Astragaloside IV protects cardiomyocytes from hypoxia-induced injury by down-regulation of lncRNA GAS5

Biomedicine & Pharmacotherapy 116 (2019) 109028

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Astragaloside IV protects cardiomyocytes from hypoxia-induced injury by down-regulation of lncRNA GAS5 Jian Du, Jia Liu, Juan Zhen, Si-Tong Yang, En-Lai Zheng, Ji-Yan Leng

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Department of Cadre Ward, The First Hospital of Jilin University, Changchun, Jilin, 130021, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Heart failure Astragaloside IV Hypoxia injury lncRNA GAS5 PI3K/mTOR

Background: Poor understanding of the regulatory mechanisms of astragaloside IV (AS-IV) in cardiovascular protection limits clinical application of AS-IV in heart failure. Hypoxia is an important stimulus in the progression of heart failure. We investigated the role of AS-IV in hypoxia-treated cardiomyoblast H9c2 cells. Methods: Cell viability and apoptotic cells in hypoxia-treated H9c2 cells were detected by CCK-8 assay and flow cytometry, respectively. Expression of proteins associated with proliferation and apoptosis was measured by Western blot. Then effects of AS-IV on hypoxia-induced cell injury were explored, and the alteration of lncRNA growth arrest specific 5 (GAS5) level under AS-IV treatment was determined by RT-qPCR. Whether AS-IV affected hypoxia-treated H9c2 cells via lncRNA GAS5 was subsequently testified. Besides, whether AS-IV regulated lncRNA GAS5 expression was via modulating the PI3K/mTOR pathway was investigated. Results: Hypoxia-induced decreasing cell viability, increasing apoptotic cells, and proteins associated with proliferation and apoptosis were all attenuated by AS-IV treatments. Then, we found that lncRNA GAS5 expression was down-regulated by AS-IV treatment, and AS-IV might affect hypoxia-stimulated H9c2 cells through lncRNA GAS5. Finally, we found that inhibition of PI3K/mTOR or mTOR could reverse the AS-IV-induced downregulation of lncRNA GAS5 in H9c2 cells. Conclusion: AS-IV protected H9c2 cells against hypoxia through down-regulating lncRNA GAS5. Besides, AS-IV might repress lncRNA GAS5 expression via activation of the PI3K/mTOR pathway.

1. Introduction Heart failure is a common problem and its incidence rate is still growing. Besides, patients who are suffering from heart failure cannot maintain a cardiac output to meet metabolic requirements and accommodate venous return [1]. It may cause serious consequence, such as disability, repeated hospitalisations, low life quality, and great economic burden for both patients themselves and healthcare system [2]. Accordingly, approximately 26 million people arereported to be affected by heart failure [3]. Although pharmacological and device therapy options have been applied for clinical treatments of heart failure, the morbidity and mortality are still far less than satisfaction [4]. Potential therapeutic drugs for heart failure as well as the underlying mechanisms are of great importance for improving the survival of patients with heart failure. Astragaloside IV (AS-IV) is a major active constituent of Astragalus membranaceus, and it has been approved by the Chinese Pharmacopeia 2010 acting as the characteristic marker for quality control purpose [5,6]. AS-IV exerts numerous pharmacological functions, such as, anti-

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inflammation [7], anti-cancer [8], neuroprotection [9], and renal protection [10]. In terms of cardiovascular protection, myocardial ischemia/reperfusion-induced injury in rats can be alleviated by AS-IV via inhibiting cell apoptosis [11]. AS-IV represses isoproterenol-induced cardiac hypertrophy via regulation of energy biosynthesis [12]. What’s more, AS-IV has proved to exert anti-apoptotic effect on hypertrophic cardiomyocytes [13]. However, the regulatory mechanisms of AS-IV in cardiovascular protection are poorly understood, which limits its clinical application in heart failure. Long non-coding RNAs (lncRNAs) which exert crucial functions in multiple biological processes, such as migration, invasion and apoptosis received considerable attention [14,15]. Increasing evidence has proved that lncRNAs function in regulating stimuli-induced cardiomyocytes injury. For example, lncRNA CARL inhibited anoxia-induced injury in cardiomyocytes [15]. Among all these identified lncRNAs, Growth Arrest-Specific 5 (GAS5) was primarily observed from isolated NIH 3T3 mouse fibroblasts and named due to its upregulated in rapamycin induced cell cycle arrest [16]. Previous study demonstrated that lncRNA GAS5 was downregulated in cardiac fibrosis tissues as well as

