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Bisdemethoxycurcumin protects cardiomyocyte mainly depends on Nrf2/HO-1 activation mediated by PI3K/AKT pathway Xing Li, Cong Huo, Yuan Xiao, Rong Xu, Yan Liu, Xin Jia, and Xiaoming Wang Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.9b00222 • Publication Date (Web): 12 Aug 2019 Downloaded from pubs.acs.org on August 14, 2019
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Chemical Research in Toxicology
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Bisdemethoxycurcumin protects cardiomyocyte mainly
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depends on Nrf2/HO-1 activation mediated by PI3K/AKT
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pathway
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Authors:
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Xing Li , Cong Huo , Yuan Xiao
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Authors Affiliations：
†
†
†,§, Rong Xu†, Yan Liu†, Xin Jia†, Xiaoming Wang,†
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† Department of Geriatrics, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, P.R China
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§ Hong-Hui Hospital, Xi'an Jiaotong University College of Medicine, Xi'an 710054, P.R China
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Corresponding author: Prof. Xiao-Ming Wang, Department of Geriatrics, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, P.R China E-mail:
[email protected] Tel: 86-029-84775543
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Corresponding author ： Prof. Xiao-Ming Wang, Department of Geriatrics, Xijing Hospital, Fourth Military
Medical University, Xi’an 710032, PR China, Tel: +86-29-84775543 E-mail address:
[email protected]（Xiao-Ming Wang）
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ABSTRACT
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Bisdemethoxycurcumin (BDMC) is one of three curcuminoids extracted from turmeric. Unlike the
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dominant ingredient curcumin with some intensive investigations, BDMC was recently reported to
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possess potent anti-tumor, anti-inflammatory, anti-atherosclerosis, anti-obesity and anti-ageing effects.
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Considering its pharmacological effects in inflammation, atherosclerosis and obesity, this study was
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designed to examine if BDMC displays cardioprotective property. In this study, staurosporine (STS) was
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used to establish cardiomyocyte injury model. Our data revealed that BDMC significantly inhibited
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myocardial apoptosis, improved cell survival, reduced caspase-3 activity and diminished reactive oxygen
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species (ROS) production. BDMC enhanced phosphorylation of protein kinase B (AKT) and extracellular
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signal-regulated kinase (ERK) and up-regulated the expression of HO-1. Inhibition of HO-1 activity by
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using tin-protoporphyrin (SnPPIX) can restrain the anti-apoptotic effect of BDMC. Furthermore,
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translocation of Nrf2 from cytoplasm to nucleus was ablated by LY294002, although only partially by
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PD98059. Up-regulation of HO-1 was weakly suppressed by PD98059 but strongly inhibited by
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LY294002. Unlike PD98059, LY294002 negated the protective effect of BDMC.
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indicated that BDMC possessed favorable cardioprotection in a Nrf2 / HO-1-dependent manner.
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Activation of Nrf2 /HO-1 mainly depended on PI3K / AKT but not MEK/ERK signaling.
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These findings
Key words: Bisdemethoxycurcumin; Nrf2; Cardiomyocytes; Apoptosis; HO-1; PI3K / Akt;
INTRODUCTION
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Cardiovascular disease remains the number one cause for the ever-rising morbidity and mortality,
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particularly the heart disease. Apoptosis is a normal physical procedure but participate in the
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development of a variety of heart diseases1-4. With more apoptotic myocardium and the less healthy
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cardiomyocytes, cardiac pump function is severely jeopardized. Therefore, the exploitation of new
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cardio-protective drug becomes the top priority of research in cardiovascular study.
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Natural compounds are commonly used in drug discovery given their structural advantage and less
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of toxic concerns compared with synthetic molecules5, 6. Natural compounds are invaluable sources in
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drug design and development. Many natural extracts from herbs have been found to possess
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cardio-protective property. Curcuminoids are natural compounds extracted from Turmeric (Curcuma
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longa L.) widely used as a dietary supplement and as an ingredient of traditional Chinese medicine. The
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natural curcuminoids consist of curcumin, demethoxycurcumin and bisdemethoxycurcumin, with a variety
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of
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Bisdemethoxycurcumin (BDMC) is a minor constituent of curcuminoids with better solubility and
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bioavailability compared with curcumin and demethoxycurcumin8, 9. Although BDMC was well known for
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its antimutagenic and antitumor effects10, BDMC was recently indicated to suppress migration and
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proliferation of vascular smooth muscle in atherosclerosis11, and to inhibit fat synthesis in adipocytes to
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retard obesity in mice12. Besides, BDMC also relieves cell aging13. Nonetheless, little information is
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available with regards to the effect of BDMC on cardiomyocyte function.
biological
properties
including
anti-tumor,
anti-oxidant,
anti-inflammatory
effects7.
