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. Author manuscript; available in PMC: 2023 Feb 1.
Published in final edited form as: Brain Res Bull. 2021 Dec 20;179:74–82. doi: 10.1016/j.brainresbull.2021.12.007

4R-cembranoid protects neuronal cells from oxygen–glucose deprivation by modulating microglial cell activation

Hefei Fu 1,2,#, Jiapeng Wang 1,3,#, Jie Wang 1, Langni Liu 1, Jianxiong Jiang 4, Jiukuan Hao 1,*
PMCID: PMC8849140  NIHMSID: NIHMS1768304  PMID: 34942325

Abstract

As major immune responsive cells in the central nervous system (CNS), activated microglia can present pro-inflammatory M1 phenotype aggravating the neuronal injury or anti-inflammatory M2 phenotype providing neuroprotection and promoting neuronal survival in neurodegenerative diseases. In this study, we demonstrated that a compound, 4R-cembranoid (4R, 1S, 2E, 4R, 6R,-7E, 11E-2, 7, 11-cembratriene-4, 6-diol cembranoids) promoted M2 phenotype while attenuated M1 phenotype in N9 cells, a microglial cell line. Following Lipopolysaccharides (LPS) or Oxygen-glucose deprivation (OGD) treatment, the N9 cells treated by 1 μM 4R showed an increased Arginase-1 (Arg1, a M2 marker) expression and a reduced inducible nitric oxide synthase (iNOS, M1 marker) expression. In addition, the conditioned medium of 4R-treated post-OGD N9 cells protected neuro2a cells, a neuronal cell line, from OGD-induced injury. The viability of neuro2a cells in OGD condition was increased by 54.5% after treated with the conditioned medium of 4R-treated post-OGD N9 cells. Furthermore, we demonstrated the protective mechanism of 4R was associated with a decreased TNF-α release and an increased IL-10 release from N9 cells. In conclusion, our study demonstrated that the neuroprotective effects of 4R were through the regulation of microglial activation by promoting the protective M2 activation and inhibiting the damaging M1 activation. Therefore, the findings of this study suggest that 4R could be a promising lead structure for the development of drugs for the treatment of ischemic stroke and other neurodegenerative diseases with an inflammatory component involved.

Keywords: 4R, cembranoid, inflammation, neuroprotection, oxygen glucose deprivation, microglia