Corresponding author at: Department of Cadre Ward, The First Hospital of Jilin University, No.71, Xinmin Street, Changchun, Jilin, 130021, China. E-mail address: [email protected] (J.-Y. Leng).

https://doi.org/10.1016/j.biopha.2019.109028 Received 2 February 2019; Received in revised form 21 May 2019; Accepted 21 May 2019 0753-3322/ © 2019 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

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activated cardiac fibroblast and overexpression of GAS5 inhibited the proliferation of cardiac fibroblast [17]. Therefore, we inferred that GAS5 might also be involved in the progress of AS-IV in hypoxia-treated cardiomyoblast H9c2 cells. Heart failure is characterized by dysregulated pumping and/or filling functions of heart, which impairs the delivery of oxygen to the body [18]. Moreover, cardiovascular diseases, myocardial infarction in particular, may cause cardiac hypertrophy and dilatation, and ultimately lead to heart failure [19]. Myocardial infarction, the major cause of heart failure, denotes the death of cardiomyocytes due to hypoxia resulting from extended ischemia [20,21]. Therefore, hypoxia is an important stimulus during progression of heart failure. H9c2 cells are derived from embryonic rat hearts and retain many cardiomyocyte phenotypes [22]. Large numbers of experiments exploring the regulatory mechanism of heart failure were performed in H9c2 cells [23,24]. Hence, in our study, we constructed in vitro hypoxia cell model using H9c2 cells to mimic the pathological process of heart failure. Then, the effects of AS-IV on hypoxia-treated H9c2 cells were verified according to the alteration of proliferation and apoptosis. Afterwards, the possible downstream targets of AS-IV as well as the associated signaling cascades were explored.

2.4. Cell apoptosis assay Apoptotic cells were identified after staining with propidium iodide (PI) and fluorescein isothiocyanate (FITC)-conjugated Annexin V. In brief, H9c2 cells were harvested and washed by phosphate buffered saline (PBS) after treatments. Then, cells were suspended in binding buffer, followed by staining in FITC-Annexin V and PI from the FITC Annexin V/Dead Cell Apoptosis Kit with FITC annexin V and PI, for Flow Cytometry (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer’s instructions. Flow cytometry analysis of stained cells was done by using a FACS can (Beckman Coulter, Fullerton, CA, USA). Data were analyzed by using FlowJo software (Tree Star, San Carlos, CA, USA). 2.5. LDH assay H9c2 cells were collected and cells were cultured for 24 h and the supernatant were obtained. Then 50 μl supernatant were transferred into 96-well tissue culture plate before adding to each well 100 μl of the Cytotoxicity Detection Kit LDH solution (Roche Diagnostics, France). After 15 min of incubation at room temperature in the darkness, the reaction was stopped by adding 50 μl stop solution. The supernatant was then diluted twofold with sterile water and its OD was measured at 490 nm. This 15-min incubation period and the twofold dilution step were established after multiple tests in various conditions.

2. Materials and methods 2.1. Cell culture and treatments

2.6. RT-qPCR H9c2 cell line was obtained from American Type Culture Collection (ATCC; ATCC® CRL-1446™, Manassas, VA, USA). H9c2 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% (v/ v) fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Inc.), 100 U/mL penicillin and 100 μg/mL streptomycin at 37 °C. Cells under normoxia were grown in an atmosphere consisting of 95% air and 5% CO2. Cells under hypoxia were incubated in a hypoxic incubator filled with an atmosphere consisting of 94% N2, 5% CO2, and 1% O2. For stimulation of AS-IV (Sigma-Aldrich, St. Louis, MO, USA), cells were incubated in DMEM containing 5–40 μg/mL AS-IV for 24 h prior to hypoxia treatments. For inhibition of PI3K and mTOR, cells were incubated in DMEM containing LY294002 (PI3K inhibitor, 50 μM, SigmaAldrich) or rapamycin (mTOR inhibitor, 50 nM, Sigma-Aldrich) for 0–24 h prior to AS-IV stimulation.

