyte apoptosis in ISO-induced HF rat models. Moreover, FA reduced the co-localization of Keap1 and nuclear factor-E2-related factor 2 (Nrf2) in heart tissues of ISO-induced HF rats, and FA alleviated the inhibitory effects of ISO on expressions of p-Nrf2, heme oxygenase-1 (HO-1) and reduced nicotinamide adenine dinucleotide phosphate quinone dehydrogenase 1 (NQO1). Additionally, Nrf2 signaling pathway inhibitor ML385 showed adverse effects. FA weakened the effects of ML385 in ISO-induced HF rat models. Collectively, FA ameliorated HF by decreasing oxidative stress and inhibiting cardiocyte apoptosis via activating Nrf2 pathway in ISO-induced HF rats. Our data elucidated the underling molecular mechanism and provided a novel insight into the cardioprotective function of FA, thus suggested the therapeutic potential of FA in HF treatment.
Heart failure (HF), resulting from ventricular contractility and/or relaxation impairment caused by cardiac structural and/or functional abnormalities, is a serious public health problem globally that can bring about substantial burden to individuals and society.1,2) Despite the improvement in the management of HF, the morbidity and mortality for patients with HF remain high, especially in low and middle income countries, with more than 37.7 million HF cases worldwide and one-year mortality rates of 34% in Africa, 23% in India and 7% in China.3–5) HF is a multifactorial and systemic disorder that can cause various architectural, biochemical, cellular and molecular changes such as excessive cellular oxidative stress and cardiocyte hypertrophy, apoptosis and necrosis.6)
Ferulic acid (FA), a natural phenolic phytochemical, has been widely found in various fruits, vegetables, cereals and grains.7,8) FA possesses plenty of biological activities such as anti-oxidant, anti-inflammatory, anti-apoptotic and cardio-protective properties.7,9) In addition, mounting evidence shows that FA has potential therapeutic effects in multiple diseases including cardiovascular diseases.7,9) For instance, FA ameliorated Angiotensin II-induced cardiocyte hypertrophy in neonatal rat ventricular cardiocytes and the introduction of sodium ferulate led to the increase of ejection fraction (EF%) and fractional shortening (FS%) and the reduction of ratio of heart/lung weight to body weight and cardiocyte cross-sectional area in transverse aortic constriction mice probably through the regulation of gut flora.10) Mahmoud et al. proved that FA prevented liver injury by reducing oxidative stress and inflammation through nuclear factor-E2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling in methotrexate-induced rats.11) In human neuroblastoma SH-SY5Y cells, FA exhibited antioxidant property by regulating Nrf2/HO-1 signaling and counteracting with trimethyltin-induced neuronal damage.12) Moreover, previous studies showed that FA could weaken cardiac toxicity and reduce cellular stress induced by multiple substances such as cyclophosphamide.13) Additionally, Jain et al. pointed out that FA protected rats from isoproterenol (ISO)-induced cardiac damage by restoration of cardiac cellular architecture, normalization of serum cardiac enzyme activities, and reduction of oxidative stress.14) Yogeeta et al. also demonstrated that FA had synergistic cardioprotective function in ISO-induced myocardial infarction when used in combination with ascorbic acid.15)
Though accumulated studies revealed that FA had cardioprotective function in ISO-induced HF model, the underlying molecular mechanism was not fully elucidated, and if FA exerted its cardioprotective function by reducing oxidative stress, inflammation, or cell apoptosis was uncertain. In this study, we built an ISO-induced HF rat model and treated them with increasing concentrations of FA. We found that the FA exerted cardioprotective effects by reducing oxidative stress and inhibiting cardiocyte cell apoptosis via activating Nrf2 pathway in ISO-induced HF rat model. Our data provided a novel insight into the cardioprotective function of FA in ISO-induced HF rat model, and proved the therapeutic potential of FA in HF treatment.
A total of 80 male Sprague-Dawley rats (7–8 weeks old, 230–280 g) were obtained from the Nanjing junke biological engineering Co., Ltd. (Nanjing, China). Rats were fed with standard rat chow and water ad libitum and were raised under the relative humidity of 50 ± 5% and temperature of 22–24 °C for 12 h light/dark cycle. The study was approved by the Animal Care and Use Committee of the First Affiliated Hospital of Soochow University and was performed following the Guide for Care and Use of Laboratory Animals. ISO (#15627; dissolved in physiological saline), and Nrf2 inhibitor ML385 (#SML1833; HPLC; ≥98%; dissolved in 10% dimethyl sulfoxide (DMSO)/90% corn oil) and FA (#128708; 99%) were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, U.S.A.).