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The nuclear factor erythroid 2-related factor 2 (Nrf2) / antioxidant responsive element (ARE)
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pathway is essential to endogenous anti-oxidative signaling14. HO-1, a critical phase II antioxidant
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downstream of Nrf2, possesses anti-oxidative, anti-inflammatory, and anti-apoptosis properties15,16.
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Therefore, the Nrf2/HO-1 signaling is deemed an important target in cardio-protection17. Compared with
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the potent Nrf2 activation property for its cardioprotection18, BDMC is known to activate Nrf2 and
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up-regulate the HO-1 levels in other cells19. Little information is available for the impact of BDMC on Nrf2
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activation in cardiomyocytes. To this end, the study was designed to examine the impact of BDMC on
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cardiomyocyte apoptosis in the setting of staurosporine challenge. Ample studies have indicated the role
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of AKT, ERK, N-terminal kinases (JNK), p38 mitogen-activated protein kinases (p38MAPK) and protein
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kinase C (PKC) in the activation of Nrf2/HO-120-23. Given that BDMC was found to activate AKT and ERK
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signaling24, we examined the role of PI3K/AKT and MEK/ERK signaling in the activation of Nrf2/HO-1 in
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myocardium.
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MATERIALS AND METHODS
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Materials. Cell culture reagents were from CORNING (Corning, NY, USA) and Gibco Life
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Technologies (New York, USA). 5-bromo-2-deoxyuridine (BrdU) and collagenaseⅡ were from MP
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Biomedicals (SantaAna, California, USA). Staurosporine, LY294002 and PD98059 were from
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MedChemExpress (Monmouth Junction, NJ, USA).
Bisdemethoxycurcumin (BDMC) was from the
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Molepedia Co., Ltd. (Shanghai, China) and its chemical structure is shown in Figure 1. HO-1 inhibitor
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SnPPIX was purchased from Frontier Scientific Inc (Logan, UT, USA). LY294002, PD98059 and BDMC
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were all dissolved with DMSO. The final concentrations of drug solutions were 50μmol·L-1 for LY294002
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and PD9805925, 26, 100μmol·L-1 for BDMC(Data see supporting information), 20μmol·L-1 for SnPPIX27 and
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4μmol·L-1 for staurosporine28. The CCK-8 cell counting kits were from Dojindo (Kumamoto, Japan).
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Reactive oxygen species (ROS) detected kit was from Beyotime Biotechnology (Shanghai, China).
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TUNEL assay kit was from the Roche (Basel, Switzerland). Caspase-3 activity assay kit was from
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Thermo Fisher Scientific (Waltham, MA, USA). Primary antibodies rabbit IgG against AKT, p-AKT, ERK,
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p-ERK and Lamin B1 were purchased from Cell Signaling Technology (Boston, MA, USA). Primary
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antibodies rabbit IgG against Nrf2 and HO-1 were purchased from the abcam (Cambridge, UK).
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Anti-β-actin mouse antibody and anti-rabbit and anti-mouse HRP conjugated IgG secondary antibodies
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were purchased from Proteintech (Rosemont, IL, USA).
O
OH
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Figure 1. Chemical structure of bisdemethoxycurcumin.
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Neonatal Mouse Cardiomyocyte Culture. The primary mouse cardiomyocyte were prepared from
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3-day-old C57 mice which were purchased from the fourth military medical university as the method
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described29. Cardiomyocytes were cultured by adding 1 × 104 cells to per wells of 96 well plate and 1 ×
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105 cells to per wells of 24 well plate and 5 × 105 cells to per wells of 6-well plate. BrdU was added to the
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culture medium to inhibit fibroblast proliferation. The cells were then divided into different groups
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according to the experimental requirements and were treated when the all cell started beating.
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Cell Viability Assay. Viability of cells was tested by CCK-8 in accordance with the manufacturer's
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instructions. Approximately 5×103 cells were seeded in a 96-well plate. Every group sets 10 repeat wells.
