Qiliqiangxin Attenuates Cardiac Remodeling via Inhibition of TGF-β1/Smad3 and NF-κB Signaling Pathways in a Rat Model of Myocardial Infarction Open Access

Myocardial infarction (MI) is a major cause of death and disability worldwide. Although the mortality rate associated with acute myocardial infarction has decreased due to early thrombolysis, percutaneous coronary intervention or coronary artery bypass grafting, patients who survive inevitably suffer from consequent left ventricular (LV) remodeling [1]. This adverse cardiac remodeling post-MI results in ventricular dysfunction and heart failure, which contribute to a poor outcome and high mortality rate [2-4]. Currently, several medicine therapies are recommended, including angiotensin-converting enzyme (ACE) inhibition, angiotensin type I receptor blocker therapy, and beta-adrenergic blockade. However, the effectiveness of these strategies to prevent LV remodeling post-MI is still limited. As a result, the mortality of patients with chronic heart failure remains high [5, 6]. Therefore, the identification of additional interventions, including treatment with novel therapeutic compounds from natural products, to protect the infarcted heart from adverse remodeling is warranted.

Interstitial fibrosis and inflammatory cell infiltration in left ventricles are common pathological features of cardiac remodeling following MI [7]. Transforming growth factor-β1 (TGF-β1) has been identified as a key regulator of cardiac fibrosis, which has wide-ranging effects that may affect cell growth, apoptosis and differentiation; increase collagen and matrix protein production; maintain fibroblast viability; and inhibit production of metalloproteinase, which facilitates collagen degradation [8, 9]. The results from myocardial infarction animal models have demonstrated that increased expression of TGF-β1 in myocardial infarction and the use of TGF-β receptor inhibitor to block the TGF-β pathway could attenuate cardiac fibrosis in MI animal models [10, 11]. In addition, nuclear factor κB (NF-κB) is increasingly recognized as a crucial player in the development of cardiac remodeling and failure after MI, which has an essential role in inflammation and innate immunity [12-14]. A member of the NF-κB family, p65, forms homo or heterodimers that are bound to inhibitor of kappa B (IκB) proteins in the cytosol. Degradation of IκB releases NF-κB dimers and enables the translocation of NF-κB into the nucleus, where it can initiate transcription of target genes. Activation of NF-κB induces the activation of genetic program that leads to the transcription of chemokines (MCP-1), cytokines (TNF-α, IL-6) and matrix metalloproteinases (MMPs) and further promotes inflammatory and fibrotic response that participate in the progression of ventricular remodeling [15-17]. Many of the models in which NF-κB inhibition showed an anti-inflammatory effect were also associated with improvements in cardiac fibrosis [17, 18]. Therefore, novel strategies specifically targeting TGF-β1 and NF-κB pathways would be of great potential therapeutic benefit for inhibiting progressive cardiac remolding in MI.

Qiliqiangxin (QL) has been demonstrated to be effective and safe for the treatment of chronic heart failure [19]. Our laboratory and others have reported that QL can attenuate myocardial remodeling and improve cardiac function in rats with experimental myocardial infarction [20-22]. Several studies have mentioned the protective effect of QL on cardiac remodeling, which are associated with inflammation regulation, energy metabolism improvement, and angiogenesis enhancement [23-26], but its mechanism has not been thoroughly elucidated to date. Therefore, using a post-myocardial infarction heart failure model, the present study evaluated the influence of QL on the heart function, cardiac fibrosis, and the expression of TGF-β1, p-Smads, collagen I, TNF-α and IL-6 to further explore whether TGF-β1 and NF-κB pathways are involved in the protective effects of QL during cardiac remodeling. The effects of QL were also compared with Valsartan, an AngII receptor antagonist commonly used in clinical practice, which is known to have cardiac protective effects against LV remodeling after myocardial infarction [27].

Qiliqiangxin consists of Ginseng, Radix Astragali, Aconite Root, Salvia miltiorrhiza, Semen Lepidii Apetali, Cortex Periplocae Sepii Radicis, Rhizoma Alismatis, Carthamus tinctorius, Polygonatum Odorati, Seasoned Orange Peel, and Ramulus Cinnamomi (Yiling Pharmaceutical Corporation, Shijiazhuang, China). The drug powder was dissolved with sterile water at the concentration of 0.1 g/mL Qiliqiangxin was prepared for the study. Valsartan (batch number X1428) was manufactured by Beijing Novartis Pharmaceutical Co. Ltd. and dissolved with sterile water.

