The Antioxidant, Anti-Inflammatory and Anti-Apoptotic Activities of the Bauhinia Championii Flavone are Connected with Protection Against Myocardial Ischemia/Reperfusion Injury

Ischemic heart disease is the leading cause of mortality, and the global incidence is rising rapidly [1]. Myocardial I/R injury is a pathophysiological process in many clinical practices, such as thrombolysis, percutaneous transluminal coronary angioplasty, coronary artery bypass grafting and other vascular surgeries. It is defined as the restoration of coronary blood flow to a previously ischemic region followed by complex pathological events causing tissue injury greater than the original ischemic insult [2]. The major clinical manifestations of this injury include arrhythmias, persistent ventricular systolic dysfunction, irreversible cardiomyocyte damage, and finally, heart failure [3]. Recent studies have demonstrated that oxidative stress, the inflammatory reaction and cell apoptosis represent key elements in the occurrence and development of myocardial I/R injury [4]. Hence, the regulation of apoptosis, inflammation and oxidative damage and the related cascade responses is considered to be a crucial therapeutic strategy for preventing myocardial I/R injury and ischemic heart disease.

Medication can trigger the release of some endogenous protective substances and has been demonstrated to be a rational approach in reinforcing cells to withstand the I/R injury environment [5]. Traditional therapeutic drugs for myocardial ischemia, such as a calcium antagonist and an angiotensin converting enzyme inhibitor, are often associated with drug resistance complications and undesirable side effects [6]. Recently, increased attention has been focused on traditional Chinese herbal treatments because of their high efficiency and low toxicity. Bauhinia championii (Benth.) is a traditional Chinese medicinal herb that is widely distributed in the Guangxi Province of China [7]. Previous studies have demonstrated that extracts of its stems promote blood circulation, reduce blood stasis, and have analgesic, anti-inflammatory, anti-oxidative, and anti-platelet aggregative effects [8,9]. Bauhinia championii flavone (BCF) is the primary active component of the stem extracts. Our previous studies have revealed that BCF alleviates hypoxia/ reoxygenation-induced myocardial injury in vitro and pituitrin-induced acute myocardial injur in vivo [10,11]. Furthermore, we demonstrated that BCF had a protective effect on MI/RI in a dose of 20 mg/kg by activating the PI3K/Akt signaling pathway [12]. In the present study, we used two doses of BCF (10, 20 mg/kg) to ascertain the anti-apoptotic and anti-inflammatory effects of BCF on myocardial I/R injuries and to further characterize the mechanisms.

BCF was identified from our lab (With rutin as a reference substance, the total flavonoid content of BCF was 82%), diluting with saline to an appropriate concentration as needed. Verapamil was obtained from Hefeng Pharmaceutical Co., Ltd. (Shanghai, China). LDH, SOD, MDA, and GSH-Px assay kits were acquired from the Jiancheng Bioengineering Institute (Nanjing, China). IL-6 ELISA kit was purchased from Yonghui Biological Technology Co., Ltd. (Beijing, China). 2, 3, 5-Triphenyltetrazoliumchloride (TTC) was obtained from Shanghai Chemical Reagent Co., Ltd. A terminal deoxynucleotidyl-transferase- mediated dUTP nick end labeling (TUNEL) apoptosis detection kit was purchased from Roche Diagnostics (Mannheim, Germany). A caspase-3 activity assay kit was obtained from the Beyotime Institute of Biotechnology (Shanghai, China). All antibodies were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA).

All experimental animals received humane care in accordance with the Guide for the Care and Use of Laboratory Animals published by the United States National Institute of Health (NIH Publication No. 85-23, revised 1996). All investigations were approved by the Bioethics Committee of Guangxi Medical University (Guangxi, China). The I/R model was developed as previously described [13]. Briefly, the rats were anesthetized with sodium pentobarbital (40 mg/kg, i.p.) and connected to a rodent ventilator. The left anterior descending (LAD) coronary artery was surgically ligated by passing a 7-0 silk suture under the LAD. Myocardial ischemia for 30 min was confirmed by visual inspection of regional cyanosis of the myocardium and ST-segment elevation on an electrocardiogram. Reperfusion for 180 min was initiated by releasing the LAD ligation and was confirmed by a color change in the ventricular surface from cyanosis to hyperemia. The rats in the sham group underwent a similar surgery without LAD occlusion.

