Pulsed Electromagnetic Fields Protect Against Brain Ischemia by Modulating the Astrocytic Cholinergic Anti-inflammatory Pathway

Abstract

Neuroinflammation is one of the most important pathological processes following brain ischemia. Pulsed electromagnetic fields (PEMFs) protect against brain ischemia, but their role in regulating neuroinflammation remains unclear. In the present study, we investigated the biological effects of PEMF exposure on brain ischemia-induced neuroinflammation through the astrocytic cholinergic anti-inflammatory pathway. PEMF exposure reduced the activation of astrocytes and neuroinflammation following brain ischemia by directly modulating astrocytic injury and inflammatory cytokine release. Inhibition of nicotinic acetylcholine receptor alpha 7 subunit (α7nAChR) by a specific antagonist reversed the regulatory effects of PEMF on astrocytes. Furthermore, negative regulation of signal transducer and activator of transcription 3 (STAT3) by α7nAChR was found to be an important downstream mechanism through which PEMF regulates astrocyte-related neuroinflammation. PEMF suppressed STAT3 phosphorylation and nuclear translocation by activating α7nAChR. These results demonstrate that PEMF exerts anti-inflammatory effects in the context of brain ischemia by modulating astrocytic α7nAChR/STAT3 signaling.

Graphical Abstract

Supplementary Information

The online version contains supplementary material available at 10.1007/s10571-022-01251-2.

Introduction

Stroke has become the leading cause of death and disability in the world, and ischemic stroke is the most important type of stroke, accounting for 60%–70% of stroke cases (Wu et al. 2019). The application of vascular recanalization therapy is limited due to its narrow time window (GBDS 2021). Neuroprotective therapy has not yet achieved ideal results in large-scale clinical research. Strategies for effectively preventing and alleviating ischemic brain injury remain the focus of current medical research. An increasing number of researchers believe that secondary injury caused by inflammation may be more severe than primary ischemic injury (Shichita et al. 2017; Zhang et al. 2021b). Therefore, the effective regulation of acute inflammation after cerebral ischemia such that it can play its normal role in maintaining and restoring function without causing tissue damage has become a new focus of research on the prevention and treatment of cerebral ischemia.

Neuroinflammation after cerebral ischemia is initiated and regulated by glial cells, but previous studies have mostly focused on the role of microglia (Werner et al. 2020). As nontraditional inflammatory cells, astrocytes, which account for approximately 90% of all neural cells, also play an important role in the initiation and regulation of the inflammatory response after cerebral ischemia (Shi et al. 2021). On the one hand, astrocytes proliferate and release proinflammatory cytokines, such as tumor necrosis factor-alpha (TNF), interleukin (IL)-6, and other inflammatory mediators, which can damage neurons (Liu et al. 2019). On the other hand, astrocytes can secrete IL-10, transforming growth factor-β (TGF-β), and other anti-inflammatory cytokines to inhibit the inflammatory reaction after ischemia (Luo et al. 2019a). Therefore, strategies for regulating the function of astrocytes in the inflammatory response after cerebral ischemia may be developed as new approaches for the prevention and treatment of cerebral ischemic injury.

Pulsed electromagnetic fields (PEMFs), as an external physical stimulus that can be applied to the human body, have been widely used for the treatment of neurological diseases, such as pathological pain, depression, and Parkinson’s disease, and the application of PEMFs has become an important method for achieving neuromodulation (Dayan et al. 2013). Previous studies have shown that PEMFs can reduce the infarct area in the brain after stroke and relieve brain tissue damage. In addition to directly inhibiting neuronal death caused by brain injury, PEMFs can promote the activation of astrocytes (Pena-Philippides et al. 2014). Furthermore, PEMFs modulate inflammatory cytokines after ischemic stroke, and this effect might be related to astrocyte dysfunction (Rasouli et al. 2012). However, the role of PEMFs in modulating astrocyte-related neuroinflammation following cerebral ischemia and the underlying mechanism have not yet been elucidated.

Nicotinic acetylcholine receptor alpha 7 subunit (α7nAChR) is widely expressed on the surface of many cells, including neurons, astrocytes, and microglia, and is the main component of the cholinergic anti-inflammatory pathway (Piovesana et al. 2021; Gamage et al. 2020). Previous studies have demonstrated that the expression and activity of α7nAChR are decreased in the ischemic area of the brain and that the activation of α7nAChR can reduce the release of inflammatory cytokines after brain ischemia to alleviate neurological deficits (Kelso and Oestreich 2012; Gatson et al. 2015). In addition, previous studies have shown that the responsiveness of brain tissues to physical stimuli is related to the modulation of α7nAChR signaling and its regulatory effects on the phenotypic conversion of microglia (Wang et al. 2012a; Ma et al. 2019). However, the biological effects of PEMFs on astrocytic α7nAChR signaling remain unclear.

Hence, our objectives were to determine the biological effects of PEMFs on astrocytes after cerebral ischemia, clarify the relationship between PEMFs and astrocyte-related neuroinflammation, and investigate the possible mechanism through which PEMFs regulate astrocytic α7nAChR signaling. Through these studies, we hope to further elucidate the therapeutic effects of PEMFs on neuroinflammation and the underlying cellular mechanism and to develop a novel strategy for the treatment of cerebral ischemia.

Materials and Methods

Animals

Male C57BL/6 mice (aged 10–12 weeks, weighing 25–28 g), which were obtained from the Experimental Center of Fourth Military Medical University, were housed in cages (six mice per cage) in an air-conditioned room at a constant temperature (approximately 27 °C) and with a 12-h light/dark cycle for at least 7 days before the study. The sample size was determined a priori for all experiments using G*Power 3.1 software (Faul et al. 2009; Charan and Kantharia 2013). Randomization of the animal groups was performed with RandoMice software (v1.1.1) according to the baseline performance in motor function tests (van Eenige et al. 2020). All animal studies were performed in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and approved by the Fourth Military Medical University Committee on Animal Care (No. 20210419).

Primary Culture of Cortical Astrocytes

Primary astrocytes were obtained from the cerebral cortices of 1- to 3-day-old neonatal C57BL/6 pups. In brief, the brains were obtained, minced, and trypsinized in 0.25% phosphate-buffered saline (PBS) to generate a cell suspension, and the resulting suspension was placed in Dulbecco’s modified Eagle’s medium (DMEM) (#11,054,001, Life Technologies, Carlsbad, CA, USA) containing 20% (v/v) fetal bovine serum (FBS) (#A3161001C, Life Technologies), cultured in poly-L-lysine (#P4707, Sigma–Aldrich, St. Louis, MO, USA)-coated T75 flasks and maintained at 37 °C in 90% relative humidity in the presence of 5% CO₂. Half of the medium was replaced every 3 days. When the cells reached confluence after 10–14 days, the flasks were shaken at 200–220 r/min for 14–16 h to remove microglia and oligodendrocytes. After shaking, more than 95% of the cells were astrocytes, as determined by immunofluorescence staining for glial fibrillary acidic protein (GFAP). After isolation, the cells were subcultured at a density of 3 ~ 4 × 10⁵ cells/well in different plates for the various experiments.

