BNC Protects H9c2 Cardiomyoblasts from H2O2-Induced Oxidative Injury through ERK1/2 Signaling Pathway

Buchang naoxintong capsule (BNC) is a traditional Chinese medicine approved for the treatment of cerebrovascular and cardiovascular diseases. However, little is known about the specific protective function or mechanism by which BNC protects against myocardial injury. This research was designed to investigate the cardioprotective effects of BNC in vitro model of hydrogen peroxide (H2O2)-induced H9c2 rat cardiomyoblasts. BNC intestinal absorption liquid was used in this study instead of drug-containing serum or extracting solution. Our study revealed that BNC preconditioning enhanced antioxidant function by increasing the activities of total-antioxygen capacity, total-superoxide dismutase, and catalase and by decreasing the production of reactive oxygen species and malondialdehyde. BNC preconditioning also activated extracellular signal-regulated kinases (ERK1/2) and inhibited apoptosis-related proteins such as poly ADP-ribose polymerase (PARP) and caspase-3. Additionally, preincubation with BNC reduced intracellular Ca2+ concentration, improved mitochondrial membrane potential, and decreased the apoptosis rate of H9c2 cells in a dose-dependent manner. These data demonstrated that BNC protects H9c2 cardiomyoblasts from H2O2-induced oxidative injury by increasing antioxidant abilities, activating ERK1/2, and blocking Ca2+-dependent and mitochondria-mediated apoptosis. Based on our results, the potency of BNC for protecting H9c2 cells from oxidative damage is comparable to that of trimetazidine.


Introduction
Oxidative stress plays a critical role in the pathophysiology of several major cardiovascular diseases such as atherosclerosis, hypertension, heart failure, and myocardial ischemical reperfusion injury [1,2]. Significant oxidative stress causes excessive production of reactive oxygen species (ROS), which is an important event in the development of cardiovascular diseases. ROS accumulation may contribute to a number of cardiovascular disorders [3,4]. Cellular sources of ROS come from the mitochondrial electron transport chain, xanthine oxidase, NADPH oxidase, lipoxygenase/cyclooxygenase, nitric oxide synthase, and autoxidation of various substances particularly catecholamines [5].
Increased ROS cause significant damage to myocardial cells, which can be neutralized by antioxidant molecules such as superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH). These antioxidant molecules counteract oxidation and play vital roles in maintaining a stable intracellular environment. Moreover, ROS damage cellular membrane by causing lipid peroxidation. Malondialdehyde (MDA) is a major lipid peroxidation product and may reflect the degree of cellular injury [6].
Extracellular signal-regulated kinases (ERKs) have been reported to be activated by oxidative stress in some cell types and play important roles in many aspects of cellular function [7]. ERK1/2 activation by ROS in cardiomyocytes mediates a wide range of activities including metabolism, migration, inflammation, cell survival, and cell death [8]. Indeed, DNA microarray data showed that nearly 100 genes were involved in cellular responses to oxidative stress [9].
Buchang naoxintong capsule (BNC) consists of 16 various kinds of traditional Chinese medicines including Astragalus membranaceus, Salvia miltiorrhiza, Ligusticum, Radix paeoniae rubra, Szechwan lovage Rhizome, Semen persicae, Carthamus tinctorius L, Frankincense, myrrh, Spatholobus suberectus, Achyranthes Root, Cassia Twig, Mulberry Twig, earthworms, scorpions, and hirudo, which is an approved 2 Evidence-Based Complementary and Alternative Medicine traditional Chinese medicine for stroke and angina [10]. BNC protected mice from atherosclerosis by reducing lipid concentrations and inhibiting maturation of dendritic cells [11]. BNC also increased the catalytic activity of the drugmetabolizing CYP2C19 enzyme [12]. The combination of BNC and clopidogrel enhanced the antiplatelet effect in patients with the CYP2C19 * 2 gene mutation [13]. BNC has been shown to have protective effects in cardiac and vascular diseases. However, the cellular and molecular mechanisms of BNC cardioprotective activity have not yet been elucidated.
In this study, oxidative stress was induced by exposing H9c2 cells to H 2 O 2 , which is a well-established model [14]. Treatments consisted BNC intestinal absorption liquids rather than extraction solutions or drug-containing sera. Trimetazidine (TMZ), a common treatment for angina in cardiac patients, served as a control [15,16].

