International Immunopharmacology 33 (2016) 83–89

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International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp

Progesterone exerts neuroprotective effects against Aβ-induced neuroinflammation by attenuating ER stress in astrocytes Yang Hong a, Xiaomin Wang a, Shuang Sun a, Gai Xue b, Jianli Li c, Yanning Hou a,b,⁎ a b c

Hebei Medical University, Shijiazhuang 050017, Hebei Province, China Department of Pharmacology, Bethune International Peace Hospital, Shijiazhuang 050082, Hebei Province, China Department of Anesthesiology, Hebei General Hospital, Shijiazhuang 050051, Hebei province, China

a r t i c l e

i n f o

Article history: Received 17 September 2015 Received in revised form 14 January 2016 Accepted 1 February 2016 Available online xxxx Keywords: Alzheimer β-Amyloid ER stress Neurosteroids Progesterone Neuroinflammation

a b s t r a c t The deposition of amyloid-β (Aβ) and neuroinflammation are critical pathological features of Alzheimer's disease (AD). Astrocytes are considered the principal immunoregulatory cells in the brain. Neurosteroid progesterone (PG) exerts neuromodulatory properties, particularly its potential therapeutic function in ameliorating AD. However, the role of PG and the neuroprotective mechanism involving in the regulation of neuroinflammation in astrocytes warrant further investigation. In this study, we found that Aβ significantly increased the processing of neuroinflammatory responses in astrocytes. The processing is induced by an increase activity of PERK/elF2ɑdependent endoplasmic reticulum (ER) stress. Additionally, the inhibition of ER stress activation by Salubrinal significantly suppressed the Aβ-induced neuroinflammatory responses in astrocytes. While the treatment of astrocytes with Aβ caused an increase of neuroinflammatory responses, PG significantly inhibited Aβ-induced neuroinflammatory cytokine production by suppressing ER stress activation together with attenuating PERK/ elF2ɑ signalling. Taken together, these results indicate that PG exerts a neuroprotective effect against Aβinduced neuroinflammatory responses, and significantly suppresses ER stress activation, which is an important mediator of the neurotoxic events occurring in Aβ-induced neuroinflammatory responses in astrocytes. These neuroprotective mechanisms may facilitate the development of therapies to ameliorate AD. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Alzheimer's disease (AD) is a progressive neurodegenerative disorder that results in a gradual decline in cognitive processes. The neuropathology of this disease is characterized by intracellular neurofibrillary tangles, senile plaques, and excessive neuronal loss [1]. The deposition of amyloid-β (Aβ) has been considered an extremely critical factor for AD development, but the mechanism underlying Aβ-induced neurotoxicity is not fully understood. It was recently recognized that neuroinflammation is a prominent feature in the modulation of AD processing and that a persistent secretion of proinflammatory cytokines is observed [2]. However, the sequence of events leading to neuroinflammation remains unclear, and the mechanism underlying Aβ-induced neuroinflammation remains an area of active investigation. Astrocytes are the most abundant glial cells in the central nervous system. Astrocytes dynamically endow neurons with trophic support and modulate information processing [3]. However, prolonged and widespread reactive astrocytes are characteristically found in the AD ⁎ Corresponding author at: Hebei Medical University, 361 East Zhongshan Road, Shijiazhuang 050017, Hebei Province, China. Bethune International Peace Hospital of Chinese PLA, 398 West Zhongshan Road, Shijiazhuang 050082, Hebei Province, China. E-mail address: [email protected] (Y. Hou).

http://dx.doi.org/10.1016/j.intimp.2016.02.002 1567-5769/© 2016 Elsevier B.V. All rights reserved.