Primary Culture of Cortical Neurons

Neuronal cortical cultures were prepared as previously described (Luo et al. 2019b). Briefly, cerebral cortices were removed from embryos at 16–18 d. Tissues were dissociated by gentle trituration with 0.25% trypsin for 15 min at 37 °C. The neurons were resuspended in neurobasal medium (#21,103,049, Thermo Fisher Scientific, Waltham, MA, USA) containing 2% B27 supplement (#A3582801, Thermo Fisher Scientific) and 0.5 mM L-glutamine and plated at a density of 3 × 10⁵ cells/cm². Before seeding, culture vessels consisting of 96-well plates, 1.5-cm glass slides, or 6-cm dishes were coated with poly-L-lysine (50 μg/mL) overnight at room temperature. The neurons were maintained at 37 °C in a humidified 5% CO₂ incubator, and half of the culture medium was changed every other day. The cultured neurons were used for in vitro studies on days 12–14 (DIV 12–14) and verified to have a viability higher than 95%.

Antibodies and Reagents

A primary antibody against GFAP (#MA5-12,023, mouse mAb) was obtained from Invitrogen (Thermo Fisher Scientific). Antibodies against signal transducer and activator of transcription 3 (STAT3) (#ab68153, rabbit mAb, knockout validation) and p-STAT3 (#ab76315, rabbit mAb) were obtained from Abcam (Cambridge, UK). An antibody against GAPDH (#5174, rabbit mAb) was obtained from Cell Signaling Technology (Danvers, MA, USA). For immunoblotting, HRP-conjugated goat anti-rabbit (#sc-2004) and goat anti-mouse (#sc-2005) secondary antibodies (Santa Cruz Biotechnology, CA, USA) were used. Alexa Fluor 488-conjugated goat anti-mouse IgG (#A28175) and Alexa Fluor 594-conjugated goat anti-rabbit IgG (#A-11012) secondary antibodies (Thermo Fisher Scientific) were used for immunostaining. α-Bungarotoxin (α-BGT, #2133) and colivelin (Tocris, #3945) were obtained from Tocris Bioscience (Bristol, UK).

Oxygen–Glucose Deprivation (OGD)

OGD was induced as described previously (Li et al. 2014). In brief, the culture medium was replaced with serum- and glucose-free DMEM, and the cells were placed in an incubator that was flushed for 5 min with 95% N₂ and 5% CO₂ at 37 °C (to mimic hypoxic conditions). Control cultures were incubated for the same period of time at 37 °C in a humidified atmosphere consisting of 95% air and 5% CO₂. After 4 h of challenge, the astrocytes or neurons were removed from the anaerobic chamber, and the medium was replaced with DMEM containing 10% FBS or neurobasal medium. The cells were maintained for an additional 24 h at 37 °C in a humidified 5% CO₂ incubator to mimic reperfusion.

Lactate Dehydrogenase (LDH) Assay

Cytotoxicity was assessed by measuring the release of LDH, which is a cytoplasmic enzyme released from cells and a marker of membrane integrity. The amount of LDH released into the culture medium was measured using an LDH cytotoxicity assay kit (#601,170) obtained from Cayman Chemical (Ann Arbor, MI, USA) according to the manufacturer’s instructions. Briefly, 100 μl of supernatant was collected from each well and placed in a new 96-well plate. LDH reaction solution was added to each well, and the mixture was incubated for 30 min at 37 °C with gentle shaking. LDH activity was determined by measuring the absorbance at a wavelength of 490 nm with a plate reader.

Cell Counting Kit-8 (CCK-8) Assay

The cell viability was evaluated using a CCK-8 assay kit (#C0037, Beyotime, Shanghai, China) following the manufacturer’s protocol. The cells were cultured in 96-well plates in a final volume of 100 μl of culture medium/well. After treatment, 10 μl of CCK-8 reagent was added to each well, and the cells were incubated for 2 h at 37 °C in 5% CO₂. The samples were shaken thoroughly for 1 min on a shaker. The absorbance of the samples was measured at a wavelength of 450 nm using a microplate reader.

Focal Cerebral Ischemia and Reperfusion

Middle cerebral artery occlusion (MCAO) was induced in 8-week-old mice following a standard protocol. Occlusion was maintained for 120 min before initiation of reperfusion, and the regional cerebral blood flow was monitored by laser Doppler flowmetry (PeriFlux 5000, Perimed AB, Järfälla, Sweden). The establishment of MCAO was considered successful if the regional cerebral blood flow showed a sharp decrease to less than 30% of the baseline (preischemia) level and recovered to more than 80% of the baseline level; animals that did not meet this requirement were excluded. Throughout the MCAO surgery, the body temperature was maintained at 37 ± 1 °C with a thermostatic blanket.

Motor Function Tests

Rotarod test: The rotarod test was performed using a rotarod device (Shanghai XinRuan Information Technology, Shanghai, China) according to a previous report. The starting speed of the rotarod was set to 5 rpm, and the acceleration was set to 20 rpm/min. Ten seconds after a mouse was placed on the rod, acceleration was initiated, and the speed at which the mouse fell off the rod was recorded. The mean speed at which the mouse fell off the rod in three attempts was recorded.

Forelimb use asymmetry (FUA) test: A mouse was placed in a cylinder (15 cm × 9 cm) and its forelimb usage during at least 15 exploratory movements (less than 20) within a period of up to 10 min was recorded. The number of times the forelimb on the same side as the brain injury was used was recorded as the ipsilateral (I) value. The number of times the forelimb on the side opposite the brain injury was used was recorded as the contralateral (C) value. The number of uses of both forelimbs was considered the bilateral (B) value. The FUA score is calculated as [I-C]/[I + C + B] × 100.

Assessment of the Infarct Volume

The mice were decapitated under anesthesia, and 2-mm-thick coronal sections of the entire brain were stained with 2% 2,3,5-triphenyltetrazolium chloride (TTC, #T8877, Sigma–Aldrich, St. Louis, MO, USA) to evaluate the infarct volume. The infarct volume percentage was calculated after TTC staining. The unstained white area was considered the lesioned area, and the red-stained area was considered the nonlesioned area. The nonlesioned area of the right hemisphere (RN) and the total area of the left hemisphere (LT) were measured by an investigator blinded to the experimental grouping using image analysis software (Adobe Photoshop CC 2019, Adobe Systems Incorporated, San Jose, CA, USA). The noninfarcted volume of the right hemisphere and the total volume of the left hemisphere are calculated as follows: V_RN = RN × slice thickness (2 mm) and V_LT = LT × slice thickness (2 mm). The infarct volume is measured as follows: %V_I = 100 × (V_LT − V_RN)/V_LT.

