European Journal of Pharmacology 732 (2014) 76–85
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Neuropharmacology and inflammation
Protective effects of puerarin against Aß40-induced vascular dysfunction in zebrafish and human endothelial cells Xi-Lin Lu a,1, Jun-Xiu Liu b,1, Qi Wu a, Si-Mei Long a, Min-Ying Zheng a, Xiao-Li Yao a, Huixia Ren c, Yong-Gang Wang d, Wei-Wei Su d, Raymond Tak Fai Cheung e, Jin-Sheng Zeng a, Huanxing Su c,n, Zhong Pei a,nn a Department of Neurology, National Key Clinical Department and Key Discipline of Neurology, Guangdong Key Laboratory for Diagnosis and Treatment of Major Neurological Diseases, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, China b Department of Obstetrics and Gynecology, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, China c State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macau 999078, China d Key Laboratory of Gene Engineering of the Ministry of Education, Guangdong Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China e Department of Medicine, The Faculty of Medicine, The University of Hong Kong, SAR, China
art ic l e i nf o
a b s t r a c t
Article history: Received 16 October 2013 Received in revised form 18 March 2014 Accepted 24 March 2014 Available online 29 March 2014
Aß40-induced vascular dysfunction has been implicated in the pathogenesis of Alzheimer's disease (AD). In the present study, we investigated the possible protective effects of puerarin against Aß40-induced vascular damage and impairment to angiogenesis in transgenic TG (fli1:EGFP) zebrafish and human endothelial cells. Aß40 peptides at 5 μM caused an obvious reduction of vessel branches in the subintestinal vein basket, induced NADPH oxidase-derived reactive oxygen species and impaired vascular endothelial growth factor (VEGF)-dependent angiogenesis. Pretreatment with puerarin attenuated Aβ40-induced vessel reduction and impairment to angiogenesis in a dose-dependent manner. In addition, Aß40 decreased VEGF-dependent phosphorylation of Akt and eNOS, whereas puerarin treatment attenuated these detrimental effects. Furthermore, the restoration of Aß40-inducedangiogenesis impairment by puerarin was abolished by either the PI3 kinase inhibitor LY294002 (10 μM) or eNOS inhibitor L-NAME. The present study suggests that puerarin exerts its protective action probably through reduction of NADPH oxidase-derived reactive oxygen species overproduction and activation of the PI3K/Akt/eNOS pathways. & 2014 Elsevier B.V. All rights reserved.
Keywords: Aß40 Zebrafish Puerarin Vascular protection
1. Introduction Alzheimer's disease (AD) is a devastating neurological affliction, and a major cause of dementia and death in the elderly population. AD has been estimated to affect more than 15 million people worldwide. Unfortunately, there are currently no treatments available to stop or even slow AD. Therefore, the development of novel therapies that can modify or stop the disease course of AD is important. AD is generally regarded as a neurodegenerative dementia. However, accumulating clinical and experimental evidence has demonstrated that vascular pathology also has an important role in the pathogenesis of AD. For example, amyloid deposition in pial
n
Corresponding author. Tel.: þ 853 83978518; fax: þ 853 28841358. Corresponding author. Tel.: þ 86 20 87755766 8282; fax: þ 86 20 87606221. E-mail addresses: [email protected] (H. Su), [email protected] (Z. Pei). 1 These authors contributed equally to this article. nn
http://dx.doi.org/10.1016/j.ejphar.2014.03.030 0014-2999/& 2014 Elsevier B.V. All rights reserved.
and intracerebral arteries was present in over 80% of AD cases. In addition, substantial structural and functional cerebromicrovascular abnormalities were detected in AD brains (Buee et al., 1994; Fischer et al., 1990). Among the deposited Aß species, Aß40 peptides are the key contributors to cerebrovascular dysfunction in AD patients because Aß40 peptides are the major species deposited in the vasculature of the AD brain. Deposition of Aß40 damages endothelium, and causes distortion and occlusion of capillaries (Thomas et al., 1996), thereby compromising the normal function of cerebral blood vessels. Cerebrovascular dysfunction further reduces cerebral blood supply and in turn exacerbates the brain's susceptibility to Aß damage in a vicious cycle. Thus, cerebrovascular dysfunction is becoming a novel target for AD treatment. Puerarin (daidzein-8-C-glucoside), the major isoflavonoid derived from the Chinese medical herb radix puerariae (kudzu root), has been used as a traditional medicine for thousand years. In modern society, the beneficial actions of puerarin have been well documented in different disease models including
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cardiovascular diseases, aging and diabetes (Wong et al., 2011). Puerarin has multiple pharmacological effects, such as antioxidant, anti-inflammatory and vessel protection effects (Liu et al., 2011; Meng et al., 2009; Zhang et al., 2011). In the cardiovascular system, this compound improves endothelial dysfunction and enhances ischemia-induced angiogenesis (He et al., 2008). Recently, puerarin has been shown to attenuate Aβ-induced cognitive impairment (Li et al., 2010). Given that oxidative stress and impairment to angiogenesis are heavily involved in the vascular pathology in AD, we hypothesize that puerarin may protect against amyloid-βinduced vascular dysfunction.
2. Materials and methods 2.1. Isolation of puerarin Puerarin was isolated and purified by the State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, China. The purity of the compound was 4 98% (Fig. 1A and B). Puerarin was dissolved in dimethyl sulfoxide (DMSO) and stored at 20 1C until ready for use. The solution form of puerarin was then diluted in phosphate buffered saline (PBS) to the required concentration. All reagents were purchased from Sigma (Sigma, Shanghai, China) unless otherwise stated. 2.2. Preparation of reagents and cell culture Aß40 was obtained from Sigma (Sigma, Shanghai, China). Aß solution was freshly prepared for each experiment as described previously (Sato et al., 2006). Briefly, 0.55 mg Aß peptide was dissolved in 130 μL of 1 mM NaOH containing 0.01% (v/v) phenol red. Next, 10 mM NaOH was added to adjust the pH to 7.5. PBS/ ddH2O was added to obtain 500 μM Aß solution. To remove the remaining undissolved Aß, centrifugation was performed at
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15,000g for 3 min before the Aß solution was aliquoted. Human umbilical vein endothelial cells (HUVECs) were obtained from Sciencell (Carlsbad, CA, USA). HUVECs (Passage 4–7) were cultured at 37 1C in M199 media (Invitrogen, Carlsbad, NM, USA) supplemented with 10% (v/v) fetal bovine serum (FBS). Cells were maintained in a humidified atmosphere of 5% (v/v) CO2. The protective effects of puerarin against Aβ-induced vascular damage on HUVECs was evaluated using a stock solution of puerarin (100 mg/mL) prepared in PBS containing 10% (v/v) DMSO (GIBCO, Langley, VA, USA). The protective effects of puerarin against Aβ-induced vascular damage on zebrafish was evaluated using a stock solution of puerarin (1 mg/mL) prepared in sterilized Milli-Q water containing 0.5% (v/v) DMSO. Vascular endothelial growth factor (VEGF) was obtained from Sigma, St. Louis, MO, USA and prepared as a stock solution of 100 μg/mL in sterilized MilliQ water.
2.3. Zebrafish angiogenesis assay and Aß peptide treatment The transgenic zebrafish line TG (fli1: EGFP), in which endothelial cells express eGFP, was kindly provided by ZFIN (Eugene, OR, USA) and maintained as previously described (Lu et al., 2012a). Zebrafish embryos were generated by natural pair-wise mating of fish that were between 3 and 12 months old. Embryos were collected as described. 5 μM Aß peptides and/or different compounds were administered from the 12-somite stages (somitogenesis) until 3 days postfertilization (dpf; larval stage) of zebrafish development. Fresh aliquots were administrated every 24 h. Control embryos were maintained in 0.1% (v/v) DMSO in fish water. Treated larvae were anesthetized with 0.04 mg/mL tricaine and observed to select images of phenotypes. To analyze Aß-induced angiogenesis impairment in zebrafish, we measured the formation of subintestinal vessels (SIV) or angiogenesis under different conditions. For quantification of SIV formation, the total length of the SIV per larvae was analyzed at
Fig. 1. Quantitative analysis of puerarin. The results of HPLC indicate that the purity of the compound is greater than 98%.