brain. Once activated, astrocytes produce several proinflammatory signal molecules, including cytokines, growth factors, complement molecules, and chemokines [4]. The released cytokines, particularly interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-ɑ, are the major effectors of neuroinflammatory signals and affect neurophysiologic mechanisms regarding cognition and memory [5]. Indeed, these reactive astrocytes revolve around senile plaques and neurofibrillary tangles in the AD brain, suggesting that Aβ deposition is a potent trigger of astrocytic activation in the AD brain. However, the mechanistic connection between Aβ deposition and astrocytic inflammation is not fully understood. ER stress has recently been regarded as a vital pathophysiological mechanism in the development of many diseases [6–8] and particularly as a relevant pathological factor of AD [9]. Multiple lines of evidence obtained from studies of post-mortem brain tissue of AD patients or animal models have led to the wide acceptable of a relationship between ER stress and Aβ-induced cytotoxicity [8, 10]. Aβ severely disrupts endoplasmic reticulum (ER) function and causes excessive activation of ER stress in neurons [11]. Excessive and prolonged ER stress activation is detrimental to neurons because a delayed defence decreases the viability of neurons and initiates an apoptotic program inside the cells [12]. Therefore, it is obvious that ER stress may be a crucial factor in the pathogenesis of AD. In addition, ER stress is linked

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with some of the major inflammation and stress signalling networks [13]. That suggests the activation of ER stress may correlate with neuroinflammatory response via a potential pathway. Steroid hormones and their metabolites within the central nervous system (CNS) are commonly defined as neuroactive steroids or neurosteroids [14]. The neuroprotective properties of neurosteroids have been recently reported in numerous studies [15–17]. One particular focus of previous studies is the neurosteroid progesterone (PG) and its neuroprotective capacity in neurodegenerative disorders [18, 19]. Our previous work found that the level of neurosteroids PG in the brain of AD rats abnormally decreased. However, subcutaneous injection of PG significantly improved cognitive abilities of AD rats [20]. Furthermore, we found that PG exhibited a neuroprotective function by inhibiting Aβ-induced mitochondrial apoptosis through mediating JNK signalling pathway in neurons [21]. PG exhibits a potential therapeutic function in the amelioration of AD, but the potential protective mechanism has not been fully elucidated. Particularly, the role of PG and the neuroprotective mechanism involving the regulation of astrocytic function warrant further investigation. In consideration of this issue, a better understanding of PG and its potential as a neuroprotective agent against AD is of great significance. Therefore, in this study, we aimed to elucidate the molecular mechanism underlying Aβ-induced inflammatory responses in astrocytes and investigate whether PG exerts a protective effect against Aβ-induced neuroinflammation and its potential neuroprotective mechanism. 2. Materials and methods 2.1. Chemicals and reagents Dimethyl sulfoxide (DMSO), poly-D-lysine, Salubrinal (Sal), Tunicamycin (TM), and Aβ1–42 fragments and progesterone were purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA). DAPI and trypsin were purchased from Solarbio Inc. (Beijing, China). DMEM, foetal bovine serum (FBS) was purchased from Gibco (Carlsbad, CA, USA). Antibodies to phospho(P)-elF2ɑ, P-PERK, total(T)-elF2ɑ, T-PERK, GRP78, GFAP and β-actin were purchased from Cell Signal Technology Inc. (Beverly, MA, USA). Goat anti-rabbit lgG/FITC was purchased from Bioss Inc. (Beijing, China). 2.2. Cell culture and treatment 2.2.1. Primary astrocyte culture Astrocytes were prepared from newborn Sprague–Dawley rat pups (b24 h). The cerebral cortices were removed from brain and digested at 37 °C for 15 min with trypsin (1.25 g/L). The cells were seeded into poly-L-lysine-coated coverslips or multiwell plates at either 1 × 105 cells/cm2 (Immunofluorescence analysis) or 1 × 106 cells/cm2 (western blotting analysis) in DMEM medium supplemented with 10% FBS, and incubated at 37 °C in a 5% CO2 incubator. The medium was exchanged every 3 days after the cultures were primarily confluent astrocytes (N90%). The confluent cultures were shaken overnight to remove microglia contamination from adherent astrocytes after 10 days incubation. More than 95% of the incubated cells were astrocytes as identified by immunofluorescent staining for GFAP. 2.2.2. Oligomeric Aβ1–42 preparation Aβ1–42 oligomers were prepared according to the methods [22]. An Aβ1–42 monomer was prepared by evaporating 2 mg Aβ1–42 dissolved in 1, 1, 1, 3, 3, 3, hexafluoro-2-propanol at room temperature for 30 min with N2 gas. The monomer was then dissolved in DMSO and diluted to 10 μM with Dulbecco's Modified Eagle's Media (DMEM, Gibco). The diluted solution was then incubated at 37 °C for 24 h to form oligomers. The Aβ1–42 oligomers preparations were centrifuged at 16,000g to remove any insoluble fibrils, and next, the supernatant