After treatments, total RNA of H9c2 cells was extracted using Trizol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) as suggested by the manufacturer. The One Step SYBR® PrimeScript™ PLUS RT-RNA PCR Kit (TaKaRa Biotechnology, Dalian, China) was employed to measure the expression levels of lncRNA GAS5 following the manufacturer’s protocol. Relative expression fold of lncRNA GAS5 was calculated on the basis of the 2−ΔΔCt method [25], and GAPDH was acted as the internal control. 2.7. Western blot analysis After treatments, H9c2 cells were harvested and lysed in RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China) containing PMSF (1 mM, Beyotime Biotechnology). Proteins in the supernatant were quantified using the Enhanced BCA Protein Assay Kit (Beyotime Biotechnology) and separated by SDS-PAGE (50 μg/lane). Afterwards, proteins in the gels were transferred to polyvinylidene difluoride (PVDF) membranes, followed by blocking in 5% bovine serum albumin in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 h at room temperature. Subsequently, PVDF membranes were incubated with appropriate primary antibodies for p53 (ab131442), p21 (ab109199), cyclinD1 (ab134175), cyclin-dependent kinase 4 (CDK4; ab199728), B cell lymphoma-2 (Bcl-2; ab196495), Bcl-2-associated X protein (Bax; ab182733), pro caspase-3 (ab90437), cleaved caspase-3 (ab49822), phosphatidylinositol-3-kinase (PI3K; ab133595), phospho (p)-PI3K (ab182651), β-actin (ab8229, all Abcam, Cambridge, UK), mechanistic target of rapamycin (mTOR; #5536), or p-mTOR (#2983, both Cell Signaling Technology, Beverly, MA, USA) at 4 °C overnight. Immune complexes were detected with a goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (ab205718, Abcam) and ECL Western blotting detection reagent (GE Healthcare, Braunschweig, Germany). The intensity of the bands was quantified by ImageJ software (National Institutes of Health, Bethesda, MA, USA).

2.2. Stable transfection LncRNA GAS5 sequence was ligated into the pcDNA3.1 plasmid (GenePharma, Shanghai, China), and the recombined plasmid was referred to as pc-GAS5. When cells were reached 70% confluence, pcDNA3.1 or pc-GAS5 was transfectedusing Lipofectamine 3000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer’s instructions. The stably transfected cells were selected after incubation in culture medium containing 0.5 mg/mL G418 (Sigma-Aldrich) for approximately 4 weeks.

2.3. Cell viability assay Cell viability was measured by using a Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Kumamoto, Japan). In brief, H9c2 cells (5 × 103 cells per well) was plated into 96-well plates and incubated overnight to allow for cell attachment. After treatments, 10 μL of CCK-8 solution was added into each well, and the cells were incubated for 1 h at 37 °C. Relative cell viability normalized to the nontreated cells was analyzed on the basis of the absorbance at 450 nm using a Microplate Reader (Bio-Rad, Hercules, CA, USA).

2.8. Statistical analysis Experiments were performed in triplicate with three repeats. The results were shown as the mean ± standard deviation (SD). Statistical 2

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Fig. 1. Hypoxia induced H9c2 cell injury. H9c2 cells were exposed to hypoxia for 3, 6, 12 and 24 h, and cells under normoxia were acted as control. (A) Cell viability was detected by CCK-8 assay. H9c2 cells were incubated under normoxia or hypoxia for 12 h, and cells under normoxia were acted as control. (B–C) Expression of proteins associated with proliferation was measured by Western blot analysis. (D) Percentage of apoptotic cells was analyzed by flow cytometry assay. (E) Expression of proteins related to apoptosis was assessed by Western blot analysis. (F) LDH was detected by Cytotoxicity Detection Kit. Data are shown as the mean ± SD of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

3.2. AS-IV attenuated hypoxia-induced H9c2 cell injury

analysis was performed using Graphpad Prism 5 software (GraphPad, San Diego, CA, USA). The P-values were calculated using the one-way analysis of variance (ANOVA) or unpaired two-tailed t-test. A P value of less than 0.05 was considered as a significant difference.

