Neurochemical Research

, Volume 39, Issue 11, pp 2189–2196 | Cite as

Puerarin Protects Against β-Amyloid-Induced Microglia Apoptosis Via a PI3K-Dependent Signaling Pathway

Original Paper

Abstract

Puerarin extracted from Radix puerariae is well-known for its anti-oxidative and neuroprotective activities. In this study, we investigated the protective effect of puerarin on amyloid-β protein (Aβ)-induced cytotoxicity and its potential mechanisms in BV-2 and primary microglial cells. We found that pretreatment with puerarin afforded protection against Aβ-induced cytotoxicity through inhibiting apoptosis in BV-2 and primary microglial cells. This result was also confirmed by the activated caspase-3 assay. Phospho-Akt and Bcl-2 expression increased after pretreatment with puerarin in BV-2 and primary microglial cells exposed to Aβ, whereas Bax expression and cytochrome c release decreased. In addition, puerarin treatment prevented the loss of mitochondrial membrane potential and reactive oxygen species production. Interestingly, these effects of puerarin against Aβ insult were abolished by LY294002, an inhibitor of PI3K phosphorylation. Taken together, these findings suggest that puerarin prevents Aβ-induced microglial apoptosis via the activation of PI3K/Akt signaling pathway, and might be a potential preventive or therapeutic agent for Alzheimer’s disease.

Keywords

Puerarin Microglia Apoptosis β-amyloid Akt 

Introduction

Alzheimer’s disease (AD) is a common neurodegenerative disorder characterized by the progressive deterioration of memory and cognition. The deposition of amyloid-β protein (Aβ) in the brain is known to play an important role in the pathogenesis of AD [1]. There have been many studies concerning the effect of Aβ on nerve cells and Aβ-induced neurotoxicity has been reported by numerous studies [2, 3]. But there is a paucity of studies on the effect of Aβ on non-neuronal brain cells.

Microglia are innate immune cells in the central nervous system (CNS) and exert multiple functions that are usually maintained in surveying states. Microglial activation can be either neurotoxic or neurotrophic; normally active microglia can phagocytose debris to promote tissue repair and regulate inflammatory responses to pathogens, but sustained and excessive activation (“overactivation”) causes release of cytotoxins that result in neurotoxicity. Alternatively, overactivation can lead to microglial apoptosis, resulting in uncontrolled inflammatory responses [4]. Puerarin, isolated from Radix puerariae, is an active component and possesses a series of pharmacological activities including anti-oxidative and neuroprotective properties [5]. Recent studies have shown that pretreatment with puerarin could provide anti-AD effect in both of in vivo and in vitro models [6, 7]. However, the role of puerarin in Aβ-induced microglia apoptosis remains unknown.

In this study, we investigate whether puerarin has a neuroprotective function in Aβ-treated microglia, and whether the effects are induced by suppressing microglia apoptosis via the activation of PI3K/Akt signaling pathway.

Experimental Procedures

Reagents

Puerarin, (MR 416.37 of purity >98 %) was obtained from Sino-Herb Bio-Technology Company (China). Aβ25–35, dimethylsulfoxide (DMSO) and LY294002 were purchased from Sigma (USA). Antibodies, including total and phospho-Akt (Ser-473), total and cleaved caspase-3, were purchased from Cell Signaling Technology (Beverly, MA). The antibodies of β-actin, Cox-IV, Bcl-2, Bax and cytochrome c (CytC) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Puerarin was dissolved in saline at a concentration of 100 mM. LY294002 was dissolved in DMSO at a concentration of 50 mM, and the concentration of the DMSO was 0.02 % in the final culture medium. The vehicles had no detectable effects in these experiments.

Cell Culture

Microglial Cultures

BALB/c mice were purchased from Experimental Animal Center of Zhengzhou University (Zhengzhou, China). Mice were housed, bred, and euthanized in accordance with protocols reviewed and approved by the Commission of Zhengzhou University for ethics of experiments on animals in accordance with international standards. Mouse primary microglial cells were isolated from mixed glial cultures, as described previously [8]. Briefly, cortices were dissected from newborn BALB/c mice and dissociated by mechanical disruption and trypsinization. Primary microglia were co-cultured with astrocytes in poly-d-lysine-coated 75-cm2 culture flasks in DMEM supplemented with 10 % fetal bovine serum (FBS) and 1 % Penicillin/Streptomycin. On days 10–14, microglial cells were harvested by shaking the cultures and collecting the floating cells. The cells were seeded into plastic tissue culture flasks. After incubation at 37 °C for 1 h, non-adherent cells were removed by replacing culture medium. The cells were grown in DMEM with 10 % FBS and maintained at 37 °C and 5 % CO2.

