Mol Cell Biochem DOI 10.1007/s11010-013-1956-4
Maslinic acid protects vascular smooth muscle cells from oxidative stress through Akt/Nrf2/HO-1 pathway Xiaofei Qin • Chunguang Qiu • Luosha Zhao
Received: 15 October 2013 / Accepted: 19 December 2013 Ó Springer Science+Business Media New York 2014
Abstract Maslinic acid (MA) is a natural triterpenoid widely distributed in edible and medicinal plants and has been demonstrated to possess bioactivity. However, its effect on vascular smooth muscle cells (VSMC) has not been explored yet. In this study, we found that heme oxygenase-1 (HO-1) expression was increased in VSMCs treated with MA. Furthermore, MA was found to induce Akt activation in a dose- and time-dependent manner. Wortmannin suppression of Akt was able to abolish HO-1 upregulation in VSMCs, suggesting the requirement of Akt activation for MA effect on HO-1. Further investigation indicated that Akt activation resulted in the elevated expression of Nrf2, a HO-1 promoter, in MA-treated VMSCs. Finally, we found that MA was able to protect VSMCs from oxidative stress induced by H2O2. Blocking the activation of Akt/Nrf2/HO-1 was able to compromise the protective effect of MA on VSMCs. Collectively, we provided evidence that MA protected VMSCs from oxidative stress through Akt/Nrf2/HO-1 pathway. Keywords stress
Maslinic acid VSMC Nrf2 Oxidative
Introduction The changes in biologic behaviors of vascular smooth muscle cells (VSMCs) are closely associated with the onset of atherosclerosis [1]. Accumulated evidences have
X. Qin C. Qiu L. Zhao (&) Department of Cardiology, The First Affiliated Hospital of Zhengzhou University, No.1 Jianshedong Road, Zhengzhou 450052, China e-mail: [email protected]
indicated that oxidative stress, which can be commonly observed in VSMCs, is highly required for migration, apoptosis, and proliferation of VSMCs [2]. Heme oxygenase-1 (HO-1) is an enzyme responsible for breakdown of heme into biliverdin, free iron, and carbon monoxide, which all can contribute to protect cells against oxidative damages. The abnormality of HO-1 has been demonstrated to be highly implicated in the onset of atherosclerosis [3]. Increasing HO-1 expression suppresses the progression of atherosclerosis in mice [4]. Consistently, HO-1 deficiency facilitates the formation of atherosclerotic disorders [5]. Furthermore, HO-1 is essential for vascular homeostasis and is closely associated with the proliferation, apoptosis, and migration of VSMCs [6, 7]. Thus, targeting HO-1 is an effective therapeutic strategy for vascular diseases [8]. Heme oxygenase-1 expression is inducible and can be elevated by many stimuli, such as oxidative stress and some compounds [9]. Maslinic acid (MA) is a natural compound extracted from olive pomace oil and virgin olive oil, and belongs to oleanane triterpene. MA possesses multiple pharmacological effects evidenced by in vitro and/or in vivo studies. MA inhibits the activities of serine proteases that are indispensable for human immunodeficiency virus (HIV) to spread [10]. MA also reduces cerebral ischemic injury by upregulating EAAT2 (GLT-1) glutamate reuptake [11]. Many evidences also demonstrate that MA has anti-tumor activity. It can suppress the proliferation of colon cancer cells [12]. In addition, MA possesses antioxidative activity on a wide range of tissues or cells. MA decreases the susceptibility of plasma and hepatocyte membrane to lipid peroxidation in rats [13]. Its ability to suppress oxidative stress was also observed in murine macrophage [14]. Furthermore, MA also prevents oxidative stress in astrocytes [15].
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However, the molecular mechanisms by MA reduces oxidative stress in cells have not been explored. In this study, we used VSMCs as model to investigate the pathways involved in MAs anti-oxidative activity.
Materials and methods Cell cultures 4-Week-old male Sprague–Dawley rats were sacrificed to obtain aortic smooth muscle cells. The protocols of isolating VSMCs have been approved by the Animal Research Committee of Zhengzhou University. The VSMCs were cultured in Dulbecco’s modified eagle medium (DMEM, Gibco) with 10 % fetal bovine serum (FBS, Gibco), 100 mg/ml streptomycin, and 100 U/ml penicillin, in a humidified 5 % CO2 condition at 37 °C. The isolated VSMCs were verified with a smooth muscle a-actin antibody (Santa Cruz). The primary VSMCs underwent 3–10 passages, and then, were used for subsequent experiments.
