Yixin-Shu Capsules Ameliorated Ischemia-Induced Heart Failure by Restoring Trx2 and Inhibiting JNK/p38 Activation

Traditional Chinese medicine has shown great safety and efficacy in the treatment of heart failure (HF), whereas the mechanism remains unclear. In this study, the protective effect of Yixin-shu (YXS) capsules, a conventional medicine for various cardiovascular diseases, against myocardial ischemia-induced HF in rats was systematically investigated by RNA-seq technology. HF rats treated with YXS (0.8 or 1.6 g/kg/d, ig) for 6 weeks had significantly decreased brain natriuretic peptide (BNP) and atrial natriuretic peptide (ANP) and collagen III and attenuated cardiac structure rupture and collagen deposition. Additionally, YXS treatment decreased the levels of interleukin-1β (IL-1β), interleukin 6 (IL-6), tumor necrosis factor-α (TNF-α), and lactate dehydrogenase (LDH) and TUNEL-positive rate and the nitrotyrosine staining, but increased levels of glutathione (GSH), total antioxidant capacity (T-AOC) activity, and mitochondrial membrane potential. Further experiments demonstrated that YXS restored Trx2 and inhibited the phosphorylation of JNK and p38, thereby improving cardiac function in the rats with HF. Silencing Trx2 decreased the protection of YXS in the response to H2O2 as evidenced by the increase of caspase-3 activity and decrease of GSH level. Thus, YXS enhanced heart function and decreased myocardial damage through restoring Trx2 and inhibiting JNK and p38 activation in ischemia-induced HF.


Introduction
Heart failure (HF), characterized by reduced heart contractility and cardiac function, is an end stage of various cardiovascular diseases, which results in serious myocardial injury at the beginning and leads to damage of many other organs in the end [1]. Multiple insults including myocardial ischemia, atherosclerosis, hypertension, cardiomyopathy, and genetic problems cause myocardial damage and result in HF. Among all these insults, myocardial ischemia has emerged as one of the major causes of HF in recent decades. Several symptoms such as cardiomyocyte hypertrophy, elevated neurohumoral level, and enhanced filling pressure happened which attempted to compensate the dysfunction of the heart and finally resulted in the acceleration of cardiac remodeling and contraction declining [2,3]. Due to the complexity of HF, multiple pharmacologic remedies were applied to halt HF progression, with unexpected side effects. For example, β-adrenergic agonists improve cardiac contractility, but also increase mortality in HF, while neurohormonal inhibition, a meaningful intervention for HF, is restricted by its side effect, such as deterioration of renal function, potassium retention, and angioedema caused by angiotensinconverting enzyme inhibitor (ACEI) [4,5]. Some other therapies such as diuretics only focus on the alleviation of the symptoms of HF, but do not prevent deterioration of HF. Thus, it is of importance to explore effective therapies for halting HF progression and revealing the underlying mechanism.
Great attention has been paid to traditional Chinese medicine (TCM) due to their effectiveness and few side effects in halting HF [6][7][8]. For example, in a randomized, multicenter placebo-controlled design with double-blind study, 640 patients with ischemia HF who were treated with Qishenyiqi dripping pills for 6 months in addition to standard treatment demonstrated improved exercise tolerance when compared to placebo [9]. Additionally, Qiliqiangxin capsule has been demonstrated to reduce important cardiac damage markers such as NT-pro-brain natriuretic peptide and improve the life quality of patients [10]. Even though considerable researches have been conducted to investigate the efficacy and the mechanism of TCM, an ample amount of researches is still needed to draw concrete conclusion whether TCM has an effect on the HF treatment. In particular, the ambiguous mechanism and poor clinical locations of TCM hinder their further application.
