Apoptosis DOI 10.1007/s10495-015-1122-4
ORIGINAL PAPER
Protective effect of berberine against myocardial ischemia reperfusion injury: role of Notch1/Hes1-PTEN/Akt signaling Liming Yu1 • Feijiang Li2 • Guolong Zhao3 • Yang Yang1,2 • Zhenxiao Jin1 • Mengen Zhai1 • Wenjun Yu4 • Lin Zhao1 • Wensheng Chen1 • Weixun Duan1 Shiqiang Yu1
•
Ó Springer Science+Business Media New York 2015
Abstract Berberine (BBR) confers cardioprotective effect against myocardial ischemia reperfusion injury (MI/ RI). Activation of Notch1/Hairy and enhancer of split 1 (Hes1) signaling also reduces MI/RI. We hypothesize that BBR may protect against MI/RI by modulating Notch1/ Hes1-Phosphatase and tensin homolog deleted on chromosome ten (PTEN)/Akt signaling. In this study, male Sprague–Dawley rats were exposed to BBR treatment (200 mg/kg/d) for 2 weeks and then subjected to MI/RI. BBR significantly improved cardiac function recovery and decreased myocardial apoptosis, infarct size, serum creatine kinase and lactate dehydrogenase levels. Furthermore, in cultured H9c2 cardiomyocytes, BBR (50 lmol/L) attenuated simulated ischemia/reperfusion-induced myocardial apoptosis. Both in vivo and in vitro study showed
Liming Yu, Feijiang Li and Guolong Zhao contributed equally to this study. & Weixun Duan [email protected] & Shiqiang Yu [email protected] 1
Department of Cardiovascular Surgery, Xijing Hospital, The Fourth Military Medical University, 127 Changle West Road, Xi’an 710032, China
2
Department of Biomedical Engineering, The Fourth Military Medical University, 169 Changle West Road, Xi’an 710032, China
3
Department of Cardiovascular Surgery, General Hospital, Ningxia Medical University, 804 Shengli South Street, Yinchuan 750004, China
4
Department of Cardiology, Xijing Hospital, The Fourth Military Medical University, 127 Changle West Road, Xi’an 710032, China
that BBR treatment up-regulates Notch1 intracellular domain, Hes1, Bcl-2 expression and p-Akt/Akt ratio, downregulates Bax Caspase-3 and cleaved Caspase-3 expression. However, the anti-apoptotic effect conferred by BBR was blocked by Notch1 siRNA, Hes1 siRNA or LY294002 (the specific inhibitor of Akt signaling) in the cultured cardiomyocytes. In summary, our results demonstrate that BBR treatment attenuates MI/RI by modulating Notch1/ Hes1-PTEN/Akt signaling. Keywords Berberine Myocardial ischemia/reperfusion Anti-apoptosis Notch1 Akt
Introduction Myocardial ischemia reperfusion injury (MI/RI) is a major cause of mortality worldwide. Although early reperfusion is necessary for myocardial salvage, reperfusion itself exacerbates myocardial injury [1, 2]. The pathogenesis of reperfusion-induced myocardial injury is apparently multifactorial, and myocardial apoptosis is one of the major pathogenic mechanisms underlying MI/RI. Enormous efforts have been made to explore the rescue approaches of ischemic/reperfused myocardium. However, further studies are needed to seek novel strategies and targets to reduce MI/RI. Berberine (BBR) is a natural isoquinoline alkaloid isolated from Coptis chinensis, Hydrastis Canadensis, etc., which has been widely used as an antibacterial, antifungal and anti-inflammatory drug, and has been used as a gastrointestinal remedy for 1000 of years [3, 4]. Because BBR also has cardioprotective properties, it has elicited much interest as a potential agent to attenuate MI/RI [5–7]. However, the specific mechanism of BBR’s cardioprotective effect remains unknown.
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Notch signaling pathway has been demonstrated to participate in a series of cellular processes such as differentiation, proliferation and apoptosis [8, 9]. In mammals, 4 Notch receptors (Notch1-4) and five Notch ligands (Deltalike1/3/4, and Jagged1/2) have been identified [9]. Notch receptor activation occurs via binding to Notch ligands. The target genes of Notch signaling include basic helix– loop–helix transcription factors Hairy and enhancer of split 1 (Hes1) and Hairy-related transcription (HRT) factor family members, which are found in both the developing and the adult heart [10]. Phosphatase and tensin homolog deleted on chromosome ten (PTEN) was first identified in 1997 as a tumor suppressor due to its high frequency mutations in various types of tumors [11–13]. As a lipid phosphatase, PTEN antagonizes phosphoinositide 3-kinase, thereby inactivating downstream protein kinases, most notably Akt and PDK1, which in turn acts on various target proteins to regulate multiple cellular functions including cell proliferation, growth and apoptosis [14]. In addition, Notch1/Hes1 signaling has been recently demonstrated to modulate PTEN/Akt to exert protective effect against MI/ RI [15]. Interestingly, another recent study has reported that BBR diminished side population in the pancreatic cancer cell lines by modulating Notch1 gene expression [16]. However, whether Notch1/Hes1 signaling and PTEN/ Akt signaling plays a role in BBR’s cardioprotective effect is unclear. In this study, we explored the action of the Notch1/Hes1 signaling pathway in BBR-induced cardioprotective effect. Furthermore, we investigated the specific role of PTEN/ Akt pathway in this circumstance.
Materials and methods Animals The present study was performed according to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (National Institutes of Health Publication No. 85-23, revised in 1996) and approved by the Ethics Committee of the Fourth Military Medical University. Healthy adult male Sprague–Dawley (SD) rats (weighing between 220 and 250 g) were obtained from the animal center of the Fourth Military Medical University. Experimental protocol Step 1 was designed to evaluate the effect of BBR on myocardial ischemia/reperfusion (MI/R) injured hearts. SD rats were orally gavaged with either 0.5 % sodium carboxymethylcellulose (CMC-Na) solution (Solarbio,
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Beijing, China) or BBR (Sigma, St. Louis, MO, USA, dissolved in 0.5 % CMC-Na solution) at a dose of 200 mg/ kg body weight per day for 2 weeks, respectively. The dosage of BBR used in vivo and in vitro were chosen based on previous researches [5, 17–21]. Two weeks after the treatment, MI/R was induced as described in our previous study [22]. Briefly, SD rats were anesthetized by 3 % pentobarbital sodium. Myocardial ischemia was produced by exteriorizing the heart through a left thoracic incision and placing a 5–0 silk suture to make a slipknot around the left anterior descending coronary artery. After 30 min of ischemia, the slipknot was released and the myocardium was reperfused for 4 h (for analysis of protein expression), 6 h (for quantification of myocardial apoptosis and infarct size) and 72 h (for cardiac function determination). Rats of sham group underwent the same surgical procedures except that the suture passed under the left coronary artery was left untied. Step 2 was designed to investigate the role of Notch1/ Hes1-PTEN/Akt signaling in the myocardial protection induced by BBR treatment in H9c2 cell lines. H9c2 embryonic rat myocardium-derived cells, a well-characterized and widely used cell line to study myocardial cell ischemia, were purchased from Tiancheng Technology (Shanghai, China). Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM, GIBCO) supplemented with 10 % inactivated fetal bovine serum (GIBCO), 500 lg/ml penicillin and 500 lg/ml streptomycin (GIBCO) at 37 °C in a humidified atmosphere with 5 % CO2. The BBR stock solution was prepared in heated PBS solution and diluted with DMEM immediately prior to use. First, we tested the toxic effect of BBR treatment at 0.5, 5, 50 and 500 lmol/L for 4 h on H9c2 cells. Based on the preliminary experiment and previous report, the dose of 50 lmol/L BBR was chosen in this study [5]. Then, the cardiomyocytes were randomly divided into the following groups (n = 8). In Group 1 (SIR), the cardiomyocytes were treated with normal DMEM for 28 h, subjected to simulated ischemic conditions for 2 h, and then incubated in normal DMEM to simulate reperfusion for 4 h. In Group 2 (SIR ? BBR), the cardiomyocytes were incubated in normal DMEM for 24 h, incubated in BBR (50 lmol/L) for 4 h, and then subjected to SIR. In Group 3 (SIR ? BBR ? Notch1/Hes1 siRNA), the cardiomyocytes were transfected with Notch1 siRNA or Hes1 siRNA (Santa Cruz Technology, USA), strictly following the manufacturer’s instructions. Then, the cardiomyocytes were incubated in DMEM with the transfection mixture for 24 h, treated with BBR for 4 h, and then subjected to SIR. In Group 4 (SIR ? Notch1/Hes1 siRNA), the cardiomyocytes were transfected with Notch1 siRNA or Hes1 siRNA, incubated in the DMEM with the transfection mixture for 24 h, incubated in normal DMEM for 4 h, and then subjected to SIR. In Group 5, the
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cardiomyocytes were treated with BBR in the presence or absence of LY294002 (10 lmol/L) for 4 h, then exposed to simulated ischemia/reperfusion (SIR) treatment. The dosage of LY294002 was chosen based on previous studies [23]. The cells were harvested after the SIR treatment for further analysis. Echocardiography Two dimensional mode and motion (M)-mode echocardiographic measurements were carried out with a Vevo 2100 high-resolution in vivo imaging system (VisualSonic, Toronto, Canada) 72 h after MI/R operation to determine the severity of the injury in vivo as described by the previous study [22, 24]. Briefly, rat were anesthetized with isoflurane and placed on the heated stage of the Vevo 2100. Parasternal long axis and short axis images were obtained in short and long axes in two-dimensional and M-mode for quantification. Left ventricular internal dimension systole (LVIDs) and left ventricular internal dimension diastole (LVIDd) were measured on the parasternal LV long axis view. All measurements represented the mean of 5 consecutive cardiac cycles. These were then analyzed on a separate work station with VevoStrain software (Vevo 2100, v1.1.1, Visualsonic, Toronto, Canada). Left