INTRODUCTION

Microglia were considered resident macrophages in the brain, but currently, they are proposed to be brain-resident immune cells but not macrophages due to different origins. Microglia participate in immune responses and play important roles in neuroinflammation. The majority of microglia exist in their resting state (M0) under normal conditions and are activated and further polarized into the M1 or M2 state upon exposure to various stimuli (Gordon 2003; Martinez et al. 2008; Town, Nikolic, and Tan 2005; Torres-Platas et al. 2014). According to previous studies, the M1 microglial state is associated with proinflammatory and pro-killing functions. After injury, M1 microglia aggravate neuronal injury and death by releasing proinflammatory factors and various toxic substances, such as tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), IL-12, major histocompatibility complex II (MHCII), cluster of differentiation 86 (CD86), CD16, and inducible nitric oxide synthase (iNOS) (Tang and Le 2016). In contrast, M2 microglia show beneficial functions, such as anti-inflammatory, tissue healing/repair, and scavenging activities (Xia et al.; Lakhan, Kirchgessner, and Hofer 2009; Wang, Tang, and Yenari 2007). The M2 microglial population can be induced upon IL-4 or IL-13 stimulation. Meanwhile, M2 microglia show upregulation of CD206 (mannose receptor), scavenger receptors (SRs), arginase-1 (Arg-1), suppressor of cytokine release 1 (SOCS1), and growth factors (Edwards et al. 2006; Martinez et al. 2013). In addition, M2 microglia secrete polyamines, IGF-1, TGF-β, and IL-10, which block the proinflammatory actions of IFN-γ, IL-6, and TNF-α to suppress inflammatory responses. M2 microglia also exhibit increased phagocytosis and promote tissue repair (Xia et al.; Lakhan, Kirchgessner, and Hofer 2009; Wang, Tang, and Yenari 2007). However, activated microglia usually exist as a continuum of M1/M2 states (Vogel et al. 2013; Murray et al. 2014; Martinez and Gordon 2014) and present mixed phenotypes in vivo (Szulzewsky et al. 2015). Although further studies are still needed to characterize M1/M2 microglia (Ransohoff 2016), some studies have shown microglial heterogeneity during disease progression (Mikita et al. 2011). While the M1 microglial population typically increases during the progressive stage of diseases, the inhibition of M1 activation often slows disease progression (King, Dickendesher, and Segal 2009; Eixarch et al. 2009; Moreno et al. 2014). Conversely, the M2 microglial population is favored in a less aggressive stage of diseases, and approaches promoting M2 activation tend to facilitate patient recovery from injury (Weber et al. 2007; Burger et al. 2009; Liu et al. 2013). Although the mechanisms of microglial activation are not fully understood, the transcription factor NF-κB participates in M1 activation by upregulating the expression of M1-associated cytokines and inflammatory mediators, such as TNF-α, iNOS, and cyclooxygenase-2 (COX-2), while the cAMP-responsive element-binding protein (CREB) and Akt pathways play important roles in regulating beneficial M2 activation (Ruffell et al. 2009). Elevated NF-κB activity upregulates M1-associated cytokines and inflammatory mediators such as TNF-α, iNOS and cyclooxygenase-2 (COX-2), leading to M1 activation. At the same time, NF-κB activity also inhibits the expression of peroxisome proliferator-activated receptor-γ (PPAR-γ), which is essential for the induction of the M2 phenotype in microglia (Herwig et al. 2013). Conversely, CREB upregulates the expression of M2-specific genes, such as IL-10 and Arg-1, promoting tissue repair (Ruffell et al. 2009). Competition between CREB and NF-κB for binding to the same coactivators has been reported (Ruffell et al. 2009; Stein, Cogswell, and Baldwin 1993), and increased CREB activity suppresses the interaction of NF-κB with coactivators for M1 activation (Martin et al. 2005).

Pathologically, reactive microglia are prevalent in response to a variety of CNS stimuli, such as ischemia, autoimmune injury, infection, and neurodegeneration (Streit, Mrak, and Griffin 2004; Kim and de Vellis 2005; Schwab and McGeer 2008; von Bernhardi, Tichauer, and Eugenin 2010). Depending on the disease stage, microglia undergo different activation patterns, including cytotoxic M1 activation, neuroprotective M2 activation (Gordon 2003; Martinez et al. 2008; Town, Nikolic, and Tan 2005), and regulatory M0 activation (Mosser and Edwards 2008). M1 microglial cells are major contributors to neurodegenerative diseases by triggering inflammatory responses through the release of various inflammatory substances (Tang and Le 2016). As illustrated in Figure 1, M1 microglial activation may be the primary or secondary event initiating inflammatory responses causing neuronal death, while M2 microglia exert neuroprotective effects by releasing anti-inflammatory cytokines and neurotrophic factors and clearing cellular debris (Cherry, Olschowka, and O’Banion 2014). When injury cannot be rescued by M2 microglia or other protective responses, neurodegeneration and neuronal death progress. For example, homeostasis between M1 and M2 microglia may shift to the M1 state in a later stage of ischemic stroke, leading to the release of M1-associated cytokines and subsequently causing more neuronal injury, increasing disability, and poor prognosis (Hu et al. 2012). Therefore, inhibition of M1 microglial inflammatory responses after ischemic stroke will exert a beneficial effect on protecting both damaged and healthy neurons from further injury and death. However, only inhibition of a single M1-associated cytokine may not be sufficient to reduce all M1-inflammatory responses because other M1-associated signaling pathways may still be active. Notably, the use of general immunosuppressants or anti-inflammatory drugs, such as NSAIDs or minocycline, for treating ischemic stroke, PD, multiple sclerosis (MS), Alzheimer’s disease (AD), or other neurodegenerative diseases may exacerbate disease progression if they are used at the wrong stage of disease (Breitner et al. 2011) where beneficial M2 anti-inflammatory responses are blocked. Therefore, an ideal treatment for brain diseases characterized by microglial dysfunction and inflammation should be administered in a subtype-specific manner by attenuating M1 and/or promoting M2 microglial responses.

Fig 1.

Fig 1.