Normal male Sprague-Dawley rats (body weight 220∼250 g) were provided by Beijing Vital River Laboratory Animal Technology Co. Ltd. (Animal license number: SCXK (Beijing) 2012-0001). The animals were fed with standard diet and water and were subject to a 12 h light and 12 h dark cycle, a temperature of 20 ± 2°C, and a humidity of 50 ± 2%. All animal experimental protocols were approved by Animal Care and Use Committee of Beijing University of Chinese Medicine and complied with laboratory animal management and use regulations. HF was induced by myocardial infarction following ligation of the left anterior descending artery (LAD). Sodium pentobarbital 1% (50 mg/kg) was administered by intraperitoneal injection. The procedure included endotracheal intubation, ventilator positive pressure ventilation, preoperative recording of 12-lead ECG, local skin disinfection, chest opening, thoracotomy device setup, and opening of the pericardium, the pulmonary cone, and the left atrial appendage 2∼3 mm from the bottom of the left anterior descending coronary artery ligation. For the rats assigned to the sham group, the same operation was performed without ligation of the left coronary artery. Twelve-lead ECG was recorded after the experiments. MI rats were fed normally for four weeks. According to transthoracic echocardiography results (Table 1), the survival rats were randomly assigned to the following groups: Model group (MI, n = 12), Sham group (Sham, n = 10), QL group (QL, n = 9), and Valsartan group (Valsartan, n = 9). QL 1.0 g/kg and Valsartan 10 mg/kg were administered, respectively, by gavage once a day during the four weeks. An equal volume of distilled water was used for model and sham group.

A noninvasive transthoracic echocardiography method was used to evaluate the morphology and function of left ventricle. Echocardiography was performed in anesthetized animals. This method consisted of a two-dimensional mode, that is, time-motion (TM) mode and blood flow measurements in pulsed Doppler mode. The left ventricular end-systolic internal diameter (LVIDs) and left ventricular end-diastolic internal diameter (LVIDd), as well as the left ventricular ejection fraction (EF) and left ventricular fractional shortening (FS), were recorded.

The hearts were harvested, weighed, washed in phosphate-buffered saline, fixed in 4% paraformaldehyde overnight, and embedded in paraffin. Each paraffin-embedded heart was cut into sections (4 µm thick) through the infarct area and stained with Masson’s trichrome. Each section was imaged under a microscope (Nikon, Tokyo, Japan). Fibrosis was calculated by computerized planimetry using ImageJ software, version 1.44 (NIH, Bethesda, MD, USA).

Immunohistochemistry staining was applied to detect a-SMA and NF-κB p65 protein expression. Tissue was prepared according to procedures as described. Each paraffin-embedded heart was cut in 4-µm-thick transverse sections across the ventricular scar area using a Microtome RM 2245 (Leica) and placed on slides. Tissue was then placed in the oven for 20 min, followed by xylene dewaxing and gradient ethanol step by step into the water. Heat mediated antigen retrieval was performed using Tris/EDTA buffer PH 9.0. Endogenous peroxidase were blocked by incubating 10 min in 3% H2O2. Slides were washed three times for 6 min in 1% PBS and incubated overnight at 4°C with primary antibodies (α-SMA 1: 500, NF-κB p65 1: 100) in 1% bovine serum albumin. Tissue slices were washed the next day three times for 6 min with 1% PBS. A prediluted HRP-polymer conjugated anti-rabbit IgG was used as the secondary antibody. Slides were washed again three times for 6 min with 1% PBS and then counterstained for 30 s with Hematoxylin. Images were visualized and captured using a digital microscope (OLYMPUS BX 50, Japan). Under ×400 magnifications.

