YiQiFuMai powder injection ameliorates chronic heart failure through cross-talk between adipose tissue and cardiomyocytes via up-regulation of circulating adipokine omentin

Biomedicine & Pharmacotherapy 119 (2019) 109418

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YiQiFuMai powder injection ameliorates chronic heart failure through crosstalk between adipose tissue and cardiomyocytes via up-regulation of circulating adipokine omentin

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Fang Li, Li-Zhi Pang, Ling Zhang, Yu Zhang, Yuan-Yuan Zhang, Bo-Yang Yu, Jun-Ping Kou

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Jiangsu Key Laboratory of TCM Evaluation and Translational Research, School of Traditional Chinese Pharmacy, China Pharmaceutical University, 639 Longmian Road, Nanjing, 211198, PR China

ARTICLE INFO

ABSTRACT

Keywords: YiQiFuMai powder injection Heart failure Cardioprotection Epicardial adipose tissue Omentin Ginsenoside Rd

YiQiFuMai Powder Injection (YQFM) is widely used in clinical practice for the treatment of heart failure (HF). However, its functional molecular mechanism remains to be fully uncovered. Our present study aimed to elucidate the impact of YQFM and underlying mechanisms on coronary artery ligation (CAL)-induced HF. Our results exhibited that YQFM significantly mitigated CAL-induced HF via meliorating the left ventricular contractile function and reducing the serum content of creatine kinase MB (CK-MB), aspartate aminotransferase (AST), interleukin-6 (IL-6), troponin (Tn), myosin, myoglobin (MYO) and myocilin (MYOC). Then, the relevance between circulating omentin level and cardiac function was investigated and we found that serum omentin levels positively associated with ejection fraction and negatively correlated with NT-proBNP content. Further, the effect of YQFM on cardiac function and omentin change in 1, 7 and 14 days CAL-induced HF mice was evaluated and the omentin secretion in isolated subcutaneous (SCAT) and epicardial adipose tissue (EAT) after YQFM treatment were detected. YQFM could increase the circulating omentin content both in 14 days CAL-induced HF mice and isolated EAT. And increased omentin in conditioned medium (CM) could inhibit simulated ischemic/ reperfusion (SI/R)-induced cardiomyocytes apoptosis. Moreover, YQFM could ameliorate myocardial apoptosis via positive regulation of AMPK, PI3 K/Akt and negative regulation of MAPKs signaling pathways. Ginsenoside Rd might partially mediated omentin-dependent protective effect of YQFM. Our findings indicated that regulation of cross-talk between adipose tissue and cardiomyocytes might be a potential target through which YQFM exerts cardioprotective effect apart from direct cardiomyocytes protection.

1. Introduction Ischemic heart disease (IHD) is the leading cause of death worldwide, and ultimately progresses toward heart failure (HF) [1,2]. HF is a complex clinical syndrome and the final stage of various cardiovascular diseases [3]. Recently, accumulating investigation indicates that myocardial ischemia (MI) has become the most essential cause of HF. MI leads to substantial left ventricular injury and causes acquired abnormalities in cardiac structure and function such as the impaired ability to fill or eject blood, and these changes may induce the further deterioration followed by HF. Clinically, statins, β-blockers and angiotensin-converting enzyme inhibitors are the mainstay of the current treatment [4]. However, these treatments provide limited symptomatic relief to a certain extent and temporarily impeding disease progression. Researchers currently focus mainly on interference in the release of inflammatory factors, energy deficit, endothelial dysfunction, ⁎

cardiomyocyte death and so on [5], but the attempts so far made are far from enough. With the development of system biology, therapeutic strategies aiming at regulation of integral metabolism and multi-target cells would possess stronger efficacies for complex multifactorial diseases. Cross-talk between the heart and adipose tissue offers new strategies for the prevention and treatment of HF [6]. Adipose tissue functions as an endocrine organ through secreting various bioactive mediators, such as adiponectin and leptin, also referred to as adipokines, which can directly affect the nearby or remote tissues [7,8]. Epicardial adipose tissue (EAT) is the visceral fat depot of the heart, an extremely active organ that produces multiple bioactive molecules, and may modulate the myocardium given its anatomical proximity to the heart [9]. Omentin is a novel adipokine that was discovered abundantly in human visceral fat tissue [10]. Low levels of circulating omentin involve in the occurrence and prevalence of coronary heart disease [11].