PEMF Application

The PEMF stimulation system was established as previously described (Li et al. 2017). In brief, this system comprised a PEMF generator (GHY-III, FMMU, Xi’an, China; China Patent No. ZL02224739.4) and a solenoid surrounded by 400 turns of enamel-coated copper wire. The waveforms generated by the PEMF system consisted of a pulse burst (burst width, 5 ms; pulse width, 0.2 ms; pulse wait, 0.02 ms; burst wait, 60 ms; pulse rise, 0.3 μs; pulse fall, 2.0 μs) repeated at 15 Hz as described in previous studies (Lei et al. 2018; Shao et al. 2021; Wang et al. 2019). The measurement accuracy of the electromagnetic field output was confirmed using a Gauss meter (Model 455 DSP Gaussmeter, Lake Shore Cryotronics). For the in vivo study, mice that underwent MCAO surgery were placed in the center of the coils 4 h after surgery for 4 h per day. Untreated MCAO mice were placed in a different chamber containing inactivated Helmholtz coils (no treatment, NT). For the in vitro study, a cell culture plate was placed in the center of the coils 4 h after OGD. Each cell culture plate belonging to the control group was placed in the center of coils that were connected to the pulse generator without generation of an output waveform (no treatment, NT).

Western Blot Analysis

After the various treatments, cell samples or brain tissue from the injury site were lysed with buffer containing PhosSTOP protease inhibitor (#4906845001) and phosphatase inhibitor (#5892970001) tablets (Roche Applied Bioscience, Indianapolis, IN, USA). The protein concentration in the supernatant was determined using a BCA protein kit. The proteins were separated on 10–15% and 10% SDS-PAGE gels and transferred onto nitrocellulose membranes (Thermo Fisher Scientific). The membranes were cut according to prestained protein ladders (#26617, Thermo Fisher Scientific) and specific molecular weights (see Fig. S1). The membranes were then soaked in 5% nonfat milk in Tris-buffered saline and 0.05% Tween 20 (TBST) for 1 h at room temperature and incubated overnight at 4 °C with the appropriate primary antibodies (GFAP, 1:1000 dilution; GAPDH, 1:1000 dilution; STAT3, 1:500 dilution; p-STAT3, 1:500 dilution). The membranes were washed with TBST and incubated for 1 h at room temperature with secondary antibodies diluted in blocking buffer. The GAPDH, STAT3, and p-STAT3 antibodies were specifically detected with a goat anti-rabbit secondary antibody. The GFAP antibody was specifically detected with a goat anti-mouse antibody. The immunoreactivity was detected with SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific). After the membranes were probed with a p-STAT3 antibody, they were stripped for 15 min at room temperature with Re-blot Plus Strong Solution (#2504, Millipore, Burlington, MA, USA) and reprobed with STAT3 antibodies for normalization of the p-STAT3 levels to control for protein loading. The optical densities of the bands were quantified using an image analysis system with ImageJ (National Institutes of Health, MA, USA). All raw data are reported in the Supplemental Materials (Fig. S1).

Inflammatory Cytokine Assessment

The release of proinflammatory cytokines and anti-inflammatory cytokines into the culture medium and brain tissue was analyzed using a mouse TNF-α ELISA kit (# CSB-E04741m, Cusabio, Houston, TX, USA), mouse TGF-β ELISA kit (#CSB-E04726m, Cusabio), mouse IL-10 ELISA kit (#CSB-E04594m, Cusabio), and mouse IL-6 ELISA kit (#E-EL-M0044c, Elabscience, Houston, TX, USA). Briefly, 100 μl of supernatant from each well of primary astrocytes or brain tissue homogenates was collected in a new 96-well plate. The levels of proinflammatory cytokines (TNF-α and IL-6) and anti-inflammatory cytokines (TGF-β and IL-10) were determined using specific quantitative sandwich ELISA kits according to the manufacturer’s instructions. The reaction was developed with streptavidin–horseradish peroxidase, and the optical density was read at a wavelength of 450 nm.

TUNEL Staining

For the quantification of cell death, TUNEL (green) staining was performed using the In Situ Apoptosis Detection Kit (#11,684,795,910, Roche) according to the manufacturer’s instructions. Brains were fixed with 4% paraformaldehyde for 4 h and then dehydrated with 30% sucrose solution. The specimens were cut into 30-μm sections for staining. The TUNEL-positive cells and 4’,6-diamidino-2-phenylindole (DAPI, #D9542, Sigma)-positive cells in images of three random areas of brain injury acquired with a 40 × objective were then counted by an investigator blinded to the experimental conditions, and the results are expressed as the number of TUNEL-positive cells relative to that of DAPI-positive cells (the cell death rate).

Immunofluorescence Staining

In vitro experiments: After fixation with 4% paraformaldehyde for 15 min at 37 °C, primary cultured cells were washed with PBS, permeabilized with 0.2% Triton X-100, and incubated with primary antibodies overnight at 4 °C. The primary antibodies were diluted as follows: GFAP, 1:500 and p-STAT3, 1:200. The cells were then incubated with secondary antibodies for 2 h. The cells were dehydrated with ethanol and mounted with DAPI for nuclear staining. Images were captured using an Olympus FV10i confocal microscope (Japan, Tokyo).

In vivo experiments: After anesthesia, the mice were perfused with ice-cold PBS and then with 4% paraformaldehyde. The brains were collected, postfixed for another 4 h, and then dehydrated with 30% sucrose. Frozen coronal slices (with a thickness of 30 mm) were obtained with a freezing microtome (Cryostat 1720, Leitz, Mannheim, Germany). The sections were washed with PBS, blocked with 5% normal goat serum for 30 min, incubated with a primary antibody against GFAP overnight at 4 °C, and washed three times with 0.01 M PBS. Appropriate secondary antibodies were applied at room temperature for 2 h. Antifade solution with or without DAPI was applied, and after the sections were completely dry, the injury site in brain tissue sections was observed with an Olympus microscope.

Statistical Analysis

Statistical evaluation was performed with GraphPad Prism software, version 9.0 (GraphPad, San Diego, CA, USA). The Shapiro–Wilk test was used to determine the normality of the data within sample groups. For the statistical analysis of two groups, the F test was used to determine the variance homogeneity, and an unpaired two-tailed Student’s t test was used to determine the significance of the difference between the two groups. For the statistical analysis of more than two groups, the Brown–Forsythe test was used to determine the variance homogeneity, and one-way ANOVA followed by Bonferroni’s multiple comparisons test was used to determine the significance of the differences among all the groups. The results from the analyses of normality and variance homogeneity are reported in Table S1. The data are expressed as the means ± SDs. A P value less than 0.05 was considered to indicate statistical significance.

