YQHX Alleviates H/R-Induced Cardiomyocyte Apoptosis by Downregulating miR-1

Yiqi Huoxue granule (YQHX) inhibits cardiomyocyte apoptosis in myocardial ischemia-reperfusion injury (MIRI); however, the underlying mechanism is unknown. In this study, hypoxia-reoxygenation (H/R) models were established using rat myocardial primary cells and H9c2 cells, lactate dehydrogenase (LDH), and creatine kinase (CK) levels and cardiomyocyte apoptosis were determined. LDH release, CK activity, caspase-3 activation, mRNA and protein ratio of Bax/Bcl-2, and miR-1 expression were significantly higher (p < 0.01) in the H/R model of rat myocardial primary cells and H9c2 cells compared with the control group and was inhibited by YQHX treatment (p < 0.01 or p < 0.05). We also found that miR-1 overexpression could enhance apoptosis in cardiomyocytes, whereas apoptosis could be reduced by YQHX treatment (p < 0.01). In conclusion, YQHX alleviates H/R-induced cardiomyocyte apoptosis by inhibiting miR-1 expression, suggesting the potential of YQHX in preventing MIRI.


Introduction
Acute myocardial infarction (AMI) has emerged as one of the major diseases endangering human health and life [1]. In clinical practice, AMI is mainly treated by percutaneous coronary intervention or drug thrombolysis; however, upon rapid restoration of coronary blood supply, myocardial ischemia-reperfusion injury (MIRI) is inevitable [2][3][4]. Cardiomyocyte apoptosis is the main pathological basis of MIRI. Anticardiomyocyte apoptosis therapy can effectively alleviate the degree of MIRI and improve prognosis [5]. Commonly used drugs, such as calcium antagonists and β-receptor blockers are clinically effective against MIRI cardiomyocyte apoptosis; however, their curative effect is not ideal [6]. erefore, developing effective anti-cardiomyocyte apoptosis drugs is of great clinical significance to improve the prognosis of AMI.
According to the theory of traditional Chinese medicine, "Qi deficiency" and "blood stasis" are the basic features of MIRI pathogenesis [7]. Interestingly, traditional Chinese medicines used to treat "Qi deficiency" and "blood stasis" in AMI have significantly alleviated MIRI [8,9]. Yiqi Huoxue granule (YQHX), composed of ginseng, astragalus, safflower, and red peony, is a common prescription made by Professor Han Lihua for the clinical treatment of cardiovascular diseases. It is known for its Qi-invigorating and blood-activation properties leading to a definite curative effect [10,11]. Previous studies have shown the anti-inflammatory properties of YQHX reducing calcium-ion and antioxidant damage [11][12][13], which are the main pathological factors of cardiomyocyte apoptosis in MIRI. YQHX has also been shown to alleviate H 2 O 2 -induced oxidative damage in cardiomyocytes and inhibit cardiomyocyte apoptosis by upregulating coupling protein 2 (UCP2) [14]. However, it remains unclear whether YQHX intervenes with cardiomyocyte apoptosis during MIRI.
miR-1 has been closely associated with ischemic heart disease, including myocardial infarction and cardiomyocyte apoptosis [15]. Its effect has been linked to the regulation of apoptosis-related genes Bax and Bcl-2 and been reported that downregulation of miR-1 improves MIRI and protects cardiac function [16]. Interestingly, several traditional Chinese medicine compounds and monomers have been found to exhibit anticardiomyocyte apoptosis effects via regulating miR-1. For example, Xinkang granules decreased myocardial injury in chronic heart failure by inhibiting miR-1 [17]. Likewise, monomers such as astragaloside IV and ligustrazine inhibit the apoptosis of ischemic myocardial cells [18,19]. However, only a few studies have evaluated the intervention of traditional Chinese medicine in MIRI-induced myocardial cell apoptosis via miR-1 regulation. Importantly, the antioxidative damage effect of safflower extract hydroxysafflower yellow, a component of YQHX, is related to the inhibition of miR-1 expression [20]. erefore, we speculated that YQHX could potentially regulate miR-1 expression.
In this study, primary rat cardiomyocytes and H9c2 cardiomyocytes were used to establish a hypoxia-reoxygenation (H/R) model to examine the intervention effect of YQHX. We found that YQHX could alleviate myocardial injury, reduce apoptosis, and regulate the expression of miR-1, thereby suggesting that miR-1 could be a potential therapeutic target for YQHX in the treatment of MIRI and other cardiovascular diseases.

