Wenyang-Yiqi Granule Suppresses Oxygen-Glucose Deprivation-Induced Cardiomyocyte Autophagy Through Mammalian Target of Rapamycin Activation in H9c2 Cells

Abstract Background  Wenyang-Yiqi Granule (WYYQ) is a four-component herbal formula, widely used to treat heart failure in China. It is known to regulate autophagy, but the mechanism(s) are unknown. Methods  H9c2 cells were treated with WYYQ for 24 hours prior to oxygen-glucose deprivation (OGD). Expressions of the autophagy markers Beclin-1 and light chain 3 (LC3) were evaluated via quantitative polymerase chain reaction analysis. Protein levels of Beclin-1, LC3, p62, and mammalian targets of rapamycin (mTOR) were determined by Western blot analysis. Transmission electron microscopy was used to explore the effects of WYYQ on autophagosome formation. Results  Treatment with WYYQ dramatically restrained OGD-induced autophagy, which was characterized by an inhibition of Beclin-1 and increased LC3 mRNA expression. In addition, WYYQ decreased the expression of Beclin-1 and the ratio of LC3-II/LC3-I; however, the abundance of p62 was enhanced at the protein level. Manipulation of the LC3-II/LC3-I ratio, p62 abundance, and autophagosome formation in response to WYYQ were associated with mTOR activity. Conclusions  These findings show that WYYQ plays a protective role during hypoxic-ischemic stress through the suppression of excessive autophagy, which may be partially explained by its effects on mTOR. These data provide novel insight into the cardioprotective effects of WYYQ during cardiomyocyte autophagy.


Introduction
Heart failure (HF) is a clinical syndrome characterized by the inability of the heart to meet the metabolic demands of the human body. 1,2 Myocardial remodeling is associated with an increased risk of HF. Previous studies have shown that autophagy, a lysosomal-dependent process that degrades intracellu-Wenyang-Yiqi Granule (WYYQ) is widely used for the prevention and treatment of HF in China, and it is a compound medication that includes Wutou (Aconiti Radix Lateralis praeparata), 6 Rougui (Cinnamomi Cortex), 7 Huangqi (Astragali Radix), 8 and Renshen (Panax Ginseng). 9 Previous studies showed that WYYQ exerts inhibitory effects on the excessive activation of the renin-angiotensin-aldosterone system (RAAS) in rats with HF. 10 However, the mechanism(s) underlying anti-RAAS over-activation were not defined. RAAS overactivation leads to cellular injury by promoting autophagic activity. 11,12 Furthermore, cardiomyocyte autophagy is modulated by angiotensin II, 13 an activator of RAAS. Therefore, this study speculated that WYYQ inhibits RAAS activity by regulating autophagy in cardiomyocytes.
Changes in the autophagy-related proteins Beclin-1, light chain 3 (LC3), and p62 reflect the status of autophagy in cells. 14,15 A higher LC3-II/LC3-I ratio promotes autophagy dysfunction. 16,17 Autophagy is regulated by the mammalian target of rapamycin (mTOR), a serine/threonine kinase. mTOR acts as a pivotal component of two distinct signaling complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2), which regulate diverse cellular behaviors. 18 Genetic inhibition of mTORC1 triggers autophagy in yeast and in Drosophila, 19 while starvation-induced autophagy can be inhibited by mTORC1 activation. 20 In addition, mTORC1 modulates autophagy at the transcriptional level by fine-tuning lysosomal genes and their activities. 21 Thus, mTOR signaling has emerged as a target to improve cardioprotection.
The relationship between WYYQ and mTOR signaling in response to HF has been poorly characterized to date. Appropriate interventions using WYYQ may limit myocardial remodeling and regulate autophagy in HF. This study established an oxygen-glucose deprivation (OGD)-mediated in vitro model to mimic autophagic conditions. The pleiotropic effects of WYYQ on the expression of the autophagy-related proteins Beclin-1, LC3, and p62 were examined. Furthermore, the underlying mechanism(s) with which WYYQ modulates mTOR signaling were explored based on autophagic dysfunction.