Preparation of BNC Intestinal Absorption Liquid and
Treatment of Rat Intestines. The powder from 100 g BNC was dissolved in 2,000 mL 95% ethanol. This solution was heated until boiling for 2 hours (h) under reflux and then filtered. Water was added in the filtrate to make a 200-mL BNC extraction solution. The 200 mL BNC extraction solution was evaporated with a rotary evaporator until almost completely dry. Tyrode's buffer solution (NaCl 8.00 g, KCl 0.28 g, NaHCO 3 1.00 g, NaH 2 PO 4 0.05 g, MgCl 2 0.10 g, CaCl 2 0.20 g, glucose 1.00 g, pH 7.4) was added to bring the volume of BNC solution to 600 mL. Rats were maintained in fasting conditions for 12 h before the experiment. Anesthesia was provided, and the intestine of each rat was quickly removed. Intestines were washed with Tyrode buffer solution (0 ∘ C) and cut into four 14 cm segments. Each segment was turned inside-out and ligated to form a sac at one end. The sac filled with Tyrode buffer was incubated in Magnus' bath for 5 minutes (min) to reach equilibration. Buffer was then exchanged with BNC solution (25 mL), which was maintained at 37 ∘ C and continuously injected with O 2 /CO 2 (95%/5%). Serosalside solutions containing absorbed constituents (2 mL) were drained into tubes after 2 h. BNC intestinal absorption liquids were filtered with a microfiltrate membrane (0.22 m) and stored at −20 ∘ C. The original concentration of BNC intestinal absorption liquid was 1 mg/mL (crude drug).

Liquid
Chromatography. BNC intestinal absorption liquid was analyzed by Ultra Performance Liquid Chromatography (UPLC) with a Waters ACQUITY C18 column (2.1 mm× 150 mm, 1.7 m). The mobile phase consisted of acetonitrile and 0.5% formic acid with gradient elution at a flow rate of 0.3 mL/min. The detection wavelengths were set at 235, 280, 324, and 400 nm. The injection volume was 2 L and the column temperature was maintained at 30 ∘ C. 2.6. MTT Assay. The optimum concentration and application time for H 2 O 2 and the protective effect of BNC on H9c2 cells were determined by MTT assay. Cells were dispersed by trypsinization and seeded at (8,000-10,000) cells/well in a 96-well plate overnight before being treated. Subsequently, 20 L MTT solution (5 mg/mL) was added to each well and incubated at 37 ∘ C for 4 h. The supernatant was removed, and the insoluble formazan product was dissolved in 150 L DMSO. Absorbance of each culture well was measured with a microplate reader (Molecular Devices, USA) at a wavelength of 570 nm.

Biochemical Analysis of H9c2
Cell Lysate. H9c2 cells were adjusted to 1 × 10 6 cell/mL after trypsinization, washed with phosphate-buffered saline (PBS) twice, and centrifugated at 1,500 rpm/min for 10 min. The supernatant were then removed. The precipitate obtained through centrifugation was crushed by ultrasonic wave, and the cell lysates were resuspended. T-AOC, T-SOD, CAT, and MDA were determined with a microplate reader (Molecular Devices, USA) according to the protocol of the detection kit. Protein content was measured with the BCA Bradford protein assay (Pierce, USA). USA). Excitation and emission wavelengths were set at 488 nm and 525 nm, respectively.

Detection of Apoptosis with Annexin V-PI.
H9c2 cells were harvested with 0.25% trypsin, washed twice with cold PBS (4 ∘ C), and resuspended in 500 L binding buffer. Cells were incubated with 10 L Annexin V for 60 min in the dark at 4 ∘ C, and then with 5 L propidium iodide for 5 min at room temperature. Fluorescence was analyzed with a FACStar Plus flow cytometer (Becton-Dickinson, USA).

Qualitative Analysis of BNC Intestinal Absorption Liquid.
Eleven constituents were detected in BNC extraction solution. These were calycosin, ligustilide, paeoniflorin, protocatechualdehyde, salvianolic acid B, senkyunolide A, tanshinone I, caffeic acid, ferulic acid, rosmarinic acid, and hydroxysafflor yellow A. Eight constituents in BNC intestinal absorption liquid were examined, which is consistent with BNC extraction solution except calycosin, ligustilide, and senkyunolide A. Results indicated that the composition of BNC intestinal absorption liquid was similar to that of BNC extraction solution (Figure 1).  (Figure 2(a)) and 52.37 ± 3.90% (Figure 2(b)). Thus, cells were treated with this concentration in subsequent experiments.  (Figure 3(b)).  Figure 4).

BNC Protected H9c2 Cells from Damage Caused by H
Oxidative stress has been implicated in the pathogenesis of myocardial injury. To determine whether BNC affects oxidative stress-related biochemical enzymes, the levels of oxidant and antioxidant enzymes, such as T-AOC, T-SOD, CAT, and MDA, were measured in H9c2 cell lysates. The activities of T-AOC, T-SOD, and CAT were increased in a dose-dependent manner in the BNC pretreatment group relative to the H 2 O 2 group, whereas MDA production was reduced ( Figure 5). These results confirmed that the in vitro antioxidant capacity of BNC was comparable to that of TMZ.  Figure 6).    (Figure 8).   Figure 10(a)). These results showed that BNC ( (Figure 10(a)). However, pretreatment with BNC induced phosphorylation of ERK1/2 more rapidly, with increased band intensity present at 1 h. Expression of p-ERK1/2/ERK1/2 was significantly increased in cells preconditioned with BNC in comparison to the H 2 O 2 group (Figure 10(c)). Pretreatment of cells with the highly selective ERK1/2 inhibitor, PD98059 (10 M), indicated that ERK1/2 inhibition blocked the protective effect of BNC in H 2 O 2treated cells (Figure 10(b)).