Male Sprague-Dawley rats (250 - 280 g) were randomly assigned into six groups (n = 8): (I) Sham group: the rats received vehicle (3  ml/kg saline); (II) BCF control group: the rats were received BCF at 20 mg/kg; (III) I/R group: the rats were subjected to I/R and received vehicle (3  ml/kg saline); (IV) I/R+Verapamil group: the rats were subjected to I/R and received verapamil at 8 mg/kg; (V) I/R+BCF L group: the rats were subjected to I/R and received BCF at 10 mg/kg; (VI) I/R+BCF H group: the rats were subjected to I/R and received BCF at 20 mg/kg.

Verapamil or BCF were administered 15 min prior to ischemia via a sublingual intravenous injection, and the sham control and I/R model rats received equal volumes of saline.

I/R-induced cardiac dysfunction was determined by invasive hemodynamic evaluation methods. A micro-catheter connected to the MS4000 organism signal quantitative analytical system (Longfeida Technology Co., Ltd.) was inserted into the left ventricle through the right common carotid artery to record the left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP) and maximum rise/down velocity of the left intraventricular pressure (± dp/dt_max) at baseline, after 30 min of ischemia, and after 30, 60, 90 and 120 min of reperfusion.

The myocardial infarct size was measured as previously described [14]. Immediately following reperfusion, the LAD was ligated completely and 4 mL of 1% Evans blue dye was injected retrogradely into the aorta to delineate the region of myocardial perfusion. The hearts were rapidly excised and cooled in saline at -80°C for 5 min. The atria were removed, and the ventricles were cut into 2-mm transverse slices from the apex to the base. The viable myocardium in these slices was then stained at 37°C for 15 min with 1% 2, 3, 5-triphenyltetrazolium chloride (TTC). The area of the white zone (unstained by Evans blue and TTC) was determined as the infarct size (IS), while the area unstained by Evans blue was estimated to be AAR. Infarct size was determined as the percentage of unstained LV in the slices, IS/AAR. The extent of ischemic myocardium was calculated as the ratio AAR/LV.

After reperfusion, blood samples were collected and the serum was separated by centrifugation at 3000 r/min at 4°C for 10 min. The levels of LDH, SOD, MDA and GSH-Px in the serum were measured by a colorimetric method using commercial kits (Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's protocols.

Blood samples were collected from the carotid artery after 3 h of reperfusion. Serum level of inflammatory cytokine IL-6 was determined by ELISA.

We conducted TUNEL with an In Situ Cell Death Detection Kit, POD (Roche, Germany) according to the manufacturer's protocol. In this method, the TUNEL- positive brown-colored cells were considered to be apoptotic cells. The results were scored semiquantitatively by averaging the number of apoptotic cells/field at 400× magnification. Five fields were evaluated per tissue sample, and the cardiomyocyte apoptosis represented the apoptosis index (AI) and was calculated as follows: AI = the number of TUNEL-positive cells/the total number of cells counted×100%.

Caspase-3 activity was measured according to the manufacturer's instructions. In brief, myocardial tissue samples were lysed with a lysis buffer on ice for 5 min after homogenization with a homogenizer. After mixing proteins from the tissue lysates, the reaction buffer and caspase-3 substrate were incubated in 96-well plates at 37°C for 4 h. After incubation, the samples were measured at 405 nm by a microplate reader.

