Quercetin attenuates cardiomyocyte apoptosis via inhibition of JNK and p38 mitogen-activated protein kinase signaling pathways

    Quercetin attenuates cardiomyocyte apoptosis via inhibition of JNK and p38 mitogen-activated protein kinase signaling pathways Chengqiu Li, Ting Wang, Chunyuan Zhang, Jichang Xuan, Changjiang Su, Yuqi Wang PII: DOI: Reference:

S0378-1119(15)01483-3 doi: 10.1016/j.gene.2015.12.012 GENE 41043

To appear in:

Gene

Received date: Revised date: Accepted date:

27 September 2015 17 November 2015 4 December 2015

Please cite this article as: Li, Chengqiu, Wang, Ting, Zhang, Chunyuan, Xuan, Jichang, Su, Changjiang, Wang, Yuqi, Quercetin attenuates cardiomyocyte apoptosis via inhibition of JNK and p38 mitogen-activated protein kinase signaling pathways, Gene (2015), doi: 10.1016/j.gene.2015.12.012

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ACCEPTED MANUSCRIPT Quercetin attenuates cardiomyocyte apoptosis via inhibition of JNK and p38

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mitogen-activated protein kinase signaling pathways

Authors

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Chengqiu Li 1,a*, Ting Wang 1,a, Chunyuan Zhang a, Jichang Xuan a, Changjiang Su a, Yuqi Wang a

a

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Affiliations

Department of Cardiovascular Medicine, The Zoucheng People's Hospital, Jining,

These authors contributed equally to this work.

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Shandong, 273500, PR China

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*Corresponding author at: Department of Cardiovascular Medicine, The Zoucheng

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People's Hospital, 59 Qianquan Road, Jining, Shandong, 273500, P.R. China. Tel.: +86 537 5250811; fax: 86 537 5250811.

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E-mail address: [email protected] (C. Li) The authors declare no competing financial interests. Author contributions: C.L. designed the study. T.W., C.Z., J.X., C.S. and Y.W. performed the experiments and collected the data. C.L. and T.W. analyzed and interpreted the experimental data. C.L. and T.W. prepared the manuscript.

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ACCEPTED MANUSCRIPT ABSTRACT Quercetin (Que), a plant-derived flavonoid, possesses various biological functions.

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Moreover, Que exerts multiple benefical actions in treatment of cardiovascular

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diseases and there are an inverse association between Que intakes and occurrence and development of various cardiovascular diseases. Some researchers have inferred that the mechanisms of Que to protect cardiomyocytes from ischemia/

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reperfusion (I/R) injury may be involved in modulation of intracellular signal pathways

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and regulation of proteins expression in vivo. The current study investigated whether Que has any protective effects on cardiomyocytes from Hypoxia/reoxygenation (H/R)

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in vitro and its potential cardioprotective mechanisms. The cell viability of Que on

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H9c2 cardiomyoblast cells was assessed by MTT. Apoptosis was evaluated by both

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Hoechst33342 staining and Flow cytometric analysis (FACS). Furthermore, the effect of Que, SP600125 (JNK inhibitor) and SB203580 (p38 inhibitor) on mitogen-activated

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protein kinases (MAPKs) and the expression of apoptosis related proteins (Bcl-2, Bax and Caspase-3) was determined by Western blotting. MTT assays showed that pretreated with Que could increase the viability of H9c2 cardiomyocytes suffered H/R. Both Hoechst33342 staining and FACS confirmed Que could remarkably suppress the H/R-induced apoptotic cardiomyocytes. In addition, Que significantly increased Bcl-2 expression, but alleviated H/R-induced the phosphorylation of JNK and p38, which further inhibited the activation of Bax and caspase-3 directly or indirectly. In summary, our results imply that Que can induce cardioprotection by inhibition of JNK and p38 mitogen-activated protein kinase signaling pathways and modulating the 2

ACCEPTED MANUSCRIPT expression of Bcl-2 and Bax proteins that provides a new experimental foundation for

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myocardial ischemia disease therapy.