BV-2 Cell Culture

The cells were purchased from Cell Center of Peking Union Medical College (Beijing, China) and cultured in DMEM medium with 5 % FBS and 1 % Penicillin/Streptomycin. Cultures were incubated at 37 °C and 5 % CO2 in a fully humidified incubator.

Preparation of Aged Aβ25–35

25–35 was solubilized in sterile water at a concentration of 1 mM, incubated in a capped vial at 37 °C for 72 h to form aggregated amyloid [9], and stored at −20 °C until use. During the experiments we added the peptide stock solution directly to the solution bathing the cells to achieve a final concentration of 20 μM.

Cell Viability Assay

BV-2 and primary microglial cells were pretreated with increasing concentrations (1–1,000 μM) of puerarin for 1 h, followed by exposure to Aβ (20 μM) for 24 h or BV-2 and primary microglial cells were pretreated with or without puerarin (100 μM) for 1 h and exposed to Aβ (20 μM) for 6, 12, 24 and 48 h respectively. Cell viability was assessed by the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-dipheny-ltetrazolium bromide (MTT). Briefly, 20 μl MTT (5 mg/ml, Sigma-Aldrich) was added to each well, and plates were incubated at 37 °C for 4 h and then quantifying the color formation by means of Elisa plate reader at 570 nm wavelength using 200 μl MTT solubilization solution.

TUNEL Assay

BV-2 and primary microglial cells were pretreated with puerarin (100 μM) for 1 h, followed by exposure to Aβ (20 μM) for 24 h. 10 μM LY294002 was added to cells 1 h prior to puerarin. The apoptotic cells were determined by terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) assay using a situ cell death detection kit (Roche Diagnostic, Indianpolis, IN) according to the manufacturer’s instructions as described in our previous studies [10]. The percentage of apoptotic cells was calculated by counting approximately 500 cells.

Western Blot Analysis

To obtain cytosolic and mitochondrial protein extracts, the cells were subfractionized in homogenization buffer. The cytosolic and mitochondrial fractions were separately isolated by centrifugation as described previously [11]. 30 μg of protein were separated by SDS–polyacrylamide gel electrophoresis and then transferred onto nitrocellulose membrane. After blocking in 5 % fat-free milk for 1 h, the membrane was incubated with the blocking solution containing the first antibody overnight at 4 °C. After washing, the blot was then incubated with a second antibody. The blot was washed again before being analyzed by the ECL system (Amersham Pharmacia). The signals were quantified by densitometric analysis using a densitometer.

Mitochondrial Membrane Potential

The mitochondrial membrane potential was detected using rhodamine 123 (Rh-123) fluorescent dye. Rh-123 can enter the mitochondrial matrix and cause photoluminescent quenching dependent on mitochondrial transmembrane potential. BV-2 and Primary microglial cells were incubated with Rh-123 for 30 min at 37 °C. After incubation, cells were rinsed with PBS. The fluorescence was measured at an excitation wavelength of 488 nm and an emission wave length of 510 nm by fluorescence microplate reader.

Intracellular ROS Generation

As described previously [12], dichlorohydrofluorescein diacetate (DCFDA) was used to measure the total level of reactive oxygen species (ROS) production. Briefly, the cells were harvested and washed in Krebs–Ringer solution, resuspended at 2 × 106 cells/ml, and loaded with DCFDA for 30 min at 37 °C in the dark. The cells were pelleted by centrifugation and resuspended in 2 ml of Krebs–Ringer solution. ROS production was determined by measuring the fluorescence resulting from the incorporation of the dye at the appropriate excitation and emission wavelengths in a spectrofluorimeter. Appropriate positive and negative controls were included in each measurement.

Statistical Analysis

Data are expressed as mean ± SD. The results were statistical analyzed using one-way ANOVA and the Newman–Keuls test. All statistical analyses were performed using SPSS version 17.0. P < 0.05 was considered statistically significant.