Rats were randomly divided into 8 groups (n = 10 in per group): Control group (normal rats without any treatment); HF group (rats with ISO treatment alone as previously described16): FA group (rats treated with 50 mg/kg of FA alone); HF + FA (5 mg/kg) group (rats treated with ISO and 5 mg/kg of FA); HF + FA (25 mg/kg) group (rats treated with ISO and 25 mg/kg of FA); HF + FA (50 mg/kg) group (rats treated with ISO and 50 mg/kg of FA); HF + ML385 (50 mg/kg) group (rats with the combination treatment of ISO and 50 mg/kg of ML385); HF + ML385 + FA (rats treated with ISO, 50 mg/kg of FA and 50 mg/kg of ML385). Drugs or agents were administrated as previously reported16): ISO (150 mg/kg body weight daily) given intraperitoneally for 2 d; FA given orally for 4 d prior to ISO treatment and then administrated for another 2 d together with ISO; ML385 given intraperitoneally (50 mg/kg body weight daily) for 4 d at 1 h before FA treatment and then administrated for another 2 d together with ISO.
Cardiac Function AssessmentA polyethylene catheter attached to the pressure transducer and bridge amplifier (AD Instruments, Sydney, Australia) was cannulated into the left common carotid artery of anesthetized rats, followed by the record of HR by PowerLab Data Acquisition System (AD Instruments). The left ventricle internal diameter thickness (LVIDs), left ventricle posterior wall thickness (LVPWs), fraction shortening (FS) and ejection fraction (EF) were measured by a Vevo 770 echocardiography (Visual Sonics Inc., Toronto, ON, Canada) with a 17.5 MHz ultrasonic probe. Blood samples were collected from inferior vena cava and immediately centrifuged at 3000 rpm for 15 min. After euthanasia, rat hearts were immediately dissected and stored at −80 °C for subsequent experiments.
Biochemical AnalysisThe plasma levels of superoxide dismutase (SOD), malondialdehyde (MDA), glutathione peroxidase (GSH-Px), lactate dehydrogenase (LDH), and N-terminal brain natriuretic peptide (NT-proBNP) were determined by corresponding enzyme-linked immunosorbent assay (ELISA) kits (Shanghai Xinfan Biotechnology Co., Ltd., Shanghai, China) following the protocols of manufacturer.
ImmunofluorescenceTen micromolar cryosections of heart tissues of ISO-induced HF rats were fixed by 100% cold acetone for 20 min, then blocked with 5% bovine serum albumin (BSA) in phosphate buffered saline (PBS) for 1 h at room temperature. Primary antibodies including KEAP1 (Cell signaling #8047, 1: 400) and Nrf2 (Cell signaling #14596, 1 : 400) were incubated overnight at 4 °C. Samples were then stained with AlexaFluor488 or AlexaFluor594 (Invitrogen, U.S.A.) and recorded images using an inverted laser-scanning confocal microscope (TCS SP5, Leica, Germany).
Terminal Deoxynucleotidyl Transferase-Mediated Deoxyuridine Triphosphate Nick-End Labeling (TUNEL) AssayIn order to evaluate cell apoptotic pattern in cardiac tissues, TUNEL assay was performed by thein situ cell death detection kit (Roche Diagnostics, Mannaheim, Germany) based on the manufacturer’s instructions. Briefly, paraffin-embedded left ventricle tissue sections (4 µm) received the sequential treatments as blow: dewaxation, rehydration, protease treatment, permineralization, and the addition of TUNEL reaction mixture, Converter-POD and substrate solution. Finally, samples were analyzed under a light microscope.