Cells were stimulated with increasing doses of AS-IV for 24 h, and cell viability was determined. As shown in Fig. 2A, cell viability stayed unchanged, indicating that there was neither pro-proliferative effect nor toxicity on H9c2 cells when the concentration of AS-IV was 0–40 μg/mL. Then, cells were exposed to hypoxia with or without AS-IV pretreatments (10, 20 and 40 μg/mL). CCK-8 assay presented that hypoxia-induced decrease of cell viability was significantly mitigated by AS-IV pretreatments, as evidenced by increased cell viability induced by 10–40 μg/mL AS-IV (all P < 0.05, Fig. 2B). Since there was no statistically significant difference between the hypoxia +20 μg/mL ASIV group and the hypoxia +40 μg/mL AS-IV group (P > 0.05), the concentration of AS-IV for subsequent experiments was 20 μg/mL. Same as what we obtained from Fig. 1C, western blot results showed that hypoxia-induced alteration of p53, p21, cyclinD1 and CDK4, while these results weresignificantly reversed by AS-IV pretreatment (P < 0.05, Fig. 2C). Similarly, hypoxia-induced increase of apoptotic cells (Fig. 1D), as well as alteration of proteins associated with apoptosis (Fig. 1E), was markedly reversed by AS-IV pretreatment (P < 0.05, Fig. 2D–E). Importantly, we detected the LDH release to determine the death level of H9c2 cells and results presented that with the administration of AS-IV, LDH releasing level was inhibited statistically to some extent which suggested that AS-IV had the ability to reduce cell death. Results collectively illustrated that AS-IV pretreatment could attenuate hypoxia-induced cell injury in H9c2 cells.

3. Results 3.1. Hypoxia induced H9c2 cell injury After hypoxia treatments, proliferation and apoptosis of H9c2 cells were measured. Compared with non-treated cells, cell viability was significantly decreased when the hypoxia treatments lasted for 6 h (P < 0.01), 12 h and 24 h (P < 0.001, Fig. 1A). As the cell viability was declined to approximately 50% when the duration of hypoxia treatment was 6 h and cell viability further decreased around 60% when hypoxia treatment time is 12 h, therefore, cells were exposed to hypoxia for 12 h in subsequent experiments. Meanwhile, expression levels of proteins associated with proliferation including p53 and p21 were notably elevated by hypoxia (P < 0.05, Fig. 1C), whereas expression levels of cyclinD1 and CDK4 were observably declined by hypoxia (P < 0.05), as relative to the control group (Fig. 1B–C). Results of flow cytometry assay showed percentage of apoptotic cells in cells exposed to hypoxia was dramatically higher than that in nontreated cells (P < 0.001, Fig. 1D). Likewise, hypoxia treatments upregulated expression of pro-apoptotic Bax and cleaved caspase-3 while down-regulated expression of anti-apoptotic Bcl-2 (Fig. 1E). On the other side, LDH assay was performed to detect the levels of cell death after cells were treated with hypoxia. It can be obviously seen from Fig. 1F that hypoxia administration significantly promoted the LDH release (P < 0.01) which indicated that hypoxia stimulated cells induced higher death level in cells. Those results indicated that in vitro hypoxia cell model was constructed successfully.

3.3. AS-IV down-regulated lncRNA GAS5 expression in H9c2 cells The expression of lncRNA GAS5 after AS-IV treatment in H9c2 cells was evaluated. In Fig. 3, we found AS-IV treatment (20 or 40 μg/mL) could prominently down-regulate lncRNA GAS5 expression (both P < 0.01), suggesting the possible involvements of lncRNA GAS5 in 3

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Fig. 2. Astragaloside IV (AS-IV) attenuated hypoxia-induced H9c2 cell injury. H9c2 cells were stimulated with 5, 10, 20 or 40 μg/mL, and non-treated cells were acted as control. (A) Cell viability was detected by CCK-8 assay. H9c2 cells were treated with 0, 10, 20 or 40 μg/mL AS-IV for 24 h, followed by hypoxia for 12 h. Nontreated cells were acted as control. (B) Cell viability was testified by CCK-8 assay. H9c2 cells were treated with 0 or 20 μg/mL AS-IV for 24 h, followed by hypoxia for 12 h. Non-treated cells were acted as control. (C) Expression of proteins associated with proliferation was measured by Western blot analysis. (D) Percentage of apoptotic cells was analyzed by flow cytometry assay. (E) Expression of proteins related to apoptosis was assessed by Western blot analysis. (F) LDH was detected by Cytotoxicity Detection Kit. Data are shown as the mean ± SD of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001. 4

