Anshen Buxin Liuwei Pill, a Mongolian Medicinal Formula, Could Protect H2O2-Induced H9c2 Myocardial Cell Injury by Suppressing Apoptosis, Calcium Channel Activation, and Oxidative Stress

Background Anshen Buxin Liuwei pill (ABLP) is a Mongolian medicinal formula which has a therapeutic effect on the symptoms such as coronary heart disease, angina pectoris, arrhythmia, depression and irritability, palpitation, and short breath. However, its bioactivity against cardiac injury remains unclear. Methods The protective effect of ABLP was evaluated using H9c2 cells. Cell viability, intracellular Ca2+, reactive oxidative indices, and mitochondrial membrane potential (∆ψ) were assessed, respectively. The mRNA levels of Ca2+ channel-related genes (DHPR, RyR2, and SCN5A) and oxidative stress-related genes (Keap1, Nrf2, and HO-1) were measured by RT-PCR. Results 0.5–50 μg/mL ABLP could significantly decrease H2O2-induced cell injury by suppressing cell necrosis/apoptosis and excess oxidative stress, ameliorating the collapse of ∆ψ, and reducing intracellular Ca2+ concentration. Furthermore, 0.5–50 μg/mL ABLP reversed H2O2-induced imbalance in the mRNA levels of DHPR, RyR2, SCN5A, Keap1, Nrf2, and HO-1 gene in H9c2 cells, which further illustrate the mechanism. Conclusion ABLP provided protective and therapeutic benefits against H2O2-induced H9c2 cell injury, indicating that this formula can effectively treat coronary disease. In addition, the present study also provides an in-depth understanding of the pharmacological functions of ABLP, which may lead to further successful applications of Mongolian medicine.


Introduction
Cardiovascular disease (CVD) is a leading cause of death globally, contributing to more than 17 million deaths in 2017, of which mortality of coronary heart disease (CHD) is the most prevalent. In China, CHD is the main threat to human death. e morbidity of CHD in China was relatively lower than in Western countries [1]. However, due to the vast population base, about 23 million CHD cases were reported in China in 2016 [1]. Advances in cardiovascular research have identified oxidative stress as an important pathophysiological pathway in the development and progression of heart failure [2].
As an important part of traditional Chinese medicine (TCM), the traditional Mongolian medicine (TMM) system is also being practiced like other ethnic or traditional minority medicines in China on the basis of philosophies and practices of Mongolian culture. e origin of TMM belongs to the early 13 th century. Later on, it evolved into a typical medicine system in Inner Mongolia and its neighboring areas [3]. TMM has accumulated a wealth of clinical experiences in the development process based on its unique theoretical systems.
Anshen Buxin Liuwei pill (ABLP), a Mongolian medicinal formula, consisted of 6 medicinal materials including Bos taurus domesticus Gmelin (named "Niu-Xin" in Chinese), Myristica fragrans Houtt. (named "Rou-Dou-Kou" in Chinese), Choerospondias axillaris (Roxb.) Burtt et Hill (named "Guang-Zao" in Chinese), Eugenia caryophyllata unb. (named "Ding-Xiang" in Chinese), Aucklandia lappa Decne. (named "Mu-Xiang" in Chinese), and Liquidambar formosana Hance (named "Feng-Xiang-Zhi" in Chinese). TMM medical records consider that ABLP has a therapeutic effect on the symptoms such as coronary heart disease, angina pectoris, arrhythmia, depression and irritability, palpitation, and short breath [4]. Zhang [5] treated 114 patients with angina pectoris with ABLP and compound Danshen tablets, respectively. e results show that compared with compound Danshen tablets, ABLP is better in clinical efficacy and ECG results, and ABLP is more effective in the treatment of angina pectoris. Anlusi [6] randomly divided 112 patients with angina pectoris of coronary heart disease into the treatment group (n � 56) and control group (n � 56). e control group was treated with metoprolol succinate sustained release tablets, and the treatment group was treated with ABLP for 4 weeks. e results showed that the total effective rate of the treatment group was 94.64%, which was significantly higher than that of the control group (85.71%). e combination of ABLP and metoprolol succinate sustained release tablets can significantly reduce the frequency and duration of angina pectoris, effectively improve the symptoms of patients, and improve the quality of life. However, the pharmacological effect and mechanism need further clarification. erefore, an ameliorative effect of ABLP on cardiomyocytes injury should be further investigated to improve the development and utilization of the formula. e present study was thus conducted to evaluate the influence of ABLP on H 2 O 2 -induced H9c2 cardiomyocytes injury. e cell apoptosis and viability, intercellular Ca 2+ , and oxidative stress indices, as well as mitochondria function, were detected to discuss the action pathways.