absorbances at 464 and 530 nm were measured, and the bilirubin concentration was then calculated. qPCR assays Total RNA was extracted from VSMCs with Trizol solution following the manufacturer’s instructions. Reverse transcriptase reaction was performed using RNA as templates. qPCR was performed according to TaqManÒ PCR kit protocol on Applied Biosystems 7300 real time PCR System. The primers specific for HO-1 and GAPDH were as follows. HO-1 (forward): 50 -GGGTGACAGAAGAGGC TAAGACC-30 ; HO-1 (reverse): 50 -AGATTCTCCCCTGC AGAGAGAAG-30 . GAPDH (forward): 50 -TCAGTGGTG GACCTGACCTG-30 ; GAPDH (reverse): 50 -TGCTGTAG CCAAATTCGTTG-30 . Immunoblot analysis
MA was purchased from Sigma-Aldrich and prepared with dimethyl sulfoxide (DMSO). VSMCs were treated with 5, 10, or 20 lM MA for 24 h, or pretreated with 20 lM MA 3 h prior to indicated concentrations of hydrogen peroxide (H2O2), were added to the cultures. 10 lM Wortmannin (WM, Sigma) or 10 lM zincprotoporphyrin (ZnPP, J&K Scientific Ltd, China) was added to the media 1 h before MA treatment. In the untreated control groups, the cells were treated with the same amount of DMSO as that in treated groups.
The cells were harvested, and then were lysed with M-PERÒ Mammalian Protein Extraction Reagent (Thermo Scientific). The isolated protein was separated on 10 % SDS-PAGE gels, transferred to 0.45 lm nitrocellulose membranes, blocked with PBS containing 5 % fat-free milk, and incubated with different primary antibodies. Overnight, the membranes were incubated with corresponding secondary antibodies. The bands were visualized using a chemiluminescene Western blotting detection kit (Amersham). The involved antibodies were purchased from Cell Signaling Technology (CST). They included HO-1 (1:1,000, #5853), p-Akt (1:1,000, #13038), Akt (1:1,000, #2938), b-tubulin (1:1,000, #2128), Nrf2 (1:1,000, #12721). The expression levels of these proteins were all quantified using ImageJ software.
Cell viability assay
Immunofluorescent staining
2,000 VSMCs were planted in each well of 96-well plates. The cells were treated with indicated chemicals or stimulations, and then, MTT (5 mg/ml) was added to the culture for 4 h, followed by adding 150 ll DMSO. The absorbance at 490 nm was read on Model 550 microplate reader (BioRad).
VSMCs were treated with or without MA (20 lM) for 24 h, fixed with 4 % polyoxymethylene, and incubated with p-Akt antibody (1:200, #13038, CST) for 2 h at 4 °C. DAPI was used to stain nuclei of VSMCs. The fluorescence was observed under a CLS-2SS confocal microscope (Thorlabs).
Chemicals and treatment
Nrf2 siRNA transfection HO-1 activity assay The procedures to detect HO activity have been described in previous publication [16]. Briefly, VSMCs-derived microsomes were added to a reaction mixture which contains NADPH (20 mM), hemin (10 mM), and 1 mg rat hepatic cytosol protein, followed by 1 h incubation at 37 °C in the dark. After chloroform extraction, the
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Chemical modified small interfering RNA (siRNA) specific for Nrf2 (50 -CCGGCATTTCACTGAACACAA-30 ) was purchased from Shanghai GenePharma Co.,Ltd. 40 nM siRNA was transfected into VSMCs using LipofectamineTM 2000 (Life Technologies Invitrogen) according to the instructions. 24 h later, cells were used for subsequent experiments.
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Fig. 1 MA increased HO-1 expression in a dose-dependent manner in VSMCs. a VSMCs were treated with 5, 10, 20 lM MA for 24 h. The expression levels of HO-1 proteins were detected by immunoblot analysis. b The HO-1 expression was quantified using ImageJ software. c mRNA level of HO-1 was also evaluated in the VSMCs treated with 5, 10, 20 lM MA for 24 h. d The activity of HO-1 was measured in the VSMCs treated with 5, 10, 20 lM MA for 24 h
Statistical analysis
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Different doses of MA (5, 10, and 20 lM) were added to the cultures of VSMCs, and 24 h later, the cells were subjected to isolations of protein and RNA. Immunoblot analysis of HO-1 expression revealed that MA was able to induce the expression of HO-1 in a dose-dependent manner (Fig. 1a, b). Similarly, HO-1 mRNA abundance was also elevated in MA-treated VSMCs, and the effect is positively correlated with the concentrations of MA (Fig. 1c). Furthermore, HO-1 activity was assessed in the MA-treated VSMCs. The data showed that MA increased the activity of HO-1 in a dose-dependent fashion (Fig. 1d). Subsequently, we evaluated the effect of MA on HO-1 expression and activity at different time points. Although there was no time-dependent increase in HO-1 proteins (Fig. 2a, b), qPCR assays indicated that HO-1 mRNA was
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Fig. 2 MA increased HO-1 expression in a time-dependent manner in VSMCs. a VSMCs were treated with 20 lM MA for 2, 6 and 24 h. The expression levels of HO-1 proteins were detected by immunoblot analysis. b The HO-1 expression was quantified using ImageJ software. c mRNA level of HO-1 was also evaluated in the VSMCs treated with 20 lM MA for 2, 6, and 24 h. d The activity of HO-1 was measured in the VSMCs treated with 20 lM MA for 2, 6, and 24 h
All the statistical analyses were double-tailed Student’s test. The difference was considered to be significant and very significant when P \ 0.05 and P \ 0.01, respectively.