Isolation and Culture of Rat Primary Cardiomyocytes.
A 10 cm cell-culture dish containing Joklik was placed on ice. Heparin sodium (0.625 U/mL) was injected intraperitoneally in SD rats (0.1 mL/100 g). After 10 min, 20% urethane solution (1 g/kg) was injected into the abdominal cavity. Next, the thoracic cavity was opened, and the heart was resected and placed in a culture dish. After the heart was removed, the rat was immediately euthanized by cervical dislocation. Redundant tissue was stripped off to free the aorta. e heart was washed with Joklik perfusate at a constant temperature of 37°C. After 2-3 min, the perfusate was replaced with collagenase circulatory perfusion. After 30-45 min, the digestion was terminated following turbidity of the circulatory perfusate and hardness of the heart. During the entire perfusion process, a continuous perfusion of pure oxygen was ensured. Residual tissue was removed using a 100-mesh filter (Missouri, USA). To obtain the supernatant, the solution was centrifuged at 2000 rpm for 20 s at room temperature (RT). Separation was facilitated by the addition of gradient recalcification solution using natural sedimentation in a water bath at 37°C, and the process was carried out three times. ree hours before the separation of primary cardiomyocytes, 1 mL of M199+ medium was added to 4% laminin precoated 6-cm cell-culture dishes. After complete separation, the culture dishes were gently shaken. Culturing of primary cardiomyocytes was carried out in a constant temperature incubator at 37°C and 5% CO 2 for 3 h. e original medium was discarded, and fresh M199+ medium was added. Cells were randomly labeled and divided into groups.

Preparation of an H/R Model of Rat Primary
Cardiomyocytes and H9c2 Cells. H9c2 cells were purchased from Shanghai Cell Bank of Chinese Academy of Sciences (Shanghai, China). H9c2 cells or rat primary cardiomyocytes were seeded in 6-well plates and cultured until 90% confluence. en, cells were randomly divided into 3 groups and subjected to the following interventions: (1) control group (cells were cultured in normal conditions without any treatment); (2) H/R group (cells were placed in a closed anoxic chamber for 3 h, the culture medium was changed, and the cells were cultured in 5% CO 2 in an incubator for 0.5 h); (3) YQHX group (cells were pretreated with YQHX for 12 h prior to H/R treatment). Lastly, the cells were harvested for subsequent experiments. Evidence-Based Complementary and Alternative Medicine 2.5. Cell Transfection. H9c2 cells (1.5 × 10 5 cells/well) were seeded into 6-well plates. At 50% confluency, the original medium was discarded, and the cells were washed with PBS. en, DMEM containing 2% fetal bovine serum was added. NC mimics (sense: UUCUCCGAACGUGUCACGUTT, antisense: ACGUGACACGUUCGGAGAATT) or miR-1 mimics (sense: UGGAAUGUAAAGAAGUAUGUAU, antisense: ACACACUUCUUUACAUUCCAUU) were transferred using riboFECT ™ CP transfection reagent (RiboBio, Guangzhou, China). Cells were harvested 48 h after transfection for the indicated experiments.
2.6. Cell Viability Assay. Primary cardiomyocytes were seeded in a 96-well plate at a density of 1.5 × 10 3 cells/well. Next, different concentrations of YQHX were added. Experiments for each YQHX concentration were performed in triplicate. After culturing for 12 h, 10 μL of 1.0 mg/mL MTT solution was added to each well. e plate was incubated at 37°C and 5% CO 2 for 4 h and mixed by slow shaking for 10 min. e absorbance of the cells in the wells was measured at 490 nm using a microplate reader ( ermo Scientific, Massachusetts, USA). Similarly, H9c2 cells were seeded in 96-well plates at a density of 7 × 10 3 cells/well and subjected to similar treatment as that used for the primary cardiomyocytes to determine cell viability.