All animals were euthanized after four weeks of drug administration, and their hearts were immediately harvested and stored in liquid nitrogen until western blot analyses were performed. The following antibodies were used: mouse monoclonal anti-TGF-β1 (1: 500, ABcam, Inc.), rabbit monoclonal anti Smad3 (phosphor C25A9) (1: 500, Cell Signaling Technology, Inc.), mouse anti human Smad7 polyclonal antibody (1: 500, Cell Signaling Technology, Inc.), rabbit polyclonal anti-TNF alpha (1: 1000, ABcam, Inc.), Anti-IL6 antibody (1: 1000, ABcam, Inc.), Anti-Collagen I antibody (1: 1000, ABcam, Inc.), mouse monoclonal antialpha SMA antibody (1: 500, ABcam, Inc.), NF-κB p65 (1: 1000, Santa Cruz, Inc.) and anti-p-IκBα antibody (1: 1000, Cell Signaling Technology, Inc.). Antibody proteins were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes, which were then incubated with antibodies at 4 °C. The membranes were further incubated with horseradish peroxidase-conjugated anti-rabbit IgG (1: 2, 000) for two hours at room temperature. ECL visualization was performed and the Gene Gnome Gel Imaging System (Syngene Co.) was used to capture the resulting images. ImageJ (NIH image, Bethesda, MD) was used to analyze the gel images.

All experimental data were presented as the mean ± SD. Single factor analysis of variance (ANOVA) was performed with the statistical software SPSS17.0. Dunnett’s T3 was used for unequal variances, and A probability of < 0.05 was considered to be statistically significant.

Cardiac function was assessed by echocardiography before and up to eight weeks after MI. No significant difference in Ejection fraction (EF) was observed between the QL treated and MI animals at baseline (Table 1). On the eight weeks following the induction of MI, ESV and LVIDs were higher, while EF and FS were lower in the rats with MI compared to the sham operated group. In contrast, ESV and LVIDs were significantly lower, and EF and FS were significantly greater in the QL and the Valsartan treated MI hearts compared with the control MI group. Following treatment for four weeks, although the measurements obtained for EDV and LVIDd displayed a decreasing trend in both the QL and the Valsartan groups versus the MI group, the difference was not statistically significant (Fig. 1A-F). Furthermore, QL and the Valsartan reduced heart weight-to-body weight ratios and attenuated adverse cardiac structural changes (Fig. 1G-H) following MI.

Interstitial fibrosis is a well-known feature of cardiac remodeling after MI. Masson’s trichrome staining for interstitial fibrosis revealed that LV collagen fractional area was highly increased in MI versus Sham rats and was reduced by QL and Valsartan treatments (Fig. 2A). Expression of α-SMA, a myofibroblast marker, was increased in the hearts of animals with MI compared with the Sham group; however, QL or Valsartan treatment significantly reduced its expression (Fig. 2B and D). The α-SMA-positive myofibroblasts actively synthesize extracellular matrix (ECM) components. Next, we measured type I collagen, the main collagen isoform produced by cardiac fibroblasts. Using western blot analysis, we observed that type I collagen deposition was significantly higher in rats in the MI group compared to those in the Sham group; however, QL significantly decreased type I collagen protein expression in the hearts (Fig. 2C). Considered together, these parameters of interstitial fibrotic remodeling were significantly reversed by QL.

As TGF-β1/Smad3 signaling pathway is well-recognized as the contributor for cardiac fibrosis after MI, we also checked their expression levels after QL treatment. MI evidently increased the protein levels of TGF-β1 in rat hearts compared with the Sham group at the endpoint of 8 weeks; however, QL and Valsartan treatment significantly reduced its expression compared with the MI group (Fig. 3A). Similarly, MI evidently increased the protein level of p-Smad3 in rat hearts compared with the Sham group, and QL or Valsartan treatment significantly reduced its expression compared with the MI group (Fig. 3B). Nevertheless, MI evidently reduced the protein level of p-Smad7 in rat hearts compared with the Sham group, and QL or Valsartan treatment significantly increased its expression compared with the MI group (Fig. 3C).

Previous studies have reported that cytokines (TNF-α and IL-6) expression plays a critical role in the development of cardiac remodeling and failure after MI. In the aforementioned studies, we evaluated the effects of QL treatment on TNF-α and IL-6 expression in post-MI heart. As shown in Fig. 4, there was minimal expression of TNF-α and IL-6 in the Sham group. Compared to the Sham group, the expression of TNF-α and IL-6 increased in MI group. By contrast, in the QL and Valsartan treatment groups, the expression of TNF-α and IL-6 was reduced. The above results indicated that QL attenuated inflammatory reactions in rats with MI.