Assessment of Cell Viability.
We followed the methods of Ma et al. [7]. Cell survival was observed with a phasecontrast microscope (Olympus, Japan). At the same time, cell viability was evaluated by the MTT method, real-time cell analyzer (RTCA) [8], and LDH contents. e amount of MTT formazan was qualified by determining the absorbance at 490 nm using a microplate reader (Tecan A-5082, Magellan, Austria). As for RTCA technology, background measurements were taken from the wells by adding 50 μl of the same medium to the E-16 plates. RTCA Software Package 1.2 was used to calibrate the plates. Cells were plated at a density of 1 × 10 5 cells/ml with the fresh medium to a final volume of 200 μl. Cells were incubated for 24 h at 37°C and 5% CO 2 in the RTCA cradle. When the cell density is sufficient, follow-up experiments are performed. According to the manufacturer's instruction, the level of LDH in cells was detected with a commercially available kit. e absorbance was measured at 450 nm with a microplate reader as described above.

Measurement of MDA Production and SOD Activity.
Cells in the logarithmic growth phase were incubated in 6-well plates for 24 h for stabilization; then, the medium was replaced with DMEM with or without 0.5-50 μg/mL ABLP for 12 h. After incubated with 50 μM H 2 O 2 for 0.5 h, the cells were harvested and sonicated with phosphate buffer saline (PBS) to obtain cell homogenates. e MDA levels and SOD activities were detected at 532 nm and 450 nm by a microplate reader, respectively, according to the manufacturer's instruction. e 2 Evidence-Based Complementary and Alternative Medicine protein concentrations of lysis buffer were measured by the BCA protein assay kit to standard the concentration of MDA and SOD.

Morphological Assessment of Apoptosis.
Hoechst staining kit is a classic and fast and easy method of detecting apoptosis. According to the manufacturer's protocol, H9c2 cells from different treatment groups were washed with PBS and incubated with Hoechst 33258 staining fluid at 37°C for 5 min. e cells' nuclear morphological changes were observed under an Olympus fluorescence microscope (Tokyo, Japan) at 461 nm. e nucleus of normal cells was normal blue fluorescence, while the nucleus of apoptotic cells was dense stained or fragmentary dense stained, with some white color.

Measurement of Mitochondrial Membrane Potential (ΔΨ).
e change in the mitochondrial membrane potential (ΔΨ) was determined using the JC-1 kit. H9c2 cells from different treatment groups were washed with DMEM and incubated with JC-1 (1 μM) in DMEM at 37°C for 20 min. After washing with DMEM, the cells were immediately detected by flow cytometry. Green fluorescence was detected by FL1 channel, red fluorescence was detected by FL2 channel, and the relative proportion of red and green fluorescence was commonly used to measure the proportion of mitochondrial depolarization.

Detection of Intracellular ROS.
DCFH-DA fluorescent probe was employed to monitor intracellular ROS. Cells in the logarithmic growth phase were incubated in 6-well plates for 24 h for stabilization; then, the medium was replaced with DMEM with or without 0.5-50 μg/mL ABLP for 12 h and then incubated with 50 μM H 2 O 2 for 0.5 h. After treatment, the cells were washed with fresh DMEM three times and then incubated with 10 μM DCFH-DA at 37°C for 30 min. Ultimately, the fluorescence intensity of cells was observed under a fluorescence microscope, and data were evaluated using Image J software (National Institutes of Health, Bethesda, MD, USA) [9, 10].