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72 hpf and 30 larvae were examined for each group. The total length of the SIV was determined using the software Image J (version 1.43) (Yu et al., 2010). For the quantification of angiogenesis, larvae with angiogenesis in SIV were counted and the percentage of embryos per group was calculated. 30 larvae in each experiment in each group were calculated. The criteria for angiogenesis included: (a) presence of vessels spiking out of the basket structure and/or (b) extension of the SIV basket into the yolk extension region with more than seven vertical branches within the basket (Raghunath et al., 2009). 2.4. Cell invasion, migration and tube formation HUVEC invasion was investigated as described (Lu et al., 2012b; Raghunath et al., 2009). Briefly, the effect of puerarin on HUVEC invasion was measured using the 10 mm tissue culture insert (transwell) with polycarbonate membrane (8 mm pores) and 24-well companion plate. The upper side and lower side of the membrane were pre-coated with 1:30 (v/v) and 1:100 (v/v) of Matrigel, respectively. The HUVECs were resuspended in low serum (1% (v/v) FBS) medium and seeded onto the culture inserts at 5 104 cells per insert in triplicate. HUVECs at 80% confluence were serum starved in medium (0.5% (v/v) FBS) for 6 h. The medium was then changed to fresh low serum (1% (v/v) FBS) medium containing vascular endothelial growth factor (VEGF; 100 ng/mL) in the presence or absence of Aß40 (5 μmol/l), LY294002 (10 μmol/l) or L-NAME (100 μmol/l). HUVEC cells were pretreated with 1 μmol/l puerarin 30 min before addition of Aß. After an additional 8-h incubation, the inserts were then removed and washed with PBS. Non-invasive cells on the upper surface of the membrane were removed by wiping with cotton swabs. The inserts were fixed in paraformaldehyde, stained with DAPI and mounted on microscope slides. Images of the invasive cells were captured at 100 magnification using a fluorescent inverted microscope and a CCD camera. Following this, HUVEC invasion was quantified by counting the number of cells per insert using the software Metamorph Imaging Series (Molecular Devices, Tokyo, Japan). The HUVEC migration assay was performed using the wound healing method as previously described (Lam et al., 2008; Lu et al., 2012a, 2012b). The HUVECs (3 105 cells) were seeded into each well of a 24-well plate and incubated with complete medium at 37 1C and 5% (v/v) CO2. After 24-h incubation, cells were starved for an additional 24 h in low serum (0.5% (v/v) FBS) medium. The HUVECs were then scraped away horizontally in each well using a P100 pipette tip. Three randomly selected views along the scraped line were photographed on each well using a fluorescent inverted microscope and the CCD camera attached to the microscope at 50 magnification. HUVECs at 80% confluence were serum starved with medium (0.5% (v/v) FBS) for 6 h. The medium was changed to fresh low serum (1% (v/v) FBS) medium with or without VEGF (100 ng/mL) in the presence or absence of Aß40 (5 μmol/l), LY294002 (10 μmol/l) or L-NAME (100 μmol/l). HUVEC cells were pretreated with 1 μmol/l puerarin 30 min before addition of Aß. After an additional 12-h incubation, another set of images were taken by using the same method. Image analysis for signs of migration was performed using Metamorph Imaging Series. The average scraped area of each well under each condition was measured and subtracted from that of the before-treatment condition. Data are expressed as percentage wound closure relative to the wound closure area in the control medium. The wound closure area of the control cells was set at 100%. Endothelial tube formation was assessed in 24-well plates using growth factor-reduced MatrigelTM as described previously (Abdel-Malak et al., 2009; Lam et al., 2011). Briefly, growth factorreduced Matrigel (250 μL) was pipetted onto 24-well culture plates
and polymerized for 30 min at 37 1C. HUVEC cells were pretreatment with 1 μmol/l puerarin 30 min before addition of Aß. HUVECs were seeded on Matrigel-coated plates at a density of 5 104 in low serum (1% (v/v) FBS) medium containing vascular endothelial growth factor (VEGF; 100 ng/mL) in the presence or absence of Aß40 (5 μmol/l), LY294002 (10 μmol/l) or L-NAME (100 μmol/l) and were then incubated at 37 1C for 18 h. The network-like structures were examined under an inverted microscope (at 50 magnification). Tube-like structures were defined as endothelial cord formations that were connected at both ends. The tube length was quantified using the software NIH Image as reported earlier (Abdel-Malak et al., 2009; Lam et al., 2011). 2.5. Western immunoblot analysis HUVECs at 80% confluence were serum starved with medium (0.5% (v/v) FBS) for 6 h. HUVEC cells were pretreated with 1 μmol/l puerarin 30 min before addition of Aß and treated with human VEGF (100 ng/mL) in the presence or absence of Aß40 (5 μmol/l), LY294002 (10 μmol/l), or L-NAME (100 μmol/l) for up to 60 min. Whole-cell protein extracts (20 μg) were separated on 10–12.5% sodium dodecylsulfate polyacrylamide gels, and transferred to nitrocellulose membranes (Amersham, Pittsburgh, PA, USA). The blotted membranes were incubated with antibodies against Akt, phosphorylated Akt (Ser473), endothelial nitric oxide synthase (eNOS), phosphorylated eNOS (Ser1177) and β-actin. Densitometrical results were calculated by NIH image software. The primary antibodies used were anti-eNOS (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-phosphorylation of eNOS at S1177 (P-eNOS S1177) (Cell Signaling Technology, Danvers, MA, USA), antiphospho-Akt (Cell Signaling), anti-Akt (Cell Signaling), and antiβ-actin (Biosynthesis Biotechnology, Beijing, China). 2.6. Respiratory burst assay in zebrafish The respiratory burst assay was performed with zebrafish embryos by measuring oxidation of H2DCFDA to fluorescent DCF (Hermann et al., 2004; Li et al., 2013). Embryos in 96-well microtiter plates (Corning Incorporated, USA) were pretreated with 0.1, 1 or 10 μM puerarin for 30 min before Aß40 incubation. Afterwards, embryos were treated with 1 μg/mL H2DCFDA. The intensity of fluorescence was measured by a Multi-Mode Microplate Reader (Molecular Device, USA), using excitation and emission filters of 480 710 and 530 7 10 nm. The intensity of wells containing embryos was normalized to those wells containing culture media only, and the results were expressed as the percentage of fluorescence (%DCF) relative to untreated controls. 2.7. Statistical analysis All data are presented as the mean 7S.E.M. Differences among test groups were analyzed by ANOVA, using Newman–Keuls multiple comparison test (Prism 4.0, GraphPad Software, Inc., San Diego, CA, USA). A P value o0.05 was considered statistically significant.
3. Results 3.1. Puerarin attenuated Aß40-induced vascular damage in zebrafish We first examined whether puerarin can attenuate Aß40induced vascular damage in a transgenic TG (fli1:EGFP) zebrafish. Zebrafish are an ideal model for vascular studies because zebrafish are transparent and have a rapidly developing vascular system during the embryo and larval period. Transgenic TG (fli1:EGFP)
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zebrafish express EGFP in endothelial cells and thus the blood vessels of TG (fli1:EGFP) zebrafish can be easily detected in zebrafish embryos and larvae. To optimize the dose of Aß40 necessary to induce vascular damage, a dose-response curve was performed. Treatment with Aß40 peptides at 5 μM caused an obvious reduction of vessel branches in the SIV basket and slightly affected the organization of the intersegmental vessels (ISVs) without severely affecting zebrafish survival (Table 1 and Fig. 2A and C), Thus, Aß40 peptide at 5 μM was selected for the entire zebrafish study. We then measured the SIV length in different groups at 72 hpf. The SIV length was 1499 732.3 μm in the control and Table 1 Effects of Aβ peptide treatment on zebrafisah survival at 72 hpf. Treatment
Survival (%)
Control 1 μM 5 μM 10 μM
917 1.3 877 1.2 827 1.4 507 2.1
Values are means 7 S.D. for 4 replicative measurements. Tg (fli1:EGFP) y1 zebrafish were treated for up to 72 hpf with Aβ-40 (1–10 μM). Survival rate was based on 35 embryos for each treatment.