The above results indicated that QL attenuated cytokines expression in rats with MI. Furthermore, previous studies have shown that proinflammatory cytokines induce the phosphorylation and degradation of IκB, and then NF-κB, which is released from the inhibitory signalosome to translocate to the nucleus and induce the transcription of a number of genes, resulting in the expression of inflammatory proteins, such as TNF-α and IL-6. Therefore, we tested whether NF-κB activation is suppressed by QL and Valsartan treatments. Immunohistochemical analysis (Fig. 5A) showed a much stronger labeling of anti-NF-κB p65 antibody than was detected in the heart of post-MI rats compared with sham-operated control rats, and labeling was considerably weaker in rats of QL treatment group. Semiquantitative immunoblotting (Fig. 5B) revealed that the expression of NF-κB p65 in the nucleus was significantly increased in the MI rats compared with sham-operated rats. Compared with the MI group, the NF-κB p65 in the nucleus was reduced in the QL Treatment group. As shown in Fig. 5C, there was minimal expression of p-IκBα in the Sham group. Compared with the Sham group, the expression of phosphorylated IκBα increased in the MI group. By contrast, in the QL group, the expression of phosphorylate IκBα was decreased. Taken together, these results indicate that QL attenuates AMI-induced inflammatory reactions in rats at least partly by suppressing NF-κB activation.

In the present study, we have investigated the protective effect of QL on cardiac remodeling and the possible mechanisms in a rat model of MI closely mimicking human anatomy physiology. The primary findings of this study include the following: 1) QL effectively attenuated myocardial fibrosis by inhibiting collagen production, cardiac fibroblast activation, and myofibroblast formation; 2) QL suppressed the expression of proinflammatory cytokine; and 3) the underlying mechanism may involve the suppression of TGF-β1/Smad3 and NF-κB signaling. Taken together, these data demonstrate that certain direct beneficial effects of QL on intramyocardial fibrosis remodeling and inflammation response following MI, culminating in better preserved cardiac structure and function.

Cardiac remodeling after AMI is a complex process with numerous continuous and overlapping events [28]. In an early phase, cardiac remodeling is a consequence of fibrotic repair of the necrotic area with scar formation. Next, the remodeling process is driven by architectural rearrangements of the surviving myocardium, including myocyte hypertrophy, myocardial fibrosis, and ultimately, progressive left ventricular dilation. LV cavity dilatation following myocardial infarction is one of the compensatory reactions of the failing heart. However, excess dilatation evokes LV systolic and diastolic dysfunction that leads to heart failure [29]. Prevention of unfavorable LV remodeling is important for improvement of morbidity and mortality rates after MI. It has been confirmed that interstitial fibrosis is a typical characteristic of cardiac remodeling following MI, which is characterized by net accumulation of extracellular matrix (ECM). In the myocardium, cardiac ECM remodeling is well-documented in post-MI hearts in the infarct zone, as well as in both ventricles remote to the infarct scar [30, 31]. Collagens I and III are the best-characterized ECM components. In models of cardiac fibrosis of myocardial infarction, type I collagen exhibits more intense and prolonged upregulation than collagen III [32]. Activated myofibroblasts are the main cellular sources of collagens in the fibrotic heart. α-SMA expression has been extensively used as a marker of fibroblast differentiation into its activated state, the myofibroblast. In this study, we observed excessive I collagen deposition and upregulated α-SMA protein expression in post-MI hearts, both of which were reversed by QL therapy, suggesting that QL may have antifibrosis effects by inhibiting fibroblast activation and collagen production.

From the perspective of traditional Chinese medicine (TCM), the fundamental problem in heart failure post-MI is the prolonged deficiency of heart qi and yang, which causes the heart to become too weak to move blood and transport fluid, leading to blood “stasis,” phlegm “stagnation,” and disposition in heart [33]. These conditions are consistent with the myocardial pathological changes of collagen deposition and interstitial fibrosis. Qiliqiangxin capsule are a specific TCM extract obtained from 11 types of herbs, including Ginseng, Radix Astragali, Aconite Root, Salvia miltiorrhiza, Semen Lepidii Apetali, Cortex Periplocae Sepii Radicis, Rhizoma Alismatis, Carthamus tinctorius, Polygonatum Odorati, Seasoned Orange Peel, and Ramulus Cinnamomi, which are well-known to have effects of invigorating the heart qi and warming yang, accelerating blood circulation, and removing phlegm and congestion. Pharmacological studies have found that QL contains a number of active substances, such as ginseng saponin, astragalus saponin, flavonoid, cardenolide, and phenolic acid, which have been demonstrated to have positive inotropic, vasodilation, anti-inflammation, and antifibrosis effects. In this study, we further demonstrated that QL improves cardiac remodeling by inhibiting excessive collagen deposition and cytokine expression.