Measurement of Intracellular Calcium Ion ([Ca 2+ ]) Levels.
Fluo-4/AM was employed as Ca 2+ indicators to measure intracellular Ca 2+ concentrations. After treatment, the cells were washed with fresh DMEM three times, and the H9c2 cells were loaded with 5 μmol/L Fluo-4/AM for 30 min at 37°C in the dark. e fluorescence intensity was analyzed by flow cytometry.

RNA Extraction and Real-Time Polymerase Chain Reaction (RT-PCR).
RT-PCR was employed to determine the gene expression. After treatment, the total RNA was extracted from H9c2 cells with the TRIzol reagent (Qiagen, Valencia, CA United States). For mRNA quantification, cDNA was synthesized from 1 μg of total RNA using the PrimeScript TM RT reagent kit (TaKaRa, Dalian, China) following the manufacturer's instructions. Reactions were performed in a 20 μL volume according to the thermal cycler manufacturer's protocol (Rox) using an ABI 7500 RT-PCR system (Applied Biosystems) under the following condition: 30 s at 95°C, followed by 40 cycles of 15 s at 95°C, and 1 min at 57°C or 60°C [11]. e primer sequences used for the amplification of target genes are given in Table 1.

Statistical
Analysis. Data analysis and calculation of standard deviation were done by SPSS 18.0 (IBM, US). e results were presented as means ± SEM. e data comparison was performed using a one-way analysis of variance (ANOVA) followed by Dunnett's test to detect intergroup differences. A probability (P) value of less than 0.05 was considered statistically significant.

ABLP Efficiently Potent H9c2 Cells against H 2 O 2 -Induced
Cytotoxicity. As shown in Figure 1(a), the cell viability was dose-dependently reduced in H9c2 cells after incubation with H 2 O 2 (50-200 μM) for 0.5 h. 50 μM was thus selected as the optimal concentrations of H 2 O 2 to discuss the influence of ABLP further. MTT assay and RTCA showed that 0.5-50 μg/mL ABLP could significantly increase H 2 O 2 -induced cell injury (Figures 1(b) and 1(c)). LDH is an important marker of cell injury. As shown in Figure 1(d), the content of LDH in cells decreased markedly in the H 2 O 2 group compared with the control group (P < 0.05), indicating apparent release of LDH due to cell damage and split. However, the cell decrease was significantly blocked by preincubation of ABLP in a dose-dependent manner (P < 0.05 vs. the H 2 O 2 group). Together, these findings indicated that ABLP could promote cell survival and reduce cell damage in H9c2 cells subjected to H 2 O 2.

ABLP Strongly Reduced H 2 O 2 -Induced Oxidative Stress and Intercellular ROS Release.
e oxidative stress and antioxidant activity markers detected in the study are shown in Figure 2. MDA contents of the H 2 O 2 group were significantly elevated in Figure 2(a) (P < 0.05 vs. the control group). Conversely, SOD activity was markedly declined in Figure 2(b) (P < 0.01 vs. the control group). However, 25 μg/ mL and 50 μg/mL ABLP pretreatment significantly reduced MDA elevation and enhanced SOD activity (P < 0.05 vs. the H 2 O 2 group). At the same time, an increase in intercellular ROS occurred in the H 2 O 2 group with a stronger DCF fluorescence signal compared with the control group, which could be attenuated by ABLP treatment (Figures 2(c) and 2(d)). On the other hand, JC-1 staining showed that ΔΨ drastically decreased in the H 2 O 2 group than in the control group, which could be significantly blunted by ABLP pretreatment (Figure 3(b)). Figure 4, the Ca 2+ concentration was significantly enhanced in the H 2 O 2 groups than the control group (P < 0.01), which could be decreased by pretreatment with ABLP (0.5-50 μg/mL). Figure 5, the mRNA expression levels of DHPR, RyR2, and SCN5A in the H 2 O 2 group increased compared with the control group, suggesting that H 2 O 2 could upregulate L-type       Evidence-Based Complementary and Alternative Medicine voltage-dependent Ca 2+ channels. Conversely, compared with the H 2 O 2 group, 0.5-50 μg/mL ABLP pretreatment significantly downregulated the mRNA expressions of DHPR, RyR2, and SCN5A (P < 0.05). e influence of ABLP on the oxidative stress pathway is shown in Figure 5. Compared with the control group, incubation of H 2 O 2 could upregulate the mRNA level of Keap1 and decrease the mRNA levels of Nrf2 and HO-1. Compared with the H 2 O 2 group, 0.5-50 μg/mL ABLP pretreatment could significantly reversely regulate the mRNA levels of the three genes (P < 0.05).