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regulation loop between mTOR and GAS5. Taken together, results suggested that AS-IV might down-regulate lncRNA GAS5 expression via the PI3K/mTOR pathway in H9c2 cells. 4. Discussion The five-year mortality rate of patients suffering heart failure is 50%, and over one third of deaths due to cardiovascular causes in the United States are attributed to heart failure [26]. Although the cardioprotection activity of AS-IV has been widely reported, the underlying mechanism is far from clear. In our study, we constructed in vitro hypoxia cell model and consolidated that AS-IV could attenuate hypoxiainduced H9c2 cell injury. Afterwards, we reported for the first time that AS-IV affected hypoxia-treated H9c2 cells through down-regulating lncRNA GAS5. Moreover, we also proved that AS-IV might down-regulate lncRNA GAS5 via activation of the PI3K/mTOR pathway. Cell death of cardiomyocytes is an important component of the cardiac remodeling which might cause heart failure [27,28]. Therefore, cell viability and apoptosis are two factors that we focused on to evaluate whether hypoxia cell model was constructed successfully. p53, a tumor suppressor, is stabilized and activated after hypoxia stimulation, resulting in cessation of cell growth and proliferation [29]. p21, a the CDK inhibitor (CKI), is a downstream target of p53 which can induce G1/S boundary cell cycle arrest and senescence [30]. Cyclin D1 is a critical promoter of the cell cycle, and it can activate CDK4, resulting in initiation of DNA replication [31]. Hypoxia treatment in our study decreased cell viability, up-regulated expression of p53 and p21, and down-regulated expression of cyclinD1 and CDK4, indicating that hypoxia repressed proliferation of H9c2 cells. Bax is a pro-apoptotic protein whereas Bcl-2 is an anti-apoptotic protein. When the ratio of Bax/Bcl-2 is increased, the expression of cleaved caspase-3 may be increased, suggesting an elevated cell apoptosis [32]. Besides, LDH releasing level could be treated as one of the standard marker for quantifying cell death [33]. In this study, hypoxia enhanced percentage of apoptotic cells, up-regulated expression of Bax and cleaved caspase-3, and down-regulated expression of Bcl-2. Besides, hypoxia also increased the releasing of LDH. Taken together, results indicated that hypoxia promoted H9c2 cell apoptosis or cell death. Those results suggested that in vitro hypoxia cell model was constructed successfully. Considering the widely reported cardioprotection activity of AS-IV, the effects of AS-IV on hypoxia-treated H9c2 cells were explored. We proved that hypoxia-induced alteration was significantly abrogated by AS-IV, presenting increased proliferation and reduced apoptosis as well as cell death. The protective roles of AS-IV against hypoxia in this study were consistent with literatures reported previously. Zhang et al. have reported that AS-IV attenuates injury of neonatal rat cardiomyocytes induced by hypoxia/reoxygenation [34]. Li et al. have proved that ASIV can promote cardiomyocyte proliferation and act as a cardiac regenerator in mice [35]. A previous study illustrated that AS-IV reduced apoptosis through down-regulation of Bax and cleaved caspase-3 as well as up-regulation of Bcl-2 in rats with acute kidney injury [36]. Moreover, AS-IV was also reported to enhance LDH release in LPS-stimulated H9C2 cells [37], which stands for the similar point with our data. However, the effects of AS-IV on expression of proteins associated with proliferation in hypoxia-treated H9c2 cells were contrary to that in cancer cells [38], which needs more investigations in the future. LncRNAs are newly discovered RNAs longer than 200 nucleotides, and they perform various functions in numerous biological processes [39]. Li and colleagues have found that AS-IV might affect cells via regulation of lncRNA. They proved that migration and viability of hepatocellular carcinoma were repressed by AS-IV through inhibition of lncRNA ATB [40]. LncRNA GAS5 is originally isolated in 1988 and has been widely reported as a tumor suppressor in various malignancies [41]. Mazar et al. have stated that lncRNA GAS5 regulates proliferation and apoptosis of human neuroblastoma cells through activation of p53 [42]. Down-regulation of lncRNA GAS5 also indicates decreased p21

Fig. 3. Astragaloside IV (AS-IV) down-regulated lncRNA GAS5 expression in H9c2 cells. H9c2 cells were stimulated with 10, 20 or 40 μg/mL, and nontreated cells were acted as control. LncRNA GAS5 expression was determined by RT-qPCR. Data are shown as the mean ± SD of three independent experiments. **, P < 0.01.