Corresponding author. E-mail address: [email protected] (J.-P. Kou).

https://doi.org/10.1016/j.biopha.2019.109418 Received 18 July 2019; Received in revised form 28 August 2019; Accepted 30 August 2019 0753-3322/ © 2019 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

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Recently, researchers have demonstrated that omentin ameliorates acute ischemic injury in the heart by suppressing myocyte apoptosis through both AMPK- and Akt-dependent mechanisms [12]. Also, in other studies, omentin was verified to attenuate the pathological process of myocardial hypertrophy via the activation of AMPK in the heart [13], and prevent doxorubicin-induced apoptosis of H9c2 cells through the inhibition of mitochondrial reactive oxygen species production [14]. In addition, plasma omentin levels significantly decreased in patients with obesity-related disorders, including atherosclerosis and type 2 diabetes [15,16]. These results indicate that omentin may represent a novel target molecule for the treatment of IHD and its serum level may appear to be a promising biomarker for cardiac prognosis. Further investigations are still needed to elucidate the association between omentin and HF. With increasing demand of drugs for the treatment of HF clinically, more and more researchers have showed their great interests in traditional Chinese medicine (TCM). YiQiFuMai powder injection (YQFM) has been re-developed derived from a famous complex prescription Sheng-Mai-San, which is composed of Ginseng Radix Et Rhizoma Rubra, Ophiopogonis Radix, and Schisandrae Chinensis Fructus [17]. Sixty-five compounds were identified from YQFM by UFLC-IT-TOF/MS, including forty-two compounds from P. ginseng, sixteen compounds from S. chinensis, and seven compounds from O. japonicus, of which there were four steroidal saponins, one borneol pyranoside and two flavonoids. Considerable clinical trials have indicated that YQFM can be widely applied for the treatment of chronic heart failure with better efficacy and fewer side effects compared with standard medical treatments. Previous studies have demonstrated that YQFM and its compounds could ameliorate HF via NF-κB inactivation and cytokines suppression [18], meanwhile, improve the tolerability of myocardium in hypoxia-induced heart injury [19]. In our previous studies, YQFM inhibited cardiomyocyte apoptosis through AMPK activation signaling pathways against myocardial ischemia/reperfusion injury [20]. Moreover, YQFM could attenuate coronary artery ligation (CAL)-induced myocardial remodeling and heart failure through modulating MAPKs signaling pathway [21]. Nevertheless, the potential molecular mechanisms of YQFM and bioactive constituents against HF remain to be elucidated. In current study, we investigated the association between the circulating omentin and HF, and the possible implication of the adipose tissue and cardiomyocytes' cross-talk modulation in the YQFM effect. Furthermore, the potential bioactive compounds present in YQFM were screened via HPLC-Q-TOF/MS analysis.

2.2. Surgical preparation and experimental protocol in vivo ICR male mice (8 weeks old) were obtained from the Experimental Animal Center of Yangzhou University (Yangzhou, China). All animal's welfare and experimental procedures were operated in accordance with National Institutes of Health Guide for the Care and Use of Laboratory Animals, and the protocols used were also consistent with the Animal Ethics Committee of China Pharmaceutical University, China Pharmaceutical University, Nanjing, China. The model of CAL-induced heart failure was produced as previously described [22]. Provisionally, after anesthetizing the mice by intraperitoneal injection of chloral hydrate, the hearts were exteriorized with a left thoracic incision followed by making a slipknot (6-0 silk) around the left anterior descending coronary artery. After ligation, the heart was immediately placed back into the intrathoracic space followed by manual evacuation of air and closure of muscle and the skin. Then survival mice were separated randomly into model or each administration groups. The sham group was given the same surgery without ligating the left anterior descending coronary artery. Animals have not divided into each groups in the beginning of surgery, except sham operated mice. After each surgery was finished, we randomly divide survival mouse into model or each administration groups. And the survival rate of animals was more than 90%. The surviving mice were seperated randomly into following groups: the sham group, the model group (CAL 1 day, 7 days, 14 days), the YQFM group (CAL 14 days) with different doses (0.13 g/kg, 0.26 g/kg, 0.53 g/kg, i.p.), the YQFM group (0.53 g/kg, i.p.) with different time of ligation (CAL 1 day, 7 days, 14 days), Metoprolol (Met) group (CAL 14 days, 5.14 mg/kg, i.g.) and Captopril (CAP) group (CAL 14 days, 0.16 g/kg, i.p.). Met and CAP were used as positive drugs. Both the sham and the model mice were received an equal volume of physiologic saline via intraperitoneal injection. The treatment maintained for 1 day to two weeks (once a day) according to the difference of each group. 2.3. Echocardiographic measurement After 1 day, 7 days and 14 days' treatment, according to previous study [23], echocardiography was performed using the Vevo2100 imaging system with a 30-MHz probe. The following parameters were detected: interventricular septum in diastole (IVSd), left ventricle interior diameter in diastole (LVIDd), left ventricle posterior wall in diastole (LVPWd), and left ventricular volume in diastole (LV Vold). Besides, data of the left ventricular pre ejection period (LVPEP) and left ventricular ejection time (LVET) were obtained in PW Doppler Mode. Based on these measurements, the following parameters were further calculated: ejection fraction (EF) = (LVEDV − LVESV) / LVEDV, left ventricular endocardial fractional shortening (FS) = (LVDd − LVDs) / LVDd, left ventricular Mass (LV Mass) = 0.8 × 1.04(IVS + LVD + LVPW)+0.6, mean velocity of circumferential fiber shortening (mVCF) = (LVIDd − LVIDs) / (LVIDd*LVET), stroke volume (SV) = LV Vold − LV Vols.