Western Blot AssayProteins were extracted from cardiac muscle tissues using radio immunoprecipitation assay (RIPA) lysis buffer (Beyotime, Shanghai, China) and quantified using bicinchoninic acid (BCA) protein assay kit (Beyotime). Next, an equal amount of protein samples was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, U.S.A.). Next, the membranes were blocked with 5% skim milk and probed with primary antibodies against cleaved caspase3 (1 : 1000, #9664, Cell Signaling Technology, Danvers, MA, U.S.A.), cleaved caspase9 (1 : 1000, #9507, Cell Signaling Technology), Nrf2 (1 : 1000, AF0639, Affinity Biosciences, Cincinnati, OH, U.S.A.), p-Nrf2(1 : 1000, DF7519, Affinity Biosciences), HO-1 (1 : 10000, ab68477, Abcam), Keap1 (1 : 1000, #8047, Cell signaling) and reduced nicotinamide adenine dinucleotide phosphate (NADPH) quinone dehydrogenase 1 (NQO1, 1 : 10000, ab80588, Abcam). After incubated with secondary antibody conjugated with horseradish peroxidase (1 : 10000, ab205718/ab205719, Abcam), the membranes were developed using Pierce™ ECL Western blotting Substrate (Thermo Scientific, Rockford, IL, U.S.A.). Protein quantitative analysis was performed using Quantity One Software (Version 4.6.6, Bio-Rad Laboratories Inc., Hercules, CA, U.S.A.). β-Actin was used as the loading control.
Statistical AnalysisThe data were analyzed by GraphPad Prism software v6.0 (San Diego, CA, U.S.A.) and expressed as mean ± standard deviation. The one way ANOVA followed by Tukey’s test was used to evaluate the differences among groups. p < 0.05 was regarded as statistically significant.
Firstly, echocardiography analysis revealed that the addition of FA (50 mg/kg) did not influence heart rate (Fig. 1A), LVIDs (Fig. 1B), LVPWs (Fig. 1C), EF% (Fig. 1D), and FS% (Fig. 1E) in normal rats. Also, there was no significant difference of NT-proBNP (an HF marker) plasma level between normal control group and FA-treated group (Fig. 1F). These data revealed that the introduction of FA (50 mg/kg) did not influence cardiac function of normal rats. However, a notable decline of heart rate (Fig. 1A), EF% (Fig. 1D), and FS% (Fig. 1E) and a remarkable up-regulation of LVIDs (Fig. 1B), LVPWs (Fig. 1C), and plasma NT-proBNP level (Fig. 1F) were observed in HF group compared to control group, suggesting that ISO-induced HF rat model were successfully established. Next, the effect of different concentrations of FA on cardiac function was assessed through the above parameters in ISO-induced rat HF rat model. Results showed that FA administration led to a dose-dependent increase of heart rate (Fig. 1A), EF% (Fig. 1D), and FS% (Fig. 1E) and a concentration-dependent reduction of LVIDs (Fig. 1B), LVPWs (Fig. 1C) and NT-proBNP plasma level (Fig. 1F) in HF rats, suggesting that FA could ameliorate the cardiac parafunction induced by ISO in rats.
(A–E) Heart rate, LVIDs, LVPWs, EF, and FS of rats were measured by echocardiography system. (F) The level of NT-proBNP in plasma samples of rats was detected by ELISA assay. * p < 0.05 compared to the control group; ** p < 0.01 compared to the control group; # p < 0.05 compared to the HF group; ## p < 0.01 compared to the HF group.
Considering the close link of oxidative stress and HF, plasma levels of oxidative stress indexes such as SOD, MDA, and GSH-Px were measured in rats with different treatment. Also, the plasma level of LDH was tested to examine the degree of cell injury. Our data presented that FA (50 mg/kg) treatment had no significant influence on SOD activity, MDA content, GSH-Px level and LDH release in plasma samples of rats (Figs. 2A–D), suggesting that FA could not trigger oxidative damage in rats. However, SOD and GSH-Px activities were remarkably reduced and MDA and LDH content were markedly increased in plasma samples of HF group relative to control group (Figs. 2A–D), suggesting that ISO-induced HF model presented excessive oxidative stress. Moreover, our data revealed that different doses of FA treatment led to the concentration-dependent increase of SOD and GSH-Px activities and dose-dependent reduction of MDA and LDH content in HF rats (Figs. 2A–D), suggesting that FA could concentration-dependently alleviated oxidative stress and cytotoxicity induced by ISO in rats.