Cell Culture
H9c2 cells, a rat cardiomyocyte cell line, were obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in an incubator at 37°C and 5% CO 2 in DMEM supplemented with 10% FBS, 100 IU/mL penicillin, and 100 IU/mL streptomycin.

Oxygen-Glucose Deprivation Treatment
OGD-induced cell injury models were established as previously described. 22 Briefly, H9c2 cells were seeded at a density of 1.0 Â 10 5 cells in 24-well culture plates under normal culture conditions. For OGD treatment, cells at 80 to 90% confluence were switched to serum-free and glucosefree DMEM and were transferred to a tri-gas incubator perfused with 95% N 2 and 5% CO 2 for 6 hours. Subsequently, cells were pretreated with WYYQ at different concentrations (0.4, 0.8, and 1.6 mg/mL) and/or rapamycin (10 nM) for 24 hours prior to establishing the OGD model. Cells that served as the control group were incubated in a standard medium.

Cell Viability Assays
For cell viability assays, H9c2 cells were seeded at a density of 0.7 Â 10 5 cells per well in 96-well plates. After 24 hours, various concentrations of WYYQ (0, 0.2, 0.4, 0.8, 1.6, 3.2, and 6.4 mg/mL) were applied. Cells were continuously cultured for 24 hours prior to the addition of 10 μL of medium containing CCK-8 reagent for each well. Plates were incubated for 30 h at 37°C in the dark, and the absorption at 450 nm was measured using a microplate reader (Thermo Fisher Scientific, Waltham, MA, United States) following the manufacturer's instructions. Data shown are the average of six wells per group.

Transmission Electron Microscopy Assessments
Transmission electron microscopy (TEM) was performed as previously described. 23 In brief, H9c2 cells were harvested and fixed in 2.5% glutaraldehyde (Sigma-Aldrich, St. Louis, MO, United States) in 0.1 M phosphate-buffered saline (pH 7.4) at 4°C for 2 hours. Cells were then fixed in 1% osmium acid at 4°C for 1 hour, dehydrated in gradient ethanol, embedded in epoxy resin 812 mixed with acetone, sliced, and stained with uranyl acetate and lead citrate. Images were obtained via TEM (JEM-1400, JEOL Ltd., Tokyo, Japan).

Quantitative Real-Time Polymerase Chain Reaction
Total RNA was extracted from H9c2 cells using the Trizol reagent, followed by subsequent cDNA synthesis through

Annexin-V FITC/PI Assay
Cellular apoptotic rates were determined using Annexin-V FITC/PI assay kits as previously described. 25 Briefly, cells pretreated with WYYQ were collected and resuspended in binding buffer (provided with the kit) at 1.0 Â 10 5 cells/mL. Cells were labeled with annexin-V-FITC and propidium iodide (PI) for 20 minutes at room temperature in the dark and were analyzed by flow cytometry (JAZZ; BD Biosciences, San Diego, CA, United States). Data were analyzed using FlowJo 7.6 software (TreeStar, Ashland, OR, United States).

Statistical Analysis
Experiments were performed in six repetitions. Data are presented as the mean AE standard deviation. Statistical differences were determined by one-way analysis of variance followed by LSD tests for two-group comparisons amongst multiple comparisons. p-Values 0.05 were considered statistically significant.

Oxygen-Glucose Deprivation Intervention Induces Autophagy in H9c2 Cells
H9c2 cells were exposed to OGD for 0, 3, 6, 9, and 12 hours, then harvested, and the expressions of autophagy-related genes or proteins were assessed. The data showed that the mRNA levels of Beclin-1 markedly increased at 6 hours of OGD compared with levels at 0 hour (►Fig. 1A). A significant enhancement in the expression of LC3 mRNA was also observed at 6 hours (►Fig. 1B). Western blot analysis showed that the LC3-II/LC3-1 ratio was significantly higher from 3 hours of OGD 3 to 12 hours (►Fig. 1C). Moreover, the expression of Beclin-1 was maximally upregulated at 6 hours of OGD (►Fig. 1C), while p62 levels were downregulated at 6 hours of OGD (►Fig. 1C). According to these data, 6 hours of OGD were selected as an appropriate time point for the induction of autophagy.