was diluted in DMEM prior to addition to cultures at the final concentrations. 2.3. Immunofluorescence assay The cultured primary astrocytes were cultured with standard conditions described above (Methods-2.2.1), and then exposed to Aβ1–42 oligomers (Methods-2.2.2) for indicated times. The cells were washed with ice-cold PBS and fixed in 4% ice-cold paraformaldehyde for 30 min. Fixed cells were permeabilized with 0.05% Triton X-100, and blocked with 3% bovine serum albumin (BSA) in PBS for 1 h. Next, the cells were incubated with primary antibody (anti-GFAP, 1:300 dilution) overnight at 4 °C in a humidified container. The next day, the cells were washed and then stained with FITC-conjugated goat anti-rat lgG secondary antibodies (1:100 dilution) for another 1 h. After washed with PBS, nuclei were subsequently stained with DAPI for 5 min. Images were visualized under fluorescence microscope (IX-81, Olympus). 2.4. Western blotting assay The cells were lysed, and proteins were extracted from cultured primary astrocytes using RIPA Lysis buffer (Beyotime Institute of Biotechnology, Jiangsu China). The supernatants were collected and total proteins were measured using a BCA protein assay kit. The total extracted proteins were separated on 10% or 15% SDS-polyacrylamide gel, and transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Burlington, MA). Membranes were blocked with 5% non-fat milk in tris-buffered saline containing 0.1% Tween for 2 h at room temperature and incubated with primary antibody (anti-GRP78, P-elF2ɑ, P-PERK, T-elF2ɑ, T-PERK and β-actin 1:1000) at 4 °C overnight. After washing with TBS-T (10 mM Tris–HCl, 150 mM NaCl, 0.05% Tween20), membranes were incubated with secondary antibody for 2 h at room temperature. Blots were developed with an ECL detection kit, and integrated densities of bands were measured by Image J software. 2.5. ELISA assay Astrocytes treatment supernatants were collected and the levels of IL-1β and TNF-α in culture supernatant were determined by ELISA according to the manufacturer's instructions. Briefly, the supernatants (100 μL) were added to the appropriate well in rat anti-IL-1β and TNFα pre-coated plates. Next, 50 μL biotin-labelled anti-IL-1β and TNF-α were added to each well. After 2 h incubation, the plates were washed 5 times with wash buffer, and 50 μL substrate A was added into each well and incubated for 1 h at 37 °C. In a following step, 50 μL substrate B was added, and the plates were kept in the dark at 37 °C for 30 min. Finally, 50 μL stop solution was added to terminate reaction. Detection and quantification of the plates were carried out with a microplate reader (Molecular Device Corporation, Sunnyvale, CA, USA) at 450 nm. The results were expressed as pg/mL for culture media. 2.6. Statistical analysis The experimental results were expressed as the mean ± SD. Statistically significant difference analysis was carried out by student's t-test and one-way ANOVA followed by the Dunnett's test with SPSS 13.0 software. Mean values were considered to be statistically significant in this study at p b 0.05 or less. 3. Results 3.1. Aβ upregulates GFAP expression and induces morphological changes in astrocytes Astrocytes are susceptible to activate in a variety of neuropathological states [23]. The upregulation of GFAP expression and morphological