Results

PEMF Treatment Reduced Brain Damage and Neurological Deficits Following Brain Ischemia

To investigate the biological effects of PEMFs on brain ischemia, we induced MCAO to mimic brain ischemia in vivo. The animals were exposed to PEMFs or control treatment 4 h per day for 7 days beginning 30 min after MCAO (after the animals recovered from anesthesia) (Fig. 1A). An analysis of motor function indicated that PEMF exposure prolonged the time spent on the rod in the rotarod test [Fig. 1B: all groups (one-way ANOVA), F(5, 66) = 76.92, P < 0.001; MCAO + NT vs. MCAO + PEMF (Bonferroni’s multiple comparisons test), t(66) = 5.550, P < 0.001] and increased the activity of the forelimb contralateral to the injury in the FUA test [Fig. 1C: all groups (one-way ANOVA), F(5, 66) = 187.9, P < 0.001; MCAO + NT vs. MCAO + PEMF (Bonferroni’s multiple comparisons test), t(66) = 6.610, P < 0.001]. Next, TTC staining [Fig. 1D: all groups (one-way ANOVA), F(2, 15) = 78.47, P < 0.001; MCAO + NT vs. MCAO + PEMF (Bonferroni’s multiple comparisons test), t(15) = 5.063, P < 0.001] and TUNEL staining [Fig. 1E: all groups (one-way ANOVA), F(2, 15) = 75.37, P < 0.001; MCAO + NT vs. MCAO + PEMF (Bonferroni’s multiple comparisons test), t(15) = 5.323, P < 0.001] showed that PEMFs attenuated the ischemic volume and brain tissue damage after MCAO. These results suggested that PEMFs exerted potential therapeutic effects on brain ischemia.

Fig. 1.

PEMF treatment reduces brain damage and alleviates neurological deficits following brain ischemia. A Schematic diagram of the study design. After MCAO, mice were exposed to PEMFs for 4 h per day for 7 days. The mice were assigned to the following experimental groups according to the study design: Sham group, Sham + NT group (without PEMF exposure), Sham + PEMF group, MCAO group, MCAO + NT group (without PEMF exposure), and MCAO + PEMF group. B The speed at which the mice fell off the rod in the rotarod test was measured 7 days after MCAO and PEMF exposure (n = 12 mice/group). C FUA scores were measured 7 days after MCAO and PEMF exposure (n = 12 mice/group). D TTC staining of brain tissue was performed 7 days after MCAO, and the infarct volume was calculated (n = 6 mice/group). E TUNEL staining of brain tissue was performed, and the percentage of TUNEL-positive cells was calculated (n = 6 mice/group). Scale bar = 200 μm. All data are presented as the means ± SDs. *P < 0.05, **P < 0.01, ***P < 0.001

PEMF Treatment Inhibited the Activation of Astrocytes and Neuroinflammation Following Brain Ischemia

To determine the ability of PEMF treatment to influence the activation of astrocytes, the expression of GFAP (an astrocyte marker) was examined in the different groups after MCAO. GFAP expression was significantly elevated following MCAO, and PEMF treatment reduced this elevation of GFAP expression, which indicated that the activation of astrocytes was inhibited by PEMF exposure [Fig. 2A-B: MCAO, MCAO + NT, and MCAO + PEMF (one-way ANOVA), F(3, 20) = 7.588, P = 0.005; MCAO + NT vs. MCAO + PEMF (Bonferroni’s multiple comparisons test), t(20) = 3.080, P = 0.008]. We then evaluated the effects of PEMF treatment on neuroinflammation following brain ischemia and found that the proinflammatory factor (TNF and IL-6) levels were significantly elevated following MCAO [Fig. 2C TNF: all groups (one-way ANOVA), F(3, 20) = 26.51, P < 0.001; sham vs. MCAO (Bonferroni’s multiple comparisons test), t(20) = 7.254, P < 0.001; Fig. 2C IL-6: all groups (one-way ANOVA), F(3, 20) = 49.48, P < 0.001; sham vs. MCAO (Bonferroni’s multiple comparisons test), t(20) = 9.055, P < 0.001]. Interestingly, MCAO also induced a significant elevation of anti-inflammatory factors (TGF-β and IL-10) and thus exerted endogenous protective effects against brain ischemia-induced neuroinflammation [Fig. 2D TGF-β: all groups (one-way ANOVA), F(3, 20) = 73.93, P < 0.001; sham vs. MCAO (Bonferroni’s multiple comparisons test), t(20) = 4.926, P < 0.001; Fig. 2D IL-10: all groups (one-way ANOVA), F(3, 20) = 34.64, P < 0.001; sham vs. MCAO (Bonferroni’s multiple comparisons test), t(20) = 6.273, P < 0.001]. After PEMF treatment, both the TNF-α levels and the IL-6 levels were decreased following MCAO [Fig. 2C TNF: MCAO + NT vs. MCAO + PEMF (Bonferroni’s multiple comparisons test), t(20) = 4.296, P = 0.002; Fig. 2C IL-6: MCAO + NT vs. MCAO + PEMF (Bonferroni’s multiple comparisons test), t(20) = 6.341, P < 0.001]. In contrast, PEMF treatment increased the expression of TGF and IL-10 following MCAO [Fig. 2D TGF-β: MCAO + NT vs. MCAO + PEMF (Bonferroni’s multiple comparisons test), t(20) = 7.028, P < 0.001; Fig. 2D IL-10: MCAO + NT vs. MCAO + PEMF (Bonferroni’s multiple comparisons test), t(20) = 5.069, P < 0.001]. These results showed that PEMF treatment attenuated brain ischemia-induced neuroinflammation.

Fig. 2.

PEMF treatment inhibits the activation of astrocytes and neuroinflammation following brain ischemia. A The expression of GFAP after MCAO was examined by immunofluorescence staining (n = 6 mice/group). Scale bar = 100 μm. B The expression of GFAP after MCAO was analyzed by western blotting (n = 6 mice/group). C The contents of TNF and IL-6 in brain tissue were examined by ELISA (n = 6 mice/group). D The contents of TGF-β and IL-10 in brain tissue were examined by ELISA (n = 6 mice/group). All data are presented as the means ± SDs. *P < 0.05, **P < 0.01, ***P < 0.001

PEMF Treatment Blocked Astrocytic Injury and the Release of Proinflammatory Cytokines from Astrocytes