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15197 43.59 μm in the puerarin alone group, indicating that puerarin alone did not affect the formation of SIV. The SIV length was 1182.7 745.1 μm in the Aß40 group. Compared to the control group, Aß40 significantly blocked the formation of SIV. In the puerarin-pretreatment plus Aß40 groups, the SIV lengths were 1201.46 737.63 μm, 1273.547 37.84 μm, and 1409.21 742.57 μm following 0.1, 1 and 10 μM puerarin treatment, respectively (Fig. 2B and D). Compared to the Aß40 group, puerarin dose-dependently reduced Aß40-induced blockage of SIV formation in zebrafish (Fig. 2B and D). Altogether, our results suggest that puerarin between 1 and 10 μM could attenuate Aß40-induced vascular damage in zebrafish. 3.2. Puerarin attenuated Aß40-induced reactive oxygen species production in zebrafish Excessive production of reactive oxygen species is a major contributor to the vascular pathogenesis of AD. Aß40-induced reactive oxygen species in the cerebral epithelium is largely generated from superoxide-producing enzyme NADPH oxidase. The protective action of puerarin against Aß40-induced oxidative stress was examined in zebrafish embryos in vivo. NADPH oxidasederived reactive oxygen species production in zebrafish embryos was measured using dichlorofluorescein diacetate (DCF-DA), a specific dye for intracellular reactive oxygen species. Aß40 induced a rapid increase in reactive oxygen species in embryos
Fig. 2. Puerarin attenuated Aß40-induced vascular damage in zebrafish. (A) Representative brightfield and fluorescence microscopy images. (B) Effects of puerarin on the formation of subintestinal vessels (SIV) in zebrafish embryos at 72 h post-fertilization. (C) The bar chart shows quantitative data. Puerarin attenuated Aß40-induced zebrafish death in a dose-dependent manner. Asterisks indicate disorganized intersegmental vessels in the presence of Aß40 (nP o 0.05 vs. control; ANOVA). (D) The bar chart shows quantification of the SIV length in 72-hpf embryos. Puerarin (1 or 10 mM) dose-dependently attenuated the Aβ40-induced reduction of SIV length. (#Po 0.01 vs. Aß40; ANOVA).
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Fig. 3. Puerarin inhibits reactive oxygen species overproduction in zebrafish. (A) Representative fluorescence microscopy images. (B) The bar chart shows quantitative data. The embryos were treated with Aß40 in the presence or absence of puerarin. After incubation, the embryos were stained with DCFH-DA and intracellular reactive oxygen species were detected by spectrofluorometry. Puerarin (1, 10 mM) dose-dependently reduced Aß40-induced reactive oxygen species production (nPo 0.01 vs. Aβ40; ANOVA).
at 72–96 hpf (Fig. 3A). In Aß40-treated embryos, fluorescence was evident and the relative fluorescence intensity was around 188 716% of the DMSO control. As shown in Fig. 3B, the fluorescence intensity was similar between DMSO-treated and puerarintreated embryos, indicating that puerarin alone did not affect basal reactive oxygen species levels. The relative reactive oxygen species levels in Aß40-stimulated embryos were 177714%, 1447 11% and 126711% in the presence of puerarin at the concentration of 0.1, 1, or 10 μM, respectively. Altogether, the present study demonstrates that treatment with puerarin at 1 and 10 μM can reduce Aß40induced reactive oxygen species production. 3.3. Puerarin restored Aß40-induced angiogenesis impairment in zebrafish Interruption of vascular regeneration is a major pathogenic process of Aß. We then examined whether puerarin is able to restore Aß40-induced angiogenesis impairment in zebrafish. As shown in Fig. 4, VEGF at 100 ng/mL induced robust formation of ectopic SIV in transgenic zebrafish while Aß40 significantly inhibited VEGFinduced development of SIV. The percentage of ectopic SIV was 8.072.7% in the control, 5.171.6% in the Aß40, 86.574.6% in the VEGF, and 32.376.2% in the VEGF plus Aß40 groups. When incubated for up to 72 h, puerarin dose-dependently restored Aβinduced angiogenesis impairment. The percentage of ectopic SIV was 35.476.7%, 63.076.8% and 71.977.8% following 0.1, 1 and 10 μM puerarin treatment, respectively. 3.4. Puerarin protected against Aß40-induced vessel damage via the PI3K/Akt/eNOS pathways in mammalian endothelial cells in vitro To test whether the zebrafish study can be translated to preclinical studies, we further tested the protective effects of puerarin
against Aß40-induced vessel damage in cultured HUVECs. Aß40 has been shown to impair the critical steps of vascular regeneration, including invasion, migration, and tube formation of HUVECs. Consistently, our in vitro capillary tube formation assay showed that HUVECs produced an organized pseudo capillary network in the presence of VEGF in the absence of Aß40, but was unable to form a proper tube in the presence of Aß40 (Fig. 5). In agreement with the results obtained from zebrafish experiments, puerarin attenuated Aß40-induced angiogenesis impairment in HUVECs. As shown in Figs. 6 and 7, Aß40-treated HUVECs were able to partially maintain VEGF-induced pseudo capillary architecture in the presence of puerarin. Consistently, the invasion and migration assay showed that puerarin significantly reduced Aß40-induced impairment to invasion and migration of HUVECs (Figs. 6 and 7). Invasion studies showed that the percentage decrease was 35.3% following Aβ-40 treatment, and 32.9%, 13.5% and 7.7% in the Aß40 plus puerarin 0.1, 1, and 10 μM groups, respectively. Similarly, migration studies showed that the percentage decrease was 24.6% in the presence of Aß40, and 20.4%, 12.9% and 7.1% following Aß40 plus puerarin at 0.1, 1 and 10 μM, respectively. Activation of PI3K/Akt/eNOS is known to mediate VEGFinduced angiogenesis while Aß40 inhibits Akt/eNOS signaling in angiogenesis. Interestingly, puerarin has been shown to enhance angiogenesis through activation of the Akt/eNOS pathway in a rat model of myocardial infarction (Zhang et al., 2006). Therefore, PI3K/Akt/eNOS inhibitors were applied to examine the possible involvement of PI3K/Akt/eNOS in the vascular protection of puerarin. As shown in Figs. 5–7, the restoration of angiogenesis processes including tube formation, migration and invasion by puerarin were abolished by either LY294002 (10 μM), a PI3 kinase inhibitor, or L-NAME(100 μM), an eNOS inhibitor, suggesting that puerarin may recover the response capability of endothelial cells to VEGF via the PI3K/Akt/eNOS pathway. During angiogenesis,
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Fig. 4. Puerarin restored Aβ40-induced angiogenesis impairment in zebrafish (A) Representative fluorescence microscopy images. (B) The bar chart shows quantitative data. VEGF at 100 ng/mL induced robust formation of ectopic subintestinal vessels (SIV) in transgenic zebrafish while Aß40 significantly inhibited VEGF-induced development of SIV. Puerarin (0.1, 1 and 10 uM) dose-dependently restored Aß40-induced angiogenesis impairment (nPo 0.01 vs. Aß40 plus VEGF; ANOVA).
Fig. 5. Puerarin reduced Aß40-induced impairments in tube formation of HUVECs. (A) Representative microscopy images. (B) The bar chart shows quantitative data for HUVEC tube formation with different treatments on tube length SIV. HUVECs produced an organized pseudo capillary network in the presence of VEGF without Aß40, but was unable to form a proper tube in the presence of Aß40. The restoration of angiogenesis by puerarin was abolished by either LY294002 or L-NAME. (nPo 0.01 vs. Aß40 plus VEGF; #P40.05 vs. Aß40 plus VEGF; ANOVA).
PI3K/Akt/eNOS is activated through phosphorylation. We further examined the effects of puerarin on the phosphorylation of Akt and eNOS in the presence of Aß40. As shown in Figs. 8 and 9, Aß40 significantly decreased the phosphorylation of Akt (Ser473-phosphorylated) induced by VEGF (100 ng/mL). Aß40 also inhibited
the active levels of eNOS (eNOS; Ser1177-phosphorylated). In contrast, puerarin significantly attenuated Aß40-induced inhibition of phosphorylation of Akt and eNOS. Altogether, these data suggest that Akt and eNOS mediate the vascular protection of puerarin.