Results
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Fig. 3 MA induced the activation of Akt pathway in VSMCs. a VSMCs were treated with 5 or 20 lM MA for 6 or 24 h. phosphorylated Akt (p-Akt) were detected by immunoblot analysis. b The p-Akt level was quantified using ImageJ software. c The p-Akt abundance in VSMCs treated with or without MA was also detected using immunofluorescent staining. Bars 50 lm
increased in VSMCs that were stimulated with 20 lM MA in a time-dependent manner (Fig. 2c). Consistently, HO-1 activity was also induced in MA-treated VSMCs in a timedependent way (Fig. 2d).
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MA activated Akt signaling in VSMCs Given that Akt activation is required for Nrf2-mediated HO-1 upregulation in cells [17], we subsequently detected if Akt signaling pathway was activated in MA-treated VSMCs. Immunoblot analysis revealed that Akt phosphorylation was heightened when MA (5 and 20 lM) was added to the cultures of VSMCs (Fig. 3a), suggesting that Akt pathway was activated by MA stimulation. The effect of MA on the Akt phosphorylation appeared to be in a time- and dosedependent manner. In addition, we used immunofluorescent staining to confirm the stimulatory effect of MA on Akt activation. Phosphorylated Akt was extensively observed in VSMCs 24 h after 20 lM MA was added to the media (Fig. 3c). Akt activation is essential for MA-induced HO-1 overexpression To further study the role of Akt signaling in MA-caused HO-1 upregulation, we employed a PI3 K inhibitor, wortmannin, to block the activation of Akt signaling in VSMCs. Wortmannin treatment revealed that HO-1 expression was greatly suppressed in MA-stimulated VSMCs when Akt activation was abolished (Fig. 4a). Also, HO-1 activity was also reduced in the cells incubated with both MA and wortmannin (Fig. 4b). The above data demonstrated that Akt activation is required for MA’s effect on HO-1 in VSMCs. MA induced Akt-dependent Nrf2 expression in VSMCs Nrf2 has been documented to be the major promoter for HO-1 expression [18, 19]. Therefore, we investigated if the effect of MA on HO-1 expression is mediated by Nrf2. Firstly, Nrf2 expression was found to be increased in
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Fig. 5 MA induced Akt-dependent Nrf2 expression in VSMCs. a VSMCs were treated with 5 or 20 lM MA for 6 or 24 h. Nrf2 expression were detected by immunoblot analysis. b The Nrf2 level was quantified using ImageJ software. c VSMCs were treated with 20 lM MA or/and 10 lM wortmannin for 24 h. Nrf2 levels were detected by immunoblot analysis. d Nrf2 level was quantified using ImageJ software
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Fig. 4 Akt activation is required for MA-caused HO-1 expression. a VSMCs were treated with 20 lM MA or/and 10 lM wortmannin for 24 h. phosphorylated Akt (p-Akt) and HO-1 were detected by immunoblot analysis. b The activity of HO-1 was measured in the VSMCs treated with 20 lM MA or/and 10 lM wortmannin for 24 h
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Fig. 6 Nrf2 is required for MA-induced HO-1 expression in VSMCs. a VSMCs were treated with 20 lM MA or/and Nrf2 siRNA for 24 h. Nrf2 and HO-1 expressions were detected by immunoblot analysis. b The activity of HO-1 was measured in the VSMCs treated with 20 lM MA or/and Nrf2 siRNA for 24 h
VSMCs treated with MA in both dose- and time-dependent manner (Fig. 5a, b). Then, we wondered if Nrf2 upregulation depended on the activation of Akt signaling. Immunoblot analysis revealed that Nrf2 overexpression was significantly suppressed in VSMCs treated with both MA and wortmannin (Fig. 5c, d). These results indicated that Akt activation is required for MA-induced Nrf2 expression in VSMCs. Nrf2 overexpression is indispensible for the effect of MA on HO-1 Next, we studied if the role of Nrf2 upregulation in the increases in HO-1 expression and activity. We employed a Nrf2-specific siRNA to inhibit the expression of Nrf2 in
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Fig. 7 Blocking Akt/Nrf2/HO1 pathway abolished the antioxidative effect of MA on VSMCs. a VSMCs were treated with different concentrations of H2O2 as well as 20 lM MA, 10 lM wortmannin, Nrf2 siRNA and 10 lM ZnPP for 24 h. The survival of cells was determined by MTT assays. b sub-G0/G1 subpopulation was quantified in VSMCs with the same treatment
