Mechanism of QSYQ on anti-apoptosis mediated by different subtypes of cyclooxygenase in AMI induced heart failure rats

Heart failure (HF) induced by acute myocardial infarction (AMI) remains the leading cause of morbidity and mortality worldwide, inspite of extensive investigations [1]. Exploration of effective prevention and therapy for HF poses a major challenge to the entire medical community. The pathogenesis of HF and new therapeutic approaches for HF need to be investigated further.

Abundant evidence indicates that inflammation and apoptosis play important roles in the development of HF [2‐4]. Previous studies found that inflammatory cytokines promote development of HF [5, 6]. In particular, arachidonic acid (AA) metabolism plays an important role in HF development [7, 8]. The key rate-limiting enzymes in AA pathway are cyclooxygenases (COXs) and they have been used as targets of non-steroidal anti-inflammatory drugs (NSAIDs) in clinical treatment of HF. Large-scale randomized clinical experiments showed that aspirin, along with other NSAIDs which target COXs, has cardio-protective effects [9]. COX1 and COX2 are the two isoenzymes of cyclooxygenases. COX1 is expressed constitutively in most tissues, whereas COX2 is the inducible form of the enzyme that is produced upon stimulation by growth factors and cytokines (e.g., inflammation) [10].

Myocardial apoptosis has been identified as another essential process in the development of HF [11]. Activation of apoptotic pathways leads to myocyte damage and eventual myocardial fibrosis. P53-dependent myocardial apoptosis is one of the apoptotic pathways that contribute to progress of HF [12]. P53 activates the extrinsic apoptotic pathway by triggering the expression of transmembrane protein FasL, whose receptor belongs to TNF receptor family (TNF-R) [13, 14]. The activation of that particular ‘death’ receptor (TNF-R) family leads to a cascade expression of caspases, including caspase-8 and caspase-3, which in turn enhances apoptosis [15]. Overexpression of the FasL antigen has been reported in myocardial infarction tissues in rats [16]. As activation of P53 pathway can lead to many significant outcomes, the expression of P53 needs to be strictly regulated. MDM2 could bind with P53 gene and down-regulate P53 level by inhibiting its transcriptional activity [17]. Numerous studies have demonstrated that AA and its metabolic intermediates can lead to apoptosis. For example, Prostaglandin E2 (PGE2) is a downstream metabolite of the AA pathway and could induce apoptosis in a dose-dependent manner [18, 19]. Specifically, PGE2 can increase P53 transcriptional activity by binding with the subtype receptor EP2, thereby inducing apoptosis [20]. Activation of EP2/EP4 receptors by PGE2 can also promote apoptosis by increasing expressions of FasL, caspase8 and caspase9 [21]. The AA pathway also exerts diverse effects on the inflammation process through promoting the mRNA expression of receptor activator of NF-κB [22]. Therefore, factors in the AA pathway would be ideal potential targets for the treatment of HF [23, 24]. A series of experimental studies have been carried out to investigate treatment of HF by inhibiting AA metabolism, specifically by suppressing activation of COXs. However, traditional NSAIDs (such as aspirin) have non-specific inhibition effects on COX1 and COX2, resulting in side effects such as gastrointestinal diseases [25, 26]. On the other hand, specific COX2 inhibitors increase the risk of cardiovascular diseases [27].

Traditional Chinese medicines (TCM) have been used in the treatment of HF for thousands of years, and many drugs have been proven effective [28]. QiShenYiQi (QSYQ) was a patent prescription of TCM approved by China’s Food and Drug Administration (CFDA) in 2003 for treating HF (Approval number of CFDA: Z20030139) [29]. Briefly, QSYQ comprises four medical herbs, namely Radix Salvia miltiorrrhiza (Danshen), Panax notoginseng (Sanqi), Radix Astragalus membranaceus (Huangqi), and Dalbergia odorifera T. Chen (Jiangxiang). The major active ingredients are ginsenosides Rg1 and Rb1 (from Sanqi), astragaloside (from Huanqi), and tanshinol (from Danshen) [30, 31]. Large-scale randomized and controlled clinical trials have proven that QSYQ has a definite effect in improving heart function [32, 33]. Animal studies also demonstrated that QSYQ could effectively attenuate myocardial fibrosis, probably through inhibiting the renin-angiotensin-aldosterone pathway [34]. Moreover, studies showed that QSYQ has an anti-inflammatory effect in rats with AMI-induced HF [35]. Our previous study confirmed that QSYQ can attenuate inflammation by inhibiting STAT3 and NF-κB signalling pathway [36]. Recently, network Pharmacology has been applied to reveal the underlying pharmacological mechanisms of QSYQ. Apoptosis was predicted to be one of the most likely targeted pathways of QSYQ [37]. However, whether the efficacy of QSYQ is exerted by direct inhibition of myocardial apoptosis or anti-inflammatory response remains to be validated.

In this study, we aim to investigate the underlying pharmacological mechanisms of QSYQ in HF model. The effects of QSYQ on AA pathways and COXs-P53-FasL mediated apoptosis pathway were studied. HF model was induced by ligation of the left anterior descending (LAD) coronary artery in rats. This study will provide insights into the clinical treatment of HF induced by AMI.

Heart failure is the ultimate consequence of a large number of cardiovascular diseases. HF remains the major cause of mortality and morbidity worldwide, despite major advances in treatment approaches. The high rates of mortality and morbidity mandate discoveries of novel therapeutic strategies for it [39]. QSYQ is a patent formula of TCM and has been prescribed for the treatment of HF for many years [33]. However, the mechanism of the therapeutic effect of QSYQ remains unclear. As inflammation and apoptosis play important roles in the pathogenesis of HF, we aimed to investigate whether QSYQ could regulate these two processes in the HF rat model.