Fig. 1 The effect of berberine treatment on cardiac function in IRinjured hearts. a Representative motion (M)-mode images by echocardiography. Cardiac function was assessed by echocardiography 72 h after MI/R operation. b Left ventricular ejection fraction (LVEF). c Left ventricular fractional shortening (LVFS). MI/R, myocardial ischemia reperfusion (30 min/72 h); V, vehicle [0.5 %
ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) were calculated by the use of computer algorithms. All of these measurements were performed in a blinded manner. Determination of myocardial infarction and apoptosis At the end of 6-h reperfusion, myocardial infarction was determined by means of a double-staining technique and a digital imaging system (infarct area/area-at-risk 9 100 %) [22]. Myocardial apoptosis was analyzed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay using an in situ cell death detection kit (Roche Molecular Biochemicals, Mannheim, Germany) as described previously [22]. The index of apoptosis was expressed by number of apoptotic cardiomyocytes/the total number of cardiomyocytes counted 9 100 %. Determination of serum CK and LDH Blood samples (1 ml) were drawn at 6 h after reperfusion. Serum creatine kinase (CK) and lactate dehydrogenase (LDH) activities were measured spectrophotometrically
sodium carboxymethylcellulose (CMC-Na) solution]; BBR, berberine 200 mg/kg/d; LVIDs, left ventricular internal dimension systole; LVIDd, left ventricular internal dimension diastole. The results are expressed as the mean ± SEM, n = 8. aaP \ 0.01 versus Sham group, bbP \ 0.01 versus MI/R ? V group, ccP \ 0.01 versus MI/R ? BBR group
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Fig. 2 The effect of berberine treatment on apoptotic index, infarct size, LDH release and CK release in IR-injured hearts. a Left representative photomicrographs of in situ detection of apoptotic cardiomyocytes by TUNEL staining. Green fluorescence shows TUNEL-positive nuclei; blue fluorescence shows nuclei of total cardiomyocytes; Original 400 9magnification; Right percentage of TUNEL-positive nuclei; b Top representative photographs of heart sections. Blue-stained portion indicates non-ischemic, normal region; red-stained portion, ischemic/reperfused but not infarcted region; and
negative-stained portion, ischemic/reperfused infarcted region. Bottom myocardial infarct size expressed as percentage of area-at-risk (AAR); c Serum lactate dehydrogenase (LDH) level; d Serum creatine kinase (CK) level. MI/R, myocardial ischemia reperfusion (30 min./6 h); V, vehicle [0.5 % sodium carboxymethylcellulose (CMC-Na) solution]; BBR, berberine 200 mg/kg/d. The results are expressed as the mean ± SEM, n = 8. aaP \ 0.01 versus Sham group, bbP \ 0.01 versus MI/R ? V group, ccP \ 0.01 versus MI/R ? BBR group (Color figure online)
(Beckman DU 640, Fullerton, CA) in a blinded manner as described previously [22]. All kits for measuring CK and LDH activities were purchased from the Institute of Jiancheng Bioengineering (Nanjing, Jiangsu, China).
was initiated by returning the cells to normal culture medium for 4 h in a humidified cell culture incubator (21 % O2, 5 % CO2, 37 °C). Cell viability analysis
Simulated ischemia/reperfusion treatment The SIR treatment was performed using physiological concentrations of potassium, hydrogen, and lactate. The procedure was performed as described previously [25]. Briefly, the cardiomyocytes were exposed to an ischemic buffer containing (in mmol/L) 137 NaCl, 12 KCl, 0.49 MgCl2, 0.9 CaCl2, and 4 HEPES. This buffer was also supplemented with (in mmol/L) 10 deoxyglucose, 0.75 sodium dithionate, and 20 lactate. The buffer pH was 6.5, and the cells were incubated for 2 h in a humidified cell culture incubator (21 % O2, 5 % CO2, 37 °C). Reperfusion
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Cells, 1 9 104 per well, were seeded in 96-well culture plates. Viability of cardiac cells was determined by 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as described previously [26]. Briefly, after the cells were treated and washed with PBS, 10 lL of MTT dye was added to each well at a final concentration of 0.5 mg/ml. After 4-h of incubation, 100 lL of DMSO, the solubilization/stop solution, was added to dissolve the formazan crystals, and the absorbance was measured using a microtiter plate reader (SpectraMax 190, Molecular Device, USA) at a wavelength of 490 nm. The cell viability
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Fig. 3 The effect of berberine treatment on Notch1/Hes1-PTEN/Akt signaling and apoptotic signaling in MI/R-injured hearts. a Representative blots; b NICD expression; c Hes1 expression; d PTEN expression; e p-Akt/Akt ratio; f Caspase-3 expression; g Bcl-2 expression; h Bax expression; I Cleaved caspase-3 expression. NICD,
Notch1 intracellular domain; MI/R, myocardial ischemia reperfusion (30 min/4 h); V, vehicle [0.5 % sodium carboxymethylcellulose (CMC-Na) solution]; BBR, berberine 200 mg/kg/d. The results are expressed as the mean ± SEM, n = 8. aaP \ 0.01 versus Sham group, bbP \ 0.01 versus MI/R ? V group
was calculated by dividing the optical density of samples with the optical density of sham control.
centrifugation at 4 °C for 15 min at 12,000 rpm. After quantitation of protein concentration, 30 lg of total protein was separated by SDS-PAGE and then transferred to a polyvinylidene difluoride membrane (Millipore, USA). The membranes were blocked for 2 h at 37 °C with 5 % non-fat dry milk, then incubated with primary antibody including p-Akt, Akt, PTEN, Hes1, Caspase-3, Bcl-2, Bax, b-actin (Santa Cruz Technology, USA), cleaved Caspase-3 (Beyotime Biotechnology, China), Notch1 intracellular domain (NICD) (Abcam, USA) (1:1000) over night at 4 °C. After three washings with TBST, the membranes were incubated with secondary antibody in TBST solution for 30 min at 37 °C, then, washed as above. The positive protein bands were developed using a chemiluminescent system, and the bands were scanned and quantified by densitometric analysis using an image analyzer Quantity One System (Bio-Rad, Richmond, CA, USA).
Determination of cellular apoptosis After the H9c2 cardiomyocytes were fixed in paraformaldehyde (4 %) for 24 h, the cellular apoptosis was also analyzed by performing a TUNEL assay using the in situ cell death detection kit according to the manufacturer’s instructions [27]. The apoptotic index was expressed as the number of positively stained apoptotic cardiomyocytes/the total number of cardiomyocytes counted 9 100 %. Western blot analysis The myocardial and cardiomyocytes samples were lysed in lysis buffer on ice for 20 min. The lysates were clarified by
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Fig. 4 The effect of berberine and Notch1 siRNA on cell viability and apoptosis in simulated ischemia/reperfusion treated cardiomyocytes. a, b Viability of cardiomyocytes was determined by MTT and was calculated by dividing the optical density of samples with the optical density of sham control. c Left representative photomicrographs of in situ detection of apoptotic cardiomyocytes by TUNEL staining. Green fluorescence shows TUNEL-positive nuclei; blue
fluorescence shows nuclei of total cardiomyocytes; Original 200 9magnification; Right percentage of TUNEL-positive nuclei. BBR, berberine 50 lmol/L treated for 4 h; SIR, simulated ischemia/ reperfusion. The results are expressed as the mean ± SEM, n = 8. dd P \ 0.01 versus Con group, eeP \ 0.01 versus SIR group, ff P \ 0.01 versus SIR ? BBR group, ggP \ 0.01 versus SIR ? BBR ? Notch1 siRNA group (Color figure online)
Statistical analysis
reperfusion was examined. Echocardiography demonstrated a significant increased LVEF and LVFS in BBR treated group compared with the MI/R ? V group (P \ 0.01, Fig. 1). To examine whether BBR decreased myocardial apoptosis, we further measured myocardial apoptotic index, myocardial infarct size, serum LDH and CK levels after 6-h of reperfusion. Compared with the MI/R ? V group, BBR treatment also attenuated cardiac apoptosis by decreasing apoptotic index and infarct size (P \ 0.01, Fig. 2a, b). In addition, serum CK and LDH levels were also significantly reduced after 6-h of reperfusion in BBR treated rats (P \ 0.01, compared with the MI/R ? V group, Fig. 2c, d). In addition, vehicle (0.5 % CMC-Na solution) or BBR (200 mg/kg/d) treatment alone had no significant effect on the cardiac function, apoptotic index, infarct size, serum LDH and CK levels compared with the Sham group
All values are presented as mean ± SEM. Differences were compared by ANOVA followed by Bonferroni correction for post hoc t test, where appropriate. Probabilities of \0.05 were considered to be statistically significant. All of the statistical tests were performed with the GraphPad Prism software version 5.0 (GraphPad Software, Inc., San Diego, CA).