modulation of AS-IV. 3.4. AS-IV alleviated hypoxia-induced H9c2 cell injury through downregulating lncRNA GAS5 In Fig. 4A, expression level of lncRNA GAS5 in cells transfected with pc-GAS5 was markedly higher than that in cells transfected with pcDNA3.1 (P < 0.01), indicating that lncRNA GAS5 was overexpressed successfully after cell transfection. Then, transfected or untransfected cells were exposed to hypoxia with or without AS-IV pretreatments. Effects of AS-IV pretreatment on hypoxia-treated cells were significantly abrogated by lncRNA GAS5 overexpression, showing decreased cell viability (P < 0.05, Fig. 4B), up-regulated expression of p53 and p21 (P < 0.05, Fig. 4C), down-regulated expression of cyclinD1 and CDK4 (P < 0.05, Fig. 4C), and increased apoptosis (P < 0.01, Fig. 4D–E). As we have concerned in the former experiments, LDH release levels were examined. It can be shown in the Fig. 4F that transfection with pc-GAS5 enhanced the releasing of LDH which indicated that overexpression of GAS5 promoted cell death in a significant level (P < 0.05). Results implied that AS-IV might attenuate hypoxia-induced H9c2 cell injury through down-regulating lncRNA GAS5. 3.5. AS-IV down-regulated lncRNA GAS5 via the PI3K/mTOR pathway in H9c2 cells The involvements of the PI3K/mTOR pathway in AS-IV-associated modulation were finally explored. Firstly, PI3K/mTOR pathway under different treatment including hypoxia, AS-IV + hypoxia and transfection with pc-GAS5 was determined. Obviously, the phosphorylation of PI3K and mTOR was enhanced expressed by hypoxia and which were then weakened by AS-IV administration. Furthermore, transfection with pc-GAS5 enhanced the phosphorylation of PI3K and mTOR (Fig. 5A). Under the hypoxia treatments, after incubation with PI3K inhibitor LY294002, phosphorylation levels of PI3K and mTOR were decreased with time gradually (Fig. 5B). Under the hypoxia treatments, after incubation with mTOR inhibitor Rapamycin, phosphorylation level of PI3K stayed unchangeable, whereas phosphorylation level of mTOR was decreased with time gradually (Fig. 5C). RT-qPCR results showed the down-regulation of lncRNA GAS5 expression induced by AS-IV was significantly bated by LY294002 pretreatment (P < 0.01, Fig. 5D) or Rapamycin pretreatment (P < 0.01, Fig. 5E). On the opposite, we detected whether lncRNA GAS5 affected mTOR expression. Interestingly, we found that lncRNA GAS5 overexpression increased the phosphorylation of mTOR, which indicated that lncRNA GAS5 activated mTOR pathway (Fig. 5F), which revealed opposite trend with mTOR to lncRNA GAS5, which suggested that there was negative 5

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Fig. 4. Overexpression of lncRNA GAS5 abrogated the effects of astragaloside IV (AS-IV) on hypoxia-induced H9c2 cells. H9c2 cells were transfected with pcDNA3.1 or pc-GAS5, and non-treated cells were acted as control. (A) Expression of lncRNA GAS5 was determined by RT-qPCR. Transfected or untransfected H9c2 cells were treated with 0 or 20 μg/mL AS-IV for 24 h, followed by hypoxia for 12 h. Non-treated cells were acted as control. (B) Cell viability was testified by CCK-8 assay. (C) Expression of proteins associated with proliferation was measured by Western blot analysis. (D) Percentage of apoptotic cells was analyzed by flow cytometry assay. (E) Expression of proteins related to apoptosis was assessed by Western blot analysis. (F) LDH was detected by Cytotoxicity Detection Kit. Data are shown as the mean ± SD of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

lncGAS5 might exert important roles in cell cycle gene expression and through which to regulating cell cycle. The regulatory mechanism of AS-IV on lncRNA GAS5 expression was preliminarily studied. mTOR was involved in a wide spectrum of biological activities, such as cell growth, differentiation, and metabolism [47]. A previous study has identified that mTOR inhibition could up-regulate lncRNA GAS5 expression in prostate cancer cells [48]. Dual PI3K/mTOR inhibition was proved to enhance lncRNA GAS5 expression level in all cell types in another literature [49]. Therefore, whether ASIV regulated lncRNA GAS5 expression via activation of the PI3K/mTOR pathway was finally investigated. Results in this study proved that ASIV-induced down-regulation of lncRNA GAS5 expression could be notably reversed by inhibition of PI3K and mTOR or inhibition of mTOR, indicating that AS-IV might down-regulate lncRNA GAS5 expression via the PI3K/mTOR pathway. On the opposite, previous study also proved that lncRNA GAS5 achieved its functions in regulating cell biological activities in a mTOR-dependent manner [50], which suggested that there was interactions between lncRNA GAS5 and mTOR pathway. Our results revealed that lncRNA GAS5 increased the phosphorylation of mTOR, which indicated that GAS5 could activate mTOR pathway. Besides, the regulation between lncRNA GAS5 and mTOR belongs to negative regulation loop. The mechanism behind why lncRNA GAS5 could