To ascertain whether PEMF treatment participated in the regulation of astrocyte-related neuroinflammation, we established an OGD model using primary astrocyte cultures and exposed the cells to PEMFs (Fig. 3A). PEMF treatment increased the viability of primary astrocytes exposed to OGD [Fig. 3B: all groups (one-way ANOVA), F(5, 42) = 102.0, P < 0.001; control vs. OGD (Bonferroni’s multiple comparisons test), t(42) = 15.04, P < 0.001; OGD + NT vs. OGD + PEMF (Bonferroni’s multiple comparisons test), t(42) = 8.396, P < 0.001] and reduced the release of LDH from these cells [Fig. 3C: all groups (one-way ANOVA), F(5, 42) = 125.0, P < 0.001; control vs. OGD (Bonferroni’s multiple comparisons test), t(42) = 16.46, P < 0.001; OGD + NT vs. OGD + PEMF (Bonferroni’s multiple comparisons test), t(42) = 7.827, P < 0.001]. Furthermore, PEMF treatment suppressed the release of proinflammatory cytokines (TNF and IL-6) from astrocytes [Fig. 3D TNF: all groups (one-way ANOVA), F(5, 42) = 29.82, P < 0.001; control vs. OGD (Bonferroni’s multiple comparisons test), t(42) = 7.940, P < 0.001; OGD + NT vs. OGD + PEMF (Bonferroni’s multiple comparisons test), t(42) = 5.902, P < 0.001; Fig. 3D IL-6: all groups (one-way ANOVA), F(5, 42) = 52.00, P < 0.001; control vs. OGD (Bonferroni’s multiple comparisons test), t(42) = 8.918, P < 0.001; OGD + NT vs. OGD + PEMF (Bonferroni’s multiple comparisons test), t(42) = 5.486, P < 0.001] and induced increases in the levels of anti-inflammatory cytokines (TGF-β and IL-10) in astrocyte-conditioned medium under normal condition or during OGD treatment [Fig. 3D TGF-β: all groups (one-way ANOVA), F(5, 42) = 57.33, P < 0.001; control + NT vs. control + PEMF (Bonferroni’s multiple comparisons test), t(42) = 4.449, P < 0.001; control vs. OGD (Bonferroni’s multiple comparisons test), t(42) = 8.803, P < 0.001; OGD + NT vs. OGD + PEMF (Bonferroni’s multiple comparisons test), t(42) = 6.782, P < 0.001; Fig. 3D IL-10: all groups (one-way ANOVA), F(5, 42) = 57.92, P < 0.001; control + NT vs. control + PEMF (Bonferroni’s multiple comparisons test), t(42) = 3.824, P = 0.006; control vs. OGD (Bonferroni’s multiple comparisons test), t(42) = 8.165, P < 0.001; OGD + NT vs. OGD + PEMF (Bonferroni’s multiple comparisons test), t(42) = 3.286, P = 0.031]. These results indicated that the PEMF treatment regulated astrocyte survival and astrocyte-related neuroinflammation.

Fig. 3.

PEMF treatment blocks astrocytic injury and the release of proinflammatory cytokines from astrocytes. A Schematic diagram of the study design. Primary astrocytes were assigned to the following experimental groups according to the study design: control group, control + NT group (without PEMF exposure), control + PEMF group, OGD group, OGD + NT group (without PEMF exposure), and OGD + PEMF group. B Cell viability was assessed by the CCK-8 assay (n = 8 cell cultures/group). C Cytotoxicity was assessed by the LDH assay (n = 8 cell cultures/group). D The contents of TNF and IL-6 in astrocyte-conditioned medium were examined by ELISA (n = 8 cell cultures/group). E The contents of TGF-β and IL-10 in astrocyte-conditioned medium were examined by ELISA (n = 8 cell cultures/group). All data are presented as the means ± SDs. *P < 0.05, **P < 0.01, ***P < 0.001

The PEMF-Mediated Release of Inflammatory Cytokines from Astrocytes protected Neurons from OGD

To investigate the role of the changes in astrocytic inflammatory cytokines mediated by PEMFs in neuronal injury, astrocyte-conditioned medium (ACM) derived from PEMF-exposed primary astrocytes was applied to normal primary neuronal cultures or neurons subjected to OGD (Fig. 4A). The cell viability and LDH release from neurons obtained in the presence of 25% ACM obtained from astrocytes (NT group) did not differ from the results obtained in normal culture medium [Fig. 4B Control: ACM NT vs. ACM PEMF (unpaired two-tailed Student’s t test), t(14) = 0.5321, P = 0.603; Fig. 4C Control: ACM NT vs. ACM PEMF (unpaired two-tailed Student’s t test), t(14) = 1.975, P = 0.068]. In contrast, 25% ACM derived from PEMF-exposed primary astrocytes (PEMF group) led to an increase in cell viability and a reduction in LDH release from neurons subjected to OGD [Fig. 4B OGD: ACM NT vs. ACM PEMF (unpaired two-tailed Student’s t test), t(14) = 6.160, P < 0.001; Fig. 4C Control: ACM NT vs. ACM PEMF (unpaired two-tailed Student’s t test), t(14) = 2.860, P = 0.013]. Similar experiments were then performed to evaluate the effects of ACM derived from PEMF-exposed astrocytes following OGD on neurons. Normal neurons in the presence of 25% ACM obtained from PEMF-exposed primary astrocytes following OGD (OGD + PEMF group) induced a significant elevation of cell viability and reduction of LDH release with respect to the results obtained with 25% ACM obtained from primary astrocytes following OGD without PEMF exposure (OGD + NT group) [Fig. 4D Control: ACM NT + OGD vs. ACM PEMF + OGD (unpaired two-tailed Student’s t test), t(14) = 3.429, P = 0.004; Fig. 4E Control: ACM NT + OGD vs. ACM PEMF + OGD (unpaired two-tailed Student’s t test), t(14) = 5.836, P < 0.001]. Similarly, the treatment of neurons incubated under OGD conditions with 25% ACM obtained from the OGD + PEMF group significantly increased cell viability and reduced LDH release in comparison to the results obtained with 25% ACM obtained from the OGD + NT group [Fig. 4D OGD: ACM NT + OGD vs. ACM PEMF + OGD (unpaired two-tailed Student’s t test), t(14) = 7.215, P < 0.001; Fig. 4E OGD: ACM NT + OGD vs. ACM PEMF + OGD (unpaired two-tailed Student’s t test), t(14) = 7.024, P < 0.001]. These results demonstrated that the PEMF-treated astrocytes protected neurons from OGD by mediating the release of inflammatory cytokines.

Fig. 4.