Yixin-shu (YXS) capsules, which originated from an ancient TCM formula named ShenMaiSan, is a standardized Chinese Materia Medicine product recorded in the Chinese Pharmacopeia and is widely used for various cardiovascular diseases in clinic [11,12]. YXS is composed of seven herbs, including Radix Ginseng, Fructus Schisandrae Chinensis, Radix Ophiopogonis, Astragalus Membranaceus, Salvia Miltiorrhiza, Ligusticum Wallichii, and Fructus Crataegi. Recent researches revealed that YXS remarkably decreased myocardial ischemia/reperfusion injury, reduced hydrogen peroxide-caused damage, and improved ET-1-induced cardiomyocyte dysfunction [6,13,14]. As early as 24 h after reperfusion, YXS significantly decreased myocardial injury and ameliorated heart function through decreasing oxidative damage and cell apoptosis via upregulating liver-X-receptor α [13]. And potential HF biomarkers such as TNNI3, GAL-3, HSP 70, FABP3, and CKAP5 were identified in the protection of YXS against HF which facilitated the clinical location of YXS [6]. In vitro, YXS protected against H 2 O 2 -induced damage through APEX1, TFCP2, and 5 other transcription factors by using an integrated strategy of transcriptome, proteomics, and pharmacological network analysis [15] and then identified schisandrin A and schisandrin B as the effective components of YXS against ET-1-induced cardiomyocyte dysfunction [14]. Additionally, YXS was proved to promote mesenchymal stem cells to differentiate into cardiac-like cells in vitro [11]. It is worth noting that although it was previously determined that YXS has a significant protective effect on HF induced by myocardial ischemia, the mechanism remains unclear. Here, taking advantage of RNA-seq technology, the protective effect of YXS on MI-induced HF was systematically investigated and the underlying mechanism was revealed, to provide reference for promoting the clinical application of YXS.

Materials and Methods
2.1. Animal Model. Male Sprague-Dawley (SD) rats with weight at 250-270 g were provided by Beijing Huafukang Bio-technology Co., Ltd., China ((certificate no. SCXK (Jing) 2014-0004)). The rats were kept in the Laboratory Animal Center of China Academy of Chinese Medical Sciences (Beijing, China) with a 12-hour alternating of day and night. Free access to food and water was allowed for the rats. All the animal experiments were approved by the Committee on the Animal Care and Use of Institute of Experimental Research Center, China Academy of Chinese Medical Sciences (Beijing, China).
The SD rats were anesthetized with 10 mg/ml pentobarbital sodium by intraperitoneal injection before the following surgery. Briefly, the left anterior descending coronary artery (LAD) of these rats was tightened for four weeks to prepare the HF rats. Briefly, the LAD was tightened with a 6-0 silk suture. The sham normal group rats received the same procedures but without tightening the silk suture at LAD. After the surgery for 4 weeks, the rats with HF were randomly divided into four groups according to the echocardiography results. The rats received different treatments such as normal (saline, ig), HF (heart failure, saline, ig), HF+YXS-H (1.6 g/kg/d, ig), HF+YXS-L (0.8 g/kg/d, ig), and HF+VST (Valsartan 8 mg/kg/d, ig  [11]. Valsartan was purchased from Hainan Aumei Pharmaceutical Co., Ltd. (Hainan, China). Visual Sonics Vevo 770 Ultrasound System (Toronto, Canada) with a 17.5 MHz probe was applied to detect the heart function at 0 and 6 weeks after drug administration, and the left ventricular fractional shortening (LVFS) and left ventricular ejection fraction (LVEF) were calculated.
2.2. RNA-Seq Analysis. TRIzol reagent (Cat# 15596-018, Life Technologies, USA) was applied to obtain RNA from the harvested samples. After the RNA integrity was assessed, the RNA was used for the following sequencing libraries based on Illumina® NEBNext® Ultra™ RNA Library Prep Kit (NEB, USA). The next step was the same to the previous researches [16]. All the sequencing and mapping were finished by Novogene Bioinformatics Technology Co., Ltd. (Beijing, China). After sequencing, the read mapping referred to rat reference genome (ensemble release 83) and the read number was obtained by using HTSeq v0.6.1. Fragments per Kilobase Million (FPKM) were used to represent the gene expression level, and edgeR software was applied to calculate differentially expressed genes (DEs) [17]. The detected genes with fold change no less than 2 and FDR < 0:05 were defined as DEs. The raw data of YXS-mediated protection against HF 2 Oxidative Medicine and Cellular Longevity has been uploaded into https://www.ncbi.nlm.nih.gov/sra/ PRJNA532999. The DEs after YXS treatment were enriched using DAVID [18]. The DEs were associated with disease targets of HF using String, and then, the network was generated by using Cytoscape 3.4.0.