Discussion
e damage of cardiomyocyte results from the combined action of multiple factors, among which oxidative stress is one of the leading and direct drivers of cell damage. During oxidative stress, the imbalance between the free radicals and ROS can be observed and lead to harmful effects on the body. Reactive oxygen plays an integral role in both myocardial injury and repair. All biological molecules are destroyed by the solid oxidative activity of ROS, especially cells damaged by lipid peroxidation [12]. Calcium is one of the most crucial intracellular messengers; it functions to translate extracellular stimuli into intracellular signaling pathways that ultimately regulate cellular development, survival, differentiation, and gene expression [13]. us, any factors or stimuli that disrupt this well-defined Ca 2+ balance give rise to heart disease, including arrhythmia [14].
It is well-known that intracellular calcium overload could enhance intracellular ROS production. e augmented ROS directly attacks SERCA2 to decrease its activity, increasing the open probability of RyRs [15,16] to give rise to [Ca 2+ ] elevation in damaged cells [17] and then causing arrhythmias by apoptosis [18][19][20].
Although previous research showed the cardioprotective effects of ABLP in a clinic, the essential mechanism of the myocardial protection of ABLP has not yet been fully explained. e present study revealed that 0.5-50 μg/mL of ABLP could protect H9c2 cells against H 2 O 2 -induced H9c2 cells injury via markedly promoting cell viability, inhibiting cell apoptosis, downregulating excessive oxidative indices, preventing calcium overload, and restoring mitochondrion functions. e protective mechanism of ABLP is shown in Figure 6. Intracellular calcium and ROS overload can lead to myocardial cells damage. Many studies have indicated that sarcoplasmic reticulum is the main calcium pool of myocardial cells, and there are two calcium release channels, RyR channel and IP3R channel, in the sarcoplasmic reticulum. Since RyR density is significantly higher than IP3R, RyR plays a key role in regulating the cardiac arousal systolic conjugate [7].  Evidence-Based Complementary and Alternative Medicine to enter into the nucleus during normal biological conditions through its binding to Keap1.
In contrast, during induced conditions, Nrf2 gets dissociated from Keap1 and enters the nucleus to activate a range of antioxidant genes and enzymes [21]. erefore, under the status of oxidative stress, Keap1 mRNA will be ascending and Nrf2 and HO-1 mRNA descending. Our study confirmed the protective effect of ABLP against myocardial injury via the Nrf2/Keap1/ARE pathway. However, myocardial injury can deteriorate to various cardiovascular diseases, and it has a relatively unique mechanism of action in some cardiac diseases, which also needs further research and exploration.

Conclusion
Taken together, ABLP provided protective and therapeutic benefits against H 2 O 2 -induced H9c2 cell injury, suggesting that this formula could be effective for the treatment of coronary disease. In addition, the present study provided a deeper understanding of the pharmacological functions of ABLP, which may lead to further successful applications of Mongolian medicine.

Data Availability
e data used to support the findings of this study are included within the article.

Disclosure
Yue Dong is the first author.

Conflicts of Interest
e authors declare that they have no conflicts of interest.

Authors' Contributions
Xian-Ju Huang and Hai-Ying Tong designed the experiment. Xian-Ju Huang supervised the project. Yue Dong and Yu-Jia Huang performed the experiments. Ghulam Murtaza helped analyze the data. Xian-Ju Huang, Muhammad Sarfaraz Iqbal, and Ghulam Murtaza wrote the manuscript. All authors have read and approved the final manuscript prior to submission.