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Role of microglia activation in the pathology of neuronal injury.

In the search for novel compounds to treat neurodegenerative diseases, we found that 4R-cembranoid, an extract from tobacco leaves, may be a lead compound with anti-inflammatory and neuroprotective properties. 4R belongs to the family of cembranoids containing 14-carbon cembrane ring cyclic diterpenoids (Hann et al. 1998; Ferchmin et al. 2001). 4R is a stable and highly lipophilic small molecule (MW: 306) that penetrates the blood–brain barrier (BBB) and remains in the brain (Velez-Carrasco et al. 2015). 4R has been reported to exert neuroprotective and anti-inflammatory effects on brain ischemia-, NMDA-, 6-OHDA- or organophosphorus insecticide-induced injury (Martins et al. 2015; Ferchmin et al. 2005; Hu et al. 2017; Ferchmin et al. 2015; Ferchmin et al. 2014; Eterovic et al. 2013; Eterovic et al. 2011). Studies have also shown that activation of the PI3-kinase/Akt cascade and inhibition of ICAM-1, VCAM-1 and NF-κB activities are involved in the neuroprotective and anti-inflammatory effects of 4R (Martins et al. 2015; Ferchmin et al. 2005). The therapeutic effects of 4R on both in vitro and in vivo models of ischemic stroke and Parkinson’s disease are associated with anti-inflammatory actions in brain endothelial cells (Hu et al. 2017; Martins et al. 2015). This knowledge has inspired us to hypothesize that 4R may also exert anti-inflammatory effects on microglia by modulating M1/M2 phenotypes to protect neurons from injury after ischemia or inflammation.

Altogether, the present study intends to (i) evaluate the effect of 4R on microglial activation in an LPS-induced inflammation model or an oxygen-glucose deprivation (OGD)-induced ischemia model and (ii) evaluate the effect of 4R-treated N9 cell-derived conditioned medium on the viability of Neuro2a cells subjected to OGD and elucidate the mechanisms underlying the protective effect of 4R.

MATERIALS AND METHODS

In vitro inflammatory model using N9 cells.

LPS and 4R preparation:

The LPS stock solution was prepared by dissolving LPS powder (Sigma–Aldrich, St. Louis, MO) in phosphate-buffered saline (PBS) to a final concentration of 20 μg/ml. The LPS stock solution was diluted with cell culture medium at 5:1000 v/v to a final concentration of 100 ng/mL LPS. The 4R stock prepared in dimethyl sulfoxide (DMSO) (Sigma–Aldrich, St. Louis, MO) was diluted in cell culture medium to final concentrations of 50 nM, 1 μM, and 8 μM. N9 cells (ATCC Inc.) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Corning, Manassas, VA) containing 10% v/v fetal bovine serum (FBS) (Atlanta Biological, Lawrenceville, GA), 100 U/mL MEM nonessential amino acids (HyClone Laboratories, South Logan, UT) and 1% v/v penicillin/streptomycin (100X) (Corning, Manassas, VA) at 37 °C with 20% O2, 5% CO2, and 95% humidity. The medium was changed every 2–3 days until cells reached 80% confluence, and then the cells were treated with culture medium containing 100 ng/ml LPS and incubated for 24 h. 4R treatments or the same volume of vehicle (DMSO) were added at the same time as the LPS challenge.

The procedures for establishing the OGD condition

OGD condition:

Briefly, N9 cells were incubated with glucose/serum-free medium (Corning, Manassas, VA) in a hypoxia incubator with a 1% oxygen level for 1 h. After 1 h of OGD, the cells were incubated with cell culture medium alone, cell culture medium supplemented with 4R, or cell culture medium supplemented with DMSO at 37 °C with 95% air and 5% CO2 for 24 h. Then, the cells were refreshed with normal medium and cultured for another 24 h. The conditioned media of N9 cells were then collected to determine its effects on the viability of Neuro2a cells cultured under OGD conditions and to detect cytokine release via ELISAs. The experiments used to determine the effects of conditioned media of N9 cells on the viability of Neuro2a cells cultured under OGD conditions were performed as described below. Briefly, Neuro2a cells were seeded into 96-well plates (1000 cells per well) and subjected to the OGD conditions described above for 2 h. Then, the cells were incubated for 24 h with conditioned media collected from OGD-stimulated N9 cells or normal N9 cells with/without 4R treatments. The viability of Neuro2a cells was determined using the MTT assay.