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Key words: Cardiomyocytes; Hypoxia/reoxygenation; Quercetin; JNK; p38

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ACCEPTED MANUSCRIPT 1. INTRODUCTION Ischemic heart disease secondary to acute myocardial infarction is a severe health

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problem in the world, which is a primary cause of morbidity and mortality (Rutschow et

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al., 2006; Golovkin et al., 2014; Liu et al., 2014a; Mardan-Nik et al., 2014). Myocardial cell death and heart failure due to ischemic heart injury are the major cause of mortality around the world (Tang et al., 2013; Liu et al., 2014b; Zaman et al., 2015). In

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the past few decades, extensive studies have been performed to exploring effective

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strategies and drugs to ameliorate or prevent ischemic heart injury(Machado-Silva et al., 2015), such as flavonoids, possess the cardioprotective effects(Ge et al., 2014;

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Liu et al., 2014a). Quercetin (Que, Figure1A) is one of the major flavonoids found

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present in various foods including apples, berries, Brassica vegetables, capers,

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grapes, onions, shallots, tea, and tomatoes, as well as red wine(Bartekova et al., 2015). Research evidences suggested that Que is nontoxic and possesses a broad of

pharmacological

and

biological

functions

including

anti-oxidative,

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range

anti-inflammatory, anti-coagulation, and oxygen radical-scavenging activities(Erden Inal and Kahraman, 2000; Yu et al., 2013; Wu et al., 2014). Several studies indicated that Que protects the myocardium from ischemic heart injury when given before ischemia (Jin et al., 2012). Recently, in another study, a significant reduction of the myocardial infarct size in both normal and diabetic animals by Que has been reported (Annapurna et

al.,

2009). However,

the molecular mechanism

Que-mediated cardioprotection is still not completely elucidated.

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underlying

ACCEPTED MANUSCRIPT Apoptosis after myocardial ischemia is one of the major pathways that lead to the process of cell death (Ding et al., 2013). It has been extensively demonstrated that the

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mitogen-activated protein kinase (MAPK) family plays an important role in induction of

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myocardial apoptosis during reperfusion. There are three main subtypes of MAPK family: death kinases[c-jun amino-terminal kinases (JNK1 and JNK2)/stress-activated protein kinase (SAPK) and the p38 (α and β) MAPKs] as well as survival kinase [(the

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extracellular signal-regulated kinases (ERK1 and ERK2)](Marczin et al., 2003). It is

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common knowledge that p38 and JNK MAPKs are pro-apoptotic whereas ERKs are the modulators of cell survival after reperfusion(Clanachan et al., 2003), even though

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there are still conflicting reports on the role of MAPKs in death or survival after

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stress(Hahn et al., 1992). Clinical evidences demonstrated that H/R-induced

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apoptotic myocardial cells death has been associated with an increased expression in p38 and JNK MAPKs(Gao et al., 2002; Yokota and Wang, 2015). Therefore,

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regulating the activity of MAPK signaling pathway, particularly the activation of JNK and P38, is vital for protecting myocardial cells after H/R injury.

Although it has been previously demonstrated that Que reduces myocardial and cardiomyocyte necrotic death by activating survival kinases, we do not know whether the protection by Que is mediated by inhibiting signaling pathways via death kinases. Therefore, using H9c2 cardiomyoblast cells in the present study, we hypothesized that reduction of cardiomyocyte apoptosis after H/R by Que is associated with inhibition in expression and activation of death kinases (JNKs and p38) and caspase activation. 5

ACCEPTED MANUSCRIPT 2. Materials and methods 2.1. Materials

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Hoechst 33342 stain, MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium

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bromide] and Que were the products of Sigma (Sigma, St. Louis, USA). The Annexin V/propidium iodide apoptosis detection kit was obtained from Invitrogen Corporation (Eugene, OR, USA). Primary antibodies against Bcl-2, Bax, Caspase-3, JNK, p-JNK,

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p38, p-p38 and β-actin were obtained from Santa Cruz Biotechnology, Inc. (CA, USA).

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JNK inhibitor (SP600125) and p38 inhibitor (SB203580) used in our experiment was

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2.2. Cell culture

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obtained from Abcam (Abcam, Cambridge, UK).