(A–D) SOD activity (A), MDA content (B), GSH-Px content (C) and LDH release level (D) were measured by ELISA assay. ** p < 0.01 compared to the control group; # p < 0.05 compared to the HF group; ## p < 0.01 compared to the HF group.
Next, the effect of FA on cardiocyte cell apoptosis was evaluated through TUNEL assay and Western blot assay. Our data revealed that there was no obvious difference in cell apoptotic percentage (Fig. 3A) and cleaved caspase3/9 protein expression (Fig. 3B) between FA-treated group and normal control group, indicating that FA had no influence on cardiocyte cell apoptosis. In other words, FA could not induce cardiocyte cell injury. However, cell apoptotic percentage (Fig. 3A) and cleaved caspase3/9 protein level (Fig. 3B) were strikingly elevated in ISO-induced HF model group than that in control group, suggesting that cardiocytes were injured in ISO-induced HF model. Moreover, our outcomes further showed that administration of FA led to a dose-dependent reduction of cell apoptotic percentage (Fig. 3A) and cleaved caspase3/9 protein expression (Fig. 3B) in ISO-induced HF model, which suggested that FA alleviated cardiocyte cell injury in ISO-induced HF rat model.
(A) Cell apoptotic pattern was detected by TUNEL assay in rat heart tissues. (B) The expression levels of apoptosis related proteins (cleaved caspase3 and cleaved caspase9) were detected by Western blot assay. ** p < 0.01 compared to the control group; # p < 0.05 compared to the HF group; ## p < 0.01 compared to the HF group.
To further investigate whether Nrf2 signaling pathway was involved in the regulation of FA-mediated cardioprotective effect in HF, expression levels of Nrf2 signaling pathway-related proteins such as Keap1, Nrf2, p-Nrf2, HO-1 and NQO1 were detected by Western blot assay. As presented in Fig. 4A, no obvious alterations in protein expression of Keap1, Nrf2, p-Nrf2, HO-1 and NQO1 were observed in FA group relative to control group, which suggested that FA did not affect Nrf2 signaling pathway in normal rats. However, protein levels of p-Nrf2, HO-1 and NQO1 were markedly down-regulated while Keap1 up-regulated in HF group than that in control group, suggesting that Nrf2 signaling pathway was inhibited in ISO-induced HF rat model. The introduction of FA led to a dose-dependent increase of p-Nrf2, HO-1 and NQO1 protein levels and decrease of Keap1 in ISO-induced HF rats, which suggested that FA could activate Nrf2 signaling pathway in ISO-induced HF rat model (Fig. 4A). Moreover, immunofluorescence staining of Keap1 and Nrf2 in heart tissues of ISO-induced HF rats suggested that there were more Keap1 and Nrf2 co-localized in HF group, but their co-localization was obviously reduced when treated with 50 mg/kg FA in the HF + 50 mg/kg FA group (Fig. 4B). This indicated that FA facilitated the disassociation of Nrf2/Keap1 complex, thus increased the nuclear translocation of Nrf2 and activated Nrf2 signaling in ISO-induced HF rat model. Collectively, our results demonstrated that FA activated Nrf2 signaling pathway in ISO-induced HF rat model.
A, Protein levels of Keap1, Nrf2, p-Nrf2, HO-1 and NQO1 were measured through Western blot assay in rat hearts. B, 10 µM cryosections of heart tissues of ISO-induced HF rats were used for immunofluorescence staining with Keap1 (Gray) and Nrf2 (Gray). Represent images were shown. Scale bar = 20 µM. ** p < 0.01 compared to the control group; # p < 0.05 compared to the HF group; ## p < 0.01 compared to the HF group.