Results The effect of BBR treatment on MI/RI To investigate whether BBR has cardioprotective effect against MI/RI in rats, left ventricular function after 72-h of
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Fig. 5 The effect of berberine and Notch1 siRNA on NICD, Hes1, PTEN expression and p-Akt/Akt ratio in simulated ischemia/reperfusion treated cardiomyocytes. a Representative blots; b NICD expression; c Hes1 expression; d PTEN expression; e p-Akt/Akt ratio.
BBR, berberine 50 lmol/L treated for 4 h; SIR, simulated ischemia/ reperfusion. The results are expressed as the mean ± SEM, n = 8. ee P \ 0.01 versus SIR group, ffP \ 0.01 versus SIR ? BBR group, gg P \ 0.01 versus SIR ? BBR ? Notch1 siRNA group
(P [ 0.05, Figs. 1 and 2). These data indicate that BBR treatment might protect myocardium from apoptosis following MI/RI, thus improving cardiac function.
These data indicated that BBR confers significant antiapoptotic effect and the Notch1/Hes1 and PTEN/Akt signaling might play a role in this action.
The effect of BBR treatment on Notch1/Hes1 and PTEN/Akt signaling pathway in MI/R hearts
The effect of BBR and Notch1 siRNA treatment on cell viability and apoptosis in SIR-treated cardiomyocytes
We further measured the levels of Notch1/Hes1 and PTEN/ Akt signaling and the expression of apoptotic related protein in MI/R injured hearts. As depicted in Fig. 3, Western blotting analysis indicated that MI/RI significantly increased the ratio of p-Akt/Akt, Caspase-3 expression, cleaved Caspase-3 expression and Bax expression compared with the sham group (P \ 0.01); conversely, MI/RI produced a significant decrease in the expression of Bcl-2 (P \ 0.01, compared with sham group). Treatment with BBR produced a significant increase in the expression of NICD, Hes1 and Bcl-2 (P \ 0.01, compared with the MI/R ? V group). Besides, BBR treatment also up-regulated the ratio of p-Akt/ Akt and down-regulated the expression of PTEN, Caspase-3 and Bax (P \ 0.01, compared with the MI/R ? V group).
To investigate whether BBR has direct protective effect against MI/RI and the underlying mechanism, we performed in vitro studies in H9c2 cardiomyocytes subjected to SIR. In order to test whether experimental dosages of BBR affect the survival of H9c2 cells, cardiomyocytes were exposed to BBR (0.5, 5, 50 or 500 lmol/L) for 4 h. Then, cell viability was determined by MTT. As shown in Fig. 4a, 500 lmol/L BBR significantly affected the cell viability compared with the control group (P \ 0.01). Based on this preliminary experiment and the previous study, we chose 50 lmol/L in our study. Next, the cells were subjected to SIR treatment in the absence or presence of BBR (50 lmol/L) or Notch1 siRNA pretreatment. BBR
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Fig. 6 The effect of berberine and Notch1 siRNA on apoptotic signaling in simulated ischemia/reperfusion treated cardiomyocytes. a Representative blots; b Caspase-3 expression; c Bcl-2 expression; d Bax expression; e Cleaved caspase-3 expression. BBR, berberine
50 lmol/L treated for 4 h; SIR, simulated ischemia/reperfusion. The results are expressed as the mean ± SEM, n = 8. eeP \ 0.01 versus SIR group, ffP \ 0.01 versus SIR ? BBR group, ggP \ 0.01 versus SIR ? BBR ? Notch1 siRNA group
treatment for 4 h resulted in a significant increase in cell viability (Fig. 4b, P \ 0.01, compared with the SIR group). However, BBR-induced cardioprotective effect was significantly blunted by Notch1 siRNA (Fig. 4b, P \ 0.01, compared with the SIR ? BBR group). As shown in Fig. 4c, BBR treatment also reduced the apoptotic index, while Notch1 siRNA administration significantly blocked this protective effect (P \ 0.01, compared with the SIR ? BBR group). Compared with the SIR group, SIR ? Notch1 siRNA treatment had no significant effect on cell viability and apoptotic index (P [ 0.05). These findings indicated that BBR had direct anti-apoptotic effect against SIR injury and Notch signaling might play a key role in its protective action.
effect. As depicted in Fig. 5, BBR treatment significantly up-regulated the expression of NICD, Hes1 and the ratio of p-Akt/Akt, down-regulated PTEN expression (P \ 0.01, compared with the SIR group). However, Notch1 siRNA administration significantly blocked these effects (P \ 0.01, compared with the SIR ? BBR group). Consistently, BBR exerted significant anti-apoptotic effect by reducing the expression of Caspase-3, Bax, cleaved Caspase-3 and increasing Bcl-2 expression (Fig. 6, P \ 0.01, compared with the SIR group). These effects were also blocked by Notch1 siRNA (Fig. 6, P \ 0.01, compared with the SIR ? BBR group). Compared with the SIR group, SIR ? Notch1 siRNA treatment had no significant effect on apoptotic signaling (Fig. 6, P [ 0.05). These results further suggested that BBR’s anti-apoptotic effect may be mediated by Notch1/Hes1 signaling.
The effect of BBR and Notch1 siRNA treatment on NICD, Hes1, PTEN expression, p-Akt/Akt ratio and apoptotic signaling in SIR-treated cardiomyocytes NICD, Hes1, PTEN expression, p-Akt/Akt ratio and apoptotic signaling were examined to further investigate the underlying mechanism of BBR’s cardioprotective
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The effect of BBR and Hes1 siRNA treatment on cell viability and apoptosis in SIR-treated cardiomyocytes To further investigate the role of Hes1 in BBR’s cardioprotective action, Hes1 siRNA was utilized in the present
Apoptosis Fig. 7 The effect of berberine and Hes1 siRNA on cell viability and apoptosis in simulated ischemia/reperfusion treated cardiomyocytes. a Representative photomicrographs of in situ detection of apoptotic cardiomyocytes by TUNEL staining. Green fluorescence shows TUNEL-positive nuclei; blue fluorescence shows nuclei of total cardiomyocytes; Original 2009 magnification; b Percentage of TUNELpositive nuclei; c Viability of cardiomyocytes was determined by MTT and was calculated by dividing the optical density of samples with the optical density of sham control. SIR, simulated ischemia/reperfusion. The results are expressed as the mean ± SEM, n = 8. hh P \ 0.01 versus SIR group, ii P \ 0.01 versus SIR ? BBR group, jjP \ 0.01 versus SIR ? BBR ? Hes1 siRNA group (Color figure online)
experiment. As expected, Hes1 siRNA administration significantly blocked BBR’s protective effect by increasing the apoptotic index (Fig. 7b, P \ 0.01, compared with the SIR ? BBR group). Hes1 siRNA also decreased cell viability compared with the SIR ? BBR group (Fig. 7c, P \ 0.01). Compared with the SIR group, SIR ? Hes1 siRNA treatment had no significant effect on apoptotic index and cell viability (P [ 0.05). These findings suggested that Hes1 played a key role in BBR’s protective action. The effect of BBR and Hes1 siRNA treatment on Hes1, PTEN expression, p-Akt/Akt ratio and apoptotic signaling in SIR-treated cardiomyocytes We further measured the Hes1, PTEN expression, p-Akt/Akt ratio and apoptotic signaling to investigate the underlying mechanism. As seen in Fig. 8, BBR’s effect on Hes1 expression, PTEN expression and p-Akt/Akt ratio was
significantly blocked by Hes1 siRNA administration (P \ 0.01, compared with the SIR ? BBR group). In addition, Hes1 siRNA treatment also blocked the anti-apoptotic effect of BBR by increasing the expression of Caspase-3, Bax, cleaved Caspase-3 and decreasing Bcl-2 expression (Fig. 9, P \ 0.01, compared with the SIR ? BBR group). Although SIR ? Hes1 siRNA treatment significantly increased the expression of PTEN and decreased the ratio of p-Akt/Akt, this treatment had no significant effect on the apoptotic signaling (P \ 0.01, compared with the SIR group). These data provided the direct evidence that Notch1/Hes1 signaling played a key role in BBR’s anti-apoptotic action (Fig. 10). The effect of BBR and LY294002 treatment on cell viability and apoptosis in SIR-treated cardiomyocytes To investigate the role of Akt in BBR’s cardioprotective action, LY294002, a specific Akt inhibitor, was introduced
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Apoptosis Fig. 8 The effect of berberine and Hes1 siRNA on Hes1, PTEN expression and p-Akt/Akt ratio in simulated ischemia/ reperfusion treated cardiomyocytes. a Representative blots; b Hes1 expression; c PTEN expression; d p-Akt/Akt ratio. BBR, berberine 50 lmol/L treated for 4 h; SIR, simulated ischemia/ reperfusion. The results are expressed as the mean ± SEM, n = 8. hhP \ 0.01 versus SIR group, iiP \ 0.01 versus SIR ? BBR group, jjP \ 0.01 versus SIR ? BBR ? Hes1 siRNA group
to the present experiment. As shown in Fig. 7b, LY294002 treatment also significantly blocked BBR’s protective effect by increasing the apoptotic index (P \ 0.01, compared with the SIR ? BBR group). Compared with the SIR ? BBR group, co-treatment with LY294002 significantly reduced cell viability (Fig. 7c, P \ 0.01). Consistently, SIR ? LY294002 treatment had no significant effect on apoptotic index and cell viability compared with the SIR group, (P [ 0.05). These findings suggested that Akt might participate in the anti-apoptotic action of BBR. The effect of BBR and LY294002 treatment on pAkt/Akt ratio and apoptotic signaling in SIR-treated cardiomyocytes Similarly, we examined the effect of BBR and LY294002 treatment on Akt signaling and apoptosis related protein to determine whether the BBR’s protective action could be mediated by activation of Akt signaling. As seen in Fig. 11b, BBR’s effect on p-Akt/Akt ratio was significantly blocked by LY294002 administration (P \ 0.01, compared with the SIR ? BBR group). In addition, LY294002 treatment also blocked the anti-apoptotic effect of BBR by decreasing Bcl-2 expression and increasing the expression of Caspase-3, Bax, cleaved Caspase-3 (Fig. 11c–f,
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P \ 0.01, compared with the SIR ? BBR group). These data provided the direct evidence that Akt signaling played a pivotal role in BBR’s anti-apoptotic action.
