Resveratrol Downregulates STAT3 Expression and Astrocyte Activation in Primary Astrocyte Cultures of Rat
Abstract
Astrocytes respond to all forms of central nervous system (CNS) insults by a process referred to as reactive astrogliosis. Inhibition of astrocyte growth and activation is an important strategy for promoting injured CNS repair. STAT3 (signal transducer and activator of transcription 3) is reported to be a critical regulator of astrogliosis, and resveratrol (RES, a dietary polyphenol) is considered to be a natural inhibitor of STAT3 expression and phosphorylation. In this study, we investigated the effects of RES on STAT3 expression and phosphorylation, and then on the proliferation and activation of astrocytes, a critical process in reactive astrogliosis, in rat primary cultured astrocytes and an in vitro scratch-wound model. RES downregulated the expression levels of STAT3, P-STAT3 and GFAP (glial fibrillary acidic protein) in cultured astro- cytes. The positive index of Ki67 was apparently reduced in cultured astrocytes after RES treatment. Meanwhile, cultured astrocyte proliferation and activation were attenuated by RES. Moreover, in the established in vitro scratch-wound model the increased expression levels of STAT3, P-STAT3 and GFAP induced by scratching injury were also clearly inhibited by RES. In addition, the inhibitory effect of RES on cell proliferation was similar to that of AG490 (a selective inhibitor of STAT3 phosphorylation) and abrogated by Colivelin (a STAT3 activator) stimuli. Taken together, our data suggest that RES is able to inhibit reactive astrocyte proliferation and activation mainly via deactivating STAT3 pathway. So RES may have a therapeutic benefit for the treatment of the injured CNS.
Keywords : Resveratrol · Astrocyte activation · STAT3 · GFAP
Introduction
Astrocytes are the most widely distributed, complex, and highly differentiated cells in the mammalian central nervous system (CNS). In addition to playing essential roles in the normal functioning of the healthy CNS, astrocytes respond to all forms of CNS insults by a process referred to as reac- tive astrogliosis[1]. Reactive astrocytes are characterized by increased glial fibrillary acidic protein (GFAP), cellular hypertrophy, and proliferation[2]. In severe cases, reactive astrocytes proliferate excessively and form glial scars in the damaged CNS [3], which are beneficial to CNS repair in the early phase. For example, glial scars can seal the lesion sites and separate the injured tissue from its surrounding micro- environment, thereby restoring homeostasis and preserving intact tissues. However, excessive glial scar formation can result in a mechanical and biochemical barrier to injured nervous tissue repair (axonal regeneration and functional recovery) in the later phase[4, 5]. Therefore, inhibition of growth and activation of astrocytes may reduce the negative effects and retain the beneficial effects of astrocyte reactiv- ity, which may be a new strategy to promote repair of the injured CNS.
Signaling by the Janus kinases-STAT3 (Jak-STAT3) path- way has been postulated to regulate astrogliogenesis dur- ing development [6], in part, because a genetic deficiency of STAT3 leads to impaired astroglial differentiation [7]. Moreover, it has been reported that STAT3 is a critical regu- lator of reactive astrogliosis and glial scar formation in CNS insults [8, 9]. Additionally, STAT3 is activated in many cell types by a number of cytokines, hormones, or growth factors implicated in injury responses, several of which, including IL-6 (interleukin-6), CNTF (ciliary neurotrophic factor), LIF (leukemia inhibitory factor), EGF (epidermal growth fac- tor), and TGFα (transforming growth factor α), have been implicated as triggers of reactive astrogliosis [10]. Thus, STAT3 is an important activator of astrocyte reactivity and its modulation affects the repair of CNS damage.