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changes in astrocytes could be considered reliable markers of astrocytic activation [24]. In this study, we assessed the level of GFAP expression and possible alterations to the astrocytic morphology in response to Aβ-induced cytotoxicity. Astrocyte-enriched cultures were treated with 5 μM Aβ for the indicated times (2, 4, and 6 h), and we found that the level of GFAP expression increased after Aβ treatment (Fig. 1A). After 6 h of exposure to Aβ, astrocytes significantly presented increase of GFAP expression and apparent morphological alterations with an enlarged body and shorter filopodium-like processes (Fig. 1B). Such upregulation of GFAP expression and morphological changes are thought to represent characteristics of astrogliosis [24]. These results suggest that Aβ-induced astrocytic morphological injury and activation are early events closely associated with Aβ cytotoxicity. 3.2. Aβ exacerbates IL-1β and TNF-ɑ release in astrocytes Previous studies have shown that the amounts of specific inflammatory cytokines correlate strongly with the pathological development of AD [25–27]. To determine whether inflammatory responses are concomitantly involved in Aβ-induced astrocytic activation, the release of inflammatory cytokines was measured by ELISA. Astrocyte-enriched cultures were treated with 5 μM Aβ for different time periods. The secretion of proinflammatory cytokines from reactive astrocytes increased in response to Aβ treatment, and a significantly elevated release of IL-1β and TNF-ɑ was observed after 6 h of exposure (compared with the non-treated group, Fig. 2A and B). These findings imply a close connection between inflammatory responses and the neurotoxic events that occur downstream of Aβ and suggest that the suppression of these inflammatory responses may represent a valid therapeutic strategy for AD. 3.3. Aβ induces ER stress by activating the PERK/eIF2ɑ pathway in astrocytes Based on our recent results, a better understanding of the mechanisms underlying the secretion of proinflammatory cytokines from reactive astrocytes may be crucial for determining their contribution to Aβ-induced neurotoxicity. To investigate whether ER stress was activated in Aβ-treated astrocytes, we treated astrocyte-enriched cultures with 5 μM Aβ for 6 h or 12 h. The results showed that Aβ treatment for 6 h or 12 h both significantly upregulated GRP78 expression compared with the non-treated group (Fig. 3A). Based on this observation, we also evaluated the changes in the phosphorylation of two representative ER stress markers that are successively synthetized in the unfolded protein response (UPR) pathway [28]. A western blotting analysis indicated that the P-PERK/T-PERK and P-eIF2ɑ/T-eIF2ɑ levels were both increased

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following Aβ treatment for 6 h or 12 h (Fig. 3B). Overall, these results indicate that Aβ induces ER stress by activating the PERK/eIF2ɑ pathway in astrocytes. 3.4. Aβ accelerates IL-1β and TNF-ɑ release by inducing ER stress activation in astrocytes To investigate whether there is a relationship between ER stress activation and the release of inflammatory cytokines in astrocytes. We treated astrocytes with the ER stress promoter TM (5 and 10 μg/mL) for 3 h. The results showed that TM significantly increased the release of both IL-1β and TNF-α in astrocytes (Fig. 4A and B). To further demonstrate this hypothesis, astrocytes were pre-treated with the ER stress inhibitor Salubrinal (5, 10, and 20 μM) for 1 h and then were exposed to 5 μM Aβ for 6 h. A statistical analysis revealed that Salubrinal (10 μM) significantly inhibited the Aβ-induced release of IL-1β and TNF-α compared with the Aβ-treated group (Fig. 4C and D). Therefore, the molecular mechanism underlying Aβ-induced proinflammation in astrocytes closely correlate with ER stress, suggesting that the suppression of ER stress activation may represent an appropriate molecular mechanism for mediating these inflammatory responses in AD. 3.5. Progesterone suppresses Aβ-induced inflammatory responses in astrocytes The neurosteroid PG exhibits a neuroprotective capacity in several neurodegenerative disorders [29]. To examine the hypothesis that PG has a potential influence on ameliorating Aβ-induced inflammation, we co-incubated astrocytes with different doses of PG (0.25, 0.5, 1, and 2 μM) and Aβ for 6 h. PG significantly attenuated Aβ-induced IL-1β and TNF-α production in a dose-dependent manner (Fig. 5A and B). 1 μM PG exhibited a protective capacity in suppressing Aβ-induced IL-1β and TNF-α release compared with the Aβ-treated group (Fig. 5A and B). In addition, we treated astrocytes with 1 μM PG for different periods of time (3, 6, 12, and 24 h) following 6 h of Aβ treatment. The results indicate that PG significantly inhibits Aβ-induced IL-1β and TNF-α production after 6 h of treatment (Fig. 5C and D). These data suggest that the neuroprotection that PG shows against Aβ-induced neurotoxicity is associated with a decreased release of inflammatory cytokines in astrocytes. 3.6. Progesterone attenuates Aβ-induced inflammatory responses by suppressing PERK/eIF2ɑ-dependent ER stress activation in astrocytes PG exhibits a potential neuroprotective function in ameliorating Aβ-induced proinflammatory responses. However, its neuroprotective