2. Materials and methods 2.1. Drugs and reagents YQFM was provided by Tasly Pharmaceutical Co., Ltd. with the batch number of 20,161,016. The quality of YQFM was confirmed by HPLC-DAD-ELSD analysis according to our previous studies (20). Ginsenoside Rd was purchased from Nanjing Zelang Bio-Technology Co., Ltd (Nanjing, China). The ELISA kits for determination of creatine kinase MB (CK-MB), aspartate aminotransferase (AST), interleukin-6 (IL-6), troponin (Tn), myosin, myoglobin (MYO) and myocilin (MYOC) were obtained from Nanjing Jin Yibai Biological Technology Co. Ltd. (Nanjing, China). The ELISA kits for determination of omentin was purchased from Kejian Biology Science and Technology Co., Ltd. (Shanghai, China). Antibody against GAPDH was from Kangchen Biotech Inc. (Shanghai, China), antibody against β-Tublin was obtained from Nanjing Sunshine Biotechnology Co., LTD. (Nanjing, China), antibody against Bcl-2, Bax, p38, p-p38, JNK, p-JNK, ERK1/2, p-ERK1/2, AMPK, p-AMPK, PI3 K, Akt, and p-Akt were purchased from Cell Signaling Technology (Boston, MA, USA).

2.4. Serum biochemical indicators and omentin determination At the end of the observation, blood was collected and serum was separated by centrifugation. The contents of CK-MB, AST, IL-6, Tn, myosin, MYO, MYOC and omentin were measured with respective ELIDA kits according to the manufacturer's protocols. In addition, the epicardial adipose tissue (EAT) and inguinal subcutaneous adipose tissues (SCAT) were isolated and chopped into small pieces. Equal amount of EAT and SCAT from normal mice were cultured in fresh DMEM with or without YQFM (400 μg/ml). The medium was collected for concentration detection of omentin after 24, 36 and 48 h. 2

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2.5. Adipose tissue-derived conditioned medium (CM) preparation

2.9. Immunohistochemistry analysis

The EAT of ligated and treated mice were isolated and cultured as above described. The isolated EAT from sham group (sham operation 14 days), model group (CAL 14 days), YQFM group (0.53 g/kg, i.p.), CAP group (0.16 g/kg, i.p.), Met group (5.14 mg/kg, i.g.) were disposed by the oxygen and glucose deprived (OGD) technique. In our study, the OGD injury was produced by incubating with none-glucose DMEM and exposed to a hypoxic environment of 94% N2, 5% CO2, and 1% O2 for 6 h at 37 °C in a humidified N2/CO2 incubator and then in a standard incubator with 5% CO2 in normal atmosphere at 37 °C for 6 h. After OGD finished, the medium from each group was collected as CM.

For immunohistochemical staining, the heart specimens were fixed in 4% paraformaldehyde and embedded in paraffin, then sectioned at 4 μm thicknesses, deparaffinized, rehydrated in PBS, and incubated with 3% hydrogen peroxide to block endogenous peroxidase activity. The sections were then incubated with blocking liquid at 37 °C for 1 h (Beyotime Institute of Biotechnology, Shanghai, China). Immunohistochemistry analysis was conducted using primary antibodies of cleaved caspase-3 (1:100, Cell Signaling Technology, Boston, CA, USA) at 4 °C for 24 h. After washing, sections were incubated with the HRP-conjugated secondary antibody (1:200, Bioworld Technology, Louis Park, MN, USA) at 37 °C for 1 h. After incubated with DAB, counterstained with hematoxylin, dehydrated sections were observed under a light microscope (DX45, Olympus Microsystems Ltd., Japanese) and imaged at 200× magnification.