Estimation of CK Activity in Rat Cardiomyocytes.
Following the instructions of CK kit (Beijing, China), cells were detached and collected using a crude enzyme solution.
en, cells were crushed in an ice bath using ultrasonication and centrifuged at 1000 g and 4°C for 10 min. e supernatants collected were placed on ice. Next, 200 μL of the supernatant, 450 μL of working solution, and 350 μL of H 2 O were added to a 1 mL cuvette. After thorough mixing, the absorbance of the reaction mixture was measured at 340 nm for 10 s. Subsequently, the cuvette containing the mixture was placed in a 37°C water bath for 3 min to dry the sample. en, the absorbance of the reaction mixture was measured for 190 s at 340 nm, and CK activity was calculated according to the instructions in the kit.

LDH Detection.
Cell culture medium was collected and centrifuged at 400 g and 4°C for 5 min. e supernatant was added to a 96-well plate (120 μL/well), and the zero-setting group was established (i.e., 120 μL fresh medium).
en, 60 μL of LDH detection working solution was added to each well. After proper mixing, the mixture was incubated in the dark RT for 30 min, and the absorbance was measured at 450 nm using a microplate reader ( ermo Scientific, Massachusetts, USA).

qRT-PCR.
To determine miR-1 expression levels, total miRNA was extracted from cells using the miRcute miRNA extraction and isolation kit. Reverse transcription was performed using an miRNA first-strand cDNA synthesis kit (cervical-loop method). To determine Bax and Bcl-2 mRNA levels, total RNA extracted from the cardiomyocytes was reverse transcribed into cDNA. Quantitative real-time PCR was performed using TB Green Premix Ex Taq GC (TaKaRa, Dalian, China). Samples were analyzed in triplicate and GAPDH (or U6) was used as the internal reference gene. e products were predenatured at 95°C for 30 s, denatured at 95°C for 5 s, annealed at 60°C for 30 s, and amplified for 36 cycles. After the reaction, Ct values were obtained determined using the 7500 software program. Data were exported for statistical analysis, and the 2 −△△Ct method was used for calculations. Primers that were used are shown in Table 1.

Western
Blotting. Rat primary cardiomyocytes and H9c2 cells were lysed in RIPA lysis buffer, and protein concentrations were measured using the BCA method. Each sample was mixed with 5×loading buffer and heated at 100°C for 5 min. After 10% SDS-PAGE gel electrophoresis, protein samples were transferred onto PVDF membranes and blocked with 5% skimmed milk powder at RT for 1 h. en, the membranes were incubated with 1 : 1000 anti-Bax, anti-Bcl-2, and 1 : 5000 anti-GAPDH primary antibodies at 37°C for 1.5 h or at 4°C overnight, washed with TBST, and incubated with secondary HRP-conjugated antibodies (diluted 1 : 1000), including anti-mouse or anti-rabbit antibodies for 1 h at RT. Next, membranes were washed three times with TBST solution for 10 min. PVDF membranes were placed in ECL solution, and images were acquired using a gel-imaging system. e gray values of the respective protein bands were analyzed using ImageJ software.
2.11. Statistical Analysis. All data are expressed as the mean ± standard deviation (x ± s).
e statistically significant differences between groups were determined using oneway ANOVA and post hoc Tukey's test. p < 0.05 or p < 0.01 indicated that the difference was statistically significant.

Screening for YQHX Cytotoxicity.
According to the Chinese Pharmacopoeia [21], nontoxic drugs' high concentration can cause toxic reactions and damage human tissues and organs. erefore, we should determine the nontoxic and effective drug concentrations. In in vitro studies performed using rat primary cardiomyocytes and H9c2 cells, the 0.75 mg/mL YQHX group exhibited an increase in cell viability compared with the control group (Figures 1(a) and 1(b)). However, when the concentration of YQHX was 1.5 mg/mL, no significant differences were observed between the two groups. Moreover, the cell viability decreased significantly in the treatment groups compared with the control group when the YQHX concentration was higher than 3 mg/mL.