Discussion
Oxidative stress in cardiomyocytes has an important role in the pathogenesis of many cardiovascular diseases, and ROS generated by oxidative stress are central to cardiac injury [17]. Studies have shown that mitochondria play prominent roles in the transduction and amplification of the apoptotic response in cardiomyocytes during oxidative stress [18,19]. ROS promote the release of cytochrome c by increasing mitochondrial permeability. Pro-caspase-3 is downstream of cytochrome c in the apoptotic cascade and is cleaved by active caspase-9 to produce active caspase-3. In addition to caspases-6 and caspases-7, caspase-3 contributes to oligonucleosomal DNA fragmentation, which ultimately leads to apoptosis [20,21]. Ca 2+ is critical for cardiomyocyte contractility and for the signaling pathways in cardiac growth and remodeling. Cytosolic Ca 2+ increases abnormally in cardiomyocytes as a consequence of ischemic reperfusion injury or other stresses, causing myocardial dysfunction and cell death. Increased Ca 2+ activates calpains, leading to disruption of plasma membrane. Calpain activates the proapoptotic protein, BID. Calpain also cleaves autophagy protein 5, shifting the balance from autophagy to apoptosis [22]. We found that BNC inhibited H 2 O 2 -induced activation of PARP and caspase-3, reduced intracellular Ca 2+ concentrations, and improved H 2 O 2 -induced impairment of the mitochondrial  MAPKs are serine/threonine protein kinases involved in cellular proliferation, differentiation, and death [23,24]. Activation of the ERK1/2 MAPK subfamily in cardiac myocytes regulates expression of many genes and relates to the development of cellular hypertrophy. Activation of ERK1/2 in response to H 2 O 2 is mediated through the Ras/Raf1/Mek pathway [25]. ERK1/2 activation may promote inflammation and result in necrosis by up-regulating interleukin-1 [26]. ERK1/2 activation may also inhibit apoptosis by down regulating Bad and up-regulating Bcl-2 [27]. Our research indicated that BNC exerted a protective effect against oxidative stress-mediated injury in H9c2 cells by inducing early phosphorylation of ERK1/2 ( Figure 11). BNC is a traditional Chinese medicine for the treatment of cerebrovascular and cardiovascular diseases. BNC intestinal absorption liquid was used in this study instead of drug-containing serum and extraction solution. Traditional Chinese medicines are usually consumed orally. Thus, the small intestine is a major absorption site for herbal medicines. However, herbal mixtures contain multiple ingredients, and complicated herb-herb interactions can occur during intestinal absorption [28]. The process for producing intestinal absorption liquids imitates the complicated absorption process of traditional Chinese medicine. Absorbed constituents from the digestive tract may produce therapeutic effects [29]. UPLC analysis further confirmed that the compounds in BNC intestinal absorption liquids are similar to the compounds present in BNC extraction solution. Our studies lay a foundation for illuminating the cardioprotective effect of BNC preconditioning on oxidative stress damage. However, further studies need to be performed to understand and develop new strategies for cardioprotection during oxidative stress.
TMZ is a clinically effective antianginal drug that has direct cytoprotective effects on the myocardium. TMZ reduces oxygen free radical production at the cellular level [30], reduces intracellular calcium overload, and improves mitochondrial metabolism [31]. Mesenchymal stem cells that were preconditioned with TMZ before implantation significantly enhanced the recovery of myocardial function by increasing gene and protein expression levels of HIF-1, survivin, p-Akt, and Bcl-2. Our data demonstrated that H 2 O 2induced impairment was attenuated in TMZ-preconditioned H9c2 cells. These results are consistent with previous studies. These data indicated that the potency of BNC for protecting H9c2 cells from oxidative damage is similar to that of the positive control, TMZ.

Conclusion
BNC protected H9c2 cardiomyocytes from H 2 O 2 -induced oxidative injury by enhancing antioxidant abilities, activating ERK1/2 signaling, inhibiting apoptosis-related signal transduction pathways, reducing intracellular Ca 2+ concentrations, and improving mitochondrial membrane potential. This research provides evidence that BNC exerted a protective effect on cardiovascular damage and the potency of BNC for protecting H9c2 cells from oxidative injury is comparable to that of trimetazidine.