PEMF-treated astrocytes protects neurons from OGD. A Schematic diagram of the study design. ACM from astrocytes were assigned to the following experimental groups according to the study design: ACM NT group, ACM PEMF group, ACM OGD + NT group, and ACM OGD + PEMF group. Primary neurons were assigned to the control group and OGD group. B Cell viability was assessed by the CCK-8 assay (n = 8 cell cultures/group). C Cytotoxicity was assessed by the LDH assay (n = 8 cell cultures/group). D Cell viability was assessed by the CCK-8 assay (n = 8 cell cultures/group). E Cytotoxicity was assessed by the LDH assay (n = 8 cell cultures/group). All data are presented as the means ± SDs. *P < 0.05, **P < 0.01, ***P < 0.001

Involvement of Astrocytic α7nAchR in the Anti-inflammatory Effects of PEMF

Previous studies have reported that α7nAchR is involved in the regulation of neuroinflammation. To clarify the involvement of astrocytic α7nAchR signaling in the effects of PEMFs on neuroinflammation, primary astrocytes were pretreated with an antagonist of α7nAchR (α-BGT, 100 nM) after OGD and then exposed to PEMFs (Fig. 5A). The inhibition of astrocytic α7nAchR by α-BGT partially reversed the effects of PEMFs on cell viability [Fig. 5B: all groups (one-way ANOVA), F(3, 28) = 55.73, P < 0.001; OGD + PEMF + vehicle vs. OGD + PEMF + α-BGT (Bonferroni's multiple comparisons test), t(28) = 4.585, P < 0.001] and LDH release [Fig. 5C: all groups (one-way ANOVA), F(3, 28) = 76.32, P < 0.001; OGD + PEMF + vehicle vs. OGD + PEMF + α-BGT (Bonferroni’s multiple comparisons test), t(28) = 4.484, P < 0.001]. Furthermore, α-BGT inhibited the regulatory effects of PEMF on the levels of proinflammatory cytokines [Fig. 5D, TNF: all groups (one-way ANOVA), F(3, 28) = 15.10, P < 0.001; OGD + PEMF + vehicle vs. OGD + PEMF + α-BGT (Bonferroni’s multiple comparisons test), t(28) = 3.408, P = 0.012; Fig. 4D IL-6: all groups (one-way ANOVA), F(3, 28) = 27.52, P < 0.001; OGD + PEMF + vehicle vs. OGD + PEMF + α-BGT (Bonferroni’s multiple comparisons test), t(28) = 5.753, P < 0.001] and anti-inflammatory cytokines [Fig. 5E, TGF-β: all groups (one-way ANOVA), F(3, 28) = 19.85, P < 0.001; OGD + PEMF + vehicle vs. OGD + PEMF + α-BGT (Bonferroni’s multiple comparisons test), t(28) = 4.397, P < 0.001; Fig. 4E, IL-10: all groups (one-way ANOVA), F(3, 28) = 37.69, P < 0.001; OGD + PEMF + vehicle vs. OGD + PEMF + α-BGT (Bonferroni’s multiple comparisons test), t(28) = 3.727, P = 0.005]. These results suggested that PEMFs protected against astrocyte injury and neuroinflammation by affecting the activity of astrocytic α7nAchR.

Fig. 5.

Involvement of astrocytic α7nAchR in the anti-inflammatory effects of PEMFs. A Schematic diagram of the study design. Primary astrocytes were assigned to the following experimental groups according to the study design: control group, OGD + PEMF group, OGD + PEMF + vehicle group, and OGD + PEMF + α-BGT group. B Cell viability was assessed by the CCK-8 assay (n = 8 cell cultures/group). C Cytotoxicity was evaluated by the LDH assay (n = 8 cell cultures/group). D The contents of TNF and IL-6 in astrocyte-conditioned medium were examined by ELISA (n = 8 cell cultures/group). E The contents of TGF-β and IL-10 in astrocyte-conditioned medium were examined by ELISA (n = 8 cell cultures/group). All data are presented as the means ± SDs. *P < 0.05, **P < 0.01, ***P < 0.001

The Modulation of α7nAchR/STAT3 Signaling Contributed to the Regulation of Astrocytic Function by PEMFs

STAT3, as an important transcription factor, has been implicated in the activation of neuroinflammation, which can be negatively modulated by α7nAchR. First, we further verified the role of α7nAchR/STAT3 signaling in the modulation of astrocytes by PEMFs. PEMFs reduced STAT3 phosphorylation in astrocytes following OGD [Fig. 6A STAT3/GAPDH: all groups (one-way ANOVA), F(4, 25) = 0.6532, P = 0.630; OGD + NT vs. OGD + PEMF (Bonferroni’s multiple comparisons test), t(25) = 0.1924, P > 0.999; Fig. 6A p-STAT3/STAT3: all groups (one-way ANOVA), F(4, 25) = 35.24, P < 0.001; OGD + NT vs. OGD + PEMF (Bonferroni’s multiple comparisons test), t(25) = 6.108, P < 0.001]. The presence of α-BGT, an antagonist of α7nAchR, reversed the PEMF-induced dephosphorylation of STAT3 in OGD-treated astrocytes [Fig. 6A STAT3/GAPDH: OGD + PEMF + vehicle vs. OGD + PEMF + α-BGT (Bonferroni’s multiple comparisons test), t(25) = 0.4809, P > 0.999; Fig. 6A p-STAT3/STAT3: OGD + PEMF + vehicle vs. OGD + PEMF + α-BGT (Bonferroni’s multiple comparisons test), t(25) = 4.739, P < 0.001]. Moreover, PEMFs suppressed the nuclear translocation of p-STAT3 following OGD, and this effect was inhibited by pretreatment with α-BGT (Fig. 6B). These results demonstrated that PEMFs blocked the STAT3-associated signaling by astrocytic α7nAchR.

Fig. 6.

The modulation of α7nAchR/STAT3 signaling contributes to the regulation of astrocyte function by PEMFs. A The expression levels of p-STAT3 and STAT3 were analyzed by western blotting (n = 8 cell cultures/group). B The expression and distribution of p-STAT3 were examined by immunofluorescence staining. Scale bar = 40 μm. All data are presented as the means ± SDs. *P < 0.05, **P < 0.01, ***P < 0.001

We subsequently clarified the role of STAT3 in the protective effects of PEMFs on astrocytes (Fig. 7A). The activation of STAT3 using colivelin (50 μg/ml), an activator of STAT3, attenuated the effects of PEMFs on cell viability [Fig. 7B: all groups (one-way ANOVA), F(2, 21) = 77.76, P < 0.001; OGD + PEMF + vehicle vs. OGD + PEMF + colivelin (Bonferroni’s multiple comparisons test), t(21) = 4.355, P < 0.001] and LDH release [Fig. 7C: all groups (one-way ANOVA), F(2, 21) = 104.0, P < 0.001; OGD + PEMF + vehicle vs. OGD + PEMF + colivelin (Bonferroni’s multiple comparisons test), t(21) = 5.422, P < 0.001]. Furthermore, colivelin suppressed the regulatory effects of PEMFs on the levels of proinflammatory cytokines [Fig. 7D, TNF: all groups (one-way ANOVA), F(2, 21) = 40.20, P < 0.001; OGD + PEMF + vehicle vs. OGD + PEMF + colivelin (Bonferroni’s multiple comparisons test), t(21) = 3.893, P = 0.003; Fig. 7D IL-6: all groups (one-way ANOVA), F(2, 21) = 96.92, P < 0.001; OGD + PEMF + vehicle vs. OGD + PEMF + colivelin (Bonferroni’s multiple comparisons test), t(21) = 7.585, P < 0.001] and anti-inflammatory cytokines [Fig. 7E, TGF-β: all groups (one-way ANOVA), F(2, 21) = 38.82, P < 0.001; OGD + PEMF + vehicle vs. OGD + PEMF + colivelin (Bonferroni’s multiple comparisons test), t(21) = 3.935, P = 0.002; Fig. 7E, IL-10: all groups (one-way ANOVA), F(2, 21) = 42.19, P < 0.001; OGD + PEMF + vehicle vs. OGD + PEMF + colivelin (Bonferroni’s multiple comparisons test), t(21) = 3.574, P = 0.005]. These results indicated that the inhibition of STAT3 activation was associated with the protective effects of PEMFs.