Results

Preventive Effect of Puerarin on Aβ-Induced Microglia Apoptosis

Cell viability detected by MTT assay revealed that puerarin significantly prevented cell death induced by Aβ in BV-2 and primary microglial cells in dose-dependent manner up to 100 μM and in time-dependent manner up to 24 h (Fig. 1). Thus, we selected 24 h time and 20 μM concentration of Aβ and 100 μM concentration of puerarin for subsequent experiments.
Fig. 1

Protective effect of puerarin on Aβ-induced cytotoxicity in microglia. a, b Puerarin reduces Aβ-induced cell death in a dose-dependent manner in BV-2 and primary microglial cells. BV-2 and primary microglial cells were pretreated with increasing concentrations (1–1,000 μM) of puerarin for 1 h, followed by exposure to Aβ (20 μM) for 24 h, and cell viability was determined by MTT. c, d Puerarin reduces Aβ-induced cell death in a time-dependent manner in BV-2 and primary microglial cells. BV-2 and primary microglial cells were pretreated with (black bars) or without (white bars) puerarin (100 μM) for 1 h and exposed to Aβ (20 μM) for the indicated times, and cell viability was determined by MTT. Data obtained from three separate experiments are expressed as mean ± SD.; *P < 0.05 versus con, #P < 0.05 versus Aβ. con control, PU puerarin, β-amyloid protein

To determine whether apoptosis played a role in Aβ-induced cell death, TUNEL assay was performed. We found that treatment of BV-2 and primary microglial cells with 20 μM Aβ for 24 h evoked marked cell apoptosis as indicated by the percentage of TUNEL-positive cells. Pretreatment of BV-2 and primary microglial cells with 100 μM puerarin, prior to Aβ exposure, significantly decreased cell apoptosis. Pretreatment with the LY294002 abolished the protective effect of puerarin against Aβ-induced apoptosis in BV-2 and primary microglial cells (Fig. 2).
Fig. 2

Puerarin prevents Aβ-induced apoptosis through a PI3K-dependent pathway in microglia. BV-2 and primary microglial cells were pretreated with puerarin (100 μM) for 1 h, followed by exposure to Aβ (20 μM) for 24 h. 10 μM LY294002 was added to cells 1 h prior to puerarin. Apoptotic cells were measured by TUNEL assay. Photographs of representative TUNEL-stained cells are shown at the left. Results represent mean ± SD. of three independent experiments. *P < 0.05 versus con, #P < 0.05 versus Aβ, **P < 0.05 versus PU. con control, PU puerarin, LY LY294002, β-amyloid protein. PU = Aβ + PU. LY = Aβ + PU +LY

Effects of Puerarin on Aβ-Induced Phosphorylation of Akt and Caspase-3 Activation

Western blot results showed that puerarin pretreatment (100 μM) increased phospho-Akt level which decreased significantly with Aβ treatment alone in BV-2 and primary microglial cells. LY294002 abolished the alteration of phospho-Akt level exerted by puerarin (Fig. 3a, b). We further determined the activation of caspase-3 by Western blot analysis. BV-2 and primary microglial cells treated with Aβ exhibited an increase in cleaved caspase-3 level, which was significantly suppressed by the puerarin pretreatment (Fig. 3c, d). Moreover, LY294002 inhibited the protective effect of puerarin against Aβ-induced caspase-3 activation.
Fig. 3

Effects of puerarin on the levels of phospho-Akt (serine 473) and activated fragment of caspase-3 in microglia. BV-2 and primary microglial cells were pretreated with puerarin (100 μM) for 1 h, followed by exposure to Aβ (20 μM) for 24 h. 10 μM LY294002 was added to cells 1 h prior to puerarin. a, b Western blots and quantitative analysis of phospho-Akt after puerarin pretreatment. c, d Western blots and quantitative analysis of caspase-3 after puerarin pretreatment. β-actin was used as an internal standard. Data were mean ± SD. of three independent experiments. *P < 0.05 versus con, #P < 0.05 versus Aβ, **P < 0.05 versus PU. pAkt phospho-Akt. con control, PU puerarin, LY LY294002, β-amyloid protein. PU = Aβ + PU. LY = Aβ + PU +LY