YQHX Attenuated the Degree of Cell H/R Damage.
Based on the above results, 0.75 mg/mL of YQHX was determined as the optimum intervention concentration that did not affect cell viability. To clarify the protective effect of YQHX, H/R models of cardiomyocyte injury were Evidence-Based Complementary and Alternative Medicine intervened with YQHX. We found that compared with the control group, the release of LDH and CK in the H/R group was significantly higher in the primary cardiomyocytes. Interestingly, compared with the H/R group, the YQHX group showed significant inhibition of LDH release and CK activity (Figures 2(a) and 2(b)). Similar results were observed when using H9c2 cells. Compared with that in the control group, LDH release in the H/R group increased and significantly decreased after YQHX intervention (Figure 2(c)).

YQHX Decreases Bax/Bcl-2 and Caspase-3 in the H/R Model of Primary Cardiomyocytes.
e Bax/Bcl-2 mRNA ratio in the H/R group was significantly higher, compared with that in the control group and was significantly decreased after YQHX intervention (p < 0.05) (Figure 3(a)). e protein expression of Bax/Bcl-2 and caspase-3 was upregulated in the H/R group than in the control group. YQHX treatment significantly ameliorated caspase-3 and Bax/Bcl-2 expression (Figures 3(b) and 3(c)). ese data suggested an inhibitory effect of YQHX in cardiomyocyte apoptosis.

YQHX Decreases Bax/Bcl-2 and Caspase-3 in the H/R Model of H9c2
Cells. In addition to primary cardiomyocytes under H/R condition, damage and apoptosis were also evaluated in the H/R model of H9c2 cells. Bax/Bcl-2 and caspase-3 expression was significantly higher in the H/R group than in the control group ( Figure 4). However, YQHX treatment significantly inhibited the increase in H/R-induced levels of Bax/Bcl-2 and caspase-3. e results were similar to those obtained for the primary cardiomyocytes.

YQHX Downregulated miR-1 Expression in the H/R Model of Cells.
To examine the apoptosis-related role of YQHX in the H/R model, the expression of miR-1 in primary cardiomyocytes and H9c2 was determined using qRT-PCR. We found that miR-1 was obviously upregulated in the H/R group than in the control group. However, compared with that in the H/R group, miR-1 was significantly decreased in the YQHX group ( Figure 5, p < 0.05).

YQHX Inhibited Cardiomyocyte Apoptosis by Inhibiting miR-1 Levels.
e above results indicated that the significantly increased expression of miR-1 in the H/R model correlated positively with myocardial injury and the apoptosis rate in cardiomyocytes. However, treatment with YQHX significantly decreased miR-1 expression and alleviated myocardial cell apoptosis and myocardial injury. erefore, we speculated that YQHX might exert an anticardiomyocyte apoptosis role by regulating the expression of miR-1. To our hypothesis, H9c2 cells were transfected with miR-1 mimics. We found that this step significantly  increased the miR-1 expression in the transfection group ( Figure 6(a)). Moreover, Bax/Bcl-2 and caspase-3 levels were significantly increased in the miR-1 mimic group than the H/R group. Notably, YQHX intervention significantly decreased Bax/Bcl-2 and caspase-3 levels in the miR-1 mimic + YQHX group compared with that in miR-1 mimic group (Figures 6(b)-6(d)). ese results indicated that the anticardiomyocyte apoptosis effect of YQHX was related to the inhibition of miR-1 levels.