The heart samples were harvested and fixed in 4% (v/v) polyformaldehyde and then dehydrated before being embedded by paraffin for further analysis. The embedded samples were sectioned, rehydrated, and stained with H&E staining and Masson's trichrome staining (D026-1-3) to evaluate heart injury and collagen deposition. As for the IF for heart tissue, the samples were rehydrated and then incubated with sodium citrate before permeabilizing with Triton X-100, which was followed by 5% BSA blocking. For IF staining, the cell samples were incubated with 10% (v/v) formalin for 20 min and then followed with 0.5% Triton X-100 for 20 min. After that, the samples were treated with 5% BSA for 30 min, before the primary antibodies such as thioredoxin 2 (Trx2, ab185544) and p-JNK (ab131499) and nitrotyrosine (sc101358) were added, respectively, and incubated at 4°C overnight. Then, samples were treated with second antibodies such as goat anti-mouse (ab6785) or goat anti-rabbit (ab150080). The nucleus was counterstained with DAPI for 10 min and observed with LSM-880 confocal microscope (Carl Zeiss, Oberkochen, Germany).
2.4. JC-1 Staining and TUNEL Staining. TUNEL detection kit (G002) purchased from Nanjing Jiancheng Bioengineering Institute and mitochondrial membrane potential assay kit with JC-1 (C2006) from Beyotime Biotechnology were used for cell apoptosis detection. All procedures were performed based on the instruction from the kits. Briefly, the cells with different treatments were incubated with JC-1 staining solution at 37°C for 20 minutes and then washed twice with JC-1 staining buffer before being observed under an LSM-880 confocal microscope (Carl Zeiss, Oberkochen, Germany). For terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining, the cells after various treatments were fixed with 4% paraformaldehyde for 30 min and then incubated with 1% BSA for 10 min. After that, the samples were incubated with 0.5% Triton X-100 for 20 min, which was followed by TdT enzyme reaction solution for 60 min treatment. Finally, DAPI was used for nucleus staining for 5 min before observing with an LSM-880 confocal microscope (Carl Zeiss, Oberkochen, Germany). And the quantitative result of TUNEL-positive cells and the fluorescence ratio of red and green were carried out through ImageJ. (ab126425) were used to detect the p-JNK/JNK and p-p38/p38, respectively. And the ELISA experiments were performed according to the instructions. Briefly, the serum or protein extract was incubated into the plates immobilized with specific antibodies and followed by incubation with a secondary antibody conjugated with a horseradish peroxidase for detection through a microplate reader (DNM-9602G).
2.6. Western Blotting. Heart tissue was lysed using RIPA lysis buffer (R0020, Solarbio), with protease inhibitor (0.1% phenylmethanesulfonyl fluoride (PMSF)). The method of protein extraction is as follows: the lysate was prepared by using 1 ml RIPA with 10 μl phenylmethanesulfonyl fluoride (PMSF, protease inhibitor), and the final concentration of PMSF is 1 mM. The myocardial tissue was cut into small fragments, and the lysate was added according to the proportion of 150-250 μl lysate per 20 mg tissue and homogenized with a glass homogenizer until it was fully cracked. The pyrolyzed sample was centrifuged at 10000-14000 g for 3-5 minutes, and the supernatant was collected. The solution was boiled after the loading buffer of 4′ sodium dodecyl sulfonate (SDS, P1015, Solarbio) was added. Then, the separation of proteins was carried out on 10% polyacrylamide gels by electrophoresis and then was transferred to polyvinylidene difluoride membranes. Bovine serum albumin was used to block, and primary antibodies such as anti-Trx2 antibody (ab185544, Abcam), anti-JNK1/2 antibody (sc-137019), anti-JNK1+JNK2 (phospho T183+Y185) antibody (ab131499), anti-collagen III antibody (Sigma C7805), and anti-β-actin antibody (cst4970) were loaded for 24 h at 4°C and then incubated with secondary antibody conjugated with HRP (Jackson 111-035-003). The signals on PVDF membranes were captured using scanning densitometry with image analysis software (Science Lab 2005 Image Gauge; Tokyo, Japan) after applying an enhanced chemiluminescence plus detection system (Pierce Biotechnology).