2.6. Cell culture and in vitro experiment Rat H9c2 cardiomyocyte cell line was obtained from Shanghai Institute of Cell Biology, Chinese Academy of Sciences (Shanghai, China). The H9c2 cells were maintained in DMEM with the addition of 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. All groups were conducted with the above CM and described OGD procedure besides the control group. Finally, the medium were collected for omentin evaluation. Furthermore, cell viability was assessed using cell counting kit-8 (CCK-8) following the instructions (Beyotime Institute of Biotechnology, Shanghai, China). H9c2 cells were replaced from normal medium to CM and then OGD was performed. 10 μl of CCK-8 solution was added to each well containing 100 μl of culture medium, and the plate was incubated for 4 h at 37 °C. The absorbance was read at 450 nm using a microplate reader, and cell viability was expressed as percentage of absorbance to control values. FITC-conjugated Annexin V and propidium iodide (PI) were applied to identify apoptotic cells and performed following the manufacturer’s instructions. Initially, H9c2 cells were cultured in CM, then the OGD was used to mimic hypoxia. After OGD procedure, H9c2 cardiomyocytes were harvested, washed with PBS, resuspended with binding buffer. Finally, H9c2 cells were incubated with Annexin-V and PI in a final concentration of 100 ng/mL at room temperature in the dark for 15 min. Cellular fluorescence was analyzed with a flow cytometer and data were analyzed by FlowJo software.

2.10. Western blot analysis As reported [21], LV samples of each group were homogenized in RIPA buffer. Equal amount of proteins were loaded and separated by denaturing SDS-PAGE. Membranes were blocked with 3% BSA and stained with primary antibodies as follows: GAPDH (dilution 1:8000), β-Tublin (dilution 1: 1000), PI3 K, Bax, Bcl-2, AMPK, p-AMPK, Akt, pAkt, p38, p-p38, JNK, p-JNK, ERK1/2, p-ERK1/2 (dilution 1: 1000). After washing, membranes were then probed with the HRP-conjugated secondary antibody (dilution 1:10000, Bioworld Technology, Louis Park, MN, USA). The complexes were finally detected with ECL reagent (Vazyme Biotech, Nanjing, China), visualized by ChemiDoc™ MP System (Bio-Rad) and analyzed using Image Lab™ software (version 4.1, Bio-Rad). 2.11. Screening of bioactive components The EAT was prepared as afore mentioned, then YQFM solution was added and removed after incubation for 24 h. Finally, the tissue was washed with PBS for 5 times to remove unbound components, denatured and extracted with 80% methanol by ultrasonic extraction to liberate components bound to the tissue. The desorption eluate was centrifugated and the supernatant was condensed for HPLC-MS analysis. 2.12. Statistical analysis

2.7. Determination of caspase-3 activities in vivo

All values in the text and figures were expressed as mean ± SEM. Statistical analysis was carried out using Student’s two-tailed t-test for comparison between two groups and one-way analysis of variance (ANOVA), followed by Dunnett’s test when the data involved three or more groups. P < 0.05 was defined as significant.

After the hearts were homogenized, samples were centrifuged twice at 12,000 rpm for 10 min at 4 °C. The supernatant was collected for the determination of protein content with Bradford protein assay kit (Nanjing Jian Cheng Biotech Co., Ltd., Nanjing, China). The rest of supernatants were obtained for analyzing the level of caspase-3 by ELISA kit (Shanghai Kejian Biotech Co., Ltd., Shanghai, China).

3. Results

2.8. TUNEL staining for apoptosis in vivo

3.1. YQFM improved the left ventricular contractile function and decreased the concentration of serum biochemical indicators in CAL-induced HF mice

Two weeks after surgery, the hearts were collected, fixed in 4% paraformaldehyde and embedded in paraffin. The specimens were sectioned at 4 μm thickness, deparaffinized and rehydrated in PBS, terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling (TUNEL) staining was performed using the TUNEL BrightGreen Apoptosis Detection Kit (Vazyme Biotech, Nanjing, China). After apoptotic nuclei were labeled with green fluorescein staining, DAPI was used for nuclear staining. For each slice, TUNEL-positive cells were counted in randomly selected three fields under a fluorescence microscope (Leica Microsystems) and expressed as the ratio of TUNEL positive nuclei over DAPI stained nuclei.