Western blotting

Protein sample preparation:

N9 cells in each well were washed with PBS buffer and lysed with RIPA lysis buffer (containing PMSF, sodium orthovanadate and protease inhibitor cocktail) and 5 nM dithiothreitol (DTT) (100 μL per well for 6-well plates). The cells were then scraped, transferred into 1.5 ml Eppendorf tubes, and incubated on ice for 30 minutes. Then, the tubes were centrifuged at 15,000 g for 10 minutes at 4 °C, and the precipitates were discarded. The total protein concentration in each sample was determined using the Pierce™ BCA protein assay kit (Thermo Scientific, Rockford, IL) according to the manufacturer’s protocol. After the BCA assay, an equal volume of 2X Laemmli sample buffer with 5% (v/v) 2-mercaptoethanol was added to each sample. The samples were then boiled at 100 °C for 10 minutes and cooled to room temperature. Then, 20 μg of protein sample was loaded into a stacking gel and subjected to electrophoresis at 120 V for an appropriate time until the proteins were well separated. The proteins in the gel were transferred to PVDF membranes (Bio-Rad, Hercules, CA) using a semi-dry transfer system at 20 V for 70 minutes, and the membrane was blocked for 1 h at room temperature with 5% (w/v) skim milk powder in 10 mM Tris, 100 mM NaCl and 0.1% Tween-20 buffer (TBST, pH=7.5). The membrane was incubated with the desired primary antibody diluted in TBST containing 3% BSA and 0.01% sodium azide overnight at 4 °C with mild shaking after a brief wash with TBST. On the second day, following 3 washes with TBST for 10 minutes each, the membrane was incubated with a secondary antibody diluted in the blocking solution for 1 h at room temperature. Then, the membrane was washed with TBST 3 times again and placed in a dark room to detect protein expression using the chemiluminescence method.

Thiazolyl blue tetrazolium bromide reagent (MTT) assay

An MTT cell proliferation assay kit (Cayman Chemical Company, Ann Arbor, MI) was used to determine cell viability. According to the manufacturer’s instructions, MTT was first dissolved to a concentration of 5 mg/ml in PBS, then 20 μl were added to each well of Neuro2a cells (96-well plate) and incubated at 37 °C for 4 h. The media were then carefully removed from each well, and the precipitated formazan crystals in each well were dissolved in 200 μl of DMSO (Fisher Scientific, Pittsburg, PA). After shaking on a plate for 5 minutes, the absorbance was measured at 560 nm and subtracted from the background absorbance at 670 nm using a SPECTRAmax plus 384 microplate reader (Molecular Devices, CA).

Enzyme-linked immunosorbent assay (ELISA)

The cytokine concentrations in conditioned media collected from N9 cells were detected using ELISA kits (Pepro Tech Inc., NJ). Briefly, a 96-well ELISA microplate was coated with the capture antibody overnight at room temperature, and then the solution was aspirated and wells were washed 4 times with wash buffer (0.05% Tween-20 in PBS). Three hundred microliters of blocking buffer (1% BSA in PBS) were added to each well, incubated for 1 h and washed 4 times. Next, 100 μl of conditioned medium were added to each well, incubated for 2 h, and the plate was washed 4 times a. After a 2 h incubation with the antibodies, the solution was aspirated and each well was washed 4 times with PBS. Then, the streptavidin-HRP conjugate was added to each well and incubated for 30 min followed by 4 washes. Finally, 100 μl of 3,3’,5,5’-tetramethylbenzidine (TMB) (SeraCare company, Gaithersburg, MD) were added to each well and incubated for 20 minutes to develop the color; 100 μl of 1 M hydrochloric acid (Fisher Scientific, Waltham, MA) were added to stop the color development. The absorbance was immediately measured at 450 nm and subtracted from the background absorbance at 620 nm using a SPECTRAmax plus 384 microplate reader (Molecular Devices, CA).

Statistical analysis

Data are presented as the means ± standard deviations. Statistical comparisons among multiple groups were performed using one-way analysis of variance (ANOVA) followed by Dunnett’s test. The threshold of the p value was set to 0.05, and p<0.05 was considered statistically significant.