Cell
Culture, Cell Viability, LDH and GSH Levels, and T-AOC Activity. Peking Union Medical Collage provided H9C2 cells for our in vitro experiments. The cells were cultured at 37°C in an incubator with culture medium containing highglucose DMEM (Gibco, USA), 10% (v/v) fetal bovine serum, 100 μg/ml penicillin, and 100 μg/ml streptomycin. The culture medium was changed every three days, and cells were passaged when 80% of cell confluence was reached. As for cell viability detection, H9C2 cells were incubated with various concentrations of YXS extract (0, 3.91, 15.625, and 62.5 μg/ml) and then incubated with 500 μm H 2 O 2 for 1 hour. After that, cell viability was measured by using Cell Counting Kit-8 (CCK-8) which was obtained from Dongren Chemical Technology Co., Ltd. The YXS extract was prepared by dissolving the powder from YXS capsule in ethanol (95%, v/v) and extracting for 2 hours, followed by rotary evaporation and then resuspending with Tyrode buffer solution (glucose 1.00 g, NaCl 8.00 g, NaHCO 3 1.00 g, MgCl 2 0.10 g, CaCl 2 0.20 g, KCl 0.28 g, NaH 2 PO 4 0.05 g, pH 7.4) at a concentration of 16% (w/v). The kits for LDH (A020-2), GSH (A006-2-1), and T-AOC (A015-2-1) were obtained 3 Oxidative Medicine and Cellular Longevity from Nanjing Jiancheng Bioengineering Institute, and the detection was performed according to their instructions.
2.10. Measurement of Caspase-3 Activity. Caspase-3 activity (G015-1-2) was obtained from Nanjing Jiancheng Bioengineering Institute, and the detection was performed according to their instructions. Briefly, the cell proteins were added with 25 μg Ac-DEVD-pNA at 37°C for 90 min, which was used as a colorimetric-specific substrate. The OD value was measured on a microplate reader (Molecular Devices, USA). One unit of caspase-3 activity is the enzyme amount that cleaves 1 nmol Ac-DEVD-pNA at 37°C per hour under saturated substrate concentrations.
2.11. Statistical Analysis. The comparison of the results was performed using SPSS V17.0 software. One-way ANOVA with LSD's post hoc multiple-comparison analysis was used for the comparison for multiple groups while the Student t -test was used to analyze data between two groups. Data was expressed as mean ± standard deviation (SD), and P < 0:05 was deemed statistically significant, P < 0:01 for highly significant, while P < 0:001 for remarkably significant.

YXS Treatment Improved MI-Induced HF.
To investigate the protective effect of YXS against HF, histology staining, cardiac contraction function, and ANP and BNP were measured. As shown in Figure 1, an increase of ANP and BNP in HF was remarkably reduced after YXS treatment, which exhibited the same protection effect as the positive drug VST. In addition, an obvious rupture and breakage in myocardial fiber and collagen deposition were observed in the failing hearts, while treatment of YXS or VST apparently ameliorated rupture and collagen deposition in myocardial tissue. Additionally, the increase of collagen III in HF was reduced by YXS or VST treatment, and this was consistent with the Masson staining result, further confirming the attenuation of myocardial fibrosis by YXS and VST. Moreover, an obvious reduction in left ventricular ejection fraction (Δ%LVEF) was found in the HF group, indicating an obvious myocardial dysfunction occurred in the HF group. However, Δ%LVEF was significantly higher with the treatment of YXS or VST. These data indicated that YXS showed an obvious protection in rats with HF.

RNA-Seq Analysis of the Potential Mechanism of YXS.