TGF-β1 is a locally generated cytokine that has been implicated as a major stimulator of tissue fibroinflammatory changes. TGF-β1 is observed in post-MI rat heart and is associated with fibroblast-to-myofibroblast phenolconversion and concomitant activation of canonical Smad signaling [34]. As a main downstream signaling transducer of TGF-β1, Smad3 can be phosphorylated by activated type I receptor of TGF-β1, followed by complex formation with Smad4 and translocation into nucleus, where it acts as a transcription factor and promotes the expression of target genes, including type I and type III collagen. The use of mice with a targeted deletion of Smad3 shows that most of the pro-fibrotic activities of TGF-β are mediated by Smad3 [35, 36]. Smad7 is an inhibitory Smad protein that is able to compete with Smad3 and TGF-β receptor complex TGF-βRI binding, thereby blocking the later signal conduction process. High expression of exogenous Smad7 could inhibit phosphorylation of Smad3 [37]. In the present study, TGF-β1/p-smad3 and the downstream collagen I expression were all inhibited by QL in post-MI rat hearts. At the same time, the α-SMA expression level was inhibited in QL treated MI rats. While the p-smad7 expression was upregulated. These results suggested that QL inhibits the signaling pathway of TGF-β1/Smad3, as well as further inhibits the myofibroblast proliferation and a marked upregulation of type I collagen expression by upregulating the protein expression of Smad7, which provides new ideas for clinically improving myocardial fibrosis in the post-MI heart.

Although the expression of proinflammatory cytokines may be involved in wound healing after MI, it is believed that the overexpression of cytokines damages cardiac tissue and evokes excess deposition of fibrotic components, even in the non-infarcted myocardium [38]. The NF-κB transcriptional activation pathway is considered to be a “master regulator” of inflammation. It is found primarily in the cytosol bound to its inhibitor IκB proteins. Upon stimulation by cytokines or other inducers, IκB proteins are targeted for proteasomal degradation by the IκB kinase. Once IκB degrades, NF-κB translocates to the nucleus and binds DNA at κB sites in the regulatory region of proinflammatory genes and promotes their transcription. Activated NF-κB increases the expression of TGF-β1, TNF-α, IL-6 and IL-1β, which subsequently activate collagen deposition and myocardial fibrosis that lead to myocardial remodeling and HF [39, 40]. It has been reported that the inhibition of NF-κB binding decreased cardiac damage following MI [41]. The NF-κB plays a pivotal role in the coordinated transactivation of cytokine and fibrosis factor that might be involved in myocardial damage after AMI. In the present study, NF-κBp65, p-IκB, and the downstream TGF-β1, TNF-α and IL-6 expression were all inhibited by QL in post-MI rat hearts. These results suggested that QL inhibits the signaling pathway of NF-κB and further suppresses proinflammatory cytokines expression, at least partly through the suppression of the IκB phosphorylation and degradation. These effects may contribute to the QL-mediated preventive effects on cardiac inflammatory and fibrotic diseases, such as acute myocardial infarction.

In the present study, we identified that the administration of QL has notable benefits, preventing cardiac remodeling following MI. The potential mechanisms may be associated with the anti-fibrotic and anti-inflammatory effects of QL on the ischemic myocardium. Based on our results, we conclude that QL carries out its anti-fibrotic effects primarily by suppressing the TGF-β1/Smad3 signaling pathway, which might contribute to its attenuation of myofibroblast proliferation and collagen deposition. The possible mechanisms may also involve inhibition of the NF-κB signaling, which might contribute to its suppression of proinflammatory cytokines overexpression. Further studies should explore the effects of QL on cardiac fibroblasts proliferation and the NF-κB and TGF-β1/Smad3 intracellular signaling pathways in vitro to ultimately offer new avenues for the prevention and treatment of this and other related diseases.

This work was supported by the National Natural Science Foundation of China (Grant no. 81273945).

All authors declare that they have no Disclosure Statements.

A. Han and Y. Lu contributed equally to this work.