Discussion Myocardial apoptosis is one of the major pathogenic mechanisms following MI/RI. Reducing apoptosis could prevent the loss of contractile cells, minimize MI/R-induced cardiac injury and therefore slow down or even prevent the occurrence of heart failure [28–30]. Previous studies have confirmed the ameliorative effect of BBR on MI/RI induced apoptosis [6, 31]. In this study, we also found that BBR treatment exerted cardioprotective effect, as evidenced by improved post-MI/R cardiac functional recovery and attenuated myocardial apoptosis. Notch signaling pathway is an important cell–cell communication system, which has indispensable functional activities to control tissue formation in cardiogenesis [32]. The binding of Notch1 ligand to its receptor triggers the csecretase-mediated proteolytic cleavage of NICD, which then translocates into the nucleus to form a transcriptionactivating complex, thereby mediating its downstream target gene Hes1 [33]. Notch1 is implicated in cardiac
Apoptosis
Fig. 9 The effect of berberine and Hes1 siRNA on apoptotic signaling in simulated ischemia/reperfusion treated cardiomyocytes. a Representative blots; b Caspase-3 expression; c Bcl-2 expression; d Bax expression; e Cleaved caspase-3 expression. BBR, berberine
50 lmol/L treated for 4 h; SIR, simulated ischemia/reperfusion. The results are expressed as the mean ± SEM, n = 8. hhP \ 0.01 versus SIR group, iiP \ 0.01 versus SIR ? BBR group, jjP \ 0.01 versus SIR ? BBR ? Hes1 siRNA group
development, where it regulates cardiomyocyte proliferation, myocardial trabeculation, and valve formation [34, 35], as well as in the maintenance of adult heart tissue integrity [36, 37]. Interestingly, as the myocardium matures to the adult hormonal and contractile state, the level of endogenous Notch signaling (e.g. Notch1/Hes1) decreases steadily in heart tissue [38], which may suggest its loss of control in tissue homeostasis during adult life stage. However, recent studies have demonstrated that re-activation of Notch signaling may constitute an adaptive response following pathological stress to increase survival rate or regenerate tissues. For example, pharmacological blockade of Notch1 leads to Caspase-3dependent apoptosis in adult islets, in contrast, over activation of Notch1 protects against apoptosis [39]. Furthermore, the critical role of Notch signaling has been established in the maintenance of functional homeostasis in adult heart. It is reported that Notch1 signaling activation contributes to cardioprotection provided by ischemic preconditioning and postconditioning by suppressing apoptosis [40]. Interestingly, another recent study showed that BBR diminishes side population in the
pancreatic cancer cell lines PANC-1 and MIA PaCa-2 by modulating Notch1 gene expression [16]. In our in vivo study, we found that the expression of NICD and Hes1 are significantly up-regulated by BBR treatment, indicating the pivotal role of Notch1/Hes1 signaling in BBR’s protective action. PI3K-Akt signaling is a critical signaling pathway involved in a broad range of biological processes and Akt/Protein kinase B possesses pro-survival and antiapoptotic activities. In addition, Akt deactivation has been well established to be a causal mediator of cell death [41]. On the other hand, PTEN is a nonredundant, plasma membrane lipid phosphatase that converts the phosphatidylinositol-3,4,5-phosphate into the inactive phosphatidylinositol-4,5-diphosphate, functioning as an endogenous antagonist of Akt signaling [13]. Several studies have reported the pivotal role of PTEN inhibition, which regulated the Akt signaling, in myocardial protection. Conditional cardiomyocyte-specific PTEN knockout mice showed reduced cell death and apoptosis in the in vivo model of MI/R [42]. Furthermore, Pei and colleagues reported that Notch1 signaling protected
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Apoptosis Fig. 10 The effect of berberine and LY294002 on cell viability and apoptosis in simulated ischemia/reperfusion treated cardiomyocytes. a Representative photomicrographs of in situ detection of apoptotic cardiomyocytes by TUNEL staining. Green fluorescence shows TUNEL-positive nuclei; blue fluorescence shows nuclei of total cardiomyocytes; Original 2009 magnification; b Percentage of TUNELpositive nuclei; c Viability of cardiomyocytes was determined by MTT and was calculated by dividing the optical density of samples with the optical density of sham control. SIR, simulated ischemia/reperfusion. The results are expressed as the mean ± SEM, n = 8. kk P \ 0.01 versus SIR group, ll P \ 0.01 versus SIR ? BBR group, mmP \ 0.01 versus SIR ? BBR ? LY294002 group (Color figure online)
against MI/RI partly though PTEN/Akt mediated anti-oxidative and anti-nitrative effect, thus reducing post-MI/R apoptosis and improving cardiac function recovery [15]. Zhao and colleagues have reported that Notch1 exhibited cardioprotection in ischemic preconditioning by crosstalk with the reperfusion injury salvage kinase (RISK) signaling pathway, possibly through PTEN inhibition [43]. Our data corroborates these previous findings and suggests an inverse relationship between activation of Hes1 signaling and inhibition of PTEN expression. Both Notch1 siRNA and Hes1 siRNA all significantly up-regulated PTEN expression and reduced Akt activity, thus abolishing BBR’s antiapoptotic effect. Next, we evaluated the role of Akt in BBR’s cardioprotective action. BBR has been tested to protect against oxidative damage via a PI3K/Akt-dependent mechanism in NSC34 motor neuron-like cells [44]. Additionally, Xiao and colleagues also demonstrated that BBR
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protected endothelial progenitor cell from damage of TNF-a via the PI3K/AKT/eNOS signaling pathway [45]. However, these effects were all abolished by LY294002, the specific Akt inhibitor [44, 45]. In this study, we also found that co-treatment with LY294002 markedly attenuated BBR’s cardioprotective effect. Although further study is still needed to investigate the underlying mechanisms, we do proved that Notch1/Hes1 signaling plays a key role in BBR’s anti-apoptotic action and PTEN/Akt, the down-stream signaling pathway, participates in this process. In summary, our findings suggest that BBR treatment exerts a profound cardioprotective effect against apoptosis induced by MI/RI. This protection appears to be largely due to the modulation of Notch1/Hes1-PTEN/Akt signaling pathway. These results revealed that BBR may be a promising candidate for the treatment of MI/RI in cardiac surgery and ischemic heart diseases.
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Fig. 11 The effect of berberine and LY294002 on p-Akt/Akt ratio and apoptotic signaling in simulated ischemia/reperfusion treated cardiomyocytes. a Representative blots; b p-Akt/Akt ratio; c Caspase3 expression; d Bcl-2 expression; e Bax expression; f Cleaved caspase-3 expression. BBR, berberine 50 lmol/L treated for 4 h; SIR,
simulated ischemia/reperfusion. The results are expressed as the mean ± SEM, n = 8. kkP \ 0.01 versus SIR group, llP \ 0.01 versus SIR ? BBR group, mmP \ 0.01 versus SIR ? BBR ? LY294002 group
Acknowledgments The work presented here was carried out in collaboration between all authors. Shiqiang Yu and Weixun Duan defined the research theme and revised the manuscript critically. Liming Yu, Feijiang Li and Guolong Zhao designed methods and experiments, carried out the laboratory experiments and wrote the paper. Yang Yang, Zhenxiao Jin, Mengen Zhai, Wenjun Yu, Lin Zhao and Wensheng Chen collected and analyzed the data, interpreted the results. The authors thank Buying Li, Xiaowu Wang and Yanyan Ma, Department of Cardiovascular Surgery, Xijing Hospital, The Fourth Military Medical University, for technical support in western blot and cell culture. This study was supported by grants from the National Natural Science Foundation of China (81470415, 81470411, 81270170) and the Natural Science Foundation of Shannxi Province (2014JM4106).
3. Remppis A, Bea F, Greten HJ, Buttler A, Wang H, Zhou Q, Preusch MR, Enk R, Ehehalt R, Katus H, Blessing E (2010) Rhizoma Coptidis inhibits LPS-induced MCP-1/CCL2 production in murine macrophages via an AP-1 and NFkappaB-dependent pathway. Mediat Inflamm 2010:194896. doi:10.1155/2010/ 194896 4. Liu F, Liang HL, Xu KH, Tong LL, Tang B (2007) Supramolecular interaction of ethylenediamine linked beta-cyclodextrin dimer and berberine hydrochloride by spectrofluorimetry and its analytical application. Talanta 74(1):140–145. doi:10.1016/j. talanta.2007.05.048 5. Chen K, Li G, Geng F, Zhang Z, Li J, Yang M, Dong L, Gao F (2014) Berberine reduces ischemia/reperfusion-induced myocardial apoptosis via activating AMPK and PI3K-Akt signaling in diabetic rats. Apoptosis Int J Program Cell Death 19(6):946–957. doi:10.1007/s10495-014-0977-0 6. Chang W, Zhang M, Li J, Meng Z, Xiao D, Wei S, Chen L, Wang C, Hatch GM (2012) Berberine attenuates ischemia-reperfusion injury via regulation of adenosine-50 -monophosphate kinase activity in both non-ischemic and ischemic areas of the rat heart. Cardiovas Drugs Therapy 26(6):467–478. doi:10.1007/s10557012-6422-0 7. Zeng XH, Zeng XJ, Li YY (2003) Efficacy and safety of berberine for congestive heart failure secondary to ischemic or idiopathic dilated cardiomyopathy. Am J Cardiol 92(2):173–176 8. Piazzi G, Bazzoli F, Ricciardiello L (2012) Epigenetic silencing of Notch signaling in gastrointestinal cancers. Cell Cycle 11(23):4323–4327. doi:10.4161/cc.22388
Conflict of interest of interest.