The high-throughput RNA-seq technology is a widely used tool for the transcriptome gene profile analysis and is suitable for the investigation of the mechanism of TCM in multiple pathological processes. To reveal the underlying mechanism of YXS-mediated protection effect, the transcriptional gene expression was analyzed by RNA-seq technology (Table S1). As indicated by Figure 2(a), there were 637 DEs with 519 upregulated and 120 downregulated in HF compared to the normal group. In the YXS-treated group, 333 DEs including 113 upregulated and 220 downregulated were found when compared to the HF group. Different from YXS treatment, 125 upregulated genes with only 74 downregulated genes were observed after VST treatment, indicating these two drugs work in a different manner. Further hierarchical cluster analysis of the RNA-seq data indicated that YXS treatment reversed the upregulated genes involved in biological process including "inflammatory response," "Tolllike receptor signaling pathway," "extracellular matrix organization," "focal adhesion," "cell migration," "cell proliferation," "dilated cardiomyopathy," and "regulation of apoptotic process" while it increased the downregulated genes involved in "heart contraction" and "regulation of blood circulation" in the HF group (Figure 2(b)).
To further reveal the key biological process in YXSmediated protection against HF, the DEs between various treatments were enriched into Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and Gene Ontology (GO) as shown in Figures 2(c) and 2(d). The enriched KEGG and GO terms in HF included "inflammatory response," "MAPK cascade," "cell growth," "PI3K-AkT signaling pathway," "cell proliferation," "extracellular matrix," "cell migration," "regulation of apoptotic process," "collagen fibril organization," and "NF-kappaB (NF-KB) import into nucleus." It is noteworthy that DEs between HF with or without YXS treatment were also enriched into terms such as "inflammatory response," "MAPK cascade," "extracellular matrix," "cell migration," "cell proliferation," and "NF-kappaB import into nucleus." Moreover, YXS also showed an obvious effect on genes involved in "leukocyte migration," "cellular response to interleukin-1," and "negative regulation of cell death." The in vitro RNA-seq analysis indicated that upregulated genes were enriched into GO terms such as "cell cycle," "DNA repair," and "DNA replication" while downregulated genes were enriched into GO terms such as "MAPK cascade," "inflammatory response," "apoptotic process," and "response to oxidative stress" (Figure S1).
Further associating the DEs with disease targets of HF demonstrated that 60 DE genes were associated with 63 disease targets, which were enriched into GO terms such as "actin cytoskeleton," "Toll-like signaling pathway," "MAPK  10x; (f) western blotting of collagen III and its semiquantitative data by ImageJ (n = 3); (g) the representative echocardiography images of rats receiving different treatments in short-axis M-mode and the increased LVEF (?%LVEF) after various treatments (n = 6). Data were expressed as mean ± SD (n = 5 -7). # P < 0:05 and ## P < 0:01 vs. normal; * P < 0:05 and * * P < 0:01 vs. HF. 5 Oxidative Medicine and Cellular Longevity signaling pathway," "NOD-like receptor signaling pathway," "cellular calcium ion homeostasis," and "cardiac muscle contraction" (Figure 3). To verify the network analysis results, the levels of Tlr4 and Myd88 which were critical targets in the Toll-like signaling pathway and changes in F-actin of heart tissue were also confirmed in Figure S2. The increased expression of Tlr4 and Myd88 in H 2 O 2 -induced cell model was significantly decreased by YXS treatment ( Figure S2A). Additionally, YXS at high dose obviously improved the Factin arrangement and size in HF rats and the F-actin became larger and regular than that in HF ( Figure S2B).

YXS Inhibited
Inflammation, Oxidative Damage, and JNK Activation, While Enhancing Trx2 Level in HF Rats. In this work, both the in vitro and in vivo RNA-seq analysis indicated MAPK cascade was involved in YXS-mediated protection, and it has been well known that MAPK cascade played a vital role in mediating reactive oxygen species (ROS) and inflammation signals in HF development. Thus, in our study, we focused on the MAPK cascade and we speculated that YXS may protect against HF through modulating MAPK cascades. Recently, Trx2 has been identified as an important upstream molecule that regulated MAPK cascade; thus, the effect of YXS on Trx2 and its downstream MAPK cascade was investigated.