Fig. 1. Aβ upregulates GFAP expression and induces morphological changes in astrocytes. A. Aβ upregulates GFAP expression in astrocytes. Astrocyte-enriched cultured were exposed to 5 μM Aβ for different periods of time (2, 4, and 6 h), and the expression of GAFP was assayed by western blotting analysis. B. Aβ induces morphological changes in astrocytes. Astrocytes were exposed to 5 μM Aβ for the indicated times (6 h), and the cells were subsequently immunostained with antibodies against glial fibrillary acidic protein (GFAP) to label astrocytes (green). DAIP was used to stain nuclei (blue). The western blotting data represent the means ± SD from three independent experiments. *p b 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 2. Aβ exacerbates IL-1β and TNF-ɑ release in astrocytes. Astrocyte-enriched cultured were treated with 5 μM Aβ for 3, 6, 12 or 24 h. The levels of IL-1β (A) and TNF-ɑ (B) in the medium of treated astrocytes were assayed by ELISA. The data represent the means ± SD from three independent determinations. **p b 0.01, ***p b 0.001.

mechanism has not been fully demonstrated. Therefore, we speculated that PG might exert a neuroprotective effect in mediating ER stress. The results showed that PG inhibited Aβ-induced ER stress in a dose- or time-dependent manner by decreasing GRP78 expression (Fig. 6A and B). Treatment with 1 μM PG for 6 h resulted in a maximal protective effect in suppressing ER stress (Fig. 6B). Consistent with this finding, the P-PERK/T-PERK and P-eIF2ɑ/T-eIF2ɑ levels were both decreased in Aβ-treated astrocytes following PG treatment (Fig. 6C). These data suggest that the PERK/elF2ɑ signalling pathway is implicated in the neuroprotective mechanism of PG. Although the degree of inflammation was increased by treatment with Aβ, pre-treatment with Salubrinal or PG both significantly attenuated the Aβ-induced release of IL-1β and TNF-α (Fig. 6D and E). Therefore, these findings demonstrate that PG plays a neuroprotective role in ameliorating Aβ-induced inflammatory responses by suppressing PERK/eIF2ɑ-dependent ER stress activation. 4. Discussion Inflammation is a crucial pathological hallmark of AD [26]. Inflammation clearly occurs in pathologically vulnerable regions of the AD brain, as demonstrated by an increased secretion of proinflammatory

cytokines [30]. Neuroinflammatory mediators affect neurophysiological mechanisms associated with cognition and memory [5]. Reactive astrocytes strongly produce high levels of inflammatory mediators, such as proinflammatory cytokines, nitric oxide, complement factors, and inflammatory proteins [31]. However, the contribution of reactive astrocytes to the pathophysiology of AD is not fully understood. In this study, we observed that treatment with Aβ altered the morphology of astrocytes, as demonstrated by terminal swelling filopodium-like processes, and induced the upregulation of GFAP expression [23]. Additionally, Aβ significantly induced the inflammatory cytokines IL1β and TNF-ɑ in cultured astrocytes. It is well recognized that astrocytic activation is accompanied by an increased production of potentially neurotoxic factors, including proinflammatory cytokines, nitric oxide and reactive oxygen species [32]. Indeed, astrocytic activation is always observed when astrocytes enter a stress state. In our study, we found that Aβ treatment significantly increased the production of IL-1β and TNF-ɑ in astrocytes. However, the ER stress inhibitor Salubrinal significantly inhibited the Aβ-induced release of IL-1β and TNF-α. Our current findings support the hypothesis that ER stress can trigger neuroinflammatory responses [33]. In fact, the ability of ER stress to induce an inflammatory response