Fig. 7.

The inhibition of STAT3 by PEMFs exhibits protective effects after OGD. A Schematic diagram of the study design. Primary astrocytes were assigned to the following experimental groups according to the study design: control group, OGD + PEMF + vehicle group, and OGD + PEMF + colivelin group. B Cell viability was assessed by the CCK-8 assay (n = 8 cell cultures/group). C Cytotoxicity was evaluated by the LDH assay (n = 8 cell cultures/group). D The contents of TNF and IL-6 in astrocyte-conditioned medium were examined by ELISA (n = 8 cell cultures/group). E The contents of TGF-β and IL-10 in astrocyte-conditioned medium were examined by ELISA (n = 8 cell cultures/group). All data are presented as the means ± SDs. *P < 0.05, **P < 0.01, ***P < 0.001

α7nAchR/STAT3 Signaling Contributed to the Neuroprotective Effects of PEMFs Following Brain Ischemia

According to previous in vitro studies, astrocytic α7nAchR signaling might be involved in the regulation of PEMF-induced neuroprotective effects. Therefore, further studies were performed to verify this hypothesis in vivo (Fig. 8A). First, the intraperitoneal administration of α-BGT (1.0 μg/kg/d) reversed the reduction in the infarct volume [Fig. 8B: MCAO + PEMF + vehicle vs. MCAO + PEMF + α-BGT (unpaired two-tailed Student’s t test), t(10) = 5.224, P < 0.001] and the recovery of neurological function [Fig. 8C: all groups (one-way ANOVA), F(3, 44) = 54.57, P < 0.001; MCAO + PEMF + vehicle vs. MCAO + PEMF + α-BGT (Bonferroni’s multiple comparisons test), t(44) = 4.891, P < 0.001; Fig. 8D: all groups (one-way ANOVA), F(3, 44) = 118.6, P < 0.001; MCAO + PEMF + vehicle vs. MCAO + PEMF + α-BGT (Bonferroni’s multiple comparisons test), t(44) = 5.773, P < 0.001] induced by PEMF exposure following MCAO. Second, astrocyte activation was elevated by an antagonist of α7nAchR (Fig. 8E), and this elevation led to increases in the levels of proinflammatory cytokines [Fig. 8F TNF: all groups (one-way ANOVA), F(3, 20) = 53.30, P < 0.001; MCAO + PEMF + vehicle vs. MCAO + PEMF + α-BGT (Bonferroni’s multiple comparisons test), t(20) = 7.251, P < 0.001; Fig. 8F, IL-6: all groups (one-way ANOVA), F(3, 20) = 58.74, P < 0.001; MCAO + PEMF + vehicle vs. MCAO + PEMF + α-BGT (Bonferroni’s multiple comparisons test), t(20) = 4.100, P = 0.003] and decreases in the levels of anti-inflammatory cytokines [Fig. 8G TGF-β: all groups (one-way ANOVA), F(3, 20) = 66.08, P < 0.001; MCAO + PEMF + vehicle vs. MCAO + PEMF + α-BGT (Bonferroni’s multiple comparisons test), t(20) = 6.993, P < 0.001; Fig. 8F IL-10: all groups (one-way ANOVA), F(3, 20) = 55.29, P < 0.001; MCAO + PEMF + vehicle vs. MCAO + PEMF + α-BGT (Bonferroni’s multiple comparisons test), t(20) = 4.959, P < 0.001]. Furthermore, PEMFs suppressed the phosphorylation of STAT3, and the inhibition of α7nAchR reversed this effect in vivo [Fig. 8H: all groups (one-way ANOVA), F(4, 25) = 35.62, P < 0.001; MCAO + NT vs. MCAO + PEMF (Bonferroni’s multiple comparisons test), t(25) = 6.319, P < 0.001; MCAO + PEMF + vehicle vs. MCAO + PEMF + α-BGT (Bonferroni’s multiple comparisons test), t(25) = 5.492, P < 0.001]. Overall, these results suggested that activation of α7nAchR signaling participated in the neuroprotective effects of PEMFs following brain ischemia and that this effect was related to the modulation of α7nAchR/STAT3 signaling.

Fig. 8.

α7nAchR/STAT3 signaling contributes to PEMF-induced neuroprotection following brain ischemia. A Schematic diagram of the study design. Mice were assigned to the following experimental groups according to the study design: sham group, MCAO + NT group, MCAO + PEMF group, MCAO + PEMF + vehicle group, and MCAO + PEMF + α-BGT group. B TTC staining of brain tissue was performed, and the infarct volume was calculated (n = 6 mice/group). C The speed at which the mice in the different groups fell off the rod in the rotarod test was measured (n = 12 mice/group). D FUA scores were measured for the different groups (n = 12 mice/group). E The expression of GFAP was examined by immunofluorescence staining. Scale bar = 100 μm. F The contents of TNF and IL-6 in brain tissue were examined by ELISA (n = 6 mice/group). G The contents of TGF-β and IL-10 in brain tissue were examined by ELISA (n = 6 mice/group). H The expression levels of p-STAT3 and STAT3 were analyzed by western blotting (n = 6 mice/group). All data are presented as the means ± SDs. *P < 0.05, **P < 0.01, ***P < 0.001

Discussion

In the present study, we explored the role of PEMFs in regulating neuroinflammation after brain ischemia and their biological effects on the astrocytic cholinergic anti-inflammatory pathway. The results showed that the neuroprotective effects of PEMFs against brain ischemia were associated with the inhibition of reactive astrocytes and inflammatory cytokines. Further in vitro studies indicated that PEMFs directly improved the viability of astrocytes and reduced the release of inflammatory cytokines from astrocytes. We found that a specific antagonist of α7nAChR reversed the anti-inflammatory effects of PEMFs. Moreover, PEMFs suppressed the phosphorylation and nuclear translocation of STAT3 by activating α7nAChR. These results demonstrate that PEMFs exert anti-inflammatory effects in the context of brain ischemia by modulating the astrocytic cholinergic anti-inflammatory pathway.