As indicated in Figure 4, to confirm the vital role of antioxidation and anti-inflammation of YXS in resisting HF, antioxidant molecules such as GSH level and T-AOC and inflammatory factors such as IL-1β, IL-6, and TNF-α were detected. As shown in Figure 4(a), only YXS at high dose   Oxidative Medicine and Cellular Longevity significantly increased GSH levels in contrast to the effect of VST, when compared with the untreated HF group. And higher T-AOC was observed after the treatment of YXS at both high and low dose as well as VST than that in the untreated HF group. As indicated in Figure 4(b), the levels of IL-1β, IL-6, and TNF-α were significantly higher in HF than in the normal group, whereas these were remarkably reduced after YXS treatment. In contrast, VST treatment only significantly decreased IL-6 and TNF-α, but had no effect on IL-1β. Taken together, YXS demonstrated an obvious protective effect against HF by inhibiting inflammation and oxidative damage.  Oxidative Medicine and Cellular Longevity viability and decreased LDH level. Additionally, the increase of inflammation factors such as IL-6, and TNF-α after H 2 O 2 induction was obviously decreased by YXS. As indicated by Figure 5(c), YXS treatment also prevented H9C2 cells from the depolarization of mitochondrial membrane, as evidenced by the obvious increase in red/green ratio by JC-1 staining. Consistent with this result, H 2 O 2 caused obvious increase of TUNEL-positive rate, whereas this was significantly decreased by YXS treatment (Figure 5(d)).
To further confirm the mechanism, we evaluated the effect of YXS on Trx2 and phosphorylation of JNK and p38 in H 2 O 2 -induced model. As indicated by Figure 6, exposure of cells to H 2 O 2 led to an obvious reduction in Trx2, which was significantly ameliorated by YXS treat-ment. Additionally, the increased phosphorylation of JNK and p38 was also reduced by YXS treatment (Figures 6(b)-6(d)). Apart from that, the oxidative damage was also reduced as indicated by the decrease of nitrotyrosine (protein oxidation marker, Figure 6(e)). Thus, YXS treatment restored Trx2 and inhibited JNK and p38 phosphorylation, causing inhibition of cell apoptosis, inflammation, and oxidative damage which were consistent with our in vivo results. Apart from Trx2, Nrf2 is an important regulator in modulating oxidative stress and the effect of YXS on Nrf2 was evaluated accordingly. As shown in Figure S2, HF and H 2 O 2 led to obvious reduction of Nrf2 expression whereas YXS enhanced the Nrf2 expression and enhanced its nucleus staining ( Figure S2B-C).   (Figure 7). Three sequences to silence Trx2 were tested and Trx2 siRNA-1 successfully downregulated Trx2 expression, which was used in the following experiments. Importantly, YXS increased cellular GSH level in a concentration-dependent manner while knockdown of Trx2 significantly decreased cellular GSH level (Figure 7(b)). Additionally, the increase of caspase-3 activity in response to H 2 O 2 was obviously decreased by different concentrations of YXS treatment. Notably, silencing Trx2 significantly increased the caspase-3 activity treated with 15.625 and 62.5 μg/ml YXS (Figure 7(c)).

Discussion
In this study, YXS treatment obviously preserved cardiac function, decreased BNP and ANP levels, and attenuated structure rupture and collagen deposition. RNA-seq analysis indicated YXS protected against HF through affecting multiple processes such as oxidative damage, inflammation, apoptosis, and MAPK cascade. Further experiments confirmed that YXS decreased inflammation factors such as IL-1β, IL-6, and TNF-α, prevented cell apoptosis as evidenced by decreased TUNEL-positive rate and caspase-3 activity, and increased mitochondrial membrane potential while reducing oxidative damage as indicated by the increased levels of GSH and T-AOC. Additionally, YXS obviously elevated Trx2 while it inhibited JNK and p38 activation, thereby attenuating cardiac damage in HF. Silencing Trx2 decreased the protection of YXS in H 2 O 2 -induced cell injury as evidenced by the increase of caspase-3 activity and decrease of GSH level. Collectively, these data indicated that YXS is an effective treatment which enhanced heart function and decreased myocardial damage by restoring Trx2 and inhibiting JNK and p38 activation in ischemia-induced HF.