Wenyang-Yiqi Granule Increases Cell Viability and Reduces Apoptosis in H9c2 Cells under Oxygen-Glucose Deprivation Challenge
To determine the optimal concentration at which WYYQ protects against OGD-triggered cell injury, the cytotoxicity of WYYQ in H9c2 cells was first assessed following treatments with 0, 0.4, 0.8, 1.6, 3.2, and 6.4 mg/mL WYYQ for 12 and 24 hours via CCK-8 assays. As shown in ►Fig. 2A, no cytotoxicity occurred with cumulative concentrations of WYYQ up to 1.6 mg/mL for 12 or 24 hours. However, an obvious decrease in cell viability was observed in cells treated with ! 3.2 mg/mL WYYQ. Therefore, for subsequent assessments, the three nontoxic doses of 0.4, 0.8, and 1.6 mg/mL WYYQ were selected as low, middle, and high dose groups, respectively.
Next, it was explored whether WYYQ protected against OGD-induced cardiomyocyte apoptosis. The apoptotic cell distribution triggered by OGD was examined in H9c2 cells using annexin V/PI staining. As shown in ►Fig. 2B, H9c2 cells were characterized as early apoptotic (annexin V þ /PI -) or late apoptotic (annexin V þ /PI þ ). The OGD challenge induced cell apoptosis in up to 45.12 AE 4.79% of cells, while the apoptotic rates of H9c2 cells treated with WYYQ at concentrations of 0.4, 0.8, and 1.6 mg/mL were 14.14 AE 2.01%, 7.21 AE 0.79%, and 5.93 AE 0.63%, respectively.

Wenyang-Yiqi Granule Downregulates Oxygen-Glucose Deprivation-Induced Autophagy in H9c2 Cells
The mRNA levels of Beclin-1 and LC3, both autophagypromoting factors, were upregulated by the OGD challenge compared with the control group (►Fig. 3A-B). WYYQ treatment for 24 hours prior to the OGD challenge significantly downregulated Beclin-1 and LC3 at the mRNA level when compared with the OGD-treated group (►Fig. 3A-B). The protein levels of Beclin-1 were enhanced compared with those of the control group (►Fig. 3C), which was consistent with the mRNA expression of autophagy-associated Beclin-1 genes. The ratio of LC3-II/LC3-I was also enhanced in the OGD-treated group. In addition, the protein levels of p62, an inhibitory of autophagy, were remarkably reduced in OGDtreated H9c2 cells when compared with those of the control and WYYQ-treated H9c2 cells that demonstrated increased p62 expression (►Fig. 3C). Taken together, these data suggested that WYYQ reduced OGD-induced autophagy in H9c2 cells and that 1.6 mg/mL WYYQ was the optimal level to inhibit autophagy.

Wenyang-Yiqi Granule Suppresses Autophagosome Production in Response to Rapamycin
TEM was used to evaluate the ultrastructure of H9c2 cells undergoing autophagy. As shown in ►Fig. 4, there was a trend toward increased autophagy induced by OGD in H9c2 cells when compared with control cells. In addition, the formation of autophagosomes was suppressed by treatment with WYYQ. In addition, rapamycin accompanied by OGD resulted in an increase in autophagy processes. The increased number of autophagosomes was suppressed by pretreatment with WYYQ.