PEMFs have been proven to exert significant regulatory effects on the central nervous system and thus directly affect neuronal synaptic plasticity (Lenz et al. 2016). Repetitive transcranial magnetic stimulation (rTMS) technology, which was developed based on PEMFs, has been applied for the clinical treatment of neurological diseases such as stroke, Parkinson’s disease, Alzheimer’s disease, and depression and has achieved good therapeutic effects (Zong et al. 2019; Lefaucheur et al. 2020; Moya Gomez et al. 2021). Previous studies have shown that PEMFs reduce the MCAO-induced infarct volume and modulate the gene expression of inflammation-associated cytokines (Pena-Philippides et al. 2014). Similar results were obtained in the present study, which indicates that PEMFs modulate neuroinflammation by elevating the levels of anti-inflammatory factors and suppressing the expression of proinflammatory cytokines following cerebral ischemia. Furthermore, the present study showed that PEMF treatment improved motor function after cerebral ischemia. However, the molecular mechanisms responsible for the therapeutic effects of PEMFs on cerebral ischemia remain unclear.

Astrocytes are the most important cell subtype in the central nervous system and play an important role in the maintenance of normal nerve function and the development of neurological diseases. Under physiological conditions, PEMFs can upregulate the expression of GFAP and the release of neurotrophic cytokines, which suggests that these fields can modulate astrocyte function (Chan et al. 1999; Vincenzi et al. 2020). In the present study, we found that PEMF exposure not only inhibited the activation of astrocytes following brain ischemia but also reduced the release of proinflammatory cytokines from astrocytes and protected neurons from OGD in vitro. These results support the hypothesis that the regulation of astrocyte-associated neuroinflammation is another target of the neuroprotective effects of PEMFs in brain ischemia. However, multiple cells are involved in the release of inflammatory factors, and the comprehensive effects of these cells affect neuroinflammation after brain ischemia (Shi et al. 2019). Our results showed increases in the levels of anti-inflammatory cytokines following MCAO but found the opposite alteration in astrocytes exposed to OGD. These results clearly indicated that the inflammatory response in vivo following MCAO likely involved other cell types, such as microglia and neurons. Similarly, the regulatory effect of PEMFs on the inflammatory response should not be limited to astrocytes.

Accumulating evidence indicates that astroglial α7nAChR has anti-inflammatory properties (Wang et al. 2012b; Patel et al. 2017). The activation of α7nAchR by agonists reduces neuroinflammation and relieves brain injury in mice with brain ischemia (Zou et al. 2017). Furthermore, a considerable amount of data suggest the involvement of α7nAChR in the response to certain physical stimuli, such as electroacupuncture and mesencephalic electrical stimulation (Zhang et al. 2021a; Ma et al. 2019; Schuhmann et al. 2021). However, the relationship between this α7nAChR-based cholinergic anti-inflammatory system and the neuroprotective effects of PEMFs remains undefined. The results of our study showed that an antagonist of α7nAChR attenuated the neuroprotective and anti-inflammatory effects of PEMFs, which demonstrated that astroglial α7nAChR could be a novel target of PEMFs. However, α7nAChR is also widely expressed in neurons and is recognized as a therapeutic target for excitotoxicity (Dajas-Bailador et al. 2000). On the one hand, excitotoxicity is another important mechanism secondary to brain ischemia (Chamorro et al. 2016), and on the other hand, our previous study reported that PEMFs protect against glutamate-induced excitotoxicity in neuronal cells (Li et al. 2017). Hence, the involvement of neuronal α7nAChR might also contribute to the protective effects of PEMFs on brain ischemia.

STAT3, as one of the major regulatory elements responsible for inflammation, plays a very important role in regulating neuroinflammation induced by brain ischemia (Cheng et al. 2019; Li et al. 2021). Previous studies have reported that the activation of α7nAChR reduces inflammation by negatively regulating STAT3 signaling and its ability to affect the expression of inflammatory cytokines in microglia and astrocytes (Cao et al. 2019; Han et al. 2020). Electromagnetic fields promote the phagocytic ability of microglial cells by modulating STAT3 signaling in microglial cells (He et al. 2014, 2016), but their role in regulating STAT3 signaling in astrocytes is not fully understood. The present study provided evidence showing that PEMF exposure decreased the phosphorylation of STAT3 after brain ischemia. This effect could be reversed by inhibition of α7nAChR. Further results showed that PEMFs also suppressed the nuclear translocation of p-STAT3, which indicated the influence of PEMFs on the transcriptional effects of STAT3 signaling. Taken together, these findings indicate that the inactivation of STAT3 signaling by α7nAChR is an essential mechanism downstream of PEMF-mediated regulation of neuroinflammation and the neuroprotective effects of PEMFs against brain ischemia.

The present study has several limitations. First, it has been reported that the biological effects of PEMFs depend on related parameters (e.g., intensity, frequency, exposure time) as well as the cell phenotypes and interactions with subcellular structure. Although the parameters of the PEMFs applied in the present study were selected based on previous studies, the effects observed in this study are limited to PEMFs with the specific parameters used. Second, although the present study showed that α7nAChR is an important target through which PEMFs regulate astrocyte function, how PEMFs interfere with α7nAChR through specific response elements remains unclear, and further research is needed to establish a more direct relationship between PEMF exposure and biological effects. Third, in addition to astrocytes, α7nAChR is also expressed in neurons and microglia. The protective effects of PEMFs on brain ischemia are possibly relevant to their role in mediating neuronal or microglial α7nAChR. These potential regulatory mechanisms should be verified in further studies.

Conclusion

Our data provide preliminary evidence showing that the cholinergic anti-inflammatory pathway is involved in the neuroprotective effects of PEMFs against brain ischemia. Notably, we also provide evidence demonstrating that astrocytes are potential targets of PEMFs and that α7nAChR mediates the anti-inflammatory effects of PEMFs. Furthermore, the inhibition of STAT3 is one of the downstream effects of PEMFs resulting from its regulatory effects on astrocytic α7nAChR. Regardless of the detailed mechanism involved in this process, these results highlight that astrocyte-related neuroinflammation may be a potential therapeutic target of PEMFs in the prevention and treatment of neurological diseases.

Supplementary Information

Below is the link to the electronic supplementary material.

Author Contributions

PL, XL, and HZ contributed to the conception and design of the study. HZ, YY, and EY designed and performed the main parts of the experiments, analyzed the data, and wrote the paper. YY and CW provided the PEMF exposure system. ZT, YH, and ZZ performed the pharmacological intervention and behavioral tests. MB, DL, and JG carried out the primary culture of cortical astrocytes and ELISAs. All authors read and approved the final manuscript.

Funding

This research was funded by the National Natural Science Foundation of China (Grant Nos. 81771322, 82171363, and 82171321) and the Youth Nova Program of Shaanxi (Grant No. 2021KJXX-19).

Data Availability

The data supporting the conclusions of this article are included within the article and its additional files.

Declarations

Competing Interests

The authors have no relevant financial or nonfinancial interests to disclose.

Ethical Approval

All animal experiments were approved by the Medical Research Ethics Committee of Fourth Military Medical University (No. 20210419) and conformed to the Chinese National Institute of Health Guide for the Care and Use of Laboratory Animals (No. 2017-166).