The dietary polyphenol RES (3,5,4ʹ-trans-trihydroxy- stilbene), which is mainly found in grapes, berries, peanuts, and red wine, has been extensively studied due to its pro- tective properties, such as antitumor, antioxidant, and anti- inflammatory activities [11]. Importantly, RES can pass the blood–brain barrier and induce neuroprotective effects [12, 13]. Our previous studies of RES have demonstrated that RES is able to effectively suppress the proliferation of sev- eral tumor cells by inhibition of STAT3 signaling [14–16]. Recently, a series of studies further confirmed these data in other cell types [17–20]. These findings indicate that STAT3 is a major molecular target of RES. However, it is unknown if RES elicits similar outcomes for astrocytes. In the pre- sent investigation, we evaluate the effects of RES on STAT3 expression and phosphorylation, and then on the prolifera- tion and activation of cultured neonatal rat astrocytes.
Materials and Methods
Primary Astrocyte Cell Culture, Purification, and Treatments
Highly enriched primary astrocytes were prepared from the cerebral cortex of one-day-old neonatal rats. The cerebral cortex tissues from one-day-old neonatal rats were dis- sected and diced into 1–2-mm3 pieces in Hanks’ balanced salt solution (HBSS) and 10 mmol/L HEPES solution, fol- lowed by enzymatic digestion in HBSS/HEPES contain- ing 0.25% trypsin and 0.01% DNase I for 15 min at 37 °C with occasional shaking. Enzymatic digestion was stopped by the addition of an equal volume of DMEM containing 20% fetal bovine serum (FBS; Gibco Life Technologies, USA). The cerebral cortex tissues were further dissociated by repeated trituration with fire-polished Pasteur pipettes and were filtered through a 70-µm nylon mesh, centrifuged, and resuspended in astrocyte medium (DMEM, 10% FBS, 2 mmol/L glutamine, 100 IU/mL penicillin, 100 µg/mL strep- tomycin). The isolated cells were plated in T75 flasks and cultured in a 37 °C, 5% CO2 incubator until cells grew to confluence after 8–10 days. The flasks were then shaken at 275 rotations/min overnight at 37°C to remove neurons, microglia, and oligodendrocytes. Then, cells were washed with pre-warmed supplemented DMEM, trypsinized, and re-seeded for the experimental procedures. Confirmation of an astrocyte phenotype was based on the cells exhibiting a characteristic morphology and positive staining for GFAP. All astrocytes used in this study were passage (P) 2 cells with a purity of > 95%.
To assess the effects of RES, astrocytes were treated with various concentrations (10, 20, 40, 60, 80, 100 µmol/L) of RES (Sigma-Aldrich, USA) dissolved in DMSO) or a DMSO control at a final DMSO concentration of 0.1%. After 48 h of incubation, the cell-bearing coverslips were fixed in 4% paraformaldehyde (pH 7.4) for morphological and immunofluorescence (IF) staining analysis, and cell numbers and viabilities were determined by cell counting and MTT assay. The experimental groups were in triplicate and the experiments were repeated three times.
Immunofluorescence (IF) Staining
The harvested cell-bearing coverslips in different experimen- tal groups were fixed with 4% paraformaldehyde for IF stain- ing. Rabbit polyclonal antibodies against GFAP (ProteinTech Group Inc., USA), STAT3 (Santa Cruz Biotechnology,USA), or Ki67 (ProteinTech Group Inc., USA) were applied over- night at 4 °C. Next, the appropriate fluorophore-conjugated secondary antibodies (1:200) were applied and the nuclei were counterstained with DAPI. Negative control experi- ments performed with the appropriate species-specific IgG or with inappropriate secondary antibodies showed negligi- ble background.