Total protein was obtained from left ventricular myocardial tissues after extraction with the RIPA lysis buffer. Protein concentration was determined using the BCA protein assay kit according the manufacturer's protocol. Equal amounts of protein from each sample were separated by electrophoresis on SDS-PAGE and then transferred onto polyvinylidene difluoride (PVDF)-plus membranes. After blocking with 5% bovine serum albumin (BSA), the membranes were incubated overnight at 4°C with the primary antibodies against TLR4 (1:1000), NF-κBp65 (1:1000), P-NF-κB p65 (1:1000), Bcl-2 (1:1000), Bax (1:1000) and GAPDH (1:500). Then, the membranes were washed three times in Tris-buffered saline with 0.1% Tween 20 (TBST) and were incubated with the corresponding secondary antibody (1:5000) conjugated to horseradish peroxidase at room temperature for 2 h. Finally, the membranes were washed three times in TBST. Relative densitometry was assessed by a computerized software package (NIH Image 1.63 software).

Each sample was assayed in triplicate. The results were averaged and expressed as the mean ± SD, and the data were evaluated using the Sigma Stat (version 21.0) statistical analysis program (SPSS Inc., Chicago, IL, USA). One-way analysis of variance followed by Bonferroni's multiple comparison test was used for statistical analysis. P values less than 0.05 were considered statistically significant.

Before ischemia, there were no apparent differences in the baseline parameters among all of the experimental groups. However, compared with the sham group, I/R injury led to left ventricular dysfunction that was characterized by a significant increase in the LVEDP and a decrease in the LVSP, +dp/dt_max and −dp/dt_max. Pretreatment with BCF or verapamil resulted in the partial attenuation of reperfusion damage by decreasing LVEDP and increasing LVSP, +dp/dt_max and −dp/dt_max. Furthermore, the BCF control group had no obvious changes compared to the sham control group (Fig. 1).

No significant difference was observed in the ratio of AAR/LV between the different groups. However, the ratio of the infarct size (IS) to AAR was significantly decreased in I/R rats treated with BCF or verapamil compared with the I/R group. There were no significant differences in the ratio of IS/AAR between BCF control group and sham control group (Fig. 2A).

As shown in Fig. 2B, compared with the sham group, remarkable increases in the serum level of LDH was observed in the I/R group. In contrast, both the BCF (particularly high-dose) as well as the verapamil treatment inhibited the elevation of LDH. In the BCF alone group, no marked changes were observed in the level of LDH in comparison with the sham group.

As shown in Fig. 3, the SOD and GSH-Px activities in the I/R group were significantly lower than those in groups pretreated with BCF or verapamil. Compared with the I/R group, the BCF or verapamil-treated group had decreased concentrations of MDA, but no significant difference was observed between the BCF L group and the I/R group. Furthermore, in the BCF alone group, no marked changes were observed in the levels of SOD, GSH-Px and MDA in comparison with the sham group.

As shown in Fig. 4A, IL-6 level was lower in the BCF or verapamil-treated groups than those in the I/R group, and no significant difference was observed for IL-6 between the BCF L group and the I/R group. Compared with the sham group, significant increases in the protein levels of TLR4 and phosphorylated NF-κBp65 were noted in the I/R group but a remarkable decrease in these proteins occurred in the BCF and verapamil groups (Fig. 4B, 4C). Consistently, pretreatment with BCF in the sham group produced no marked changes.

TUNEL staining showed the absence of apoptosis in the sham group. The hearts of the animals that were subjected to I/R exhibited severe tissue damage and appeared to have dramatically increased numbers of TUNEL-positive cells (P < 0.01). In contrast, the BCF and verapamil groups demonstrated a significant reduction of TUNEL-positive cells compared with the I/R model group (P < 0.01). In addition, the caspase-3 activity was significantly increased in the model group compared with the sham group (P < 0.01), but EGCG or verapamil treatment led to a decline in the activity of caspase-3 (P < 0.01). Additionally, the BCF alone group had no obvious changes in comparison with the sham group (Fig. 5).

As shown in Fig. 6, compared with the sham group, the Bcl-2 protein levels were decreased while the Bax protein levels were increased in the I/R group. Importantly, the Bcl-2/Bax ratio was markedly decreased in the I/R group. However, pretreatment with BCF or verapamil evidently attenuated the I/R-induced increase in Bax and the decrease in Bcl-2 expression. Correspondingly, the ratio of Bcl-2 to Bax was significantly increased in the BCF or verapamil-treated group. Moreover, pretreatment with BCF in the sham group showed no obvious changes.