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The H9c2 cardiomyocyte line was obtained from the Chinese Academy of Sciences Cell Bank (Shanghai, China) and cultured as previously described. Briefly, H9c2 cells

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were cultured in high glucose DMEM supplemented with 10 % (v/v) foetal bovine serum, 1 % penicillin/streptomycin (v/v), and 2 mM L-glutamine. The cells were maintained at 37 °C with 100 % relative humidity in a CO2 incubator containing 5 % CO2 at 37 °C.

2.3. Hypoxia/reoxygenation The H/R procedures were modified from a previous study(Sun et al., 2013). High glucose DMEM medium was changed to DMEM with no glucose to mimic ischemia. Then, the H9c2 cardiomyocytes were incubated at 37 °C in an anaerobic glove box 6

ACCEPTED MANUSCRIPT (Coy Laboratory, USA), from which normal air was removed by a vacuum pump and replaced with 5 %CO2, 5 %H2, and 90 % N2. The H9c2 cardiomyocytes were cultured

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under hypoxia for 4 h. Then, the cells were removed from the anaerobic glove box

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and the medium was replaced with high glucose medium and maintained in the regular incubator to mimic reperfusion. For all experiments, cells were plated at an appropriate density according to the experimental design and were grown for 24 h to

2.4. Experimental protocols

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reach 70 to 80 % confluence before experimentation.

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The cultured H9c2 cardiomyocytes were randomly divided into different groups. In the

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control (Con) group, the H9c2 cardiomyocytes were incubated under normoxic

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conditions for equivalent durations with high glucose DMEM. The H/R group was conducted as described in the preceding section. In the Que-treated group (H/R + E),

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the H9c2 cardiomyocytes subjected to 4 hours of hypoxia and 6 h of reoxygenation and pretreated with various concentrations of Que (10, 20, 40, 80, 160μM). QUE was dissolved in glucose-free DMEM medium (Wang et al., 2011).

2.5. Cell viability analysis Cell viability was determined by the MTT assay as previously described (Sun et al., 2012). Briefly, H9c2 cells were plated on 96-well plates at a density of 1 × 104 cells/well. After designated treatment, 20 μL MTT (5 mg/mL) was added to each well and incubated for 4 h. The medium was then removed, and the formazan crystals 7

ACCEPTED MANUSCRIPT were dissolved with dimethyl sulphoxide. Absorbance was read at 570 nm on a

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microplate reader (TECAN Infinite M1000, Austria).

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2.6. Hoechst staining

H9c2 cardiomyocytes were seeded in 24-well culture plates. After designated treatment, cells were stained with Hoechst 33342 at 37°C for 20 minutes in the dark,

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washed with PBS, and observed by fluorescence inverted microscopy (IX73; Olympus,

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Tokyo, Japan).

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2.7. Flow cytometric detection of apoptosis

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The percentages of early apoptosis and necrosis were measured using an Annexin

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V-FITC/PI apoptosis kit for flow cytometry according to the manufacturer’s instructions (Invitrogen). After treatment, the cells were harvested and washed twice with cold

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PBS, and then incubated with 5 μL FITC-Annexin V and 1 μL PI working solution (100 μg/mL) for 15 min in the dark at room temperature. Cellular fluorescence was measured by flow cytometry analysis (FACS CaliburTM, BD Biosciences, CA, USA).

2.8. Western blot analysis Cell lysate preparation and western blot analysis were performed as previously described (Wang et al., 2014a). Proteins were separated on polyacrylamide gels and then

electrotransferred

onto

a

nitrocellulose

membrane

(Amersham,

Buckinghamshire, United Kingdom). The membranes were blocked for 3 h in 8

ACCEPTED MANUSCRIPT Tris-buffered saline with 0.1% Tween 20 (TBST) and 3% bovine serum albumin (BSA) and then incubated overnight at 4 °C with primary antibodies in TBST containing 1% membranes

were

then

washed

and

incubated

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The

with

alkaline

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BSA.

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phosphatase-conjugated secondary antibodies in TBST for 2 h and developed with NBT/BCIP color substrate (Promega, Madison, WI). The densities of the bands on the membrane were scanned and analyzed using an image analyzer (LabWorks Software,

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Upland, CA).