RESULTS

Effects of 4R on the expression of Arg-1 and iNOS in N9 cells.

4R upregulates Arg-1 expression in naive N9 cells (resting condition). The level of Arg-1 expression was significantly increased to 250.5% ± 65.9, 305.6% ± 52.1 and 284.2% ± 37.2 of the levels in the control group after treatment with 50 nM, 1 μM, and 8 μM 4R, respectively. The DMSO vehicle did not exert any significant effect on Arg-1 expression compared with the control group (Figure 2). Furthermore, naive N9 cells did not express iNOS; however, 100 ng/mL LPS treatment significantly upregulated iNOS expression in N9 cells (Figure 3a). Although 50 nM 4R did not exert significant effects on LPS-induced iNOS expression, 1 μM 4R treatment downregulated LPS-induced iNOS expression to 69.4% ± 8.5 of that in the LPS group (Figure 3a). In addition, LPS treatment also suppressed Arg-1 expression to 79.3% ± 8.7 of the control group, and both 50 nM and 1 μM 4R treatment restored Arg-1 expression to 122.2% ± 10.9 and 150.4% ± 22.6 of the level in the control group, respectively (Figure 3b). The vehicle (DMSO) did not significantly affect iNOS and Arg-1 expression in N9 cells (Figure 3). These findings suggest that 4R suppresses LPS-induced M1 polarization and stimulates M2 polarization in N9 cells.

Fig 2. 4R increases Arg-1 expression in N9 cell.

Fig 2.

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N9 cells were treated with no treatment (control), vehicle (DMSO), 50 nM, 1μM, 8μM 4R for 24 hours respectively, and then Arg-1 expression was evaluated by western blot. The relative protein expression of Arg-1 were analyzed via image J software and normalized to β-actin. Data are represented as mean ± SD (n=6), * p<0.05 compared to vehicle. Ctrl, control; Veh, vehicle.

Fig 3. 4R regulates LPS stimulated M1 and M2 maker expression in N9 cell.

Fig 3.

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N9 cells were treated with 100 ng/mL LPS addition with vehicle (DMSO) or 4R for 24 hours. The protein expression of M1 marker a) iNOS and M2 marker b) Arg-1 were analyzed by western blot. The relative protein expression was analyzed via image J software and normalized to β-actin. Data are represented as mean ± SD (n=6), * p<0.05 compared to LPS (a) and control (b). # p<0.05 compared to LPS with vehicle. Ctrl, control; Veh, vehicle.

4R attenuates OGD-induced M1 polarization but promotes M2 polarization in N9 cells.

N9 cells were subjected to OGD conditions for 1 h and then treated with 4R for 24 h. One hour of OGD-induced M1 activation in N9 cells resulted in the upregulation of iNOS. As shown in Figure 4a, 1 h of OGD significantly upregulated the expression of iNOS to 311.5% ± 24.7% of the level in the control group of N9 cells. Furthermore, the high iNOS expression level induced by OGD was attenuated after 1 μM 4R treatment. The iNOS level was attenuated to 50% of that in the OGD group, which was 168.5% ± 50.3 of that of the control after 1 μM 4R treatment. iNOS expression was not affected by the vehicle (Figure 4a). Notably, the expression of the M2 marker Arg-1 was not affected by OGD conditions and was 99.7% ± 11.8% of that in the control group, while 1 μM 4R treatment increased Arg-1 expression in N9 cells cultured under OGD conditions to 188.0% ± 9.9% of that in the vehicle (DMSO) group after OGD treatment (Figure 4b).

Fig. 4: The effect of 4R on M1 and M2 markers expression in N9 cells in N9 cells under OGD condition.

Fig. 4:

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The cells were subjected to OGD for 1 hour then treated with vehicle (DMSO) or 1 μM 4R for 24 hours. The protein expression of M1 marker a) iNOS and M2 marker, b) Arg-1 were analyzed by western blot. The relative protein expression was analyzed via image J software and normalized to β-actin. Data are represented as mean ± SD (n=3), * p<0.05 compared to control. # p<0.05 compared to OGD with vehicle. Ctrl, control; Veh, vehicle.