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VSMCs. Both the expression level and activity of HO-1 were identified to be decreased in VSMCs when Nrf2 siRNA was transfected into the cultures (Fig. 6a, b), demonstrating that Nrf2 induction is essential for MAinduced HO-1 overexpression. The activation of Akt/Nrf2/HO-1 pathway is required for the protective effect of MA against oxidative stress on VSMCs We were subsequently interested in the effect of MA on the oxidative damages to VSMCs. H2O2 was used to induce oxidative stress in VSMCs in the subsequent experiments. MTT assays showed that MA was able to protect the cells from the cell death induced by H2O2 (Fig. 7a). Consistently, the percentages of sub-G0/G1 population was also reduced in VSMCs that was simultaneously subjected to H2O2 (400 lM) and MA (20 lM) (Fig. 7b), suggesting that MA could prevent cells from H2O2-induced apoptosis. We also aimed to confirm the role of Akt/Nrf2/HO-1 pathway in the protective role of MA in oxidative stress against VSMCs. Wortmannin, Nrf2 siRNA and ZnPP (a HO-1 inhibitor) were used to suppress the activation of Akt/Nrf2/HO-1 pathway. MTT assays and cytometrical analysis both revealed that the activation of this molecular pathway is required for the anti-oxidative effect of MA, evidenced by lower survival and higher apoptotic rates in the cells treated with H2O2 and MA as well as these inhibitors (Fig. 7b).
Discussion MA has been shown to protect against cardiotoxicity in rat [20]. However, the involved mechanisms are still
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unknown. HO-1, whose enzymatic activity contributes to anti-oxidative defense in cells, is an ideal therapeutic target for vascular disease treatment. In this study, we provided the evidences that MA was able to induce the expression level of HO-1 in VSMCs. To our knowledge, it was the first time to provide the evidence that the change in HO-1 expression is associated with the anti-oxidative effect of MA. Interestingly, MA also affects the ROS accumulation in cancer cells. MA has been demonstrated to reduce ROS level in MCF7 breast cancer cells [21]. However, ROS was generated, but not eliminated in MA-treated astrocytoma cells [22]. The molecular mechanisms underlying the controversial responses of cancer cells to MA stimulation are still unknown. Many studies have proved that specific molecular pathway is important for compound-caused HO-1 expression. For example, phosphatidylinositol 3-kinase (PI3 K)/ Akt pathway has been demonstrated to be associated with the increase in HO-1 expression induced by eckol [18]. Also, kahweol elevates HO-1 expression in dopaminergic neurons through inducing PI3 K/Akt signaling pathway [23]. However, some other pathways were also found to be involved in the HO-1 upregulation induced by natural compounds, such as ERK, JNK, and p38 MAPK pathways [24, 25]. The pathways associated with HO-1 expression induced by compounds appear to depend on the types of cells and stimuli. Nrf2 is a key regulator of HO-1 expression in cells [26]. In fact, some evidences have shown that Nrf2 expression can be induced by many natural compounds. Oridonin and curcumin have been verified to protect against arsenicinduced toxicity by increasing Nrf2 expression, according to the data from in vitro and in vivo studies [27, 28]. Resveratrol was found to induce Nrf2 expression to
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alleviate endotoxin-related toxicity [29]. In this study, we presented evidences that MA possessed anti-oxidative property in VSMCs exposed with H2O2 by inducing Nrf2 expression. Consistently, a kavalactone derivative has been shown to promote Nrf2 expression in H2O2-exposed PC12 cells [30]. In conclusion, our studies showed that Akt/Nrf2/HO-1 pathway is involved in anti-oxidative activity of MA to VSMCs. The insight into the molecular mechanism underlying the anti-oxidative effect of MA may contribute to the reasonable development of its derivatives that have more potent bioactivity. We believe that MA and its derivatives may be a promising agent for vascular disease treatment.