2.9. Statistical evaluation

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Values were expressed as the means ± SD of at least nine independent experiments

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in duplicate. Statistical analysis of the results was carried out by one-way ANOVA

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followed by Newman-Keuls test. P-values of P<0.05 were considered significant. All

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statistical analyses were performed by using the SPSS15.0.

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3. Results

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3.1. Que promoted the viability of H9c2 cardiomyocytes exposed to H/R

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MTT assay showed that Que could enhance the viability of H9c2 cardiomyocytes at relatively low concentrations (from10 to 80 μ M) (Fig. 1B); However, higher concentrations of Que(160μM) didn't significantly harm or enhance the viability of

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H9c2 cardiomyocytes. Previous study has proved that exposure of H9c2 cells to 600μ

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M H2O2 led to a decrease in cell viability, we further investigated the effect of Que on H2O2-induced injury in H9c2 cells. Pretreatment with 20, 40 and 80μM Que could

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enhance the viability of H9c2 cardiomyocytes(Fig. 1C). Moreover, the positive effects

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of Que on the viability of H9c2 cardiomyocytes exhibited a dose-dependent manner.

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Compared with the counterpart from the H/R+/Que group, the viability of the Que-treated H9c2 cardiomyocytes was the strongest at the concentration of 40μM;

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Below or beyond the concentration, the viability of these cells gradually weakened (Fig. 1B and C). All in all, the concentration of Que (40μM) determined by MTT assay was used throughout the whole experiment.

3.2. Que protected H9c2 cardiomyocytes from apoptosis induced by H/R As shown in Figure 2, after treatment with Que, morphological characteristics indicative of apoptotic cells were observed in Hoechst 33342-stained H9c2 cardiomyocytes. In control groups, H9c2 cells showed regular and round nuclei as observed under a microscope (Fig.2A,arrows), whereas condensation and 10

ACCEPTED MANUSCRIPT fragmentation of nuclei were evident after cells being treated with H/R, which was the characteristic of apoptotic cells (Fig.2B, arrowheads). However, pretreatment with

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Que significantly decreased H9c2 cardiomyocytes apoptosis (Fig.2C). In addition,

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vehicle had no effect on cell death or cell survival during H/R (Fig.2D and E).

Annexin V-FITC/PI assay supplied further evidence of the protective effect of Que

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against H/R-induced injury (Fig.2F-K). FACS showed a massive apoptosis of H9c2 in

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the H/R group, whereas, Que could rescue these cells by considerably reducing the

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apoptosis rate about 21.4±0.56% (Fig.2L).

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3.3. Que altered the activation of JNK and p38

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In view of the significant impact of Que on H/R injury in cardiomyocytes, we next sought to examine the potential impact of Que on the major MAPK signal transduction

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mechanisms linked to the recruitment of cell death pathways. Compared with control group, significant increases the phosphorylation of JNK and p38 were observed in H/R group (Fig.3A and B; p < 0.05). However, pretreatment with Que significantly decreased the phosphorylation of JNK and p38 induced by H/R (Fig.3A and B; p < 0.05). In addition, pretreatment with SP600125 significantly decreased the phosphorylation of JNK induced by H/R and pretreatment with SB203580 significantly decreased the phosphorylation of JNK p38 induced by H/R (Fig.3A and B; p < 0.05). Those results suggested that Que could remarkably improve the survival of H9c2 cardiomyocytes in the context of H/R induced injury through inhibiting JNK and p38 11

ACCEPTED MANUSCRIPT signaling pathway.

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3.4. Que altered the expressions of bcl-2 and bax

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Fig. 4 illustrates the results of western blotting with the monoclonal anti-Bcl-2 antibody and anti-Bax antibody in cardiomyocytes after H/R injury. Compared with the H/R group, treatment with SP600125, SB203580 or Que reversed the decreased amount

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of Bcl-2 and increased amount of Bax that were detected in H/R group to the incline in

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bcl-2 expression but the decline in Bax expression (Fig.4A and B; p < 0.05). The relative ratio of Bcl-2/Bax protein also was enhanced in SP600125, SB203580 or Que

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groups as compared with the H/R group (Fig.4C; p < 0.05).