Discussion
In this study, we showed that the release of LDH and CK activity was significantly increased in the H/R models of primary rat cardiomyocytes and H9c2. However, YQHX intervention significantly reduced the release of LDH and CK activity, thereby alleviating H/R-induced myocardial injury. Furthermore, YQHX reduced the Bax/Bcl-2 ratio and caspase-3 levels and downregulated miR-1 expression. ese Evidence-Based Complementary and Alternative Medicine findings suggested that the anti-MIRI cardiomyocyte apoptosis effect of YQHX could be related to the decreased expression of miR-1, which may be a potential therapeutic target against MIRI. In AMI, after the occluded blood vessel is reopened, various degrees of myocardial damage can affect the patients' quality of life and long-term prognosis [22]. During myocardial injury, the myocardial cell membrane is damaged and a large amount of LDH is released. In cardiomyocytes, CK, an important kinase involved in energy transport and ATP regeneration, mainly existing in the cytoplasm and mitochondria. Cell damage leads to an increase in CK activity [23]. In this study, an increase in LDH release and CK activity confirmed cardiomyocyte damage in H/R. However, intervention with YQHX alleviated these effects, thereby suggesting a protective effect of YQHX via a reduction of MIRI-induced myocardial injury. Cardiomyocyte apoptosis is the main cause of myocardial injury in MIRI. erefore, attenuating cardiomyocyte apoptosis has emerged as an important strategy to improve MIRI. Various apoptosis-related factors, including Bcl, Bax, and caspase-3, are involved in cardiomyocyte apoptosis. e protein levels of these factors are closely related to the degree of cardiomyocyte apoptosis [24]. In several studies, it has been reported that prescriptions for Qi-invigorating blood-activation (similar to the principle of traditional Chinese medicine used in this study) exhibit anticardiomyocyte apoptosis effects and improve heart function [25][26][27]. Here, we showed that YQHX reduced the levels of Bax/Bcl-2 and caspase-3 in H/R and, thereby, exhibited an antiapoptotic effect in cardiomyocytes. is is similar to the Qi-invigorating results, suggesting certain anticardiomyocyte apoptosis effects of YQHX. ese effects were examined in both H9c2 cells and primary cardiomyocytes; therefore, the data are more comprehensive. miR-1 is highly expressed in the heart and plays an important regulatory role in MIRI during myocardial infarction [28,29]. e regulatory role of miR-1 in cardiomyocyte apoptosis is closely related to the downregulation of Bcl-2, upregulation of caspase-3, and the aggravation of mitochondrial injury [30][31][32][33]. Reports on the intervention of traditional Chinese medicines or the use of monomer on miR-1 in MIRI are rare. For example, picroside which inhibits apoptosis in cardiomyocytes is also known to reduce miR-1 expression [34]. e active components of YQHX, including hydroxysafflower yellow A, astragaloside IV, paeoniflorin, and ginsenoside-Rb1 can inhibit cardiomyocyte apoptosis by downregulating miRNA-1 expression [20,25,35,36]. Our findings suggested that miR-1 expression was increased under H/R conditions and could be significantly reduced after treatment with YQHX. Overexpression of miR-1 antagonized the antiapoptotic effect of YQHX. Overall, these results suggest that YQHX alleviates H/R-induced cardiomyocyte apoptosis by downregulating miR-1 (Figure 7). Bax and Bcl-2 mRNA levels were using qRT-PCR, and the Bax/Bcl-2 ratio was calculated. (c, d) Protein levels of Bax, Bcl-2, and caspase-3 were determined using western blot, and the Bax/Bcl-2 ratio was calculated. Data are expressed as mean ± SD of three independent experiments. * * p < 0.01 was considered statistically significant.

Limitation.
Our study had a few limitations. We mainly focused on in vitro experiments and did not extensively conduct in vivo studies. Also, we only explored the mechanism of the antiapoptosis effects of YQHX and did not study the mechanism of the antiapoptosis effects of the components in YQHX. us, in future studies, we will conduct in vivo experiments to observe the protective effects of YQHX in rat H/R injury and detect caspase activity and the Bax/Bcl-2 ratio to determine the extent of apoptosis. And, how the components in YQHX collectively regulate myocardial apoptosis through miR-1 will be further investigated in the future.

Conclusion
YQHX protects H9c2 cells and primary cardiomyocytes against H/R injury by downregulating miR-1. Moreover, it inhibits Bax/Bcl-2 expression and caspase-3 activation and alleviates cardiomyocyte apoptosis. ese findings show the potential of YQHX as a novel strategy for the treatment of MIRI and related conditions in a clinical setting.

Data Availability
e datasets used and/or analyzed in the current study are available from the corresponding author upon reasonable request.

Authors' Contributions
Luandie Ge, Yaqi Fan contributed equally to this work.