Wenyang-Yiqi Granule Reduces Cardiomyocyte Autophagy Through Regulating the Mammalian Targets of Rapamycin Pathway
As shown in ►Fig. 4, WYYQ suppressed the rapamycininduced increase of autophagosomes in response to OGD. Then, it was further investigated whether WYYQ could reduce autophagy-related gene or protein expression through regulation of the mTOR pathway. The results showed that rapamycin, an autophagy activator, increased the mRNA levels of Beclin-1 and LC3, which were significantly reduced by treatment with 1.6 mg/mL WYYQ (►Fig. 5A and B). Similarly, WYYQ at 1.6 mg/mL decreased the expression of Beclin-1 and the ratio of LC3-II/LC3-I at the protein level (►Fig. 5C). WYYQ also increased the expression of p62, which was decreased in H9c2 cells treated with rapamycin and OGD. Moreover, OGD resulted in a significant decrease in the phosphorylation of mTOR, which was abrogated by WYYQ (►Fig. 5C). Rapamycin also decreased mTOR phosphorylation; however, WYYQ did not reverse the decrease in phosphorylation in H9c2 cells treated with rapa-mycin, indicating that WYYQ mediated its inhibitory effects on autophagy through the mTOR pathway (►Fig. 5C).

Discussion
This study employed an OGD-induced cell model, which is an accepted model of autophagy. OGD exposure significantly induced autophagy and apoptosis in H9c2 cells. Treatment with WYYQ inhibited the increase in Beclin-1 and LC3, reduced the LC3-II/LC3-I ratio, decreased autophagosome formation, and enhanced p62 abundance through activating the mTOR pathway. For the first time, these results showed the inhibitory effects of WYYQ on cardiomyocyte autophagy under OGD exposure, thus indicating that WYYQ exerted protective effects against HF, typically accompanied by cardiomyocyte damage driven by hypoxic-ischemic stress. Autophagy has evolved as a process in which the body degrades and absorbs its own components, including longlived proteins and damaged cellular components. Autophagy is disturbed in pathological conditions. HF is characterized by cardiac remodeling, which includes the accumulation of compensatory structural and functional alterations to alleviate ventricular wall stress. 26 Autophagy occurs in hypoxic cells 27 and organs during cardiovascular diseases, such as HF, 28 suggesting its stimulation under ischemic conditions. The data of the present study showed that autophagy are activated following OGD exposure in H9c2 cells. WYYX played a positive role in cell protection through the downregulation of autophagic activity. A previous study reported that excessive autophagic activity triggers the release of apoptotic-related factors, resulting in apoptotic responses, 29 in which Beclin-1 and Bcl-2 play a role in the crosstalk between autophagy and apoptosis. The present study found enhanced apoptotic rates under OGD conditions, which was consistent with these studies. WYYQ also lowered apoptotic rates upon the comparison of the effects under OGD conditions. This suggested that WYYQ manipulated the cellular metabolic demands by regulating both the autophagic and apoptotic activity induced by nutrient deficiency. mTOR plays a central role in diverse cellular processes, including cell growth and metabolism. 30 mTOR is actively associated with physiological and pathological responses that occur during cardiovascular diseases, such as HF, 30 with its downregulation triggering the pathological development of cardiomyopathy with HF. 31 The present study showed that mTOR activity was downregulated, while autophagy was upregulated following OGD exposure in H9c2 cells. Furthermore, the impaired activity of mTOR and the enhanced autophagy induced by OGD was reversed by WYYQ. WYYQ had an inhibitory effect on OGD-induced autophagy, suggesting that it played a role in the protection of cardiomyocytes against pathological lesions because of starvation or hypoxia. A challenging view indicated that mTOR activation showed diverse dysfunctional effects, including exasperated pathological hypertrophy or misfolded protein accumulation 32 ; however, the inhibition of mTOR activity, particularly the selective inhibition of mTORC1, in addition to the enhancement of autophagy, resulted in reduced cardiovascular damage in response to pressure overload or chronic ischemic injury. 33,34 Autophagy plays a dual role in impaired cardiac tissue, namely, autophagy is cardioprotective, but its attenuation or over-stimulation is harmful to cardiomyocytes, depending on the timing and degree of autophagic activity. 35 Therefore, therapeutic strategies focused on manipulating autophagy in HF patients should consider these effects to fine-tune the autophagic process for optimal benefit.