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Maslinic acid protects vascular smooth muscle cells from oxidative stress through Akt/Nrf2/HO-1 pathway Xiaofei Qin • Chunguang Qiu • Luosha Zhao
Received: 15 October 2013 / Accepted: 19 December 2013 Ó Springer Science+Business Media New York 2014
Abstract Maslinic acid (MA) is a natural triterpenoid widely distributed in edible and medicinal plants and has been demonstrated to possess bioactivity. However, its effect on vascular smooth muscle cells (VSMC) has not been explored yet. In this study, we found that heme oxygenase-1 (HO-1) expression was increased in VSMCs treated with MA. Furthermore, MA was found to induce Akt activation in a dose- and time-dependent manner. Wortmannin suppression of Akt was able to abolish HO-1 upregulation in VSMCs, suggesting the requirement of Akt activation for MA effect on HO-1. Further investigation indicated that Akt activation resulted in the elevated expression of Nrf2, a HO-1 promoter, in MA-treated VMSCs. Finally, we found that MA was able to protect VSMCs from oxidative stress induced by H2O2. Blocking the activation of Akt/Nrf2/HO-1 was able to compromise the protective effect of MA on VSMCs. Collectively, we provided evidence that MA protected VMSCs from oxidative stress through Akt/Nrf2/HO-1 pathway. Keywords stress
Maslinic acid VSMC Nrf2 Oxidative
Introduction The changes in biologic behaviors of vascular smooth muscle cells (VSMCs) are closely associated with the onset of atherosclerosis [1]. Accumulated evidences have
X. Qin C. Qiu L. Zhao (&) Department of Cardiology, The First Affiliated Hospital of Zhengzhou University, No.1 Jianshedong Road, Zhengzhou 450052, China e-mail: [email protected]
indicated that oxidative stress, which can be commonly observed in VSMCs, is highly required for migration, apoptosis, and proliferation of VSMCs [2]. Heme oxygenase-1 (HO-1) is an enzyme responsible for breakdown of heme into biliverdin, free iron, and carbon monoxide, which all can contribute to protect cells against oxidative damages. The abnormality of HO-1 has been demonstrated to be highly implicated in the onset of atherosclerosis [3]. Increasing HO-1 expression suppresses the progression of atherosclerosis in mice [4]. Consistently, HO-1 deficiency facilitates the formation of atherosclerotic disorders [5]. Furthermore, HO-1 is essential for vascular homeostasis and is closely associated with the proliferation, apoptosis, and migration of VSMCs [6, 7]. Thus, targeting HO-1 is an effective therapeutic strategy for vascular diseases [8]. Heme oxygenase-1 expression is inducible and can be elevated by many stimuli, such as oxidative stress and some compounds [9]. Maslinic acid (MA) is a natural compound extracted from olive pomace oil and virgin olive oil, and belongs to oleanane triterpene. MA possesses multiple pharmacological effects evidenced by in vitro and/or in vivo studies. MA inhibits the activities of serine proteases that are indispensable for human immunodeficiency virus (HIV) to spread [10]. MA also reduces cerebral ischemic injury by upregulating EAAT2 (GLT-1) glutamate reuptake [11]. Many evidences also demonstrate that MA has anti-tumor activity. It can suppress the proliferation of colon cancer cells [12]. In addition, MA possesses antioxidative activity on a wide range of tissues or cells. MA decreases the susceptibility of plasma and hepatocyte membrane to lipid peroxidation in rats [13]. Its ability to suppress oxidative stress was also observed in murine macrophage [14]. Furthermore, MA also prevents oxidative stress in astrocytes [15].
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However, the molecular mechanisms by MA reduces oxidative stress in cells have not been explored. In this study, we used VSMCs as model to investigate the pathways involved in MAs anti-oxidative activity.
Materials and methods Cell cultures 4-Week-old male Sprague–Dawley rats were sacrificed to obtain aortic smooth muscle cells. The protocols of isolating VSMCs have been approved by the Animal Research Committee of Zhengzhou University. The VSMCs were cultured in Dulbecco’s modified eagle medium (DMEM, Gibco) with 10 % fetal bovine serum (FBS, Gibco), 100 mg/ml streptomycin, and 100 U/ml penicillin, in a humidified 5 % CO2 condition at 37 °C. The isolated VSMCs were verified with a smooth muscle a-actin antibody (Santa Cruz). The primary VSMCs underwent 3–10 passages, and then, were used for subsequent experiments.