As shown in Fig. 1, the significant differences in the mVCF (Fig. 1B), SV (Fig. 1C), LVPEP (Fig. 1D) and LVPEP/ LVET (Fig. 1E) between the model and sham group indicated reduced cardiac contractile function. While compared with the model group, YQFM with the dosage of 0.26 g/kg and 0.53 g/kg significantly enhanced mVCF and SV, meanwhile, YQFM (0.53 g/kg) obviously reduced LVPEP and LVPEP/LVET ratio, indicating that YQFM could improve the cardiac contractile function of HF mice. Images of M-mode echocardiogram were shown in Fig. 1A. In order to further estimate the myocardial damage induced by CAL, the content of CKeMB, AST, IL-6, Tn, myosin, MYO and MYOC were 3

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Fig. 1. YQFM improved the cardiac contractile function in CAL-induced HF mice. (A) Representative images of M-mode echocardiograms. Effect of YQFM on mVCF (B), SV (C), LVPEP (D), LVPEP/ LVET ratio (E) changes after 14 days of CAL in mice. Results were presented as mean ± SEM. #P < 0.05, ##P < 0.01 vs. Sham group, *P < 0.05, **P < 0.01 vs. Model group. n=10.

measured in serum. CKeMB, AST, IL-6, Tn, myosin, MYO and MYOC were significantly increased in model group (Fig. 2A–G). Whereas, the mice treated with YQFM distinctly decreased serum levels of these cardiac biochemical indicators.

omentin level of the same mouse was recorded, and then established corresponding relationship with its EF and NT-proBNP by correlational analysis. As illustrated in Fig. 3A–B, plasma omentin levels in serum of HF mice were positively associated with the recovery of myocardial function and injury. Further, as shown in Fig. 3C-E, there were no significant differences in the LVEF and LVFS between the 1 days’ CAL model group and sham group. Until 7 days passed, the LVEF and LVFS of model group significantly decreased. By contrast, YQFM markedly attenuated the gradual deterioration of contractile capability of HF mice. Similarly, YQFM treatment significantly improved the exacerbation of IVSd, LVIDd, LVPWd, LV Vold, and LV Mass in relatively later period of HF (generally

3.2. Correlation analysis of omentin with cardiac function in CAL-induced HF mice Then, we examined whether the circulating omentin level is associated with myocardial function and injury in HF mice. The EF has been assessed by echocardiography. In addition, the concentration of NTproBNP in serum has also been measured. Meanwhile, the circulating 4

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Fig. 2. YQFM decreased the CKeMB activity (A), AST activity (B), IL-6 content (C), Tn content (D), myosin content (E), MYO content (F), MYOC content (G) in CALinduced HF mice. Results were presented as mean ± SEM. #P < 0.05, ##P < 0.01 vs. Sham group, *P < 0.05, **P < 0.01 vs. Model group. n=10.

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Fig. 3. Association of plasma omentin levels with LVEF (A) and changes in NT-proBNP content (B) after 14 days' CAL, and the effect of YQFM on cardiac function and omentin content in 1, 7, 14 days' CAL-induced HF mice. (C) Representative echocardiograph from the various groups; (D) LV ejection fraction; (E) LV fractional shortening; (F) IVSd; (G) LVIDd; (H) LV Vold; (I) LVPWd; (J) LV Mass; (K) Omentin content. Results were presented as mean ± SEM. #P < 0.05, ##P < 0.01 vs. Sham group, *P < 0.05, **P < 0.01 vs. Model group. n=10.

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Fig. 4. The time-dependent change of omentin secretion (A) and YQFM's effect (B) on normal mice's isolated epicardial adipose tissue (EAT) and subcutaneous adipose tissue (SCAT). (C) The change of omentin content in EAT medium from each treated mice group. (D) The effect of conditioned medium on SI/R-induced cardiomyocytes injury. (E) The effect of conditioned medium on SI/R-induced cardiomyocytes apoptosis. (F) Quantitative analysis of apoptotic cells in indicated groups. Results were presented as mean ± SEM. #P < 0.05, ##P < 0.01 vs. Control group, *P < 0.05, **P < 0.01 vs. Model group. n=10.

after 14 days of CAL, Fig. 3F–J). Collectively, these data suggest that the protective effect of YQFM on the development of pathological hypertrophy and cardiac remodeling needs a relatively long time for treatment.

3.4. YQFM increased the omentin concentration in the isolated EAT of CALinduced HF mice Although YQFM could increase the serum omentin level in HF mice, it is still unclear whether the increase of omentin was secreted by adipose tissue and which kind of adipose tissue that the omentin mainly came from. To elucidate these problems, the EAT and SCAT have been isolated and cultured. As shown in Fig. 4A, the omentin level in the medium of EAT and SCAT was closer after 24 h cultured in vitro. While with the increase of time, the omentin level of these two groups’ medium gradually became lager, which indicated that the 24 h is an appropriate time point to verify the effect of YQFM on adipose tissue. As presented in Fig. 4B, after incubating YQFM for 24 h, omentin level