Protein Preparation and Western Blotting
The astrocytes in different experimental groups were har- vested in ice-cold lysis buffer (50 mmol/L Tris, 150 mmol/L NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, sodium orthovanadate, sodium fluoride, EDTA, leu- peptin, Beyotime Institute of Biotechnology, China). Equiva- lent amounts of total protein from each sample were mixed with sample buffer, boiled, and loaded onto SDS polyacryla- mide gels. Electrophoretic separation of the extracts was typ- ically performed on 7.5–15% discontinuous acrylamide gels (depending on the molecular weight of the protein of inter- est) under denaturing conditions. Proteins were then trans- ferred to polyvinylidene fluoride membrane (Amersham, Buckinghamshire, UK) and probed with primary antibod- ies against GFAP (ProteinTech Group Inc., USA), STAT3, and p-STAT3 (phosphorylation at Tyr705, Santa Cruz Bio- technology, USA). Meanwhile, an antibody against β-actin (ProteinTech Group Inc., USA) was used to stain β-actin as a loading control. Appropriate secondary HRP-conjugated antibodies were used for detection with chemiluminescent ECL reagents (Roche GmbH, Mannheim, Germany).
Cell Viability and Proliferation Assay
Cell viability was evaluated using a 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyl- tetrazolium bromide (MTT) assay. Cells (6 × 103/well) were plated in 96-well flat-bottomed culture plates (Falcon, Becton–Dickinson Labware, Franklin Lakes, NJ, USA) and routinely cultured in 200 µL DMEM medium for 24 h. The cultures were then treated with 10, 20, 40, 60, 80, and 100 µmol/L of RES for 48 h. After treatment, MTT reagent was added to each well and the plate was incubated for 4 h at 37 °C. The plate was left at room temperature until completely dry. DMSO was added and the optical den- sity (OD) of the sample plate was measured at 492 nm in a microplate reader. The result for each experimental condi- tion was verified a minimum of three times.
Cell counting was performed using an automated cell counter (Bio-Rad, Singapore) to evaluate cell growth after treatment. To detect the effect of RES on astrocyte prolifera- tion, Ki67 IF staining was performed on astrocytes with and without RES treatment and its positive index was obtained by counting Ki67-positive versus 1000 observed GFAP- positive cells under 200 × magnification. GFAP- and Ki67- positive cell numbers in the DMSO control and RES-treated groups were counted under a fluorescence microscope by two independent researchers.
Model of Reactive Astrocytes and Treatment
In order to confirm the effects of RES on reactive astro- cytes, a reactive astrocyte model was established by mechanically scratching primary cultured astrocytes to induce astrocyte activation. Astrocyte scratch injury was performed as previously reported [24]. Astrocytes were plated in poly-d-lysine (PDL)-coated culture dishes and grown to confluence. The cell monolayer was scratched with a sterile 200-µL plastic pipette tip, resulting in the formation of a 1-mm-wide gap. Immediately after scratching, cultured cells were washed twice with ster- ile 1 × PBS (phosphate buffer saline). Then, the cultured cells were divided into three experimental groups: con- trol group (primary astrocytes without lesion), scratched group (scratched astrocytes with DMSO treatment), and scratch + RES group (scratched astrocytes treated with 80 µM RES in DMSO for 24 h). After 24-h treatment, IF of Ki67 was performed on the cell-bearing coverslips, and the cells in the different experimental groups were har- vested to determine STAT3, p-STAT3, and GFAP levels by Western blotting.
Ethics Statement
Prior to the animal experiments, the research protocols were reviewed and approved by the Animal Care and Use Committee of Dalian Medical University, China. All work involving experimental animals was performed in full compliance with the NIH (National Institutes of Health) Guidelines for the Care and Use of Laboratory Animals. All experiments were performed under chloral hydrate anesthesia and all efforts were made to minimize suffering.