Accumulating evidence has shown that Chinese herbs used for the treatment of cardiovascular diseases function through the adjustment of autophagic responses. Qiliqiangxin has been shown to inhibit myocardial inflammation and cardiomyocyte death and abrogate autophagy in mouse models of pressure overload. 36 Similarly, both Xuefu Zhuyu decoction and modified Yi Qi decoction showed beneficial effects on cardiac function by lowering the expression of the autophagic proteins Beclin-1 and LC3. 37,38 In contrast, the traditional Chinese medicine Tongxinluo promoted autophagy through the activation of the mitogen-activated protein kinase/ERK, thus showing a protective role in cardiac microvascular endothelial cells during ischemic injury. 39 Orientin extracted from Polygonum orientale L protected myocardial cells against hypoxic injury through the induction of autophagy. 40 It remains a matter of debate whether the fine-tuning of autophagic responses regulated by Chinese herbs is beneficial. This study demonstrated that WYYQ protected against cell injury and inhibited OGD-induced autophagy enhancement through the activation of mTOR. Future studies will be required to accurately investigate the autophagic activity and mechanism(s) underlying the function of WYYQ on cardiomyocyte autophagy during HF progression.
Various ingredients of WYYQ have been shown to protect cardiomyocytes in vivo. Cinnamaldehyde, extracted from Lauraceae, can reduce the high glucose-induced oxidative damage of cardiomyocytes through the TRPA1/Nrf2 pathway in a db/db diabetic mouse model and inhibit the production of ROS and autophagy through the TLR4-NOX4 pathway to ameliorate LPS-induced cardiac dysfunction in rats. 41 Astragaloside IV, obtained from Huangqi (Astragali Radix), attenuated the ischemia-reperfusion injury of rat myocardial cells via energy regulation mechanism or CaSR/ERK1/2 and related apoptosis signaling pathways. 42 It has also been reported that Astragalus polysaccharide regulates oxidative stress through the MAPK pathway or down-regulated the expression ATF6-and PERK-related factors during ER stress, thus reducing cardiomyocyte apoptosis during diabetic cardiomyopathy. 43,44 Ginsenoside Rb3, an active component of Renshen (Panax Ginseng), regulates the energy metabolism of cardiomyocytes by activating the PPARα pathway in a mouse HF model to reduce apoptosis. 45 The present study (C) levels of p-mTOR, mTOR, LC3-II/LC3-I, Beclin-1, and p62 assessed through Western blot analysis. Data are the mean AE SD. n ¼ 6. ## p < 0.01 versus control, ÃÃ p < 0.01 versus OGD, and && p < 0.01 versus OGD and rapamycin. mTOR, mammalian targets of rapamycin; OGD, oxygenglucose deprivation; qPCR, quantitative polymerase chain reaction; SD, standard deviation; WYYQ, Wenyang-Yiqi Granule.
investigated the antiautophagy effects of WYYQ on cardiomyocytes in vitro. Interactions of WYYQ with the myocardium in vivo warrant further investigations in future studies.

Conclusions
In summary, this study showed that WYYQ exerts an inhibitory effect on OGD-induced autophagy. WYYQ significantly decreased the expressions of Beclin-1 and LC3 but increased the abundance of p62 following OGD exposure, in addition to the formation of autophagosomes, which were associated with the activation of the mTOR pathway. Pharmacological manipulation of autophagy represents a potential therapeutic intervention for the treatment of cardiovascular diseases.
CRediT Authorship Contribution Statement L.H. and H.W. were responsible for conceptualization, methodology, and formal analysis. S.G., X.Y., and H.W. were responsible for funding acquisition, writing original draft, and writing-review and editing.

Funding
This work was funded by the National Natural Science Foundation of China (81373610, 81673800).

Conflict of Interest
The authors declare no conflict of interest.