3.3. YQFM increased the plasma omentin level in CAL-induced HF mice As noticed in Fig. 3K, omentin markedly decreased in the 1 and 14 days' CAL-induced model group compared with the sham group. Whereas, the mice treated with YQFM, clinical drugs CAP or Met significantly improved serum levels of omentin compared with 14 days CAL-induced HF mice. 7

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Fig. 5. Effect of YQFM on CAL-induced myocardial apoptosis in HF mice. (A) Representative photomicrographs of TUNEL staining images. n = 3. (B) Quantitative analysis of apoptotic cells in indicated groups. (C) Activity of caspase-3 was measured through the specific cleavage of substrates in each group. n = 6. (D) Protein expression levels of Bcl-2 and Bax were determined by Western blot analysis. n = 3. (E) The cleaved caspase-3 production was measured by immunohistochemistry (Bar =50 μm). n = 3. Results were presented as mean ± SEM. #P < 0.05, ##P < 0.01 vs. Sham group, *P < 0.05, **P < 0.01 vs. Model group.

in the medium of EAT significantly increased compared with control group, indicating YQFM directly improved the omentin secretion in isolated EAT.

We further observed SI/R-induced cardiomyocytes apoptosis in the presence of CM. And we found that CM from drug treated mice’s EAT markedly decreased the apoptosis of cardiomyocytes (Fig. 4E-F). Clearly, these data indicated that CM collected from drug treated mice’s EAT could protect SI/R-induced cardiomyocytes injury and apoptosis. And the effect of CM might due to the increased omentin secretion level of EAT in mice.

3.5. Isolated EAT conditioned medium from YQFM attenuated SI/Rinduced injury and apoptosis in H9c2 cardiomyocytes To further verify the effect of YQFM whether depend on the change of omentin level, the CM of isolated EAT has been prepared. Initially, the omentin level in the CM of isolated EAT was measured, which cultured in vitro for 24 h. After 14 days CAL, isolated EAT of model mice secreted lower omentin into CM (Fig. 4C). While after treatment, the level of omentin, which came from EAT of YQFM, CAP and Met treated mice, significantly increased. In addition, the CM collected from different groups was applied to culture H9c2 cells respectively. Exposure of H9c2 cardiomyocytes to OGD led to a decrease in cell viability. Whereas treatment with CM, which came from isolated EAT of YQFM, CAP, and Met treated mice, maintained cell viability (Fig. 4D).

3.6. YQFM inhibited myocardial apoptosis associate with omentin increase in CAL-induced HF mice On the basis of previous results, the effect of YQFM on apoptosis in vivo was further elucidated. As shown in Fig. 5A,B, following 14 days CAL, HF mice exhibited obvious TUNEL-positive (apoptosis) cells in myocardium compared to sham treated mice. By contrast, a significant lower proportion of apoptotic cells was observed in heart slices from YQFM-treated mice. Moreover, as presented in Fig. 5C–E, 14 days CAL resulted in a noticeable increase in caspase-3 activity and expression, 8

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and the decrease of Bcl-2/Bax ratio. While YQFM treatment suppressed caspase-3 activity and the level of cleaved caspase-3 in cardiac myocytes. As previously reported [14], omentin could protect myocytes from apoptosis against ischemia/reperfusion injury, thus, the effect of YQFM on CAL-induced apoptosis in HF mice might depend on omentin increase, which came from EAT.