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Fig. 6. Puerarin reduced Aß40-induced impairment to the invasion of HUVECs. (A) Representative fluorescence microscopy images. (B) The bar chart shows quantitative data for HUVEC invasion with different treatments. Puerarin reduced Aß40-induced impairment to invasion. Restoration of the angiogenesis process by puerarin was abolished by either LY294002 or L-NAME. (nPo 0.01 vs. Aß40 plus VEGF; #P40.05 vs. Aß40 plus VEGF; ANOVA).
Fig. 7. Puerarin reduced Aß40-induced impairment to migration of HUVECs. (A) Representative fluorescence microscopy images. (B) The bar chart shows quantitative data for HUVEC migration with different treatments. Puerarin reduced Aß40-induced impairment to migration. Restoration of the angiogenesis process by puerarin was abolished by either LY294002 or L-NAME. (nPo 0.01 vs. Aß40 plus VEGF; #P40.05 vs. Aß40 plus VEGF; ANOVA).
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Fig. 8. Puerarin attenuated Aß40-induced inhibition of Akt phosphorylation Representative western blot and densitometric analysis of the phosphorylation of Akt in HUVECs after (A, B) VEGF, (C, D) Aß40 plus VEGF, (E, F) and Aß40 plus VEGF plus puerarin stimulation. nP o0.01 vs. Control (0 min). Aß40 significantly decreased the phosphorylation of Akt (Ser473-phosphorylated) induced by VEGF (100 ng/mL) whereas puerarin significantly attenuated Aß40-induced inhibition of Akt phosphorylation.
4. Discussion In the present study, we examined the protective effects of puerarin against Aß40-induced vessel dysfunction in zebrafish and cultured HUVECs. We found that puerarin significantly attenuated Aß40-induced vessel damage in vitro and in vivo. The protective effects of puerarin may be associated with its antioxidant action and activation of the Akt/eNOS-dependent signal pathway. These findings suggest that puerarin may be a potential therapeutic agent in Aß40-associated brain diseases. It is generally believed that the production of reactive oxygen species is a major contributor to the vascular pathogenesis of AD. Reactive oxygen species in brain blood vessels is largely generated from the superoxide-producing enzyme NADPH oxidase, whereas suppression of NADPH oxidase can reduce Aß40induced reactive oxygen species production and improve cerebrovascular dysfunction. Puerarin is a potent antioxidant and its antioxidant effects have been confirmed in different disease models. However, it is not clear whether puerarin can attenuate NADPH oxidase-derived reactive oxygen species. Zebrafish are an idea model to study oxidative stress in vivo because the genes and pathways involved in oxidative stress are well conserved between zebrafish and humans. Recently, the activity of NADPH oxidase has been successfully measured in vivo using the respiratory burst assay in zebrafish embryos (Li et al., 2013). In the present study, DCFDA staining was evident in Aß40-treated zebrafish embryos, suggesting that Aß40 induces excessive
reactive oxygen species generation through activation of NADPH oxidase. In contrast, puerarin significantly reduced Aß40induced reactive oxygen species generation in zebrafish embryos. The suppressive effects of puerarin on reactive oxygen species were in parallel to its protection against Aß40-induced vessel damage. Thus, suppression of NADPH oxidase-derived reactive oxygen species may, at least partially, explain its vascular protection. Soluble Aß peptides have been reported to have antiangiogenic properties and suppress blood vessel growth. Physiologically, angiogenesis is initiated by angiogenesis activators. Among endogenous angiogenesis activators, VEGF and its receptors are the major angiogenesis inducers. Interestingly, several studies have found that microvessel densities are decreased in AD brains despite elevated VEGF secretion (Sanchez et al., 2013). This paradox phenomenon suggests a failure of a reparative angiogenic mechanism in AD patients. Consistently, we found that VEGF induced robust microvascular formation in vivo using zebrafish embryos and in vitro using Matrigel assays, while Aß40 almost completely blocked VEGF-induced vessel formation in both zebrafish embryos and endothelial cells. Previously, puerarin has been shown to induce therapeutic angiogenesis in the myocardium of rats with myocardial infarction (Zhang et al., 2006). In the present study, puerarin alone did not induce normal angiogenesis. Instead, puerarin could restore VEGF-dependent angiogenesis in Aß40-treated zebrafish embryos and HUVECs. Thus, the present data suggests that the molecular mechanism
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Fig. 9. Puerarin attenuated Aß40-induced inhibition of eNOS phosphorylation. Representative western blot and densitometric analysis of eNOS phosphorylation in HUVECs after (A, B) VEGF, (C, D) Aß40 plus VEGF, (E, F) and Aß40 plus VEGF plus puerarin stimulation. nP o0.01 vs. Control (0 min). Aß40 significantly decreased the phosphorylation of eNOS (Ser1177-phosphorylated) induced by VEGF (100 ng/mL) whereas puerarin significantly attenuated the Aß40-induced inhibition of eNOS phosphorylation.
behind these effects is associated with the VEGF-dependent angiogenesis pathway. Activation of PI3K/Akt/eNOS signaling is believed to play a central role in VEGF-mediated angiogenesis. During angiogenesis, VEGF binds to its cognate receptor VEGFR and promotes PI3K/Akt activation. The activation of PI3K/Akt in turn further phosphorylates eNOS and enhances nitric oxide production, a potent mediator of angiogenesis. On the other hand, disruption of PI3K/Akt/eNOS signaling blocks VEGFdependent angiogenesis. We found that treatment with either the PI3K inhibitor almost completely abolished the restoration of angiogenesis by puerarin, suggesting that puerarin may rescue Aß40-induced angiogenesis impairment through the PI3K/Akt/ eNOS/NO signaling pathways.
Acknowledgments This study was supported by grants from the National Key Clinical Department, National Key Discipline, Guangdong Key Laboratory for Diagnosis and Treatment of Major Neurological Diseases, the National Natural Science Foundation of China (Nos. 81371255 and 81100936), Doctoral Program of Higher Education of China (No. 20110171110058), Guangdong Technological grant (Nos. 2010B050700024, 2011B050400031 and 2012B031800107), and the Natural Science Foundation of Guangdong Province (No. S2011010004860). This study was also supported by the multi year research grant University of Macau, MYRG122 (Y1-L3)ICMS12-SHX.
References 5. Conclusions In summary, the present study demonstrates the vascular protective action of puerarin against Aß40 injury in Zebrafish and HUVECs. Puerarin exerts its protective action probably through reduction of NADPH oxidase-derived reactive oxygen species overproduction and activation of PI3K/Akt/eNOS pathways. Therefore, puerarin could be used as a potent vascular protective agent in AD after further in vivo confirmation.