absorbances at 464 and 530 nm were measured, and the bilirubin concentration was then calculated. qPCR assays Total RNA was extracted from VSMCs with Trizol solution following the manufacturer’s instructions. Reverse transcriptase reaction was performed using RNA as templates. qPCR was performed according to TaqManÒ PCR kit protocol on Applied Biosystems 7300 real time PCR System. The primers specific for HO-1 and GAPDH were as follows. HO-1 (forward): 50 -GGGTGACAGAAGAGGC TAAGACC-30 ; HO-1 (reverse): 50 -AGATTCTCCCCTGC AGAGAGAAG-30 . GAPDH (forward): 50 -TCAGTGGTG GACCTGACCTG-30 ; GAPDH (reverse): 50 -TGCTGTAG CCAAATTCGTTG-30 . Immunoblot analysis
MA was purchased from Sigma-Aldrich and prepared with dimethyl sulfoxide (DMSO). VSMCs were treated with 5, 10, or 20 lM MA for 24 h, or pretreated with 20 lM MA 3 h prior to indicated concentrations of hydrogen peroxide (H2O2), were added to the cultures. 10 lM Wortmannin (WM, Sigma) or 10 lM zincprotoporphyrin (ZnPP, J&K Scientific Ltd, China) was added to the media 1 h before MA treatment. In the untreated control groups, the cells were treated with the same amount of DMSO as that in treated groups.
The cells were harvested, and then were lysed with M-PERÒ Mammalian Protein Extraction Reagent (Thermo Scientific). The isolated protein was separated on 10 % SDS-PAGE gels, transferred to 0.45 lm nitrocellulose membranes, blocked with PBS containing 5 % fat-free milk, and incubated with different primary antibodies. Overnight, the membranes were incubated with corresponding secondary antibodies. The bands were visualized using a chemiluminescene Western blotting detection kit (Amersham). The involved antibodies were purchased from Cell Signaling Technology (CST). They included HO-1 (1:1,000, #5853), p-Akt (1:1,000, #13038), Akt (1:1,000, #2938), b-tubulin (1:1,000, #2128), Nrf2 (1:1,000, #12721). The expression levels of these proteins were all quantified using ImageJ software.
Cell viability assay
Immunofluorescent staining
2,000 VSMCs were planted in each well of 96-well plates. The cells were treated with indicated chemicals or stimulations, and then, MTT (5 mg/ml) was added to the culture for 4 h, followed by adding 150 ll DMSO. The absorbance at 490 nm was read on Model 550 microplate reader (BioRad).
VSMCs were treated with or without MA (20 lM) for 24 h, fixed with 4 % polyoxymethylene, and incubated with p-Akt antibody (1:200, #13038, CST) for 2 h at 4 °C. DAPI was used to stain nuclei of VSMCs. The fluorescence was observed under a CLS-2SS confocal microscope (Thorlabs).
Chemicals and treatment
Nrf2 siRNA transfection HO-1 activity assay The procedures to detect HO activity have been described in previous publication [16]. Briefly, VSMCs-derived microsomes were added to a reaction mixture which contains NADPH (20 mM), hemin (10 mM), and 1 mg rat hepatic cytosol protein, followed by 1 h incubation at 37 °C in the dark. After chloroform extraction, the
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Chemical modified small interfering RNA (siRNA) specific for Nrf2 (50 -CCGGCATTTCACTGAACACAA-30 ) was purchased from Shanghai GenePharma Co.,Ltd. 40 nM siRNA was transfected into VSMCs using LipofectamineTM 2000 (Life Technologies Invitrogen) according to the instructions. 24 h later, cells were used for subsequent experiments.
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Fig. 1 MA increased HO-1 expression in a dose-dependent manner in VSMCs. a VSMCs were treated with 5, 10, 20 lM MA for 24 h. The expression levels of HO-1 proteins were detected by immunoblot analysis. b The HO-1 expression was quantified using ImageJ software. c mRNA level of HO-1 was also evaluated in the VSMCs treated with 5, 10, 20 lM MA for 24 h. d The activity of HO-1 was measured in the VSMCs treated with 5, 10, 20 lM MA for 24 h
Statistical analysis
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Different doses of MA (5, 10, and 20 lM) were added to the cultures of VSMCs, and 24 h later, the cells were subjected to isolations of protein and RNA. Immunoblot analysis of HO-1 expression revealed that MA was able to induce the expression of HO-1 in a dose-dependent manner (Fig. 1a, b). Similarly, HO-1 mRNA abundance was also elevated in MA-treated VSMCs, and the effect is positively correlated with the concentrations of MA (Fig. 1c). Furthermore, HO-1 activity was assessed in the MA-treated VSMCs. The data showed that MA increased the activity of HO-1 in a dose-dependent fashion (Fig. 1d). Subsequently, we evaluated the effect of MA on HO-1 expression and activity at different time points. Although there was no time-dependent increase in HO-1 proteins (Fig. 2a, b), qPCR assays indicated that HO-1 mRNA was
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Fig. 2 MA increased HO-1 expression in a time-dependent manner in VSMCs. a VSMCs were treated with 20 lM MA for 2, 6 and 24 h. The expression levels of HO-1 proteins were detected by immunoblot analysis. b The HO-1 expression was quantified using ImageJ software. c mRNA level of HO-1 was also evaluated in the VSMCs treated with 20 lM MA for 2, 6, and 24 h. d The activity of HO-1 was measured in the VSMCs treated with 20 lM MA for 2, 6, and 24 h
All the statistical analyses were double-tailed Student’s test. The difference was considered to be significant and very significant when P \ 0.05 and P \ 0.01, respectively.