Statistical Analysis
Data are presented as mean ± standard deviation of the mean (SD). The data with two groups were analyzed by Student’s t test (when the data distributed normally) or nonparametric Mann-Whitney U test (when the data did not distribute normally). The data with three or more groups were analyzed by one-way ANOVA followed by Tukey post-hoc test to determine whether there were significant differences between individual groups. All experiments were repeated at least three times. Values of P < 0.05 were considered statistically significant. Results RES Decreases STAT3 Transcription and Activation in Cultured Astrocytes The effect of RES on STAT3 transcription and activation in primary astrocytes was analyzed by real-time PCR using RNA samples extracted from the astrocytes without and with RES treatment (80 µmol/L) for 48 h. As shown in Fig. 1a, RES treatment decreased STAT3 (0.52 ± 0.09-fold change, P < 0.05) transcription compared with control astrocytes. Immunofluorescence staining of the same experimental groups revealed strong STAT3 staining in the nuclei of con- trol astrocytes, which was weakened after 48 h of 80 µmol/L RES treatment (Fig. 1). In accordance with the results of real-time PCR and IF, Western blotting showed that RES treatment caused a reduction of STAT3 expression, and P-STAT3 was significantly diminished after RES treatment (Fig. 1). RES Inhibits Cultured Astrocyte Activation To examine the effect of RES on astrocyte activation, pri- mary astrocyte cultures from the cerebral cortex of neonatal rats were treated with various concentrations (10, 20, 40, 60, 80, and 100 µmol/L) of RES. After RES treatment for 48 h, astrocyte activation were determined using the MTT assay, cell counting, and IF staining of Ki67 and GFAP. Immunofluorescence staining showed that over 95% of the cultured cells were GFAP positive (Fig. 1c). The MTT assay showed that astrocyte growth was suppressed by 80 and 100 µmol/L RES after 48-h treatment (Fig. 2a, P < 0.05). The effect of 80 µmol/L RES on astrocyte growth was also evalu- ated by cell counting at 24 h, 48 h and 72 h. The results showed that the number of cells was decreased by RES (80 µmol/L) treatment by 6.7% at 24 h, 26.9% at 48 h and 33.1% at 72 h compared to control cells (Fig. 2b, P < 0.05). Ki67 IF staining revealed that the Ki67-positive index of the RES group (59.84 ± 6.74%) was lower than that of the DMSO control group (78.6 ± 2.49%; Fig. 3, P < 0.01). In addition, flow cytometry demonstrated that RES treatment caused distinct G1-phase arrest of the cultured astrocytes. We observed that the G1 fraction was 65.76 ± 1.96% in the control cells and 78.15 ± 5.08% in the RES-treated cells (P < 0.05). Meanwhile, the proportion of S phase decreased from 32.71 ± 2.71% to 20.8 ± 4.95% when the cultured astrocytes were treated with RES (Fig. 3c). These results suggest a slower rate of proliferation in RES-treated astrocytes com- pared to control cells. The effect of RES on GFAP expression in cultured astro- cytes was analyzed by using real-time PCR using RNA samples extracted from the astrocytes without and with RES treatment (80 µmol/L) for 48 h. As shown in Fig. 1a, RES treatment decreased GFAP (0.51 ± 0.11-fold change, P < 0.05) transcription compared with control astrocytes. Immunofluorescence staining of the same experimen- tal groups revealed strong GFAP staining in the cytosolic space of control astrocytes, which was weakened after 48 h of 80 µmol/L RES treatment (Fig. 1c). In accordance with the results of real-time PCR and IF, Western blotting showed that RES treatment caused GFAP reduction (Fig. 1). Together, all these data suggest that RES inhibits astrocyte activation. RES Suppresses Scratch‑Induced STAT3 Expression and Astrocyte Activation In order to confirm the effects of RES on reactive astro- cytes, an in vitro scratch-wound model was established. In the model, primary astrocytes were scratched with a pipette tip and the astrocyte responses to injury were analyzed by Western blotting. After injury (24 h), the levels of GFAP, STAT3, and p-STAT3 were upregulated in the lesioned astrocytes, demonstrating that reactive astrocytes formed after scratching (Fig. 4). We then tested whether STAT3 activation was suppressed by RES in the reactive astrocyte model. The activation of astrocytes by injury