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He, H., Shi, M., Yang, J., Zeng, X., Qiao, H., Wu, L., Li, L., 2008. The correlation between angiogenesis and abnormal expression of SERCA2a, phospholamban and the endothelin pathway in heart failure, and improvement by puerarin. Phytother. Res. 22, 948–956. Hermann, A.C., Millard, P.J., Blake, S.L., Kim, C.H., 2004. Development of a respiratory burst assay using zebrafish kidneys and embryos. J. Immunol. Methods 292, 119–129. Lam, H.W., Lin, H., Lao, S., Gao, J., Hong, S., Leong, C., Yue, P., Kwan, Y., Leung, A., Wang, Y., Lee, S., 2008. The angiogenic effects of Angelica sinensis extract on HUVEC in vitro and zebrafish in vivo. J. Cell. Biochem. 103, 195–211. Lam, K.H., Alex, D., Lam, I.K., Tsui, S.K., Yang, Z.F., Lee, S.M., 2011. Nobiletin, a polymethoxylated flavonoid from citrus, shows anti-angiogenic activity in a zebrafish in vivo model and HUVEC in vitro model. J. Cell. Biochem. 112, 3313–3321. Li, J., Wang, G., Liu, J., Zhou, L., Dong, M., Wang, R., Li, X., Li, X., Lin, C., Niu, Y., 2010. Puerarin attenuates amyloid-beta-induced cognitive impairment through suppression of apoptosis in rat hippocampus in vivo. Eur. J. Pharmacol. 649, 195–201. Li, Z.X., Chen, J.W., Yuan, F., Huang, Y.Y., Zhao, L.Y., Li, J., Su, H.X., Liu, J., Pang, J.Y., Lin, Y.C., Lu, X.L., Pei, Z., Wang, G.L., Guan, Y.Y., 2013. Xyloketal B exhibits its antioxidant activity through induction of HO-1 in vascular endothelial cells and zebrafish. Mar. Drugs 11, 504–522. Liu, C.M., Ma, J.Q., Sun, Y.Z., 2011. Protective role of puerarin on lead-induced alterations of the hepatic glutathione antioxidant system and hyperlipidemia in rats. Food Chem. Toxicol. 49, 3119–3127. Lu, X.L., Xu, Z.L., Yao, X.L., Su, F.J., Ye, C.H., Li, J., Lin, Y.C., Wang, G.L., Zeng, J.S., Huang, R.X., Ou, J.S., Sun, H.S., Wang, L.P., Pang, J.Y., Pei, Z., 2012a. Marine cyclotripeptide X-13 promotes angiogenesis in zebrafish and euman endothelial cells via PI3K/Akt/eNOS signaling pathways. Mar. Drugs 10, 1307–1320. Lu, X.L., Luo, D., Yao, X.L., Wang, G.L., Liu, Z.Y., Li, Z.X., Li, W., Chang, F.J., Wen, L., Lee, S.M., Zhang, Z.J., Li, L., Zeng, J.S., Huang, R.X., Pei, Z., Ou, J.S., 2012b. Dl-3n-
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Neuropharmacology and inflammation
Protective effects of puerarin against Aß40-induced vascular dysfunction in zebrafish and human endothelial cells Xi-Lin Lu a,1, Jun-Xiu Liu b,1, Qi Wu a, Si-Mei Long a, Min-Ying Zheng a, Xiao-Li Yao a, Huixia Ren c, Yong-Gang Wang d, Wei-Wei Su d, Raymond Tak Fai Cheung e, Jin-Sheng Zeng a, Huanxing Su c,n, Zhong Pei a,nn a Department of Neurology, National Key Clinical Department and Key Discipline of Neurology, Guangdong Key Laboratory for Diagnosis and Treatment of Major Neurological Diseases, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, China b Department of Obstetrics and Gynecology, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, China c State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macau 999078, China d Key Laboratory of Gene Engineering of the Ministry of Education, Guangdong Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China e Department of Medicine, The Faculty of Medicine, The University of Hong Kong, SAR, China
art ic l e i nf o
a b s t r a c t
Article history: Received 16 October 2013 Received in revised form 18 March 2014 Accepted 24 March 2014 Available online 29 March 2014
Aß40-induced vascular dysfunction has been implicated in the pathogenesis of Alzheimer's disease (AD). In the present study, we investigated the possible protective effects of puerarin against Aß40-induced vascular damage and impairment to angiogenesis in transgenic TG (fli1:EGFP) zebrafish and human endothelial cells. Aß40 peptides at 5 μM caused an obvious reduction of vessel branches in the subintestinal vein basket, induced NADPH oxidase-derived reactive oxygen species and impaired vascular endothelial growth factor (VEGF)-dependent angiogenesis. Pretreatment with puerarin attenuated Aβ40-induced vessel reduction and impairment to angiogenesis in a dose-dependent manner. In addition, Aß40 decreased VEGF-dependent phosphorylation of Akt and eNOS, whereas puerarin treatment attenuated these detrimental effects. Furthermore, the restoration of Aß40-inducedangiogenesis impairment by puerarin was abolished by either the PI3 kinase inhibitor LY294002 (10 μM) or eNOS inhibitor L-NAME. The present study suggests that puerarin exerts its protective action probably through reduction of NADPH oxidase-derived reactive oxygen species overproduction and activation of the PI3K/Akt/eNOS pathways. & 2014 Elsevier B.V. All rights reserved.
Keywords: Aß40 Zebrafish Puerarin Vascular protection
1. Introduction Alzheimer's disease (AD) is a devastating neurological affliction, and a major cause of dementia and death in the elderly population. AD has been estimated to affect more than 15 million people worldwide. Unfortunately, there are currently no treatments available to stop or even slow AD. Therefore, the development of novel therapies that can modify or stop the disease course of AD is important. AD is generally regarded as a neurodegenerative dementia. However, accumulating clinical and experimental evidence has demonstrated that vascular pathology also has an important role in the pathogenesis of AD. For example, amyloid deposition in pial
n
Corresponding author. Tel.: þ 853 83978518; fax: þ 853 28841358. Corresponding author. Tel.: þ 86 20 87755766 8282; fax: þ 86 20 87606221. E-mail addresses: [email protected] (H. Su), [email protected] (Z. Pei). 1 These authors contributed equally to this article. nn
http://dx.doi.org/10.1016/j.ejphar.2014.03.030 0014-2999/& 2014 Elsevier B.V. All rights reserved.
and intracerebral arteries was present in over 80% of AD cases. In addition, substantial structural and functional cerebromicrovascular abnormalities were detected in AD brains (Buee et al., 1994; Fischer et al., 1990). Among the deposited Aß species, Aß40 peptides are the key contributors to cerebrovascular dysfunction in AD patients because Aß40 peptides are the major species deposited in the vasculature of the AD brain. Deposition of Aß40 damages endothelium, and causes distortion and occlusion of capillaries (Thomas et al., 1996), thereby compromising the normal function of cerebral blood vessels. Cerebrovascular dysfunction further reduces cerebral blood supply and in turn exacerbates the brain's susceptibility to Aß damage in a vicious cycle. Thus, cerebrovascular dysfunction is becoming a novel target for AD treatment. Puerarin (daidzein-8-C-glucoside), the major isoflavonoid derived from the Chinese medical herb radix puerariae (kudzu root), has been used as a traditional medicine for thousand years. In modern society, the beneficial actions of puerarin have been well documented in different disease models including
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cardiovascular diseases, aging and diabetes (Wong et al., 2011). Puerarin has multiple pharmacological effects, such as antioxidant, anti-inflammatory and vessel protection effects (Liu et al., 2011; Meng et al., 2009; Zhang et al., 2011). In the cardiovascular system, this compound improves endothelial dysfunction and enhances ischemia-induced angiogenesis (He et al., 2008). Recently, puerarin has been shown to attenuate Aβ-induced cognitive impairment (Li et al., 2010). Given that oxidative stress and impairment to angiogenesis are heavily involved in the vascular pathology in AD, we hypothesize that puerarin may protect against amyloid-βinduced vascular dysfunction.
2. Materials and methods 2.1. Isolation of puerarin Puerarin was isolated and purified by the State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, China. The purity of the compound was 4 98% (Fig. 1A and B). Puerarin was dissolved in dimethyl sulfoxide (DMSO) and stored at 20 1C until ready for use. The solution form of puerarin was then diluted in phosphate buffered saline (PBS) to the required concentration. All reagents were purchased from Sigma (Sigma, Shanghai, China) unless otherwise stated. 2.2. Preparation of reagents and cell culture Aß40 was obtained from Sigma (Sigma, Shanghai, China). Aß solution was freshly prepared for each experiment as described previously (Sato et al., 2006). Briefly, 0.55 mg Aß peptide was dissolved in 130 μL of 1 mM NaOH containing 0.01% (v/v) phenol red. Next, 10 mM NaOH was added to adjust the pH to 7.5. PBS/ ddH2O was added to obtain 500 μM Aß solution. To remove the remaining undissolved Aß, centrifugation was performed at
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15,000g for 3 min before the Aß solution was aliquoted. Human umbilical vein endothelial cells (HUVECs) were obtained from Sciencell (Carlsbad, CA, USA). HUVECs (Passage 4–7) were cultured at 37 1C in M199 media (Invitrogen, Carlsbad, NM, USA) supplemented with 10% (v/v) fetal bovine serum (FBS). Cells were maintained in a humidified atmosphere of 5% (v/v) CO2. The protective effects of puerarin against Aβ-induced vascular damage on HUVECs was evaluated using a stock solution of puerarin (100 mg/mL) prepared in PBS containing 10% (v/v) DMSO (GIBCO, Langley, VA, USA). The protective effects of puerarin against Aβ-induced vascular damage on zebrafish was evaluated using a stock solution of puerarin (1 mg/mL) prepared in sterilized Milli-Q water containing 0.5% (v/v) DMSO. Vascular endothelial growth factor (VEGF) was obtained from Sigma, St. Louis, MO, USA and prepared as a stock solution of 100 μg/mL in sterilized MilliQ water.