Fig. 3. Aβ induces ER stress by activating the PERK/eIF2ɑ pathway in astrocytes. A. Aβ induces ER stress activation by accelerating GRP78 expression. Astrocyte-enriched cultures were treated with 5 μM Aβ for 6 or 12 h. The cells were subsequently lysed, and the protein expression level of the ER-stress marker GRP78 was determined by western blotting analysis. B. Aβ activates the PERK/elF2ɑ pathway in Aβ-treated astrocytes. Astrocytes were treated with 5 μM Aβ for 6 or 12 h, and the protein expression levels of P-PERK, T-PERK, P-elF2ɑ, and T-elF2ɑ were determined by western blotting analysis. The data represent the means ± SD from three independent experiments. *p b 0.05.

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Fig. 4. Aβ accelerates IL-1β and TNF-ɑ release by inducing ER stress activation in astrocytes. A, B. The ER stress promoter TM accelerates IL-1β and TNF-ɑ release from astrocytes. Astrocyteenriched cultured were treated in the absence or presence of the ER stress promotor TM (5 and 10 μg/mL) for 3 h. The levels of IL-1β and TNF-ɑ in the medium of treated astrocytes were assayed by ELISA. C, D. The ER stress inhibitor Sal suppresses the Aβ-induced IL-1β and TNF-ɑ release from astrocytes. Astrocyte-enriched cultured were pretreated with or without the ER stress inhibitor Sal (5, 10, and 20 μM) for 1 h and then treated with 5 μM Aβ for 6 h. The levels of IL-1β and TNF-ɑ in the medium of treated astrocytes were assayed by ELISA. The data represent the means ± SD from three independent determinations. **p b 0.01. Sal: Salubrinal.

is observed in several diseases [13, 34, 35]. For example, obesityinduced ER stress can trigger the inflammatory response and generate peripheral insulin resistance [35]. Intestinal inflammation in inflammatory bowel disease (IBD) emerges from dysfunction in intestinal epithelium cells due to excessive ER stress [34]. The ER stress was reported to be involved in regulating NF-κB signalling, which is a probably common proinflammatory mechanisms regarding immunity responses [36]. It is suggesting that Aβ-induced ER stress is linked with the activation of NF-κB-mediating inflammation. ER stress has also been shown to increase the activity of PERK/elF2ɑ signalling. Moreover, PERK/elF2ɑ signalling may play a vital role in mediating NF-κB activation in vitro and in vivo [37]. This finding implies that PERK/elF2ɑ signalling may be involved in the NF-κB-mediated inflammatory response. Several lines of evidence have demonstrated that Aβ can induce an excessive activation of ER stress in neurons, resulting in a wide range of intracellular damages, including the disruption of intracellular homeostasis, and apoptosis [8, 11, 38]. Our current

findings aimed to establish a connection between ER stress and Aβ-induced inflammation in astrocytes and revealed that PERK/elF2ɑ signalling was activated in Aβ-treated astrocytes and involved in the regulation of the release of IL-1β and TNF-ɑ in response to ER stress. In astrocytes, the inhibition of PERK/elF2ɑ activation with the ER stress inhibitor Salubrinal significantly reduced the Aβ-induced release of IL-1β and TNF-ɑ, suggesting that PERK/elF2ɑ activation was a regulatory step in mediating Aβ-induced inflammation. This finding indicates that these proinflammatory cytokines are the neurotoxic events downstream of Aβ-induced ER stress activation. Therefore, this is a logical approach to preventing Aβ-induced neuroinflammation by inhibiting ER stress activation in astrocytes. Early findings demonstrated a neuromodulatory function of the neurosteroid PG, particularly its potential therapeutic function in ameliorating AD [20]. However, the exact mechanism in mediating AD remains poorly understood. Numerous recent studies show the neuroprotective mechanism of PG in cases of ischemia and brain injury, and

Fig. 5. Progesterone suppresses Aβ-induced inflammatory responses in astrocytes. A, B. PG suppresses the Aβ-induced release of IL-1β and TNF-ɑ in a dose-dependent manner. Astrocyteenriched cultured were treated with 5 μM Aβ in the absence or presence of different doses of PG (0.25, 0.5, 1, and 2 μM) for 6 h. The levels of IL-1β and TNF-ɑ in the medium of the treated astrocytes were assayed by ELISA. C, D. PG suppresses the Aβ-induced release of IL-1β and TNF-ɑ in a time-dependent manner. Astrocyte-enriched cultured were exposed to 5 μM Aβ for 6 h and further treated with or without PG (1 μM) for different periods of time (3, 6, 12, and 24 h). The levels of IL-1β and TNF-ɑ in the medium of the treated astrocytes were assayed by ELISA. The data represent the means ± SD from three independent determinations. **p b 0.01.