Next, Nrf2 signaling pathway inhibitor ML385 was used to further investigate whether FA exerted its functions by regulating Nrf2 signaling pathway in ISO-induced HF rat model. Firstly, Western blot assay validated that ML385 could further inactivate Nrf2 signaling pathway in ISO-induced HF rats, as evidenced by the conspicuous reduction of protein levels of p-Nrf2, HO-1 and NQO1, and increasing of Keap1 in ISO-induced HF rats following the addition of ML385 (Fig. 5A). FA weakened the inhibitory effect of ML385 on Keap1, p-Nrf2, HO-1 and NQO1 expression in ISO-induced HF rats (Fig. 5A). Functional analyses showed that ML385 treatment led to a notable reduction of heart rate, EF% and FS% and conspicuous increase of LVIDs, LVPWs and NT-proBNP plasma level in HF rats (Figs. 5B–G), suggesting that inhibition of Nrf2 signaling pathway aggravated cardiac functional disturbance in HF rats. Moreover, FA treatment markedly weakened the influences of ML385 on heart rate, LVIDs, LVPWs, EF%, FS% and NT-proBNP plasma level in HF rats (Figs. 5B–G). Moreover, the introduction of ML385 led to the noticeable increase of MDA content and conspicuous reduction of SOD activity (Figs. 5H, I), suggesting that ML385 could enhance oxidative stress and myocardial injury in HF rats. Additionally, FA markedly mitigated the inhibitory effect of ML385 on SOD activity and inhibited the increase of MDA level induced by ML385 in HF rats (Figs. 5H, I), suggesting that FA weakened oxidative stress responses by activating Nrf2 signaling pathway in HF rats. Moreover, ML385 accelerated cardiocyte cell injury in HF rats, as evidenced by the increase of cell apoptotic percentage in ML385-treated HF rats versus untreated HF rats (Fig. 5J). Furthermore, FA weakened ML385-mediated pro-apoptotic effects in cardiac tissues of HF rats (Fig. 5J). In summary, these data presented that FA alleviated cardiac dysfunction by reducing oxidative stress and inhibiting cardiocyte cell apoptosis via activating Nrf2 signaling pathway in ISO-induced HF rat model.
(A) Protein levels of Keap1, Nrf2, p-Nrf2, HO-1 and NQO1 were measured through Western blot assay in rat hearts. (B–F) Heart rate, LVIDs, LVPWs, EF, and FS of rats were measured by echocardiography system. (G) The level of NT-proBNP in plasma samples of rats was detected by ELISA assay. (H, I) SOD activity and MDA content in plasma samples of rats were measured by ELISA assay. (J) Cell apoptotic pattern in rat hearts was analyzed by TUNEL assay. * p < 0.05 compared to the control group; ** p < 0.01 compared to the control group; ##p < 0.01 compared to the HF group; # p < 0.05 compared to HF group; & p < 0.05 compared to the HF + ML385 (50 mg/kg) group.
In this text, we firstly demonstrated that ISO treatment led to a notable reduction of heart rate, EF% and FS% and a remarkable increase of LVIDs, LVPWs and NT-proBNP level in rats. These data suggested that ISO-induced rat HF model were successfully established. In addition, our data revealed that there were no significant differences in heart rate, EF%, FS%, LVIDs, LVPWs and NT-proBNP level between rats received FA treatment alone and normal rats, suggesting that FA did not influence cardiac function of normal rats. Subsequently, we further demonstrated that FA could notably ameliorate ISO-induced cardiac dysfunction in rats, as evidenced by the increased heart rate, EF% and FS% and reduced LVIDs, LVPWs and NT-proBNP level in FA-treated HF rats relative to HF rat group.
Oxidative stress has been found to be closely related with ISO-induced cardiac dysfunction.17,18) Moreover, increasing studies showed that FA exerted cardioprotective effect by reducing oxidative stress in ISO-induced cardiac injury model.15) Oxidative stress has been defined as the overproduction of reactive oxygen species (ROS) beyond body antioxidant defense ability. In antioxidant defense systems, SOD and GSH-Px can scavenge ROS and catalyze hyperoxide or superoxide into low-toxic or non-toxic substances.19,20) MDA is a product of lipid peroxidation and MDA level was positively associated with oxidative stress.21) In this text, SOD, GSH-Px and MDA were used as oxidative stress markers to measure the effect of FA on ISO-induced oxidative stress. Our data revealed that FA inhibited the increase of MDA level induced by ISO and weakened the inhibitory effects of ISO on SOD and GSH-Px activities in rats, which was in line with previous studies.15)
It has been proved that excessive oxidative stress can lead to cellular injury such as cell hypertrophy and cell apoptosis, contractile function impairment, extracellular matrix remodeling, maladaptive cardiocyte remodeling and failure in HF.19) LDH, a stable cytoplasmic enzyme, is rapidly released to extracellular matrix and blood when cells are injured.22) Hence, LDH functions as an indicator to assess the degree of cytotoxicity. Consistent with the previous studies,15) our outcomes also presented that LDH release content was strikingly elevated in ISO-induced HF rats than that in normal rats, and FA led to the notable reduction of LDH level in ISO-induced HF rats. As mentioned above, cardiocyte apoptosis is a common feature of HF.6) In this text, TUNEL assay and caspase3/9 expression analysis revealed that FA inhibited cardiocyte apoptosis in ISO-induced HF rat model. These data suggested that FA could reduce cytotoxicity and curbed cardiocyte apoptosis in ISO-induced HF rat model.