2.3. Zebrafish angiogenesis assay and Aß peptide treatment The transgenic zebrafish line TG (fli1: EGFP), in which endothelial cells express eGFP, was kindly provided by ZFIN (Eugene, OR, USA) and maintained as previously described (Lu et al., 2012a). Zebrafish embryos were generated by natural pair-wise mating of fish that were between 3 and 12 months old. Embryos were collected as described. 5 μM Aß peptides and/or different compounds were administered from the 12-somite stages (somitogenesis) until 3 days postfertilization (dpf; larval stage) of zebrafish development. Fresh aliquots were administrated every 24 h. Control embryos were maintained in 0.1% (v/v) DMSO in fish water. Treated larvae were anesthetized with 0.04 mg/mL tricaine and observed to select images of phenotypes. To analyze Aß-induced angiogenesis impairment in zebrafish, we measured the formation of subintestinal vessels (SIV) or angiogenesis under different conditions. For quantification of SIV formation, the total length of the SIV per larvae was analyzed at
Fig. 1. Quantitative analysis of puerarin. The results of HPLC indicate that the purity of the compound is greater than 98%.
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72 hpf and 30 larvae were examined for each group. The total length of the SIV was determined using the software Image J (version 1.43) (Yu et al., 2010). For the quantification of angiogenesis, larvae with angiogenesis in SIV were counted and the percentage of embryos per group was calculated. 30 larvae in each experiment in each group were calculated. The criteria for angiogenesis included: (a) presence of vessels spiking out of the basket structure and/or (b) extension of the SIV basket into the yolk extension region with more than seven vertical branches within the basket (Raghunath et al., 2009). 2.4. Cell invasion, migration and tube formation HUVEC invasion was investigated as described (Lu et al., 2012b; Raghunath et al., 2009). Briefly, the effect of puerarin on HUVEC invasion was measured using the 10 mm tissue culture insert (transwell) with polycarbonate membrane (8 mm pores) and 24-well companion plate. The upper side and lower side of the membrane were pre-coated with 1:30 (v/v) and 1:100 (v/v) of Matrigel, respectively. The HUVECs were resuspended in low serum (1% (v/v) FBS) medium and seeded onto the culture inserts at 5 104 cells per insert in triplicate. HUVECs at 80% confluence were serum starved in medium (0.5% (v/v) FBS) for 6 h. The medium was then changed to fresh low serum (1% (v/v) FBS) medium containing vascular endothelial growth factor (VEGF; 100 ng/mL) in the presence or absence of Aß40 (5 μmol/l), LY294002 (10 μmol/l) or L-NAME (100 μmol/l). HUVEC cells were pretreated with 1 μmol/l puerarin 30 min before addition of Aß. After an additional 8-h incubation, the inserts were then removed and washed with PBS. Non-invasive cells on the upper surface of the membrane were removed by wiping with cotton swabs. The inserts were fixed in paraformaldehyde, stained with DAPI and mounted on microscope slides. Images of the invasive cells were captured at 100 magnification using a fluorescent inverted microscope and a CCD camera. Following this, HUVEC invasion was quantified by counting the number of cells per insert using the software Metamorph Imaging Series (Molecular Devices, Tokyo, Japan). The HUVEC migration assay was performed using the wound healing method as previously described (Lam et al., 2008; Lu et al., 2012a, 2012b). The HUVECs (3 105 cells) were seeded into each well of a 24-well plate and incubated with complete medium at 37 1C and 5% (v/v) CO2. After 24-h incubation, cells were starved for an additional 24 h in low serum (0.5% (v/v) FBS) medium. The HUVECs were then scraped away horizontally in each well using a P100 pipette tip. Three randomly selected views along the scraped line were photographed on each well using a fluorescent inverted microscope and the CCD camera attached to the microscope at 50 magnification. HUVECs at 80% confluence were serum starved with medium (0.5% (v/v) FBS) for 6 h. The medium was changed to fresh low serum (1% (v/v) FBS) medium with or without VEGF (100 ng/mL) in the presence or absence of Aß40 (5 μmol/l), LY294002 (10 μmol/l) or L-NAME (100 μmol/l). HUVEC cells were pretreated with 1 μmol/l puerarin 30 min before addition of Aß. After an additional 12-h incubation, another set of images were taken by using the same method. Image analysis for signs of migration was performed using Metamorph Imaging Series. The average scraped area of each well under each condition was measured and subtracted from that of the before-treatment condition. Data are expressed as percentage wound closure relative to the wound closure area in the control medium. The wound closure area of the control cells was set at 100%. Endothelial tube formation was assessed in 24-well plates using growth factor-reduced MatrigelTM as described previously (Abdel-Malak et al., 2009; Lam et al., 2011). Briefly, growth factorreduced Matrigel (250 μL) was pipetted onto 24-well culture plates
and polymerized for 30 min at 37 1C. HUVEC cells were pretreatment with 1 μmol/l puerarin 30 min before addition of Aß. HUVECs were seeded on Matrigel-coated plates at a density of 5 104 in low serum (1% (v/v) FBS) medium containing vascular endothelial growth factor (VEGF; 100 ng/mL) in the presence or absence of Aß40 (5 μmol/l), LY294002 (10 μmol/l) or L-NAME (100 μmol/l) and were then incubated at 37 1C for 18 h. The network-like structures were examined under an inverted microscope (at 50 magnification). Tube-like structures were defined as endothelial cord formations that were connected at both ends. The tube length was quantified using the software NIH Image as reported earlier (Abdel-Malak et al., 2009; Lam et al., 2011). 2.5. Western immunoblot analysis HUVECs at 80% confluence were serum starved with medium (0.5% (v/v) FBS) for 6 h. HUVEC cells were pretreated with 1 μmol/l puerarin 30 min before addition of Aß and treated with human VEGF (100 ng/mL) in the presence or absence of Aß40 (5 μmol/l), LY294002 (10 μmol/l), or L-NAME (100 μmol/l) for up to 60 min. Whole-cell protein extracts (20 μg) were separated on 10–12.5% sodium dodecylsulfate polyacrylamide gels, and transferred to nitrocellulose membranes (Amersham, Pittsburgh, PA, USA). The blotted membranes were incubated with antibodies against Akt, phosphorylated Akt (Ser473), endothelial nitric oxide synthase (eNOS), phosphorylated eNOS (Ser1177) and β-actin. Densitometrical results were calculated by NIH image software. The primary antibodies used were anti-eNOS (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-phosphorylation of eNOS at S1177 (P-eNOS S1177) (Cell Signaling Technology, Danvers, MA, USA), antiphospho-Akt (Cell Signaling), anti-Akt (Cell Signaling), and antiβ-actin (Biosynthesis Biotechnology, Beijing, China). 2.6. Respiratory burst assay in zebrafish The respiratory burst assay was performed with zebrafish embryos by measuring oxidation of H2DCFDA to fluorescent DCF (Hermann et al., 2004; Li et al., 2013). Embryos in 96-well microtiter plates (Corning Incorporated, USA) were pretreated with 0.1, 1 or 10 μM puerarin for 30 min before Aß40 incubation. Afterwards, embryos were treated with 1 μg/mL H2DCFDA. The intensity of fluorescence was measured by a Multi-Mode Microplate Reader (Molecular Device, USA), using excitation and emission filters of 480 710 and 530 7 10 nm. The intensity of wells containing embryos was normalized to those wells containing culture media only, and the results were expressed as the percentage of fluorescence (%DCF) relative to untreated controls. 2.7. Statistical analysis All data are presented as the mean 7S.E.M. Differences among test groups were analyzed by ANOVA, using Newman–Keuls multiple comparison test (Prism 4.0, GraphPad Software, Inc., San Diego, CA, USA). A P value o0.05 was considered statistically significant.