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After overnight culture, the drugs were added in and the plate was continued to culture for 8 hours.
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Cultural media was abandoned and 10 µl CCK-8 reagent was added to each well. The plates were then
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incubated for 1.5 hour at 37°C. Absorbance value [optical density (OD)] was tested at 450 nm by a
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microplate reader (Bio-Rad, Hercules, CA, USA). The result was showed as percentage (100%).
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Caspase-3 Activity Assay. Caspase-3 activity was measured by kit at the 6 hours after drugs
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were added, and the protocol was performed according to the manufacturer's instructions. The change in
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caspase-3 activity was expressed as fluorescence intensity value (A.U.).
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ROS Detection. Intracellular ROS production was detected using an ROS assay kit. Briefly, cells
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were seeded in 96-well plates and treated with drugs. After 8 hours, the cells were incubated with 10μM
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DCFH-DA, the fluorescent probe, for 30 minutes. Then, the cells were washed and then measured at
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500/530nm using a microplate reader (Bio-Rad, Hercules, CA, USA). The ROS generation increment
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rate was expressed as fluorescence intensity value (A.U.).
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TUNEL Staining. The terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)
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assay was conducted using a TUNEL kit according to the manufacturer's instructions. Briefly, cells on the
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coverslips were fixed with 4% paraformaldehyde for 1 hour and then treated with 0.1% Triton X-100 for
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30 minutes at room temperature. The coverslip was washed with PBS and was incubated with 5% BSA
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for 30 minutes at room temperature. After removing the blocking reagent, TUNEL reaction liquid was
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added to the coverslip and the incubation was kept at 37°C for 1 hour, and the coverslip was then
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washed with PBS. The nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (Beyotime
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Biotechnology, Shanghai, China) for 10minutes. Photos were taken using a fluorescence microscope
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(Olympus, Tokyo, Japan). On the randomly chosen chose 5 fields for further analyze.
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Western Blot. Cells were lysed in RIPA with protease inhibitor and phosphoproteinase inhibitors
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(Roche, Basel, Switzerland). The protein concentration was measured using the bicinchoninic acid (BCA)
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protein assay by Thermo Fisher Scientific kit (Waltham, MA, USA). The protein samples were diluted with
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5x loading buffer and boiled for 10 minutes, it was loaded equally on a sodium dodecyl sulfate-(SDS) 10%
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polyacrylamide gel and electrophoresed, and then the proteins were transferred onto polyvinylidene
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fluoride (PVDF) membrane. The PVDF membrane was blocked with 5% skim milk powder for 1 hour and
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incubated with primary antibodies against Nrf2 (1:1000), HO-1 (1: 1000), AKT(1:1000), ERK (1:1000),
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p-AKT(1:1000), p-ERK (1:1000), β-actin (1:1000), Lamin B1 (1:1000) at 4°C overnight. After three
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washes with Tris-buffered saline plus Tween (TBST), the PVDF membrane was incubated with
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HRP-conjugated anti-mouse and anti-rabbit antibodies (1:5000) for 2 hours. The membrane was
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visualized by chemiluminescence using the ChemiDoc XRS+ imaging system (Bio-Rad, Hercules, CA,
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USA). All the blot Images were analyzed and relatively quantified by Image J 1.50i (Wayne Rasband,
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USA). Western blot was repeated in triplicates.
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Immunofluorescence. Cells were fixed with 4% paraformaldehyde for 30 minutes, followed by
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permeabilization with 0.5% Triton X-100 for 30 minutes and blocking with 5% BSA for 30 minutes. After
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washing with phosphate-buffered saline (PBS), the cells were incubated with anti-Nrf2 antibody (1:500)
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at 4°C overnight and then were probed with anti-rabbit IgG labeled with Alexa-Fluor 594 for 1 hour at
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25°C. The cells were washed with PBS and then treated with DAPI (Beyotime Biotechnology, Shanghai,
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China) for 10 minutes. Finally the images of cells were taken with microscope (Olympus, Tokyo, Japan).
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Statistical Analysis. Data were presented as means ± standard deviation (SD) and were analyzed
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by using GraphPad Prism software (La Jolla, CA, USA). For comparisons of more than two groups,
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one-way analysis of variance (ANOVA) with the Tukey post hoc test was used. For all comparisons, P