The authors declare that they have no conflict
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ORIGINAL PAPER
Protective effect of berberine against myocardial ischemia reperfusion injury: role of Notch1/Hes1-PTEN/Akt signaling Liming Yu1 • Feijiang Li2 • Guolong Zhao3 • Yang Yang1,2 • Zhenxiao Jin1 • Mengen Zhai1 • Wenjun Yu4 • Lin Zhao1 • Wensheng Chen1 • Weixun Duan1 Shiqiang Yu1
•
Ó Springer Science+Business Media New York 2015
Abstract Berberine (BBR) confers cardioprotective effect against myocardial ischemia reperfusion injury (MI/ RI). Activation of Notch1/Hairy and enhancer of split 1 (Hes1) signaling also reduces MI/RI. We hypothesize that BBR may protect against MI/RI by modulating Notch1/ Hes1-Phosphatase and tensin homolog deleted on chromosome ten (PTEN)/Akt signaling. In this study, male Sprague–Dawley rats were exposed to BBR treatment (200 mg/kg/d) for 2 weeks and then subjected to MI/RI. BBR significantly improved cardiac function recovery and decreased myocardial apoptosis, infarct size, serum creatine kinase and lactate dehydrogenase levels. Furthermore, in cultured H9c2 cardiomyocytes, BBR (50 lmol/L) attenuated simulated ischemia/reperfusion-induced myocardial apoptosis. Both in vivo and in vitro study showed
Liming Yu, Feijiang Li and Guolong Zhao contributed equally to this study. & Weixun Duan [email protected] & Shiqiang Yu [email protected] 1
Department of Cardiovascular Surgery, Xijing Hospital, The Fourth Military Medical University, 127 Changle West Road, Xi’an 710032, China
2
Department of Biomedical Engineering, The Fourth Military Medical University, 169 Changle West Road, Xi’an 710032, China
3
Department of Cardiovascular Surgery, General Hospital, Ningxia Medical University, 804 Shengli South Street, Yinchuan 750004, China
4
Department of Cardiology, Xijing Hospital, The Fourth Military Medical University, 127 Changle West Road, Xi’an 710032, China
that BBR treatment up-regulates Notch1 intracellular domain, Hes1, Bcl-2 expression and p-Akt/Akt ratio, downregulates Bax Caspase-3 and cleaved Caspase-3 expression. However, the anti-apoptotic effect conferred by BBR was blocked by Notch1 siRNA, Hes1 siRNA or LY294002 (the specific inhibitor of Akt signaling) in the cultured cardiomyocytes. In summary, our results demonstrate that BBR treatment attenuates MI/RI by modulating Notch1/ Hes1-PTEN/Akt signaling. Keywords Berberine Myocardial ischemia/reperfusion Anti-apoptosis Notch1 Akt
Introduction Myocardial ischemia reperfusion injury (MI/RI) is a major cause of mortality worldwide. Although early reperfusion is necessary for myocardial salvage, reperfusion itself exacerbates myocardial injury [1, 2]. The pathogenesis of reperfusion-induced myocardial injury is apparently multifactorial, and myocardial apoptosis is one of the major pathogenic mechanisms underlying MI/RI. Enormous efforts have been made to explore the rescue approaches of ischemic/reperfused myocardium. However, further studies are needed to seek novel strategies and targets to reduce MI/RI. Berberine (BBR) is a natural isoquinoline alkaloid isolated from Coptis chinensis, Hydrastis Canadensis, etc., which has been widely used as an antibacterial, antifungal and anti-inflammatory drug, and has been used as a gastrointestinal remedy for 1000 of years [3, 4]. Because BBR also has cardioprotective properties, it has elicited much interest as a potential agent to attenuate MI/RI [5–7]. However, the specific mechanism of BBR’s cardioprotective effect remains unknown.
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Notch signaling pathway has been demonstrated to participate in a series of cellular processes such as differentiation, proliferation and apoptosis [8, 9]. In mammals, 4 Notch receptors (Notch1-4) and five Notch ligands (Deltalike1/3/4, and Jagged1/2) have been identified [9]. Notch receptor activation occurs via binding to Notch ligands. The target genes of Notch signaling include basic helix– loop–helix transcription factors Hairy and enhancer of split 1 (Hes1) and Hairy-related transcription (HRT) factor family members, which are found in both the developing and the adult heart [10]. Phosphatase and tensin homolog deleted on chromosome ten (PTEN) was first identified in 1997 as a tumor suppressor due to its high frequency mutations in various types of tumors [11–13]. As a lipid phosphatase, PTEN antagonizes phosphoinositide 3-kinase, thereby inactivating downstream protein kinases, most notably Akt and PDK1, which in turn acts on various target proteins to regulate multiple cellular functions including cell proliferation, growth and apoptosis [14]. In addition, Notch1/Hes1 signaling has been recently demonstrated to modulate PTEN/Akt to exert protective effect against MI/ RI [15]. Interestingly, another recent study has reported that BBR diminished side population in the pancreatic cancer cell lines by modulating Notch1 gene expression [16]. However, whether Notch1/Hes1 signaling and PTEN/ Akt signaling plays a role in BBR’s cardioprotective effect is unclear. In this study, we explored the action of the Notch1/Hes1 signaling pathway in BBR-induced cardioprotective effect. Furthermore, we investigated the specific role of PTEN/ Akt pathway in this circumstance.
Materials and methods Animals The present study was performed according to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (National Institutes of Health Publication No. 85-23, revised in 1996) and approved by the Ethics Committee of the Fourth Military Medical University. Healthy adult male Sprague–Dawley (SD) rats (weighing between 220 and 250 g) were obtained from the animal center of the Fourth Military Medical University. Experimental protocol Step 1 was designed to evaluate the effect of BBR on myocardial ischemia/reperfusion (MI/R) injured hearts. SD rats were orally gavaged with either 0.5 % sodium carboxymethylcellulose (CMC-Na) solution (Solarbio,
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Beijing, China) or BBR (Sigma, St. Louis, MO, USA, dissolved in 0.5 % CMC-Na solution) at a dose of 200 mg/ kg body weight per day for 2 weeks, respectively. The dosage of BBR used in vivo and in vitro were chosen based on previous researches [5, 17–21]. Two weeks after the treatment, MI/R was induced as described in our previous study [22]. Briefly, SD rats were anesthetized by 3 % pentobarbital sodium. Myocardial ischemia was produced by exteriorizing the heart through a left thoracic incision and placing a 5–0 silk suture to make a slipknot around the left anterior descending coronary artery. After 30 min of ischemia, the slipknot was released and the myocardium was reperfused for 4 h (for analysis of protein expression), 6 h (for quantification of myocardial apoptosis and infarct size) and 72 h (for cardiac function determination). Rats of sham group underwent the same surgical procedures except that the suture passed under the left coronary artery was left untied. Step 2 was designed to investigate the role of Notch1/ Hes1-PTEN/Akt signaling in the myocardial protection induced by BBR treatment in H9c2 cell lines. H9c2 embryonic rat myocardium-derived cells, a well-characterized and widely used cell line to study myocardial cell ischemia, were purchased from Tiancheng Technology (Shanghai, China). Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM, GIBCO) supplemented with 10 % inactivated fetal bovine serum (GIBCO), 500 lg/ml penicillin and 500 lg/ml streptomycin (GIBCO) at 37 °C in a humidified atmosphere with 5 % CO2. The BBR stock solution was prepared in heated PBS solution and diluted with DMEM immediately prior to use. First, we tested the toxic effect of BBR treatment at 0.5, 5, 50 and 500 lmol/L for 4 h on H9c2 cells. Based on the preliminary experiment and previous report, the dose of 50 lmol/L BBR was chosen in this study [5]. Then, the cardiomyocytes were randomly divided into the following groups (n = 8). In Group 1 (SIR), the cardiomyocytes were treated with normal DMEM for 28 h, subjected to simulated ischemic conditions for 2 h, and then incubated in normal DMEM to simulate reperfusion for 4 h. In Group 2 (SIR ? BBR), the cardiomyocytes were incubated in normal DMEM for 24 h, incubated in BBR (50 lmol/L) for 4 h, and then subjected to SIR. In Group 3 (SIR ? BBR ? Notch1/Hes1 siRNA), the cardiomyocytes were transfected with Notch1 siRNA or Hes1 siRNA (Santa Cruz Technology, USA), strictly following the manufacturer’s instructions. Then, the cardiomyocytes were incubated in DMEM with the transfection mixture for 24 h, treated with BBR for 4 h, and then subjected to SIR. In Group 4 (SIR ? Notch1/Hes1 siRNA), the cardiomyocytes were transfected with Notch1 siRNA or Hes1 siRNA, incubated in the DMEM with the transfection mixture for 24 h, incubated in normal DMEM for 4 h, and then subjected to SIR. In Group 5, the
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cardiomyocytes were treated with BBR in the presence or absence of LY294002 (10 lmol/L) for 4 h, then exposed to simulated ischemia/reperfusion (SIR) treatment. The dosage of LY294002 was chosen based on previous studies [23]. The cells were harvested after the SIR treatment for further analysis. Echocardiography Two dimensional mode and motion (M)-mode echocardiographic measurements were carried out with a Vevo 2100 high-resolution in vivo imaging system (VisualSonic, Toronto, Canada) 72 h after MI/R operation to determine the severity of the injury in vivo as described by the previous study [22, 24]. Briefly, rat were anesthetized with isoflurane and placed on the heated stage of the Vevo 2100. Parasternal long axis and short axis images were obtained in short and long axes in two-dimensional and M-mode for quantification. Left ventricular internal dimension systole (LVIDs) and left ventricular internal dimension diastole (LVIDd) were measured on the parasternal LV long axis view. All measurements represented the mean of 5 consecutive cardiac cycles. These were then analyzed on a separate work station with VevoStrain software (Vevo 2100, v1.1.1, Visualsonic, Toronto, Canada). Left