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3.5. Que reduced the expression of apoptosis related protein: Caspase-3 Apoptosis after ischemia is one of the major pathways that lead to the process of cell

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death (Zhang et al., 2015). Therefore, the expression of cleaved caspase-3 also was analyzed by western blotting. In this study, we examined the expression changes of caspase-3 after H/R in each group (p < 0.05; Fig. 5A and B). The data shows that H/R significantly induced the protein expression of cleaved caspase-3, whereas treatment with SP600125, SB203580 or Que blocked the effect of H/R on the caspase-3 (p < 0.05; Fig. 5A and B).

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4. Discussion

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The mechanism involved in the cardioprotective action of Que is still open to First,

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discussion (Liu et al., 2014a). There are two major findings in the current study.

we confirmed that Que postconditioning significantly reduced H9c2 cardiomyocyte apoptosis following H/R injury in vitro. Second, we confirmed the cardioprotective

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effects of Que postconditioning through inhibiting JNK and p38 signaling pathway,

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which further abolish the activation of apoptosis related protein directly or indirectly.

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As we known, myocardial ischemia is usually unpredictable clinically(Ramkaran et al.,

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2015). Therefore, exploring effective agents that confer cardioprotection after

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myocardial ischemia provide a promising approach to attenuate myocardial I/R injury (Wang et al., 2013). Many experimental studies have focused on the proposed

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beneficial effects of flavonoids on the cardiovascular system (Bartekova et al., 2010). Que, one kinds of flavonoid, is widely present in the plant kingdom, which is currently used in the treatment of myocardial ischemia diseases and its application has shown positive results. For example, an increased intake of Que has been suggested to reduce risk of cardiovascular diseases(Perez-Vizcaino and Duarte, 2010). Previous studies have also indicated that Que could also decrease infarct size and further improve functional recovery in an acute myocardial I/R model (Brookes et al., 2002; Punithavathi and Prince, 2010). In addition, it has also been shown that Que is powerful in other several I/R models, such as renal, hepatic, or cerebral ischemia, 13

ACCEPTED MANUSCRIPT with strong protective effects in all of them (Inal et al., 2002; Dajas et al., 2003; Su et al., 2003). Our data are consistent with the above studies, and we further

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demonstrated that Que postconditioning exerted remarkable cardioprotective effects

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against cardiomyocyte apoptosis following H/R injury in vitro through inhibiting JNK and p38 signaling pathway, which was associated with a reduction in an imbalance

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between pro-apoptotic Bax and anti-apoptotic Bcl-2.

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Recent studies have showed that mitogen-activated protein (MAP) kinases play a pivotal role in signaling transduction from cell surface to the nucleus and in mediating

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cell survival and cell death(Gu et al., 2001). Previous reports in cultured rat

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cardiomyocytes subjected to a prolonged H/R demonstrated that blockade in

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activation of JNK and p38 with the specific inhibitors was associated with an attenuation of both necrosis and apoptosis (Mackay and Mochly-Rosen, 1999; Kaiser

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et al., 2004). In agreement with these reports, in the present study the phosphorylation levels of JNK and p38 elevated significantly in H/R group. Whereas, concomitant with a reduction in apoptotic cardiomyocytes by Que, activation of JNKs and p38 was significantly inhibited. These data provided an alternative explanation for Que-mediated protection by inhibiting death kinases in addition to activating survival kinases (PI-3K/Akt) as previously reported(Wang et al., 2013).

The intrinsic mitochondrion-dependent pathway, the extrinsic death receptor pathway, and the intrinsic endoplasmic reticulum stress pathway are three different death 14

ACCEPTED MANUSCRIPT signaling pathways leading to apoptosis(Qi et al., 2012). Mitochondria-dependent apoptotic pathway represents the main mechanism of programmed cell death and is

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well known to be involved in H/R-induced cellular damage (Wang et al., 2014b). As

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mitochondrial membrane-associated proteins, the members of the Bcl-2 family play vital roles in inducing anti-apoptotic effects, which regulate apoptosis by modulating mitochondrial membrane permeability(Cao et al., 2015). The anti-apoptotic protein