expression in stomach cancer cells [43]. Moreover, decreased ratio of Bax/Bcl-2 was observed in LPS-induced ATDC5 cells overexpressing lncRNA GAS5 [44]. Considering the effects of AS-IV on expression levels of p53, p21, Bax and Bcl-2, we hypothesized that there might be a correlation between AS-IV and lncRNA GAS5. In the present study, lncRNA GAS5 expression was down-regulated in H9c2 cells under AS-IV stimulation. More experiments reflected that effects of AS-IV on hypoxia-treated H9c2 cells could be bated by lncRNA GAS5 overexpression, indicating that AS-IV might function through down-regulation of lncRNA GAS5. Overexpression of GAS5 significantly decreased cell viability as along with downregulation of CyclinD1, CDK4 and upregulation of p21 and p53. It is well-known that the mammalian cell cycle is controlled by CDKs and their corresponding pathways [45]. CDKs are activated via binding to their selected cyclins in specific phases of the cell cycle, through which they phosphorylate their target proteins [31]. On the other side, the CKIs negatively regulate the activities of CDKs and control the cell cycle [30]. The p53 pathway plays a role in DNA damage response as a gatekeeper of the genome. Several lncRNAs control the expression of cyclins-CDKs, CKIs, and p53, and participate in cell cycle regulation. Some of these lncRNAs are induced by DNA damage and inhibit cell cycle progression by regulating these cell cycle regulators [46]. Therefore, we inferred that 6

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Fig. 5. Astragaloside IV (AS-IV) down-regulated lncRNA GAS5 via the PI3K/mTOR pathway in H9c2 cells. H9c2 cells were treated without inhibitor (A) or stimulated with 50 μM PI3K inhibitor LY294002 (B) or 50 nM mTOR inhibitor Rapamycin (C) for 0 h, 6 h, 12 h and 24 h. Expression of key kinases in the PI3K/mTOR pathway was tested by Western blot analysis. H9c2 cells were stimulated with LY294002 (D) or Rapamycin (E) for 24 h, followed by treatment with AS-IV for 24 h. H9c2 cells were transfected with pcDNA3.1 or pc-GAS5, and non-treated cells were acted as control. (F) The expression of phosphorylation of mTOR was detected by western blot. Non-treated cells were acted as control. Expression of lncRNA GAS5 was determined by RT-qPCR. Data are shown as the mean ± SD of three independent experiments. **, P < 0.01.

alter the phosphorylation of mTOR has not been clearly illuminated. Former studies pointed out that lncRNA GAS5 could modulated mTOR signal pathway through targeting miR-103 in prostate cancer cells [51]. This provided a potential explanation about the roles of lncRNA on the phosphorylation of mTOR. There might be AS-IV reduced injury through activation of mTOR which inhibited the expression of lncRNA GAS5. In conclusion, we verified that AS-IV could protect H9c2 cells against hypoxia injury. Then, we reported for the first time that AS-IV affected hypoxia-exposed H9c2 cells through down-regulating lncRNA GAS5 expression. Moreover, we also proved that lncRNA GAS5 might down-regulate lncRNA GAS5 expression through activation of the PI3K/mTOR pathway in H9c2 cells. This study enriched the regulatory mechanism of AS-IV in hypoxia-induced cardiomyocyte injury, and provided innovative therapeutic strategies for treatments of heart failure.

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Funding This research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. Conflict of interest The authors declare no conflict of interest. Acknowledgements None. References [1] C.D. Kemp, J.V. Conte, The pathophysiology of heart failure, Cardiovasc. Pathol. 21 (5) (2012) 365–371. [2] S.C. Inglis, R.A. Clark, R. Dierckx, D. Prieto-Merino, J.G. Cleland, Structured telephone support or non-invasive telemonitoring for patients with heart failure,

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