Effect of Puerarin on Aβ-Induced Mitochondrial Membrane Potential Alteration

It has been indicated that microglia apoptosis induced by Aβ was accompanied by the disruption of mitochondrial membrane potential [13], so we examined the effect of puerarin on the mitochondrial transmembrane potential by Rh-123 staining. When BV-2 and primary microglial cells were treated with 20 μM Aβ for 24 h at 37 °C, a decrease in the rentention of Rh-123 was observed. However, pretreatment with puerarin prevented the decrease in the retention of Rh-123 induced by Aβ, which was abolished by LY294002 (Fig. 4c, d).
Fig. 4

Puerarin significantly attenuates Aβ-induced ROS production and mitochondrial membrane potential alteration in microglia. BV-2 and primary microglial cells were pretreated with puerarin (100 μM) for 1 h, followed by exposure to Aβ (20 μM) for 24 h. 10 μM LY294002 was added to cells 1 h prior to puerarin. a, b The mitochondrial membrane potential was measured by fluorescence microplate reader using rhodamine 123 staining. c, d The production of ROS was measured by fluorescence microplate reader using DCFDA staining. Data were mean ± SD. of three independent experiments. *P < 0.05 versus con, #P < 0.05 versus Aβ, **P < 0.05 versus PU. con control, PU puerarin, LY LY294002, β-amyloid protein. PU = Aβ + PU. LY = Aβ + PU +LY

Effect of Puerarin on Aβ-Induced ROS Production

To determine whether puerarin attenuates cell death by blocking ROS generation, the intracellular ROS concentration was measured using the fluorescent dye DCFDA. Our results showed that treatment of BV-2 and primary microglial cells with 20 μM Aβ significantly increased intracellular ROS generation, but this was significantly reduced by puerarin pretreatment. LY294002 significantly abolished the protective effect of puerarin against Aβ-induced ROS production (Fig. 4a, b).

Effects of Puerarin on the Levels of Bcl-2, Bax Expression and CytC Release

Given that Bcl-2 family proteins may be the functional downstream targets of Akt [14], we determined the effects of puerarin on Bcl-2, Bax protein levels and CytC release, and the role of PI3K signal pathway. Western blot results showed that puerarin pretreatment (100 μM for 24 h) increased Bcl-2 protein level in BV-2 and primary microglial cells which decreased significantly with Aβ treatment alone, whereas Bax were significantly decreased. The LY294002 abolished the alteration of Bcl-2 and Bax levels exerted by puerarin (Fig. 5a, b). Moreover, BV-2 and primary microglial cells treated with Aβ for 24 h showed an increase in the cytosolic CytC levels. Pretreatment with puerarin attenuated the Aβ-induced increase in CytC levels. LY294002 abolished the protective effect of puerarin against Aβ-induced CytC release (Fig. 5c, d).
Fig. 5

Effects of puerarin on the levels of Bcl-2, Bax expression and CytC release in microglia. BV-2 and primary microglial cells were pretreated with puerarin (100 μM) for 1 h, followed by exposure to Aβ (20 μM) for 24 h. 10 μM LY294002 was added to cells 1 h prior to puerarin. a, b Western blots and quantitative analysis of Bcl-2 and Bax levels after puerarin pretreatment. c, d Western blots and quantitative analysis of CytC release after puerarin pretreatment. Cyclooxygenase (COX)-IV and β-actin were used as internal controls of mitochondrial and cytosolic subfractions respectively. Data were mean ± SD. of three independent experiments. *P < 0.05 versus con, #P < 0.05 versus Aβ, **P < 0.05 versus PU. CytC cytochrome c; mito mitochondrial, cyto cytosolic, con control, PU puerarin, LY LY294002, β-amyloid protein. PU = Aβ + PU. LY = Aβ + PU +LY

Discussion

Therapeutic strategies that target the accumulation and toxicity of Aβ in the CNS may offer promise for the treatment of AD. In particular, focus upon CNS microglia, that promotes neuronal regeneration and sequester toxic Aβ deposition [15], may offer significant promise for new therapies. In deed, there is increasing evidence that microglia can restrict the spread of neurodegeneration and promote neuronal regeneration. Alternatively, microglial overactivation or cell death may aggravate pre-existing neuropathology [16]. In this study, we found that Aβ exerts cytotoxic effects on microglia in a dose- and time-dependent manner, which is consistent with the finding of Jang et al. [17]. In addition, we found that puerarin exerted protection against Aβ-induced cytotoxicity through inhibiting apoptosis in BV-2 and primary microglial cells. Thus, it seems that puerarin might be a potential preventive or therapeutic agent for AD.