3. Results 3.1. Puerarin attenuated Aß40-induced vascular damage in zebrafish We first examined whether puerarin can attenuate Aß40induced vascular damage in a transgenic TG (fli1:EGFP) zebrafish. Zebrafish are an ideal model for vascular studies because zebrafish are transparent and have a rapidly developing vascular system during the embryo and larval period. Transgenic TG (fli1:EGFP)
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zebrafish express EGFP in endothelial cells and thus the blood vessels of TG (fli1:EGFP) zebrafish can be easily detected in zebrafish embryos and larvae. To optimize the dose of Aß40 necessary to induce vascular damage, a dose-response curve was performed. Treatment with Aß40 peptides at 5 μM caused an obvious reduction of vessel branches in the SIV basket and slightly affected the organization of the intersegmental vessels (ISVs) without severely affecting zebrafish survival (Table 1 and Fig. 2A and C), Thus, Aß40 peptide at 5 μM was selected for the entire zebrafish study. We then measured the SIV length in different groups at 72 hpf. The SIV length was 1499 732.3 μm in the control and Table 1 Effects of Aβ peptide treatment on zebrafisah survival at 72 hpf. Treatment
Survival (%)
Control 1 μM 5 μM 10 μM
917 1.3 877 1.2 827 1.4 507 2.1
Values are means 7 S.D. for 4 replicative measurements. Tg (fli1:EGFP) y1 zebrafish were treated for up to 72 hpf with Aβ-40 (1–10 μM). Survival rate was based on 35 embryos for each treatment.
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15197 43.59 μm in the puerarin alone group, indicating that puerarin alone did not affect the formation of SIV. The SIV length was 1182.7 745.1 μm in the Aß40 group. Compared to the control group, Aß40 significantly blocked the formation of SIV. In the puerarin-pretreatment plus Aß40 groups, the SIV lengths were 1201.46 737.63 μm, 1273.547 37.84 μm, and 1409.21 742.57 μm following 0.1, 1 and 10 μM puerarin treatment, respectively (Fig. 2B and D). Compared to the Aß40 group, puerarin dose-dependently reduced Aß40-induced blockage of SIV formation in zebrafish (Fig. 2B and D). Altogether, our results suggest that puerarin between 1 and 10 μM could attenuate Aß40-induced vascular damage in zebrafish. 3.2. Puerarin attenuated Aß40-induced reactive oxygen species production in zebrafish Excessive production of reactive oxygen species is a major contributor to the vascular pathogenesis of AD. Aß40-induced reactive oxygen species in the cerebral epithelium is largely generated from superoxide-producing enzyme NADPH oxidase. The protective action of puerarin against Aß40-induced oxidative stress was examined in zebrafish embryos in vivo. NADPH oxidasederived reactive oxygen species production in zebrafish embryos was measured using dichlorofluorescein diacetate (DCF-DA), a specific dye for intracellular reactive oxygen species. Aß40 induced a rapid increase in reactive oxygen species in embryos
Fig. 2. Puerarin attenuated Aß40-induced vascular damage in zebrafish. (A) Representative brightfield and fluorescence microscopy images. (B) Effects of puerarin on the formation of subintestinal vessels (SIV) in zebrafish embryos at 72 h post-fertilization. (C) The bar chart shows quantitative data. Puerarin attenuated Aß40-induced zebrafish death in a dose-dependent manner. Asterisks indicate disorganized intersegmental vessels in the presence of Aß40 (nP o 0.05 vs. control; ANOVA). (D) The bar chart shows quantification of the SIV length in 72-hpf embryos. Puerarin (1 or 10 mM) dose-dependently attenuated the Aβ40-induced reduction of SIV length. (#Po 0.01 vs. Aß40; ANOVA).
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Fig. 3. Puerarin inhibits reactive oxygen species overproduction in zebrafish. (A) Representative fluorescence microscopy images. (B) The bar chart shows quantitative data. The embryos were treated with Aß40 in the presence or absence of puerarin. After incubation, the embryos were stained with DCFH-DA and intracellular reactive oxygen species were detected by spectrofluorometry. Puerarin (1, 10 mM) dose-dependently reduced Aß40-induced reactive oxygen species production (nPo 0.01 vs. Aβ40; ANOVA).
at 72–96 hpf (Fig. 3A). In Aß40-treated embryos, fluorescence was evident and the relative fluorescence intensity was around 188 716% of the DMSO control. As shown in Fig. 3B, the fluorescence intensity was similar between DMSO-treated and puerarintreated embryos, indicating that puerarin alone did not affect basal reactive oxygen species levels. The relative reactive oxygen species levels in Aß40-stimulated embryos were 177714%, 1447 11% and 126711% in the presence of puerarin at the concentration of 0.1, 1, or 10 μM, respectively. Altogether, the present study demonstrates that treatment with puerarin at 1 and 10 μM can reduce Aß40induced reactive oxygen species production. 3.3. Puerarin restored Aß40-induced angiogenesis impairment in zebrafish Interruption of vascular regeneration is a major pathogenic process of Aß. We then examined whether puerarin is able to restore Aß40-induced angiogenesis impairment in zebrafish. As shown in Fig. 4, VEGF at 100 ng/mL induced robust formation of ectopic SIV in transgenic zebrafish while Aß40 significantly inhibited VEGFinduced development of SIV. The percentage of ectopic SIV was 8.072.7% in the control, 5.171.6% in the Aß40, 86.574.6% in the VEGF, and 32.376.2% in the VEGF plus Aß40 groups. When incubated for up to 72 h, puerarin dose-dependently restored Aβinduced angiogenesis impairment. The percentage of ectopic SIV was 35.476.7%, 63.076.8% and 71.977.8% following 0.1, 1 and 10 μM puerarin treatment, respectively. 3.4. Puerarin protected against Aß40-induced vessel damage via the PI3K/Akt/eNOS pathways in mammalian endothelial cells in vitro To test whether the zebrafish study can be translated to preclinical studies, we further tested the protective effects of puerarin
against Aß40-induced vessel damage in cultured HUVECs. Aß40 has been shown to impair the critical steps of vascular regeneration, including invasion, migration, and tube formation of HUVECs. Consistently, our in vitro capillary tube formation assay showed that HUVECs produced an organized pseudo capillary network in the presence of VEGF in the absence of Aß40, but was unable to form a proper tube in the presence of Aß40 (Fig. 5). In agreement with the results obtained from zebrafish experiments, puerarin attenuated Aß40-induced angiogenesis impairment in HUVECs. As shown in Figs. 6 and 7, Aß40-treated HUVECs were able to partially maintain VEGF-induced pseudo capillary architecture in the presence of puerarin. Consistently, the invasion and migration assay showed that puerarin significantly reduced Aß40-induced impairment to invasion and migration of HUVECs (Figs. 6 and 7). Invasion studies showed that the percentage decrease was 35.3% following Aβ-40 treatment, and 32.9%, 13.5% and 7.7% in the Aß40 plus puerarin 0.1, 1, and 10 μM groups, respectively. Similarly, migration studies showed that the percentage decrease was 24.6% in the presence of Aß40, and 20.4%, 12.9% and 7.1% following Aß40 plus puerarin at 0.1, 1 and 10 μM, respectively. Activation of PI3K/Akt/eNOS is known to mediate VEGFinduced angiogenesis while Aß40 inhibits Akt/eNOS signaling in angiogenesis. Interestingly, puerarin has been shown to enhance angiogenesis through activation of the Akt/eNOS pathway in a rat model of myocardial infarction (Zhang et al., 2006). Therefore, PI3K/Akt/eNOS inhibitors were applied to examine the possible involvement of PI3K/Akt/eNOS in the vascular protection of puerarin. As shown in Figs. 5–7, the restoration of angiogenesis processes including tube formation, migration and invasion by puerarin were abolished by either LY294002 (10 μM), a PI3 kinase inhibitor, or L-NAME(100 μM), an eNOS inhibitor, suggesting that puerarin may recover the response capability of endothelial cells to VEGF via the PI3K/Akt/eNOS pathway. During angiogenesis,
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Fig. 4. Puerarin restored Aβ40-induced angiogenesis impairment in zebrafish (A) Representative fluorescence microscopy images. (B) The bar chart shows quantitative data. VEGF at 100 ng/mL induced robust formation of ectopic subintestinal vessels (SIV) in transgenic zebrafish while Aß40 significantly inhibited VEGF-induced development of SIV. Puerarin (0.1, 1 and 10 uM) dose-dependently restored Aß40-induced angiogenesis impairment (nPo 0.01 vs. Aß40 plus VEGF; ANOVA).