Fig. 1 The effect of berberine treatment on cardiac function in IRinjured hearts. a Representative motion (M)-mode images by echocardiography. Cardiac function was assessed by echocardiography 72 h after MI/R operation. b Left ventricular ejection fraction (LVEF). c Left ventricular fractional shortening (LVFS). MI/R, myocardial ischemia reperfusion (30 min/72 h); V, vehicle [0.5 %
ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) were calculated by the use of computer algorithms. All of these measurements were performed in a blinded manner. Determination of myocardial infarction and apoptosis At the end of 6-h reperfusion, myocardial infarction was determined by means of a double-staining technique and a digital imaging system (infarct area/area-at-risk 9 100 %) [22]. Myocardial apoptosis was analyzed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay using an in situ cell death detection kit (Roche Molecular Biochemicals, Mannheim, Germany) as described previously [22]. The index of apoptosis was expressed by number of apoptotic cardiomyocytes/the total number of cardiomyocytes counted 9 100 %. Determination of serum CK and LDH Blood samples (1 ml) were drawn at 6 h after reperfusion. Serum creatine kinase (CK) and lactate dehydrogenase (LDH) activities were measured spectrophotometrically
sodium carboxymethylcellulose (CMC-Na) solution]; BBR, berberine 200 mg/kg/d; LVIDs, left ventricular internal dimension systole; LVIDd, left ventricular internal dimension diastole. The results are expressed as the mean ± SEM, n = 8. aaP \ 0.01 versus Sham group, bbP \ 0.01 versus MI/R ? V group, ccP \ 0.01 versus MI/R ? BBR group
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Fig. 2 The effect of berberine treatment on apoptotic index, infarct size, LDH release and CK release in IR-injured hearts. a Left representative photomicrographs of in situ detection of apoptotic cardiomyocytes by TUNEL staining. Green fluorescence shows TUNEL-positive nuclei; blue fluorescence shows nuclei of total cardiomyocytes; Original 400 9magnification; Right percentage of TUNEL-positive nuclei; b Top representative photographs of heart sections. Blue-stained portion indicates non-ischemic, normal region; red-stained portion, ischemic/reperfused but not infarcted region; and
negative-stained portion, ischemic/reperfused infarcted region. Bottom myocardial infarct size expressed as percentage of area-at-risk (AAR); c Serum lactate dehydrogenase (LDH) level; d Serum creatine kinase (CK) level. MI/R, myocardial ischemia reperfusion (30 min./6 h); V, vehicle [0.5 % sodium carboxymethylcellulose (CMC-Na) solution]; BBR, berberine 200 mg/kg/d. The results are expressed as the mean ± SEM, n = 8. aaP \ 0.01 versus Sham group, bbP \ 0.01 versus MI/R ? V group, ccP \ 0.01 versus MI/R ? BBR group (Color figure online)
(Beckman DU 640, Fullerton, CA) in a blinded manner as described previously [22]. All kits for measuring CK and LDH activities were purchased from the Institute of Jiancheng Bioengineering (Nanjing, Jiangsu, China).
was initiated by returning the cells to normal culture medium for 4 h in a humidified cell culture incubator (21 % O2, 5 % CO2, 37 °C). Cell viability analysis
Simulated ischemia/reperfusion treatment The SIR treatment was performed using physiological concentrations of potassium, hydrogen, and lactate. The procedure was performed as described previously [25]. Briefly, the cardiomyocytes were exposed to an ischemic buffer containing (in mmol/L) 137 NaCl, 12 KCl, 0.49 MgCl2, 0.9 CaCl2, and 4 HEPES. This buffer was also supplemented with (in mmol/L) 10 deoxyglucose, 0.75 sodium dithionate, and 20 lactate. The buffer pH was 6.5, and the cells were incubated for 2 h in a humidified cell culture incubator (21 % O2, 5 % CO2, 37 °C). Reperfusion
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Cells, 1 9 104 per well, were seeded in 96-well culture plates. Viability of cardiac cells was determined by 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as described previously [26]. Briefly, after the cells were treated and washed with PBS, 10 lL of MTT dye was added to each well at a final concentration of 0.5 mg/ml. After 4-h of incubation, 100 lL of DMSO, the solubilization/stop solution, was added to dissolve the formazan crystals, and the absorbance was measured using a microtiter plate reader (SpectraMax 190, Molecular Device, USA) at a wavelength of 490 nm. The cell viability
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Fig. 3 The effect of berberine treatment on Notch1/Hes1-PTEN/Akt signaling and apoptotic signaling in MI/R-injured hearts. a Representative blots; b NICD expression; c Hes1 expression; d PTEN expression; e p-Akt/Akt ratio; f Caspase-3 expression; g Bcl-2 expression; h Bax expression; I Cleaved caspase-3 expression. NICD,
Notch1 intracellular domain; MI/R, myocardial ischemia reperfusion (30 min/4 h); V, vehicle [0.5 % sodium carboxymethylcellulose (CMC-Na) solution]; BBR, berberine 200 mg/kg/d. The results are expressed as the mean ± SEM, n = 8. aaP \ 0.01 versus Sham group, bbP \ 0.01 versus MI/R ? V group
was calculated by dividing the optical density of samples with the optical density of sham control.
centrifugation at 4 °C for 15 min at 12,000 rpm. After quantitation of protein concentration, 30 lg of total protein was separated by SDS-PAGE and then transferred to a polyvinylidene difluoride membrane (Millipore, USA). The membranes were blocked for 2 h at 37 °C with 5 % non-fat dry milk, then incubated with primary antibody including p-Akt, Akt, PTEN, Hes1, Caspase-3, Bcl-2, Bax, b-actin (Santa Cruz Technology, USA), cleaved Caspase-3 (Beyotime Biotechnology, China), Notch1 intracellular domain (NICD) (Abcam, USA) (1:1000) over night at 4 °C. After three washings with TBST, the membranes were incubated with secondary antibody in TBST solution for 30 min at 37 °C, then, washed as above. The positive protein bands were developed using a chemiluminescent system, and the bands were scanned and quantified by densitometric analysis using an image analyzer Quantity One System (Bio-Rad, Richmond, CA, USA).
Determination of cellular apoptosis After the H9c2 cardiomyocytes were fixed in paraformaldehyde (4 %) for 24 h, the cellular apoptosis was also analyzed by performing a TUNEL assay using the in situ cell death detection kit according to the manufacturer’s instructions [27]. The apoptotic index was expressed as the number of positively stained apoptotic cardiomyocytes/the total number of cardiomyocytes counted 9 100 %. Western blot analysis The myocardial and cardiomyocytes samples were lysed in lysis buffer on ice for 20 min. The lysates were clarified by
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Fig. 4 The effect of berberine and Notch1 siRNA on cell viability and apoptosis in simulated ischemia/reperfusion treated cardiomyocytes. a, b Viability of cardiomyocytes was determined by MTT and was calculated by dividing the optical density of samples with the optical density of sham control. c Left representative photomicrographs of in situ detection of apoptotic cardiomyocytes by TUNEL staining. Green fluorescence shows TUNEL-positive nuclei; blue
fluorescence shows nuclei of total cardiomyocytes; Original 200 9magnification; Right percentage of TUNEL-positive nuclei. BBR, berberine 50 lmol/L treated for 4 h; SIR, simulated ischemia/ reperfusion. The results are expressed as the mean ± SEM, n = 8. dd P \ 0.01 versus Con group, eeP \ 0.01 versus SIR group, ff P \ 0.01 versus SIR ? BBR group, ggP \ 0.01 versus SIR ? BBR ? Notch1 siRNA group (Color figure online)
Statistical analysis
reperfusion was examined. Echocardiography demonstrated a significant increased LVEF and LVFS in BBR treated group compared with the MI/R ? V group (P \ 0.01, Fig. 1). To examine whether BBR decreased myocardial apoptosis, we further measured myocardial apoptotic index, myocardial infarct size, serum LDH and CK levels after 6-h of reperfusion. Compared with the MI/R ? V group, BBR treatment also attenuated cardiac apoptosis by decreasing apoptotic index and infarct size (P \ 0.01, Fig. 2a, b). In addition, serum CK and LDH levels were also significantly reduced after 6-h of reperfusion in BBR treated rats (P \ 0.01, compared with the MI/R ? V group, Fig. 2c, d). In addition, vehicle (0.5 % CMC-Na solution) or BBR (200 mg/kg/d) treatment alone had no significant effect on the cardiac function, apoptotic index, infarct size, serum LDH and CK levels compared with the Sham group
All values are presented as mean ± SEM. Differences were compared by ANOVA followed by Bonferroni correction for post hoc t test, where appropriate. Probabilities of \0.05 were considered to be statistically significant. All of the statistical tests were performed with the GraphPad Prism software version 5.0 (GraphPad Software, Inc., San Diego, CA).