Results
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Fig. 3 MA induced the activation of Akt pathway in VSMCs. a VSMCs were treated with 5 or 20 lM MA for 6 or 24 h. phosphorylated Akt (p-Akt) were detected by immunoblot analysis. b The p-Akt level was quantified using ImageJ software. c The p-Akt abundance in VSMCs treated with or without MA was also detected using immunofluorescent staining. Bars 50 lm
increased in VSMCs that were stimulated with 20 lM MA in a time-dependent manner (Fig. 2c). Consistently, HO-1 activity was also induced in MA-treated VSMCs in a timedependent way (Fig. 2d).
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MA activated Akt signaling in VSMCs Given that Akt activation is required for Nrf2-mediated HO-1 upregulation in cells [17], we subsequently detected if Akt signaling pathway was activated in MA-treated VSMCs. Immunoblot analysis revealed that Akt phosphorylation was heightened when MA (5 and 20 lM) was added to the cultures of VSMCs (Fig. 3a), suggesting that Akt pathway was activated by MA stimulation. The effect of MA on the Akt phosphorylation appeared to be in a time- and dosedependent manner. In addition, we used immunofluorescent staining to confirm the stimulatory effect of MA on Akt activation. Phosphorylated Akt was extensively observed in VSMCs 24 h after 20 lM MA was added to the media (Fig. 3c). Akt activation is essential for MA-induced HO-1 overexpression To further study the role of Akt signaling in MA-caused HO-1 upregulation, we employed a PI3 K inhibitor, wortmannin, to block the activation of Akt signaling in VSMCs. Wortmannin treatment revealed that HO-1 expression was greatly suppressed in MA-stimulated VSMCs when Akt activation was abolished (Fig. 4a). Also, HO-1 activity was also reduced in the cells incubated with both MA and wortmannin (Fig. 4b). The above data demonstrated that Akt activation is required for MA’s effect on HO-1 in VSMCs. MA induced Akt-dependent Nrf2 expression in VSMCs Nrf2 has been documented to be the major promoter for HO-1 expression [18, 19]. Therefore, we investigated if the effect of MA on HO-1 expression is mediated by Nrf2. Firstly, Nrf2 expression was found to be increased in
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Fig. 5 MA induced Akt-dependent Nrf2 expression in VSMCs. a VSMCs were treated with 5 or 20 lM MA for 6 or 24 h. Nrf2 expression were detected by immunoblot analysis. b The Nrf2 level was quantified using ImageJ software. c VSMCs were treated with 20 lM MA or/and 10 lM wortmannin for 24 h. Nrf2 levels were detected by immunoblot analysis. d Nrf2 level was quantified using ImageJ software
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Fig. 4 Akt activation is required for MA-caused HO-1 expression. a VSMCs were treated with 20 lM MA or/and 10 lM wortmannin for 24 h. phosphorylated Akt (p-Akt) and HO-1 were detected by immunoblot analysis. b The activity of HO-1 was measured in the VSMCs treated with 20 lM MA or/and 10 lM wortmannin for 24 h
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Fig. 6 Nrf2 is required for MA-induced HO-1 expression in VSMCs. a VSMCs were treated with 20 lM MA or/and Nrf2 siRNA for 24 h. Nrf2 and HO-1 expressions were detected by immunoblot analysis. b The activity of HO-1 was measured in the VSMCs treated with 20 lM MA or/and Nrf2 siRNA for 24 h
VSMCs treated with MA in both dose- and time-dependent manner (Fig. 5a, b). Then, we wondered if Nrf2 upregulation depended on the activation of Akt signaling. Immunoblot analysis revealed that Nrf2 overexpression was significantly suppressed in VSMCs treated with both MA and wortmannin (Fig. 5c, d). These results indicated that Akt activation is required for MA-induced Nrf2 expression in VSMCs. Nrf2 overexpression is indispensible for the effect of MA on HO-1 Next, we studied if the role of Nrf2 upregulation in the increases in HO-1 expression and activity. We employed a Nrf2-specific siRNA to inhibit the expression of Nrf2 in
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Fig. 7 Blocking Akt/Nrf2/HO1 pathway abolished the antioxidative effect of MA on VSMCs. a VSMCs were treated with different concentrations of H2O2 as well as 20 lM MA, 10 lM wortmannin, Nrf2 siRNA and 10 lM ZnPP for 24 h. The survival of cells was determined by MTT assays. b sub-G0/G1 subpopulation was quantified in VSMCs with the same treatment