A substantial amount of evidence has elucidated that oxidative stress, which is associated with a burst of reactive oxygen species (ROS) production and a reduction of antioxidant capacity, plays a crucial role in the pathogenesis of I/R injuries [15,16]. As a key factor contributing to oxidative damage, excessive oxygen free radicals affect antioxidant synthesis, inflammation and apoptosis and then cause various alterations of the myocardium [17]. MDA is an unsaturated fatty acid in free radicals and lipid peroxidation metabolites. As one of the end-products in the lipid peroxidation process, MDA has been used to evaluate the severity of oxygen-derived free radical-mediated injuries [18]. On the other hand, the body has an endogenous antioxidant system, which is composed of various antioxidant enzymes, to defend against the potential deleterious consequences of ROS and to protect against various forms of peroxidative injuries [19]. It is commonly accepted that SOD and GSH-Px are two dominant anti-oxidative enzymes. Acting as free radical scavengers, they inhibit the formation of ROS and prevent oxidative stress [20]. Administration of BCF elevated the activities of SOD and GSH-Px and reduced the MDA content in I/R rats. Strong evidence supports the idea that flavonoid exhibits a variety of biological effects in the cardiovascular system as a result of its potential antioxidant properties, such as quercetin and Yulangsan flavonoid [21,22]. Our present study was in line with these findings. The results demonstrated that the suppression and peroxidation of free radicals and the enhancement of the antioxidase activity in the myocardium might be, at least in part, involved in the cardioprotective mechanisms of BCF in myocardial I/R injury.

When myocardial cells are damaged or destroyed, the cardiac membrane becomes permeable or may rupture, leading to the release of intracellular enzymes into the blood stream, such as LDH. The elevation of the serum levels of these enzymes due to myocardial necrosis is considered to be a definitive diagnostic criterion for the assessment of myocardial damage [23]. In this study, the prior administration of BCF prevented the depletion of LDH enzymes in ischemic heart tissues, suggesting its membrane-stabilizing ability.

Acute myocardial infarction can trigger immediate decreases in cardiac function and ventricular dilation [24]. The impairment of ventricular function is the most common fatal complication secondary to ischemic heart diseases. Thus, improved cardiac function and the attenuation of ventricular dilation are crucial for treating ischemic heart diseases. It is well established that the LVEDP and −dp/dt_max represent ventricular diastolic function, while the LVSP and +dp/dt_max refer to ventricular systolic function [25]. These indices were abnormal during myocardial I/R. Interestingly, BCF pretreatment concurrently increased the LVSP and ±dp/dt_max, while it decreased the LVEDP, implying that it improved cardiac function via both systolic and diastolic functions.

Myocardial cell damage induced by I/R could be aggravated by the secondary intense inflammatory response. It is believed that the generation of ROS provokes an inflammatory reaction, which releases a large number of inflammatory cytokines, such as TNF-α and IL-6 [26]. TNF-α induces the inflammatory cascade by augmenting the release of other pro-inflammatory cytokines and affecting neutrophil recruitment [27]. IL-6 is a pleiotropic cytokine that holds a key position in acute inflammation and immune regulation [28]. High concentrations of IL-6 activate neutrophils, lymphocytes and monocytes/macrophages at the inflammatory site and then trigger the oxidative pathways responsible for local tissue damage [29]. TNF-α and IL-6 are pivotal in regulating the severity of I/R injuries [30]. In our previous study, TNF-α was a significant dicrease in I/R rats pretreated with BCF [12]. Moreover, the present study showed that BCF pretreatment attenuated I/R-induced IL-6 increase, suggesting that BCF restrained the extent of inflammation and limited the extent of I/R injury by preventing pro-inflammatory cytokine release.