PI3K/Akt signal pathway, one of the cell pro-survival pathways, was proposed as an essential way in preventing cell death [18]. Hence, we hypothesize that PI3K/Akt activated by puerarin possibly contributes to its neuroprotective function in Aβ-treated microglia. In this study, we indeed displayed that puerarin activated Akt phosphorylation in Aβ-treated microglia, which could be abolished by inhibition of PI3K with LY294002. Furthermore, LY294002 abolished the neuroprotective effect of puerarin on Aβ-induced apoptosis in BV-2 and primary microglial cells, suggesting that PI3K/Akt signaling pathway contributing to the neuroprotective effect of puerarin in Aβ-treated microglia. Numerous components of the PI3K/Akt pathway play a critical role in cell survival pathway. Studies have also reported that neuroprotection against oxidative stress may be via activation of Akt survival signaling [19]. In the present study, we showed that the presence of Aβ in BV-2 and primary microglial cells cultures induced ROS overproduction and reduced the mitochondrial membrane potential, leading to apoptosis and cell death. Furthermore, puerarin pretreatment of the cells decreases ROS overproduction, restores mitochondrial membrane potential, and increases cell viability. In addition, LY294002 abolished the inhibitory effect of puerarin on Aβ-induced ROS production and mitochondrial membrane potential alteration. Thus, puerarin may contribute to the cellular defense against oxidative stress during the neurodegenerative process in AD.

Bcl-2 is an anti-apoptotic protein which resides on the mitochondrial outer membrane and inhibits channel formation of Bax, a pro-apoptotic protein of the same Bcl-2 family, through which CytC leaks to the cytoplasm [20]. In this study, we also found that puerarin downregulated Aβ-induced expression of the proapoptotic proteins Bax and release of CytC, but upregulated Bcl-2 expression. Pretreatment with LY294002 abolished the protective effects of puerarin on these apoptotic-related protein changes, suggesting that the antiapoptotic effect of puerarin is mediated via the activation of PI3K/Akt signaling. It is well known that ROS-induced DNA damage can subsequently cause cell cycle arrest or even apoptosis. Furthermore, previous studies have demonstrated that ROS are involved in the apoptotic mechanisms triggered in the process of Aβ-mediated neurotoxicity and therefore may contribute to the regulation of apoptosis proteins in some AD models. Bcl-2 family members such as Bax and Bcl-2 are implicated in the process of apoptotic cell death induced by ROS-generating agents such as Aβ [21]. Thus, scavenging of ROS, inhibition of Bax expression, and upregulation of Bcl-2 by puerarin could be related to puerarin’s antioxidant effect. In addition, LY294002 abolished the protective effect of puerarin against Aβ-induced ROS production, Bcl-2 and Bax expression, CytC release and caspase-3 activation. Thus, the PI3K/Akt signaling pathway may play an important role in the neuroprotective effect of puerarin in Aβ-treated microglia.

In conclusion, the present study demonstrates that puerarin attenuates Aβ-induced microglia apoptosis by activating a PI3K-dependent pathway in association with Akt phosphorylation. Akt phosphorylation in turn increased Bcl-2 expression and mitochondrial membrane potential, decreased Bax expression and ROS production, thus inhibiting CytC release and caspase-3 activation. Our findings suggest that puerarin might be a potential drug for the AD to suppress microglial apoptosis.

Notes

Acknowledgments

This study was supported by a Grant from National Natural Science Foundation of China (No. 81201012) and China Postdoctoral Science Foundation Special Funded Project (No. 2013T60707) and The Key Science and Technique Foundation of Henan province (No. 132102310110).

Conflict of interest

There are no conflicts of interest in this article.

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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Department of Internal MedicineThe First Affiliated Hospital of Zhengzhou UniversityZhengzhouPeople’s Republic of China
  2. 2.Department of NeurologyThe First Affiliated Hospital of Zhengzhou UniversityZhengzhouPeople’s Republic of China
  3. 3.College of BioengineeringHenan University of TechnologyZhengzhouPeople’s Republic of China
  4. 4.Henan Bioengineering Research CenterZhengzhouPeople’s Republic of China

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