caused increased expression of GFAP, STAT3 and p-STAT3 in these cells but RES treatment decreased the levels of GFAP, STAT3 and p-STAT3 in reac- tive astrocytes (Fig. 4a–d, P < 0.05). Meanwhile, the positive index of Ki67 was decreased in RES group ( 47.2 ± 7.3%) comparing with scratch group ( 73.7 ± 8.1%, P < 0.01; Fig. 4e). These data suggest that RES effectively decreases scratch-induced STAT3 and p-STAT3 expression, and then astrocyte activation. Effects of Colivelin and AG490 on Astrocyte Proliferation To further clarify that inhibitory effect of RES on astro- cyte proliferation was mediated by a decreased level of P-STAT3, Colivelin (a STAT3 activator) and AG490 (a selective inhibitor of STAT3 phosphorylation) were used to stimulate or inhibit STAT3 phosphorylation in our sys- tem [14, 25]. The Western blot assay showed that Colivelin significantly increased the phosphorylation level of STAT3, while both AG490 and RES inhibited it. Moreover, RES also suppressed Colivelin-stimulated STAT3 phosphoryla- tion, but the level of p-STAT3 in Colivelin + RES group was still higher than that in RES group (Fig. 5a, b). Next, IF staining results showed that Ki67-positive index in Colivelin group (88.87 ± 7.97%) was much higher than that in con- trol group (63.18 ± 9.23%, P < 0.05), while Ki67-positive index in AG490 (28.51 ± 17.71%) or RES (27.47 ± 11.57%) group was lower than that in control group (63.18 ± 9.23%, P < 0.05, Fig. 5c, d). Furthermore, compared with RES group (27.47 ± 11.57%), the Ki67-positive index of Colive- lin + RES group was higher (67.41 ± 15.37%, P < 0.05, Fig. 5c, d ) Discussion This study showed that RES was able to suppress cultured astrocyte growth with decreased positive index of Ki67 (a marker of the cell proliferation) and G1 arrest. These results indicate that RES can inhibit the proliferation of non-tumor cells, which is consistent with previous studies regarding inhibitory effects of RES on tumor cell growth. Moreo- ver, our results indicated that RES could downregulate the expression of GFAP (a major intermediate filament protein in reactive astrocytes) in the cultured astrocytes. Notably, astrocytes are ubiquitous throughout all regions of the CNS and, in response to several neuropathological conditions of the CNS, astrocytes become reactive [26–28]. Astrocytes and reactive astrocytes are increasingly recognized as poten- tial targets for therapeutic strategies for CNS diseases. In this sense, the results in the present study suggest that RES has a potential role as a neuroprotective agent in central nervous system diseases. Many signaling cascades have been associated with astrocyte reactivity, but among them, the STAT3 signaling is emerging as a central regulator and hub to orchestrate numerous molecular and functional changes in reactive astroyctes [29]. Reactive astrogliosis can lead to astrocyte proliferation and scar formation in the injured CNS [30]. STAT3 pathway activation may induce transcriptional changes of certain genes in astrocytes, leading to the growth and proliferation of these cells [29, 31]. Gene expression microarray studies showed that the cell cycle genes were down-regulated in STAT3-CKO astrocytes, which may con- trol astrocyte proliferation [32]. Our previous studies have demonstrated that RES arrested the cells at G1 or S phase of cell cycle and induced apoptosis of malignant cells through directly suppressing STAT3 transcription, thereby altering the expression of downstream genes such as CyclinD1 and Bcl-2 [14–16]. These findings provide evidence for the inhi- bition of STAT3 biological function and activity by RES. In the present study, our results show that RES attenuated STAT3 signaling and caused arrest of cultured astrocyte proliferation in G1 phase, but a typical sub-G1 fraction in RES-treated astrocytes was not detected (Fig. 3c). So the growth inhibitory activity of RES in cultured astrocytes was driven by the induction of cell cycle arrest, not by apoptosis. These different results suggest that the response to RES is cell type-specific. In addition to cell proliferation, STAT3 also regulates the expression of GFAP in reactive astrocytes, as the gfap pro- moter has the STAT3 consensus binding sites required for induction of GFAP [33]. Thus, the STAT3 signaling pathway may serve as a target for pharmacological