TLR4 is one of the Toll-like receptor (TLR) pattern recognition receptors, and its activation induces the release of NF-κB from IκB and translocates from the cytoplasm into the nucleus, where it regulates the transcriptional activation of target genes. It is well established that the upregulation of the TLR4/NF-κB-mediated signaling pathway significantly contributes to the synthesis of pro-inflammatory cytokines, such as TNF-α, and initiates the inflammatory response [31]. Additionally, the TLR4/NF-κB pathway is also considered to be a main signal transduction pathway that is involved in oxidative stress and apoptosis. TLR4/NF-κB activation and its induction of the inflammatory cascade are key factors in myocardial I/R injury [32]. In this study, we provide evidence that BCF inhibits the expression of TLR4 and NF-κB in hearts subjected to I/R and reduces the pro-inflammatory cytokine IL-6 in the serum. This implies that BCF probably elicits an anti-inflammatory effect against myocardial I/R injury via the regulation of the TLR4/NF-κB signaling pathway.

It is apparent that oxidative stress and inflammatory reactions are early events in the development and progression of myocardial I/R injuries. Additionally, oxidative stress events are recognized as part of the processes that ultimately cause cell apoptosis and mitochondrial dysfunction, but the mechanisms are not fully understood [33,34]. Apoptosis, as a complex series of ordered cell-autonomous biochemical events, significantly contributes to cell death during myocardial I/R injury [25]. In mammalian cells, apoptosis can be executed though a death receptor-dependent (extrinsic) pathway and a mitochondria- dependent (intrinsic) pathway. Cell apoptosis through the mitochondria pathway is mediated by Bcl-2 family proteins [35]. The Bcl-2 family is commonly classified into two groups: anti-apoptotic members, such as Bcl-2 and Bcl-xL, and pro-apoptotic members, such as Bax, Bak, Bid and Bad [36]. These pro- and anti-apoptotic proteins are different with regard to their structure and tissue distribution and exert different functional effects. The major function of Bcl-2 is to stabilize the mitochondrial membrane potential, block the release of cytochrome c and inhibit caspase activity. However, Bax plays an opposing role relative to Bcl-2 [37]. Hence, the Bax/Bcl-2 ratio may serve as a useful marker for determining cell susceptibility to apoptosis. Caspase-3 is the central executer of apoptosis, and its activation is modulated by a series of signaling transduction cascades, among which the interaction between Bcl-2 and Bax proteins plays a critical role [32]. Our data revealed that pretreatment with BCF up-regulates Bax and suppresses the activation of caspase-3 after ischemia injury, while Bcl-2 is enhanced, which is in agreement with the reduced apoptotic cardiomyocytes counting results.

In conclusion, the results presented herein indicate that BCF exhibits protective effects in I/R-induced myocardial injuries in rats. The protective mechanisms of BCF may rely on its anti-oxidative activity by inhibiting lipid peroxidation and recruiting the anti-oxidative defense system, its anti-inflammatory activity by restricting the release of inflammatory mediators and the inhibition of the TLR4/NF-κB signaling pathway, and its anti-apoptotic activity, probably involving the depression of elevated Bax/Bcl-2 ratios and caspase-3 activation. Thus, BCF should be regarded as a new and promising drug that may be useful for the prevention of myocardial I/R injury and ischemic heart disease.

LDH (lactate dehydrogenase); MDA (malondialdehyde); SOD (superoxide dismutase); GSH-Px (glutathione perox- idase); IL-6 (interleukin-6); LVSP (left ventricular systolic pressure); LVEDP (left ventricular end-diastolic pressure); +dp/dt_max (the maximum rise velocity of the left ventricular pressure); -dp/dt_max (the maximum down velocity of the left ventricular pressure); TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling); BCF (Bauhinia championii flavone).

This work was supported by the National Natural Science Foundation of China (NO. 81360041); Project of Natural Science Foundation of Guangxi, China (2012 GXNSFAA053148).

The authors have no conflicts of interest.

J. Jian and F. Xuan contributed equally to this work.