Fig. 5. Puerarin reduced Aß40-induced impairments in tube formation of HUVECs. (A) Representative microscopy images. (B) The bar chart shows quantitative data for HUVEC tube formation with different treatments on tube length SIV. HUVECs produced an organized pseudo capillary network in the presence of VEGF without Aß40, but was unable to form a proper tube in the presence of Aß40. The restoration of angiogenesis by puerarin was abolished by either LY294002 or L-NAME. (nPo 0.01 vs. Aß40 plus VEGF; #P40.05 vs. Aß40 plus VEGF; ANOVA).
PI3K/Akt/eNOS is activated through phosphorylation. We further examined the effects of puerarin on the phosphorylation of Akt and eNOS in the presence of Aß40. As shown in Figs. 8 and 9, Aß40 significantly decreased the phosphorylation of Akt (Ser473-phosphorylated) induced by VEGF (100 ng/mL). Aß40 also inhibited
the active levels of eNOS (eNOS; Ser1177-phosphorylated). In contrast, puerarin significantly attenuated Aß40-induced inhibition of phosphorylation of Akt and eNOS. Altogether, these data suggest that Akt and eNOS mediate the vascular protection of puerarin.
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Fig. 6. Puerarin reduced Aß40-induced impairment to the invasion of HUVECs. (A) Representative fluorescence microscopy images. (B) The bar chart shows quantitative data for HUVEC invasion with different treatments. Puerarin reduced Aß40-induced impairment to invasion. Restoration of the angiogenesis process by puerarin was abolished by either LY294002 or L-NAME. (nPo 0.01 vs. Aß40 plus VEGF; #P40.05 vs. Aß40 plus VEGF; ANOVA).
Fig. 7. Puerarin reduced Aß40-induced impairment to migration of HUVECs. (A) Representative fluorescence microscopy images. (B) The bar chart shows quantitative data for HUVEC migration with different treatments. Puerarin reduced Aß40-induced impairment to migration. Restoration of the angiogenesis process by puerarin was abolished by either LY294002 or L-NAME. (nPo 0.01 vs. Aß40 plus VEGF; #P40.05 vs. Aß40 plus VEGF; ANOVA).
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Fig. 8. Puerarin attenuated Aß40-induced inhibition of Akt phosphorylation Representative western blot and densitometric analysis of the phosphorylation of Akt in HUVECs after (A, B) VEGF, (C, D) Aß40 plus VEGF, (E, F) and Aß40 plus VEGF plus puerarin stimulation. nP o0.01 vs. Control (0 min). Aß40 significantly decreased the phosphorylation of Akt (Ser473-phosphorylated) induced by VEGF (100 ng/mL) whereas puerarin significantly attenuated Aß40-induced inhibition of Akt phosphorylation.
4. Discussion In the present study, we examined the protective effects of puerarin against Aß40-induced vessel dysfunction in zebrafish and cultured HUVECs. We found that puerarin significantly attenuated Aß40-induced vessel damage in vitro and in vivo. The protective effects of puerarin may be associated with its antioxidant action and activation of the Akt/eNOS-dependent signal pathway. These findings suggest that puerarin may be a potential therapeutic agent in Aß40-associated brain diseases. It is generally believed that the production of reactive oxygen species is a major contributor to the vascular pathogenesis of AD. Reactive oxygen species in brain blood vessels is largely generated from the superoxide-producing enzyme NADPH oxidase, whereas suppression of NADPH oxidase can reduce Aß40induced reactive oxygen species production and improve cerebrovascular dysfunction. Puerarin is a potent antioxidant and its antioxidant effects have been confirmed in different disease models. However, it is not clear whether puerarin can attenuate NADPH oxidase-derived reactive oxygen species. Zebrafish are an idea model to study oxidative stress in vivo because the genes and pathways involved in oxidative stress are well conserved between zebrafish and humans. Recently, the activity of NADPH oxidase has been successfully measured in vivo using the respiratory burst assay in zebrafish embryos (Li et al., 2013). In the present study, DCFDA staining was evident in Aß40-treated zebrafish embryos, suggesting that Aß40 induces excessive
reactive oxygen species generation through activation of NADPH oxidase. In contrast, puerarin significantly reduced Aß40induced reactive oxygen species generation in zebrafish embryos. The suppressive effects of puerarin on reactive oxygen species were in parallel to its protection against Aß40-induced vessel damage. Thus, suppression of NADPH oxidase-derived reactive oxygen species may, at least partially, explain its vascular protection. Soluble Aß peptides have been reported to have antiangiogenic properties and suppress blood vessel growth. Physiologically, angiogenesis is initiated by angiogenesis activators. Among endogenous angiogenesis activators, VEGF and its receptors are the major angiogenesis inducers. Interestingly, several studies have found that microvessel densities are decreased in AD brains despite elevated VEGF secretion (Sanchez et al., 2013). This paradox phenomenon suggests a failure of a reparative angiogenic mechanism in AD patients. Consistently, we found that VEGF induced robust microvascular formation in vivo using zebrafish embryos and in vitro using Matrigel assays, while Aß40 almost completely blocked VEGF-induced vessel formation in both zebrafish embryos and endothelial cells. Previously, puerarin has been shown to induce therapeutic angiogenesis in the myocardium of rats with myocardial infarction (Zhang et al., 2006). In the present study, puerarin alone did not induce normal angiogenesis. Instead, puerarin could restore VEGF-dependent angiogenesis in Aß40-treated zebrafish embryos and HUVECs. Thus, the present data suggests that the molecular mechanism
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Fig. 9. Puerarin attenuated Aß40-induced inhibition of eNOS phosphorylation. Representative western blot and densitometric analysis of eNOS phosphorylation in HUVECs after (A, B) VEGF, (C, D) Aß40 plus VEGF, (E, F) and Aß40 plus VEGF plus puerarin stimulation. nP o0.01 vs. Control (0 min). Aß40 significantly decreased the phosphorylation of eNOS (Ser1177-phosphorylated) induced by VEGF (100 ng/mL) whereas puerarin significantly attenuated the Aß40-induced inhibition of eNOS phosphorylation.
behind these effects is associated with the VEGF-dependent angiogenesis pathway. Activation of PI3K/Akt/eNOS signaling is believed to play a central role in VEGF-mediated angiogenesis. During angiogenesis, VEGF binds to its cognate receptor VEGFR and promotes PI3K/Akt activation. The activation of PI3K/Akt in turn further phosphorylates eNOS and enhances nitric oxide production, a potent mediator of angiogenesis. On the other hand, disruption of PI3K/Akt/eNOS signaling blocks VEGFdependent angiogenesis. We found that treatment with either the PI3K inhibitor almost completely abolished the restoration of angiogenesis by puerarin, suggesting that puerarin may rescue Aß40-induced angiogenesis impairment through the PI3K/Akt/ eNOS/NO signaling pathways.
Acknowledgments This study was supported by grants from the National Key Clinical Department, National Key Discipline, Guangdong Key Laboratory for Diagnosis and Treatment of Major Neurological Diseases, the National Natural Science Foundation of China (Nos. 81371255 and 81100936), Doctoral Program of Higher Education of China (No. 20110171110058), Guangdong Technological grant (Nos. 2010B050700024, 2011B050400031 and 2012B031800107), and the Natural Science Foundation of Guangdong Province (No. S2011010004860). This study was also supported by the multi year research grant University of Macau, MYRG122 (Y1-L3)ICMS12-SHX.
References 5. Conclusions In summary, the present study demonstrates the vascular protective action of puerarin against Aß40 injury in Zebrafish and HUVECs. Puerarin exerts its protective action probably through reduction of NADPH oxidase-derived reactive oxygen species overproduction and activation of PI3K/Akt/eNOS pathways. Therefore, puerarin could be used as a potent vascular protective agent in AD after further in vivo confirmation.
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