The ambiguous mechanism and poor clinical location hinder the further precise application of TCM in clinic, whereas systematical investigation could guide the use of TCM and benefit the treatment of patients with HF. Evidence has been accumulated that YXS is an effective treatment in the intervention of various cardiovascular diseases including myocardial ischemia/reperfusion injury, H 2 O 2 -caused damage, and ET-1-induced cardiomyocyte dysfunction [6,13,14]. Recent research showed YXS decreased oxidative damage and cell apoptosis and ameliorated heart function via upregulating liver-X-receptor α [13] while it decreased H9C2 cell apoptosis and oxidative damage by regulating APEX1, TFCP2, and 5 other transcription factors in vitro [15]. Our research was consistent with these results that YXS can decrease oxidative damage and apoptosis. However, the inhibition of inflammation was also proved in our study as indicated by the decreased inflammation factors such as IL-1β, IL-6, and TNF-α. Moreover, previous research indicated that YXS may improve heart function in HF with the elevation of the biomarkers such as TNNI3, GAL-3, HSP 70, FABP3, and CKAP5 [6], which benefited the clinical location of YXS. In this present study, the mechanism of YXS against HF was systematically investigated by using RNAseq analysis and multiple processes such as oxidative damage, inflammation, apoptosis, and MAPK cascade were proved to be affected by YXS, indicating a multicomponent and multitarget way of YXS in treating HF. We also proved that YXS enhanced heart function and decreased myocardial damage by restoring Trx2 and inhibiting JNK and p38 activation against HF. All these results facilitated the understanding of the mechanism and the usage of YXS in clinic.
A large amount of studies demonstrated that increased oxidative stress and persistent inflammation are associated with the development of HF [19][20][21][22]. Oxidative stress can  -5). The data were expressed as mean ± SD. # P < 0:05 and ## P < 0:01 vs. normal cells; * P < 0:05, * * P < 0:01, and * * * P < 0:001 vs. H 2 O 2 -treated cells. 10 Oxidative Medicine and Cellular Longevity also lead to myocardial tissue injury and inflammation, which leads to the progress of HF [23]. Oxidative stress during HF or ischemia/reperfusion is caused by excessive production or accumulation of free radicals or their oxidative products [24]. High ROS impair myocardial contractile function, cause DNA damage and cell apoptosis, and activate matrix metalloproteinases (MMPs) which induced cardiac remodeling, augmenting the progress of HF. Moreover, ROS also induced inflammation response through regulating proinflammatory pathways such as nuclear enzyme PARP-1 and NF-KB [25]. Inhibiting oxidative damage benefits and halts HF [26], consistent with these studies that HF or H 2 O 2 led to an increase of oxidative stress as evidenced by the lower GSH and T-AOC and higher ROS level.

11
Oxidative Medicine and Cellular Longevity Importantly, an obvious inhibition of oxidative stress was observed as indicated by the increased GSH and T-AOC as well as lower ROS after YXS treatment. Additionally, it is worth mentioning that persistent inflammation after MI deteriorates ischemic damage and exaggerates cardiac remodeling in the following HF [27,28]. Targeting inflammation has been proved to be a promising strategy in resisting HF [29][30][31][32]. Notably, a remarkable inhibition of inflammation by YXS was observed as indicated by the decrease of IL-1β, IL-6, and TNF-α level in vivo and in vitro. In addition, cell apoptosis plays an important role in cardiomyocyte loss after acute myocardial infarction and participates in the subsequent development of left ventricular remodeling and symptomatic HF [33]. Apart from that, cell apoptosis induced by inflammatory cytokines also participates in the occurrence and development of chronic HF [34]. Related studies have found clusters of biomarkers related to the mechanism of HF in the aspects of inflammation, apoptosis, vascular function, and so on [35]. Our results showed that YXS decreased the TUNEL-positive rate and caspase-3 activity while it increased the mitochondrial membrane potential, which demonstrated an obvious inhibition of cell apoptosis by YXS. Thus, YXS treatment showed a beneficial protection on HF through inhibiting oxidative damage, inflammation, and apoptosis.