Results The effect of BBR treatment on MI/RI To investigate whether BBR has cardioprotective effect against MI/RI in rats, left ventricular function after 72-h of
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Fig. 5 The effect of berberine and Notch1 siRNA on NICD, Hes1, PTEN expression and p-Akt/Akt ratio in simulated ischemia/reperfusion treated cardiomyocytes. a Representative blots; b NICD expression; c Hes1 expression; d PTEN expression; e p-Akt/Akt ratio.
BBR, berberine 50 lmol/L treated for 4 h; SIR, simulated ischemia/ reperfusion. The results are expressed as the mean ± SEM, n = 8. ee P \ 0.01 versus SIR group, ffP \ 0.01 versus SIR ? BBR group, gg P \ 0.01 versus SIR ? BBR ? Notch1 siRNA group
(P [ 0.05, Figs. 1 and 2). These data indicate that BBR treatment might protect myocardium from apoptosis following MI/RI, thus improving cardiac function.
These data indicated that BBR confers significant antiapoptotic effect and the Notch1/Hes1 and PTEN/Akt signaling might play a role in this action.
The effect of BBR treatment on Notch1/Hes1 and PTEN/Akt signaling pathway in MI/R hearts
The effect of BBR and Notch1 siRNA treatment on cell viability and apoptosis in SIR-treated cardiomyocytes
We further measured the levels of Notch1/Hes1 and PTEN/ Akt signaling and the expression of apoptotic related protein in MI/R injured hearts. As depicted in Fig. 3, Western blotting analysis indicated that MI/RI significantly increased the ratio of p-Akt/Akt, Caspase-3 expression, cleaved Caspase-3 expression and Bax expression compared with the sham group (P \ 0.01); conversely, MI/RI produced a significant decrease in the expression of Bcl-2 (P \ 0.01, compared with sham group). Treatment with BBR produced a significant increase in the expression of NICD, Hes1 and Bcl-2 (P \ 0.01, compared with the MI/R ? V group). Besides, BBR treatment also up-regulated the ratio of p-Akt/ Akt and down-regulated the expression of PTEN, Caspase-3 and Bax (P \ 0.01, compared with the MI/R ? V group).
To investigate whether BBR has direct protective effect against MI/RI and the underlying mechanism, we performed in vitro studies in H9c2 cardiomyocytes subjected to SIR. In order to test whether experimental dosages of BBR affect the survival of H9c2 cells, cardiomyocytes were exposed to BBR (0.5, 5, 50 or 500 lmol/L) for 4 h. Then, cell viability was determined by MTT. As shown in Fig. 4a, 500 lmol/L BBR significantly affected the cell viability compared with the control group (P \ 0.01). Based on this preliminary experiment and the previous study, we chose 50 lmol/L in our study. Next, the cells were subjected to SIR treatment in the absence or presence of BBR (50 lmol/L) or Notch1 siRNA pretreatment. BBR
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Fig. 6 The effect of berberine and Notch1 siRNA on apoptotic signaling in simulated ischemia/reperfusion treated cardiomyocytes. a Representative blots; b Caspase-3 expression; c Bcl-2 expression; d Bax expression; e Cleaved caspase-3 expression. BBR, berberine
50 lmol/L treated for 4 h; SIR, simulated ischemia/reperfusion. The results are expressed as the mean ± SEM, n = 8. eeP \ 0.01 versus SIR group, ffP \ 0.01 versus SIR ? BBR group, ggP \ 0.01 versus SIR ? BBR ? Notch1 siRNA group
treatment for 4 h resulted in a significant increase in cell viability (Fig. 4b, P \ 0.01, compared with the SIR group). However, BBR-induced cardioprotective effect was significantly blunted by Notch1 siRNA (Fig. 4b, P \ 0.01, compared with the SIR ? BBR group). As shown in Fig. 4c, BBR treatment also reduced the apoptotic index, while Notch1 siRNA administration significantly blocked this protective effect (P \ 0.01, compared with the SIR ? BBR group). Compared with the SIR group, SIR ? Notch1 siRNA treatment had no significant effect on cell viability and apoptotic index (P [ 0.05). These findings indicated that BBR had direct anti-apoptotic effect against SIR injury and Notch signaling might play a key role in its protective action.
effect. As depicted in Fig. 5, BBR treatment significantly up-regulated the expression of NICD, Hes1 and the ratio of p-Akt/Akt, down-regulated PTEN expression (P \ 0.01, compared with the SIR group). However, Notch1 siRNA administration significantly blocked these effects (P \ 0.01, compared with the SIR ? BBR group). Consistently, BBR exerted significant anti-apoptotic effect by reducing the expression of Caspase-3, Bax, cleaved Caspase-3 and increasing Bcl-2 expression (Fig. 6, P \ 0.01, compared with the SIR group). These effects were also blocked by Notch1 siRNA (Fig. 6, P \ 0.01, compared with the SIR ? BBR group). Compared with the SIR group, SIR ? Notch1 siRNA treatment had no significant effect on apoptotic signaling (Fig. 6, P [ 0.05). These results further suggested that BBR’s anti-apoptotic effect may be mediated by Notch1/Hes1 signaling.
The effect of BBR and Notch1 siRNA treatment on NICD, Hes1, PTEN expression, p-Akt/Akt ratio and apoptotic signaling in SIR-treated cardiomyocytes NICD, Hes1, PTEN expression, p-Akt/Akt ratio and apoptotic signaling were examined to further investigate the underlying mechanism of BBR’s cardioprotective
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The effect of BBR and Hes1 siRNA treatment on cell viability and apoptosis in SIR-treated cardiomyocytes To further investigate the role of Hes1 in BBR’s cardioprotective action, Hes1 siRNA was utilized in the present
Apoptosis Fig. 7 The effect of berberine and Hes1 siRNA on cell viability and apoptosis in simulated ischemia/reperfusion treated cardiomyocytes. a Representative photomicrographs of in situ detection of apoptotic cardiomyocytes by TUNEL staining. Green fluorescence shows TUNEL-positive nuclei; blue fluorescence shows nuclei of total cardiomyocytes; Original 2009 magnification; b Percentage of TUNELpositive nuclei; c Viability of cardiomyocytes was determined by MTT and was calculated by dividing the optical density of samples with the optical density of sham control. SIR, simulated ischemia/reperfusion. The results are expressed as the mean ± SEM, n = 8. hh P \ 0.01 versus SIR group, ii P \ 0.01 versus SIR ? BBR group, jjP \ 0.01 versus SIR ? BBR ? Hes1 siRNA group (Color figure online)
experiment. As expected, Hes1 siRNA administration significantly blocked BBR’s protective effect by increasing the apoptotic index (Fig. 7b, P \ 0.01, compared with the SIR ? BBR group). Hes1 siRNA also decreased cell viability compared with the SIR ? BBR group (Fig. 7c, P \ 0.01). Compared with the SIR group, SIR ? Hes1 siRNA treatment had no significant effect on apoptotic index and cell viability (P [ 0.05). These findings suggested that Hes1 played a key role in BBR’s protective action. The effect of BBR and Hes1 siRNA treatment on Hes1, PTEN expression, p-Akt/Akt ratio and apoptotic signaling in SIR-treated cardiomyocytes We further measured the Hes1, PTEN expression, p-Akt/Akt ratio and apoptotic signaling to investigate the underlying mechanism. As seen in Fig. 8, BBR’s effect on Hes1 expression, PTEN expression and p-Akt/Akt ratio was
significantly blocked by Hes1 siRNA administration (P \ 0.01, compared with the SIR ? BBR group). In addition, Hes1 siRNA treatment also blocked the anti-apoptotic effect of BBR by increasing the expression of Caspase-3, Bax, cleaved Caspase-3 and decreasing Bcl-2 expression (Fig. 9, P \ 0.01, compared with the SIR ? BBR group). Although SIR ? Hes1 siRNA treatment significantly increased the expression of PTEN and decreased the ratio of p-Akt/Akt, this treatment had no significant effect on the apoptotic signaling (P \ 0.01, compared with the SIR group). These data provided the direct evidence that Notch1/Hes1 signaling played a key role in BBR’s anti-apoptotic action (Fig. 10). The effect of BBR and LY294002 treatment on cell viability and apoptosis in SIR-treated cardiomyocytes To investigate the role of Akt in BBR’s cardioprotective action, LY294002, a specific Akt inhibitor, was introduced
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Apoptosis Fig. 8 The effect of berberine and Hes1 siRNA on Hes1, PTEN expression and p-Akt/Akt ratio in simulated ischemia/ reperfusion treated cardiomyocytes. a Representative blots; b Hes1 expression; c PTEN expression; d p-Akt/Akt ratio. BBR, berberine 50 lmol/L treated for 4 h; SIR, simulated ischemia/ reperfusion. The results are expressed as the mean ± SEM, n = 8. hhP \ 0.01 versus SIR group, iiP \ 0.01 versus SIR ? BBR group, jjP \ 0.01 versus SIR ? BBR ? Hes1 siRNA group
to the present experiment. As shown in Fig. 7b, LY294002 treatment also significantly blocked BBR’s protective effect by increasing the apoptotic index (P \ 0.01, compared with the SIR ? BBR group). Compared with the SIR ? BBR group, co-treatment with LY294002 significantly reduced cell viability (Fig. 7c, P \ 0.01). Consistently, SIR ? LY294002 treatment had no significant effect on apoptotic index and cell viability compared with the SIR group, (P [ 0.05). These findings suggested that Akt might participate in the anti-apoptotic action of BBR. The effect of BBR and LY294002 treatment on pAkt/Akt ratio and apoptotic signaling in SIR-treated cardiomyocytes Similarly, we examined the effect of BBR and LY294002 treatment on Akt signaling and apoptosis related protein to determine whether the BBR’s protective action could be mediated by activation of Akt signaling. As seen in Fig. 11b, BBR’s effect on p-Akt/Akt ratio was significantly blocked by LY294002 administration (P \ 0.01, compared with the SIR ? BBR group). In addition, LY294002 treatment also blocked the anti-apoptotic effect of BBR by decreasing Bcl-2 expression and increasing the expression of Caspase-3, Bax, cleaved Caspase-3 (Fig. 11c–f,
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P \ 0.01, compared with the SIR ? BBR group). These data provided the direct evidence that Akt signaling played a pivotal role in BBR’s anti-apoptotic action.