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Bcl-2 is located in the mitochondrial wall and prevents mitochondrial release of

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cytochrome c, while the pro-apoptotic protein Bax resides in the cytosol but can be translocated to mitochondria to induce cytochrome c release. Therefore, the

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caspase-3 apoptotic pathway could be regulated by the Bcl-2/Bax ratio. The present

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results show that H/R significantly decreased Bcl-2 expression and dramatically

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increased Bax and cleaved-caspase- 3 expression, and treatment with Que partially reversed these changes in protein expression. These results suggest that Que could

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inhibit caspase-3 mediated apoptosis. Therefore, Que may have potential therapeutic effects on hypoxia-related diseases; this protective effect of Que on H/R might be related to changes in the expression of Bcl-2 and Bax.

5. Conclusion In conclusion, our data show that Que did protect H9c2 cardiomyocyte against H/R-induced injury in vitro through attenuating JNK and p38 signaling pathway, which further modulating Bcl-2 and Bax expression directly or indirectly. Our results indicate that administration of Que might be a potential therapeutic strategy for the prevention 15

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Figure Legends

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Figure 1. Effects of Que on H9c2 cells viability. (A) The chemical structure of Que. (B)

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H9c2 cardiomyoblast cells were treated with various concentrations (0–160 μM) of Que after H/R injury. (C) H9c2 cardiomyoblast cells were treated with various

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concentrations (0–160 μM) of Que after H2O2 injury. Cell viabilities were measured

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p<0.05 relative to H/R group.

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by MTT assay. Data were presented as mean ± SD. *p<0.05 relative to control group,

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Figure 2. Effects of Que on H/R-induced cell death and apoptosis in H9c2 cells. (A-E) Cells were treated with Que (40μM) and the nuclei were stained by Hoechst 33342.

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(F-K) cell apoptosis was analyzed using flow cytometry. (L) Statistical analysis of the

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Figure 3. Effects of Que on the phosphorylation of JNK and p38 in H/R treated cardiomyocytes. (A) Immunoblot bands were scanned. The phosphorylation of JNK and p38 were significantly increased by H/R compared with control group, while Que could inhibit phosphorylation of JNK and p38 induced by H/R. (B) The intensity of the bands were expressed as optical density (O.D.) analysis. Data were presented as mean ± SD. *p<0.05 relative to sham group. #p<0.05 relative to H/R.

Figure 4. Effects of Que on the expression of Bcl-2 and Bax in H/R treated 19

ACCEPTED MANUSCRIPT cardiomyocytes. (A) Immunoblot bands were scanned. H/R significantly decreases the relative ratio of Bcl-2/Bax protein level, the protein expression of Bcl-2 but

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increases the expression of Bax protein, while Que can reverse this result. (B) The

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intensity of the bands were expressed as optical density (O.D.) analysis. (C) Que can also reverse the relative ratio of Bcl-2/Bax protein level that is increased by H/R. Data were presented as mean ± SD. *p<0.05 relative to sham group. #p<0.05 relative to

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H/R.

Figure 5. Effects of Que on the protein expression of cleaved caspase-3. (A)

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Immunoblot bands were scanned. H/R significantly increases the protein expression

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of cleaved caspase-3, whereas Que can reverse this result. (B) The intensity of the

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bands were expressed as optical density (O.D.) analysis. Data were presented as

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mean ± SD. *p<0.05 relative to sham group. #p<0.05 relative to H/R.

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ACCEPTED MANUSCRIPT Abbreviations list

Quercetin (Que);

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Ischemia/ reperfusion (I/R);

Flow cytometric analysis (FACS);

Stress-activated protein kinase (SAPK);

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Mitogen-activated protein kinases (MAPKs);

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Hypoxia/reoxygenation (H/R);

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3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT);

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ACCEPTED MANUSCRIPT Research Highlights Que reduces the H9c2 cardiomyocytes apoptosis induced by H/R.

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Que induces cardioprotection by inhibition of JNK and p38 during H/R.

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Que increases Bcl-2 expression and inhibits the activation of Bax and caspase-3

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during H/R.

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