modification of reactive astrocytes in CNS disorders. Genetic invalidation or pharmacological inhibition of STAT3, consistently reduces or prevents the increase in gfap mRNA and/or protein levels in astrocytes following induction of reactivity. In our study, real-time PCR, Western blotting, and IF analyses showed that RES imposed led to lower levels of STAT3, p-STAT3, and GFAP in primary rat astrocyte cultures. These results show that levels of GFAP are reduced by STAT3 inhibition in cultured non-lesioned astrocytes, suggesting that STAT3 may regulate the basal expression of GFAP. Moreover, in the astrocyte culture scratch-injury model, STAT3 and p-STAT3 levels were reduced in parallel with the decrease in the GFAP immunoreactivity and Ki67 positive index after RES treatment. These results demonstrate the inhibitory effects of RES on STAT3 signaling in cultured astrocytes. Interest- ingly, we observed changes in astrocyte morphology after RES treatment (Fig. 1). RES-treated astrocytes in our cur- rent study displayed an elongated morphology in most cases. Considering that GFAP is a major IF (intermediate filament) protein expressed in reactive astrocytes, the morphological changes may be related to the reduction in GFAP accumula- tion due to RES. The STAT3 pathway is a universal inducer of astrocyte reactivity. For example, prior work from Sofroniew’s lab demonstrates that STAT3 pathway plays a dominant role in traumatic and neurotoxicity-induced astrogliosis using both a conditional gene deletion strategy (STAT3-CKO) and pharmacological inhibition of STAT3 in vivo and in vitro [8, 32, 34]. Notably, they also looked for potential effects of STAT3-CKO on other signaling pathways and found no detectable differences in the levels of p-p38, pErk and pJnk MAP kinases between control and STAT3- CKO astrocytes [32]. These findings support a pivotal role of STAT3 in reactive astrocytes. In addition, their results show that p-STAT3 tyr705 appears to be a key sign- aling event in astrogliosis resulting from divergent types of neural damages [8, 34]. Our present study indicates that RES significantly reduced the levels of STAT3 and p-STAT3, suggesting that RES can inhibit the activation of the STAT3 pathway in cultured astrocytes. In this study, we promoted or inhibited STAT3 phosphorylation by using Colivelin or AG490 and found that the stimulated growth of Colivelin-treated astrocytes and the suppressed growth of AG490-treated cells support the critical roles of STAT3 phosphorylation in promoting astrocyte proliferation. Fur- thermore, Colivelin did increase basal and RES-inhibited STAT3 phosphorylation in parallel with abrogating the inhibitory effects of RES on astrocyte proliferation. These evidences suggest that RES exerts anti-proliferation effect mainly though deactivating STAT3 signaling pathway.
RES, a dietary polyphenol found in grapes, berries, peanuts, and red wine, exhibits a wide range of biological effects, such as anti-inflammatory, antioxidant, and chemo- preventive activities [35]. More importantly, the central nervous system is a target of RES, which can pass the blood–brain barrier and induce neuroprotective effects that modulate glial functions [12, 13]. For example, RES could modulate glutathione (GSH) system in C6 astroglial cells [36]. Additionally, our findings further prove that RES can effectively inhibit the growth and GFAP expression of astrocytes, suggesting that RES does have the potential to repress reactive astrogliosis. RES obstructs the prolifera- tion of a variety of tumor cells via the inhibition of STAT3 signaling [37]. Here, our data show that 80 µmol/L RES causes inhibition of STAT3 in primary cultured astrocytes, which adds to the knowledge of its anti-proliferative poten- tial in non-tumor cells. However, Abdalla F et al reported that pre-treatment with 50 µmol/L RES did not decrease the HIV1-gp120-induced phosphorylation of STAT3 in SVG astrocytes [38]. This discrepancy may be related to the concentration of RES and the characteristics of the target cell.
In summary, RES effectively diminishes reactive astrocyte growth and activation, therefore, may regulate glial scar formation in the repair of the injured CNS. Thus, RES is a potential and promising agent that may translate into an effective clinical treatment for CNS-injured patients.