Discussion Myocardial apoptosis is one of the major pathogenic mechanisms following MI/RI. Reducing apoptosis could prevent the loss of contractile cells, minimize MI/R-induced cardiac injury and therefore slow down or even prevent the occurrence of heart failure [28–30]. Previous studies have confirmed the ameliorative effect of BBR on MI/RI induced apoptosis [6, 31]. In this study, we also found that BBR treatment exerted cardioprotective effect, as evidenced by improved post-MI/R cardiac functional recovery and attenuated myocardial apoptosis. Notch signaling pathway is an important cell–cell communication system, which has indispensable functional activities to control tissue formation in cardiogenesis [32]. The binding of Notch1 ligand to its receptor triggers the csecretase-mediated proteolytic cleavage of NICD, which then translocates into the nucleus to form a transcriptionactivating complex, thereby mediating its downstream target gene Hes1 [33]. Notch1 is implicated in cardiac
Apoptosis
Fig. 9 The effect of berberine and Hes1 siRNA on apoptotic signaling in simulated ischemia/reperfusion treated cardiomyocytes. a Representative blots; b Caspase-3 expression; c Bcl-2 expression; d Bax expression; e Cleaved caspase-3 expression. BBR, berberine
50 lmol/L treated for 4 h; SIR, simulated ischemia/reperfusion. The results are expressed as the mean ± SEM, n = 8. hhP \ 0.01 versus SIR group, iiP \ 0.01 versus SIR ? BBR group, jjP \ 0.01 versus SIR ? BBR ? Hes1 siRNA group
development, where it regulates cardiomyocyte proliferation, myocardial trabeculation, and valve formation [34, 35], as well as in the maintenance of adult heart tissue integrity [36, 37]. Interestingly, as the myocardium matures to the adult hormonal and contractile state, the level of endogenous Notch signaling (e.g. Notch1/Hes1) decreases steadily in heart tissue [38], which may suggest its loss of control in tissue homeostasis during adult life stage. However, recent studies have demonstrated that re-activation of Notch signaling may constitute an adaptive response following pathological stress to increase survival rate or regenerate tissues. For example, pharmacological blockade of Notch1 leads to Caspase-3dependent apoptosis in adult islets, in contrast, over activation of Notch1 protects against apoptosis [39]. Furthermore, the critical role of Notch signaling has been established in the maintenance of functional homeostasis in adult heart. It is reported that Notch1 signaling activation contributes to cardioprotection provided by ischemic preconditioning and postconditioning by suppressing apoptosis [40]. Interestingly, another recent study showed that BBR diminishes side population in the
pancreatic cancer cell lines PANC-1 and MIA PaCa-2 by modulating Notch1 gene expression [16]. In our in vivo study, we found that the expression of NICD and Hes1 are significantly up-regulated by BBR treatment, indicating the pivotal role of Notch1/Hes1 signaling in BBR’s protective action. PI3K-Akt signaling is a critical signaling pathway involved in a broad range of biological processes and Akt/Protein kinase B possesses pro-survival and antiapoptotic activities. In addition, Akt deactivation has been well established to be a causal mediator of cell death [41]. On the other hand, PTEN is a nonredundant, plasma membrane lipid phosphatase that converts the phosphatidylinositol-3,4,5-phosphate into the inactive phosphatidylinositol-4,5-diphosphate, functioning as an endogenous antagonist of Akt signaling [13]. Several studies have reported the pivotal role of PTEN inhibition, which regulated the Akt signaling, in myocardial protection. Conditional cardiomyocyte-specific PTEN knockout mice showed reduced cell death and apoptosis in the in vivo model of MI/R [42]. Furthermore, Pei and colleagues reported that Notch1 signaling protected
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Apoptosis Fig. 10 The effect of berberine and LY294002 on cell viability and apoptosis in simulated ischemia/reperfusion treated cardiomyocytes. a Representative photomicrographs of in situ detection of apoptotic cardiomyocytes by TUNEL staining. Green fluorescence shows TUNEL-positive nuclei; blue fluorescence shows nuclei of total cardiomyocytes; Original 2009 magnification; b Percentage of TUNELpositive nuclei; c Viability of cardiomyocytes was determined by MTT and was calculated by dividing the optical density of samples with the optical density of sham control. SIR, simulated ischemia/reperfusion. The results are expressed as the mean ± SEM, n = 8. kk P \ 0.01 versus SIR group, ll P \ 0.01 versus SIR ? BBR group, mmP \ 0.01 versus SIR ? BBR ? LY294002 group (Color figure online)
against MI/RI partly though PTEN/Akt mediated anti-oxidative and anti-nitrative effect, thus reducing post-MI/R apoptosis and improving cardiac function recovery [15]. Zhao and colleagues have reported that Notch1 exhibited cardioprotection in ischemic preconditioning by crosstalk with the reperfusion injury salvage kinase (RISK) signaling pathway, possibly through PTEN inhibition [43]. Our data corroborates these previous findings and suggests an inverse relationship between activation of Hes1 signaling and inhibition of PTEN expression. Both Notch1 siRNA and Hes1 siRNA all significantly up-regulated PTEN expression and reduced Akt activity, thus abolishing BBR’s antiapoptotic effect. Next, we evaluated the role of Akt in BBR’s cardioprotective action. BBR has been tested to protect against oxidative damage via a PI3K/Akt-dependent mechanism in NSC34 motor neuron-like cells [44]. Additionally, Xiao and colleagues also demonstrated that BBR
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protected endothelial progenitor cell from damage of TNF-a via the PI3K/AKT/eNOS signaling pathway [45]. However, these effects were all abolished by LY294002, the specific Akt inhibitor [44, 45]. In this study, we also found that co-treatment with LY294002 markedly attenuated BBR’s cardioprotective effect. Although further study is still needed to investigate the underlying mechanisms, we do proved that Notch1/Hes1 signaling plays a key role in BBR’s anti-apoptotic action and PTEN/Akt, the down-stream signaling pathway, participates in this process. In summary, our findings suggest that BBR treatment exerts a profound cardioprotective effect against apoptosis induced by MI/RI. This protection appears to be largely due to the modulation of Notch1/Hes1-PTEN/Akt signaling pathway. These results revealed that BBR may be a promising candidate for the treatment of MI/RI in cardiac surgery and ischemic heart diseases.
Apoptosis
Fig. 11 The effect of berberine and LY294002 on p-Akt/Akt ratio and apoptotic signaling in simulated ischemia/reperfusion treated cardiomyocytes. a Representative blots; b p-Akt/Akt ratio; c Caspase3 expression; d Bcl-2 expression; e Bax expression; f Cleaved caspase-3 expression. BBR, berberine 50 lmol/L treated for 4 h; SIR,
simulated ischemia/reperfusion. The results are expressed as the mean ± SEM, n = 8. kkP \ 0.01 versus SIR group, llP \ 0.01 versus SIR ? BBR group, mmP \ 0.01 versus SIR ? BBR ? LY294002 group
Acknowledgments The work presented here was carried out in collaboration between all authors. Shiqiang Yu and Weixun Duan defined the research theme and revised the manuscript critically. Liming Yu, Feijiang Li and Guolong Zhao designed methods and experiments, carried out the laboratory experiments and wrote the paper. Yang Yang, Zhenxiao Jin, Mengen Zhai, Wenjun Yu, Lin Zhao and Wensheng Chen collected and analyzed the data, interpreted the results. The authors thank Buying Li, Xiaowu Wang and Yanyan Ma, Department of Cardiovascular Surgery, Xijing Hospital, The Fourth Military Medical University, for technical support in western blot and cell culture. This study was supported by grants from the National Natural Science Foundation of China (81470415, 81470411, 81270170) and the Natural Science Foundation of Shannxi Province (2014JM4106).
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Conflict of interest of interest.
The authors declare that they have no conflict
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