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VSMCs. Both the expression level and activity of HO-1 were identified to be decreased in VSMCs when Nrf2 siRNA was transfected into the cultures (Fig. 6a, b), demonstrating that Nrf2 induction is essential for MAinduced HO-1 overexpression. The activation of Akt/Nrf2/HO-1 pathway is required for the protective effect of MA against oxidative stress on VSMCs We were subsequently interested in the effect of MA on the oxidative damages to VSMCs. H2O2 was used to induce oxidative stress in VSMCs in the subsequent experiments. MTT assays showed that MA was able to protect the cells from the cell death induced by H2O2 (Fig. 7a). Consistently, the percentages of sub-G0/G1 population was also reduced in VSMCs that was simultaneously subjected to H2O2 (400 lM) and MA (20 lM) (Fig. 7b), suggesting that MA could prevent cells from H2O2-induced apoptosis. We also aimed to confirm the role of Akt/Nrf2/HO-1 pathway in the protective role of MA in oxidative stress against VSMCs. Wortmannin, Nrf2 siRNA and ZnPP (a HO-1 inhibitor) were used to suppress the activation of Akt/Nrf2/HO-1 pathway. MTT assays and cytometrical analysis both revealed that the activation of this molecular pathway is required for the anti-oxidative effect of MA, evidenced by lower survival and higher apoptotic rates in the cells treated with H2O2 and MA as well as these inhibitors (Fig. 7b).
Discussion MA has been shown to protect against cardiotoxicity in rat [20]. However, the involved mechanisms are still
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unknown. HO-1, whose enzymatic activity contributes to anti-oxidative defense in cells, is an ideal therapeutic target for vascular disease treatment. In this study, we provided the evidences that MA was able to induce the expression level of HO-1 in VSMCs. To our knowledge, it was the first time to provide the evidence that the change in HO-1 expression is associated with the anti-oxidative effect of MA. Interestingly, MA also affects the ROS accumulation in cancer cells. MA has been demonstrated to reduce ROS level in MCF7 breast cancer cells [21]. However, ROS was generated, but not eliminated in MA-treated astrocytoma cells [22]. The molecular mechanisms underlying the controversial responses of cancer cells to MA stimulation are still unknown. Many studies have proved that specific molecular pathway is important for compound-caused HO-1 expression. For example, phosphatidylinositol 3-kinase (PI3 K)/ Akt pathway has been demonstrated to be associated with the increase in HO-1 expression induced by eckol [18]. Also, kahweol elevates HO-1 expression in dopaminergic neurons through inducing PI3 K/Akt signaling pathway [23]. However, some other pathways were also found to be involved in the HO-1 upregulation induced by natural compounds, such as ERK, JNK, and p38 MAPK pathways [24, 25]. The pathways associated with HO-1 expression induced by compounds appear to depend on the types of cells and stimuli. Nrf2 is a key regulator of HO-1 expression in cells [26]. In fact, some evidences have shown that Nrf2 expression can be induced by many natural compounds. Oridonin and curcumin have been verified to protect against arsenicinduced toxicity by increasing Nrf2 expression, according to the data from in vitro and in vivo studies [27, 28]. Resveratrol was found to induce Nrf2 expression to
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alleviate endotoxin-related toxicity [29]. In this study, we presented evidences that MA possessed anti-oxidative property in VSMCs exposed with H2O2 by inducing Nrf2 expression. Consistently, a kavalactone derivative has been shown to promote Nrf2 expression in H2O2-exposed PC12 cells [30]. In conclusion, our studies showed that Akt/Nrf2/HO-1 pathway is involved in anti-oxidative activity of MA to VSMCs. The insight into the molecular mechanism underlying the anti-oxidative effect of MA may contribute to the reasonable development of its derivatives that have more potent bioactivity. We believe that MA and its derivatives may be a promising agent for vascular disease treatment.
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