MYO, myosin and Tn reflect myocardial damage and infarcted myofilaments [28–31]. Besides, Tn, myosin and MYOC are important contractile proteins in myocardial tissue [32], thus, elevated bloodstream concentrations of them indicate that cardiac systolic function is damaged. And these results of echocardiography and determining biomarkers all illuminated the beneficial effects of YQFM on CAL-induced cardiac contractile dysfunction in mice. Recently, clinical studies have demonstrated that increased levels of plasma omentin in post-acute myocardial infarction patients were in association with improvement in myocardial damage and function after successful reperfusion therapy [33,34]. Previous investigations have indicated that omentin could regulate endothelial cell function and accelerate revascularization processes in response to ischemia [35]. Moreover, omentin was reported to promote vasodilation in isolated blood vessels [36], and systemic administration of omentin could ameliorate acute ischemic injury in the heart [14]. Thus, it is conceivable that omentin acts as a novel potential molecular target of cardiovascular disorders. However, no studies have shown that drugs could attenuate HF through modulating omentin levels. In our previous study, YQFM was demonstrated to attenuate CALinduced myocardial remodeling and heart failure through modulating MAPKs signaling pathway [21]. In addition, we have reported that YQFM could ameliorate ischemia/reperfusion-induced myocardial apoptosis via AMPK activation [20]. Similarly, omentin also prevents myocardial ischemic injury through AMPK and Akt dependent mechanisms [14], and AMPK-mediated reduction of the Ras/ERK signaling cascade appears to participate in the anti-hypertrophic effect of omentin in the heart [15]. Nevertheless, nothing is known about whether the impact of YQFM on CAL-induced HF associated with omentin. In present study, we first demonstrated that YQFM could increase circulating omentin content in CAL-induced HF mice. Meanwhile, echocardiography analysis showed that YQFM treatment improved cardiac performance and the serum omentin levels positively associated with LVEF, while negatively associated with NT-proBNP levels, which indicates that omentin functions to improve the LV function in CAL-induced HF mice. Our previous studies pointed out that YQFM could directly attenuate SI/R-induced cardiomyocytes injury [20], and exert myocardial protection effect in a non omentin-dependent way. Whereas, the omentin-dependent cardioprotection remains to be further elucidated due to the fact that YQFM could increase circulating omentin levels in CAL-induced HF mice. Omentin, as a circulating adipokine, is closely associated with adipose tissue [37]. Adipose tissue has functions beyond energy storage, such as acting as an endocrine organ to contribute to inflammatory and metabolic responses [38]. Mammalian adipose comprises two types of fat, white fat (subcutaneous and visceral fat) and brown fat. Our results exhibited that YQFM could significantly improve the ability of omentin secretion by EAT, while exert no effect on SCAT. EAT, the adipose tissue surrounding the heart, is deposited under the visceral layer of the pericardium and is regarded as a source of adipocytokines [39]. Due to its close proximity, it is not surprising that EAT plays an essential role in the progress of cardiovascular disease. And the regulation of omentin secretion from EAT might serve as an important pathway of myocardial protection. We further treated HF mice with YQFM and collected the EAT's medium as CM to treat H9c2 cardiomyocytes injured by SI/R. The ability of EAT to produce omentin decreased in HF mice, while YQFM treatment improved the ability. In addition, CM derived from HF mice could not attenuate SI/R-induced cardiomyocytes injury due to the low content of omentin, nevertheless, CM from HF mice after YQFM treatment markedly ameliorated SI/R-induced cardiomyocytes injury and apoptosis. Interestingly, we also found that two commonly used drugs for the treatment of HF in clinic, named metoprolol and captopril, could also improve myocardial injury and apoptosis through up-regulation of omentin release, which suggested that angiotensin converting enzyme inhibitors and beta receptor blockers could also increase the omentin

3.7. YQFM enhanced PI3 K/Akt, AMPK activation and inhibited MAPKs activation correlating to omentin increase in CAL-induced HF mice To further elucidate the potential mechanism of YQFM, the effects of YQFM on AMPK, PI3 K-Akt and MAPKs signaling pathway have been measured. The western blot results revealed that treatment with YQFM increased PI3 K expression and Akt phosphorylation and also increased AMPK phosphorylation (Fig. 6A–C). These results indicated that YQFM increased the activation of the AMPK and PI3 K-Akt signaling pathways after at least 7 days of treatment in HF mice. Interestingly, the change of the activation of AMPK, PI3 K, and Akt were similar as the secretion of omentin in serum of CAL mice, which indicated that YQFM might increase omentin secretion, and then activate AMPK and PI3 K-Akt signaling pathway. In addition, the activation of p38, JNK, and ERK1/2 significantly increased when 7 days after CAL performed. However, YQFM treatment significantly inhibited phosphorylation of p38, JNK, and ERK1/2 (Fig. 6D–F). This effect of YQFM might result from the increase of serum omentin, because of that omentin could inhibit the over activation of MAPKs signaling pathway to attenuate cardiac hypertrophic response, which is common in HF patients [15]. 3.8. Ginsenoside Rd in YQFM increased the release of omentin EAT and SCAT were administrated with YQFM for 24 h in vitro and then HPLC-Q-TOF/MS was applied to detect the binding ingredients. As shown in supplementary Fig. 1A-C, there were three different peaks between adipose tissue and control samples, and the retention time of peak were similar with ginsenoside Rd and ginsenoside Rg3. Further, ginsenoside Rd was validated via the MS spectra of HPLC-Q-TOF/MS analysis (Supplementary Fig. 1D). Finally, the isolated EAT were administrated with YQFM, ginsenoside Rd and schisandrin A for 6 h hypoxia and then 6 h reoxygenation. Schisandrin A is one of the main components in YQFM, and we applied it as a negative control. We found that YQFM and its mainly component ginsenoside Rd significantly increased the content of omentin, while schisandrin A showed no effect on omentin secretion (Fig. 6G). 4. Discussion The present study revealed that YQFM could improve the left ventricular function and ameliorated cardiac structural pathological changes in CAL-induced HF mice. Moreover, circulating omentin level positively associated with CAL-induced HF, and could ameliorate HF. Importantly, YQFM could improve the secretion of omentin from EAT to attenuate CAL-induced HF via the cross-talk between adipose tissue and cardiomyocytes. Ginsenoside Rd, which is an ingredient of YQFM, could increase the omentin secretion from HF mice's EAT. Echocardiography is clinically applied to detect cardiac structure and function. LVEF and LVFS were used to evaluate cardiac contractile capabilities both in basic and clinical researches [24,25]. In addition, mVCF and SV could also response to contractile function [26]. The activities of certain cardiac marker enzymes reflect the pathological process of myocardial diseases. Myocardial enzymes such as CK-MB and Tn are the most common biomarkers for MI and HF. In addition, the quantification of NT-proBNP is recommended by the European Society of Cardiology guidelines as a test to rule out HF. Our present studies showed that YQFM could maintain the cardiac function. CK-MB is found mainly in myocardium, but serum CK-MB content increase following myocardial injury [27]. Further, an increase of serum AST, 9