Nrf2, a transcription factor, has been found to be involved in regulating various biological responses or processes such as antioxidation (e.g., GSH production and regeneration), detoxification (e.g., ROS and xenobiotic detoxification), cell proliferation, metabolism (e.g., heme and iron metabolism), and organ development.23,24) Nrf2 activation can weaken the detrimental effects of ROS on cells and protect cells from oxidative stress-induced damage.24,25) Nrf2 exerts its antioxidant functions through promoting the expression of antioxidant and detoxification genes such as HO-1 and NQO1 and inducing the activation or expression of antioxidant enzymes.23) Moreover, previous studies showed that the activation of Nrf2 pathway could ameliorate cardiac function in HF and protect heart and cardiocytes from oxidative damages.23,26,27) For instance, Nrf2 overexpression in the rostral ventrolateral medulla ameliorated sympatho-excitation in mice with chronic HF.28) The administration of bardoxolone methyl (Nrf2 activator) led to the increase of stroke volume and cardiac output and reduction of left ventricle end-diastolic pressure in chronic HF rats by reducing oxidative stress and activating Nrf2 and downstream antioxidant protein (NQO1, HO1 and Catalase) signaling.29) Moreover, previous studies showed that FA could counteract cytotoxicity, ototoxicity, nephrotoxicity, hepatotoxicity and oxidative stress by activating Nrf2 pathway.30,31) Moreover, FA protected cardiocytes from high glucose-induced cell injury by increasing HO-1, glutathione S-transferase (GST) and Nrf2 expression.32) In addition, Nrf2 overexpression could ameliorate ISO-induced cardiocyte dysfunction and counteract ISO-induced oxidative stress in mouse heart.33) However, it remains unknown whether Nrf2 signaling pathway is involved in the regulation of FA-mediated cardioprotective effects in ISO-induced HF rat model. In the present study, our data revealed that p-Nrf2/Nrf2 ratio and HO-1 and NQO1 expressions were markedly reduced in ISO-induced HF rat model versus control group, and FA led to a remarkable elevation of p-Nrf2/Nrf2 ratio and HO-1 and NQO1 expression in ISO-induced HF rat model. Besides, FA facilitated the disassociation of Nrf2/Keap1 complex, thus promoted the nucleus translocation of Nrf2. These data suggested that FA might exert its cardioprotective effects through activating Nrf2 pathway in ISO-induced HF rat model. To further validate this conclusion, the effects of Nrf2 signaling pathway inhibitor ML385 on cardiac function/oxidative stress-related indexes and cardiocyte apoptosis were measured in ISO-induced HF rat model. Results showed that inhibition of Nrf2 signaling pathway by ML385enhanced cardiac dysfunction, improved oxidative stress and promoted cardiocyte injury in ISO-induced HF rat model, while these effects of ML385 were markedly weakened by FA.
Taken together, our data revealed that FA exerted cardioprotective effect by reducing oxidative stress and inhibiting cardiocyte apoptosis via activating Nrf2 signaling pathway in ISO-induced HF rat model, deepening our understanding on cellular and molecular basis of FA in the development of HF and hinting the potential values of FA in the prevention and treatment of HF.
Ling Wang, Xi-juan Zhang, Xiu-wen Liang, Zhong-hua Cui, who produced the study concepts; Ling Wang, Xi-juan Zhang who designed this study researched the literature; Xi-juan Zhang and Zhong-hua Cui who did the clinical studies; Xi-juan Zhang, Yan-xin Zhao and Ting-ting He who performed the experimental studies; Xi-juan Zhang who did the statistical analysis, and wrote the manuscript; Ling Wang and Xiu-wen Liang reviewed the manuscript.
The authors declare no conflict of interest.