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Fig. 6. Progesterone attenuates Aβ-induced inflammatory responses by suppressing PERK/eIF2ɑ-dependent ER stress activation in astrocytes. A. Progesterone suppresses Aβ-induced GRP78 expression in a dose-dependent manner. Astrocyte-enriched cultured were treated with 5 μM Aβ in the absence or presence of different doses of PG (0.5, 1, and 2 μM) for 6 h. After the cells were lysed, the protein expression of GRP78 was determined by western blotting analysis. B. Progesterone suppresses Aβ-induced GRP78 expression in a timedependent manner. Astrocyte-enriched cultured were exposed to 5 μM Aβ for 6 h and further treated with or without PG (1 μM) for different periods of time (3, 6, and 12 h). After the cells were lysed, the protein expression of GRP78 was determined by western blotting analysis. C. Progesterone suppresses the PERK/elF2ɑ signalling pathway in Aβ-treated astrocytes. Astrocytes were pre-treated with or without Sal for 1 h and then further exposed to 5 μM Aβ in the presence or absence of PG for an additional 6 h of treatment. The protein expression of P-PERK, T-PERK, P-elF2ɑ, and T-elF2ɑ was determined by western blotting analysis. D, E. Progesterone attenuates Aβ-induced IL-1β and TNF-ɑ release by suppressing ER stress activation in Aβ-treated astrocytes. The levels of IL-1β and TNF-ɑ in the medium of treated astrocytes were assayed by ELISA. The data represent the means ± SD from three independent experiments. *p b 0.05, ** p b 0.01. Sal: Salubrinal.

indicate this mechanism includes improving neuronal survival, reducing swelling, and inhibiting apoptosis [18, 19, 39]. While investigating the hypothesis that PG has a potential influence on ameliorating Aβ-induced inflammation, we illuminated that PG neuroprotection against Aβ-induced neurotoxicity was associated with a decrease in the release of the inflammatory cytokines IL-1β and TNF-ɑ in astrocytes. Interestingly, PG inhibited Aβ-induced ER stress activation, resulting in a decreased expression of GRP78. Consistent with these results, PG also significantly inhibited Aβ-induced PERK/elF2ɑ signalling upregulation, which was closely correlated with ER stress activation. Therefore, it is possible that the PERK/elF2a signalling pathway can be implicated in the neuroprotective mechanism of PG against Aβ-induced inflammation. Our previous study connected the neuroprotection of PG with the inhibition of Aβ-induced neuronal apoptosis [21]. With a heightened discussion of the neuroprotective role of PG in mediating Aβ-induced inflammation, our current findings demonstrate that ER stress is highly suppressed by PG in Aβ-treated astrocytes. Therefore, these findings provide a new understanding of the potential mechanism

contributing to the neuroprotection of PG against Aβ-induced neurotoxicity. In summary, the principal findings of this study indicate that 1) PERK/elF2ɑ-dependent ER stress activation is involved in regulation of Aβ-induced neuroinflammation in astrocytes, 2) PG exerts a neuroprotective effects against Aβ-induced neuroinflammatory responses in astrocytes, and 3) PG neuroprotection against Aβ-induced neuroinflammation is mediated by suppressing ER stress activation in astrocytes. Consistent with these findings, all of these data support the potential neuroprotective mechanism of PG in regulating astrocytes and demonstrate its neuroprotective capacity for the treatment of certain neurodegenerative conditions that have been associated with Aβ-induced neuroinflammation. Acknowledgments We are grateful to Dr. Haishui Shi for the help given with the writing of the manuscript and the scientific guidance provided.

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