The main findings of this study showed that QSYQ may exert cardio-protective effects by regulating COXs-PGE2-P53/FasL pathway. Studies have shown that high levels of serum inflammatory markers, such as TNF-α and IL-6, were correlated with severity and prognosis of patients with HF [40, 41]. Such inflammation markers can activate the AA metabolism pathway [8], which plays an important role in the development of HF [42]. Under normal circumstances, AA resides in the cell membrane as the constituent of membrane phospholipids. When exposed to various stimuli (such as inflammation), cell membrane releases a large amount of AA from the phospholipid pools. AA can be converted to biologically active metabolites, and this process is catalyzed by enzymes COX1 and COX2. COX1 is constitutively expressed and performs physiological functions, whereas COX2 is induced by inflammatory stimuli [43]. The major metabolites, such as PGD2 and PGE2, contribute to pro-myocardial fibrosis occurring in the left ventricle. Fibrosis will reduce cardiac compliances, induce apoptosis and ultimately lead to HF [11].

In addition to inflammation, myocardial apoptosis also contributes to the progression of HF [11]. The apoptotic pathway can be divided into caspase-dependent and caspase-independent ones. The former includes both extrinsic and intrinsic apoptosis pathways. The P53-mediated intrinsic myocardial apoptosis is considered to be an important feature in HF [12]. P53 is an important regulator in cell proliferation, apoptosis and DNA repair processes in ventricular remodeling and HF [44]. Expression of P53 could be down-regulated by MDM2, which combines with P53 gene promoter and inhibits its transcription. Moreover, P53 can activate the extrinsic apoptotic pathway by inducing the expression of FasL [13]. Fas is a transmembrane protein and belongs to TNF receptor family, which mediates extrinsic apoptosis pathway.

Persistent COXs activation is also able to promote myocardial apoptosis. Prostanoids, generated by COX1, contribute to cardiac cell loss by inducing myocardial apoptosis in myocardial infarction tissues [38]. For example, PGE2 could induce apoptosis by increasing p53 gene transcriptional activity and expression of FasL. Combination of EP2/EP4 receptors with PGE2 can also promote apoptotic pathway by activating expressions of both caspases eight and nine [18]. COX2 has also been reported to induce apoptosis by inflammatory mechanisms. In the development of HF, COX2 promotes myocardial apoptosis and impairs cardiac function, which induces further secretion of COX2. This vicious cycle causes a change in heart structure as well as hemodynamic abnormalities, leading to irreversible deterioration of heart function [45].

Accumulating evidence suggests that ischemia and hypoxia, coupled with activation of various inflammatory and apoptosis pathways, play a pivotal role in HF. We studied the effects of QSYQ in the AMI-induced HF rat model and several of the findings were particularly interesting. Our research evaluated the effects of QSYQ on myocardial dysfunction, inflammation, and apoptotic pathways in the HF rat model. Ultrasonic testing after 28 days of surgery showed that values of EF and FS in the model group were significantly reduced compared with those in the sham group, indicating that HF model was successfully induced. Rats in the model group were characterized by declined diastolic and systolic myocardial performance. Our results also demonstrated that inflammatory markers, such as TNF-α and IL-6, were up-regulated in the model group. The AA-mediated apoptotic pathway was thus activated by those inflammatory factors. COX1 and COX2 are the key enzymes in the AA pathway and the levels of these two enzymes were significantly increased. COXs convert AA to PGE2 and the binding of PGE2 with EP2/4 receptors actives expression of P53 and FasL, thereby initiating apoptosis process. Our results showed that levels of EP2, EP4, P53 and FasL were all increased in the model group, indicating that the apoptotic pathway was activated in rats with HF.

After administration of QSYQ for 28 days, we found that value of ejection fraction at 28 days after surgery was increased. FS was also up-regulated by QSYQ treatment, though the difference was not statistically significant. QSYQ could also increase arterial pressure to basal level in QSYQ-treated rats. The cardiac-protective effects of QSYQ were further manifested by improved values of Max dP/dt and Min dP/dt, which are indicators of systolic and diastolic capacities. These results suggested that QSYQ can improve cardiac function in HF model rats. In the molecular level, expression of MDM2 was increased and expression of P53 was reduced in QSYQ group, compared with those in the model group. Down-regulation of P53 in QSYQ group may be mediated by increased expression of MDM2, as MDM2 is a direct inhibitor of P53 transcription. Expression of FasL in QSYQ group was also restored to normal levels, suggesting that QSYQ has anti-apoptotic effect. Furthermore, QSYQ attenuated inflammation by down-regulating expressions of TNF-α, IL-6 and COXs. The anti-inflammatory effect of QSYQ is also mediated by down-regulating EP2 and EP4, which are the receptors of PGE2. Aspirin was applied as a positive control drug in our study and showed similar effect as QSYQ. Interestingly, compared with Aspirin, QSYQ had milder inhibitory effects on both COX1 and COX2. Inhibition of COX1 is considered to be the major cause of the side-effects of Aspirin [46]. Therefore, treatment with QSYQ might be an ideal alternative for patients who develop adverse reactions when treated with Aspirin. On the whole, QSYQ is a safer medicine with fewer side effects [33].

In summary, administration of QSYQ could attenuate inflammation and apoptosis in LAD-induced HF rats. One of the probable underlying therapeutic mechanisms of QSYQ is that it inhibits AA and P53/FasL pathway in a synergistic way. The simplified mechanism is shown in Fig. 9. Our study provides experimental evidence for the beneficial effects of QSYQ in the clinical application for HF.