Thioredoxin system, including Trx, peroxidase Prx, and thioredoxin reductase (TrxR), plays a key role in modulating redox hemostasis and gene knockout of Trxs and TrxRs led to embryonic lethality due to elevation of oxidative stress [36][37][38]. Specifically, Trx2 is located in mitochondria and is characterized by a dithiol motif Cys-Gly-Pro-Cys, which serves a critical function in cell survival and apoptosis in cardiomyocytes [26,39]. Mitochondrial Trx2 deficiency led to extensive apoptosis and embryonic lethality [40]. In contrast, the overexpression of Trx2 preserved cardiac function and prevented cell apoptosis through inhibiting ROS generation in dilated cardiomyopathy and HF [26]. Recent studies indicated decreased Trx2 attenuated inflammation after liver ischemia/reperfusion injury through mitochondrial quality [41], and PGC-1 can upregulate Trx2 expression in the protection    The data were expressed as mean ± SD. (d) Schematic diagram indicated the potential mechanism of YXS in the protection against HF. ### P < 0:01 vs. normal cells; * P < 0:05, * * P < 0:01, and * * * P < 0:001 vs. H 2 O 2 -treated cells; Δ P < 0:05, ΔΔ P < 0:01, and ΔΔΔ P < 0:001 compared to the related control siRNA group. 12 Oxidative Medicine and Cellular Longevity against transverse aortic constriction-induced oxidative damage [42], whereas TXNIP shuttled to bind to Trx2 and caused inflammation and apoptosis [43]. In the present study, the expression of Trx2 was decreased in both H 2 O 2 -treated H9C2 and MI-induced HF which was associated with increased oxidative damage, inflammation, and apoptosis, whereas increased Trx2 by YXS obviously attenuated these damages and halted progress of HF. Additionally, Trx2 silencing attenuated the protection of YXS in H 2 O 2 -induced cell injury as evidenced by decreased GSH and increased caspase-3 activity. These data indicated that Trx2 played an important role in the protection of YXS against MI-induced HF. Additionally, Nrf2 played a critical role in regulating oxidative stress and a close relationship between Nrf2 and Trx2 in modulating cellular oxidative stress was observed. For example, Nrf2 activation prevented Trx2 oxidation while Trx2 overexpression affected Nrf2 activity [44][45][46]. In our research, YXS enhanced Nrf2 activation both in vitro and in vivo, which protected myocardiocytes against damage from myocardial ischemia or H 2 O 2 . MAPK is a common pathway of extracellular signalinduced cell response, which participates in many biological processes such as cell proliferation, differentiation, apoptosis, and cytoskeleton reorganization. In several subfamilies of MAPK cascade signaling pathway, p38 and JNK are associated with oxidative stress and inflammation. The p38 MAPK signal pathway plays a vital role in maintaining normal heart function. However, p38 is also involved in the pathogenesis of HF by affecting the heart contraction and survival of cardiomyocytes [47]. Recent research suggested that inhibition of p38 MAPK activation showed a beneficial effect on HF [48]. Our study indicated that YXS has an inhibitory effect on p38 activation, which helped to halt HF progression. Apart from that, JNK activation resulted in cell apoptosis [49]. Growing evidence shows that increased Trx levels prevent ASK1 activation and subsequently inhibit the activation of the downstream p38 and JNK pathways [50,51]. However, it indicated that the inhibition of Trx2 on cell apoptosis was not in a JNK-dependent manner in dilated cardiomyopathy [26,52]. In contrast to these studies, we found that increased Trx2 was associated with decreased p-JNK/JNK and p-p38/p38 in YXS-mediated protection against HF. This may be caused by the multicomponent and multitarget characteristics of YXS, which maintained cardiac function through affecting multiple pathways. Recent studies indicated activated JNK caused cell apoptosis through phosphorylating c-Jun and upregulating Fas ligand (FasL) [53,54]. Therefore, YXS inhibited the activation of JNK and p38, resulting in an important protective effect in resisting HF.

Conclusions
In summary, YXS enhanced myocardial function, decreased BNP and ANP levels, and attenuated structure rupture and collagen deposition, through restoring Trx2 and inhibiting JNK and p38 activation in ischemia-induced HF. This study clarified the important role of YXS in the management of HF and its potential mechanism, facilitating the clinical usage of YXS. Additionally, this also provided a better understand-ing of the pathogenesis of HF, highlighting the importance of restoring Trx2 in HF.