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Fig. 6. Effect of YQFM on PI3 K/Akt, AMPK and MAPKs signaling pathways in 1, 7, 14 days' CAL-induced HF mice. The PI3 K (A), p-Akt, Akt (B), p-AMPK, AMPK (C), p-JNK, JNK (D), p-p38, p38 (E), p-ERK1/2, ERK1/2 (F) expression were detected using Western blot analysis. n = 3. (G) Ginsenoside Rd increased omentin secretion from EAT. Results were presented as mean ± SEM. #P < 0.05, ##P < 0.01 vs. Sham group, *P < 0.05, **P < 0.01 vs. Model group, &&P < 0.01 vs. ginsenoside Rd group.

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secretion of ETA during the treatment of HF. However, the potential mechanism of YQFM and positive drugs in up-regulation of the adipokine omentin content in serum of heart failure mice remains to be further explored. Our results indicated that myocardial protection via omentin up-regulation from EAT is not a unique pathway for YQFM. Moreover, our present data showed that YQFM inhibited myocardial apoptosis in CAL-induced HF mice, associated with enhanced PI3 K/ Akt, AMPK activation and inhibited MAPKs activation. Above-observed results and previous studies indicated at least two ways involved in the cardioprotective effect of YQFM, omentin-dependent and non omentin-dependent (direct myocardial protection). In view of the complexity of YQFM ingredients, therefore, HPLC-Q-TOF/ MS was applied to better understanding the mechanism whereby the compound works. Present studies exhibited ginsenoside Rd in YQFM could be absorbed in isolated EAT and improve the secretion of omentin. Numerous literatures have reported that ginsenoside Rd could attenuate myocardial ischemic injury through multiple pathways [40–42], however, it was first demonstrated to exert cardioprotective effect by increased omentin secretion. These findings argued that the omentin-dependent protective effect of YQFM was at least partially mediated by ginsenoside Rd. The present study still has some limitations. Firstly, more studies are required to better understanding the mechanism whereby the ingredients functions due to the complexity of YQFM. Secondly, the experiments focused on other adipose tissue and released cytokines in the regulation of cardiovascular diseases are needed in further study. Finally, the underlying mechanism of increased omentin secretion induced by YQFM remains to be elucidated. In brief, our works show that YQFM modulates omentin secretion in EAT, then inhibits myocardial apoptosis via positive regulation of AMPK, PI3 K/Akt and negative regulation of MAPKs signaling pathways, and thereby ameliorates CAL-induced HF. The findings suggest that the regulation of cross-talk between adipose tissue and cardiomyocytes might be a potential target through which YQFM and its bioactive components ginsenoside Rd exerts cardioprotective effect. Our researches provide essential insights for the understanding of the molecular mechanisms of TCM in the treatment of cardiovascular diseases, and offer a reference for investigating the relationship between adipose tissue and cardiovascular diseases.

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Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This research work was supported by the National Natural Science Foundation of China (No. 81973506, No. 81603328, No. 81774150, No. 81573719), Natural Science Foundation of Jiangsu Province (BK20160761), Project funded by China Postdoctoral Science Foundation (2016M600456, 2017T100425). And we have confirmed that the mentioned received funding did not lead to any conflict of interests regarding the publication of this manuscript. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.biopha.2019.109418. References [1] Y. Jiang, Y. Bi, J. He, Y. Xu, L. Wang, M. Xu, Status of cardiovascular health in Chinese adults, J. Am. Coll. Cardiol. 65 (2015) 1013–1025. [2] J. Ampadu, J.E. Morley, Heart failure and cognitive dysfunction, Int J Cardio 178

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