Phosphorylation of NF-κκB was involved in the 4R-mediated polarization of N9 cells.

As shown in Figure 5, the level of p65 phosphorylation in N9 cells was increased by LPS treatment. LPS treatment (100 ng/mL) significantly increased the level of p65 phosphorylation to 364.4% ± 32.8 that in the control group, and vehicle (DMSO) did not exert any significant effect on p65 phosphorylation. Although 50 nM 4R treatment did not exert a significant effect on p65 phosphorylation, 1 μM 4R treatment significantly reduced LPS-induced p65 phosphorylation to 145.5% ± 32.0 of that in the control group of N9 cells (Figure 5a). After 1 h of OGD treatment, the p65 phosphorylation level was increased to 238.4% ± 65.4 of the control and then attenuated to 109.9% ± 27.8 of the control with 1 μM 4R treatment during the recovery period (Figure 5b). Notably, 4R treatment alone did not affect p65 phosphorylation in non-OGD-treated N9 cells, which was 114.1% ± 33.4 of the level in the control group. Vehicle (DMSO) also had no significant effect on p65 phosphorylation in either non-OGD- or post-OGD-treated N9 cells (Figure 5b).

Fig 5. 4R attenuates LPS and OGD stimulated phosphorylation of NF-kB in N9 cell.

Fig 5.

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N9 cells were treated with 4R 24 hours together with 100 ng/mL LPS or after 1 hour OGD. The levels of NF-kB phosphorylation under a) LPS stimulation and b) OGD condition were evaluated by western blot. The relative protein expression was analyzed via image J software and normalized to β-actin. Data are represented as mean ± SD (n=3), * p<0.05 compared to control. # p<0.05 compared to LPS with vehicle (a) or OGD with vehicle (b). Ctrl, control; Veh, vehicle.

4R regulates OGD-induced cytokine release, which exerts a protective effect on the OGD-induced decrease in cell viability.

We applied conditioned medium from N9 cells cultured under different conditions to Neuro2a cells to study its effect on neuronal cell viability under OGD conditions. Briefly, Neuro2a cells were first subjected to OGD for 2 h. Then, conditioned medium from N9 cells cultured under OGD or normal conditions and treated with 4R or vehicle was added to Neuro2a cells and incubated for 24 h. The viability of Neuro2a cells was measured using the MTT assay. As shown in Figure 6, 2 h of OGD stimulation (Group 2) decreased cell viability to 70.3% ± 2.0 of that in the control group of normal Neuro2a cells (Group 1). The conditioned medium from N9 cells cultured under normal conditions with vehicle (Group 3) or with 1 μM 4R (Group 4) did not affect Neuro2a cell viability after exposure to OGD, which was 71.3% ± 2.6% and 68.6% ± 4.4% of that in the control group, respectively. Furthermore, the conditioned medium from 1 h OGD-stimulated N9 cells (Group 5) further exacerbated the reduction in Neuro2a cell viability after OGD exposure to 35.2% ± 5.9 of that in the control group. However, the conditioned medium from the 1 μM 4R-treated OGD-stimulated N9 cells (Group 7) significantly increased the post-OGD viability of Neuro2a cells to 52.8% ± 2.6 of that in the control vehicle-treated cells (Group 6).

Fig. 6. The effect of conditioned medium from 4R-treated N9 cells in OGD condition on viability of neuro2a cell subjected to OGD.

Fig. 6.

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The neuro2a cells were subjected to OGD for 2 h first, and then the conditioned medium from N9 cells in 1h OGD or normal conditions with 4R or DMSO was added to the neuro2a cells and incubated for 24 h. The cell viabilities of neuro2a cells were measured by MTT assay. (1) conditioned medium from normal N9 cells+ normal neuro2a cells; (2) conditioned medium from normal N9 cells + post-OGD neuro2a cells; (3) conditioned medium from normal N9 cells (DMSO)+ post-OGD neuro2a cells; (4) conditioned medium from normal N9 cells (4R) + post-OGD neuro2a cells; (5) conditioned medium from 1h post-OGD N9 cells + post-OGD neuro2a cells; (6) conditioned medium from 1h post-OGD N9 cells (DMSO) + post-OGD neuro2a cells; (7) conditioned medium from 1h post-OGD N9 cells (4R) + post-OGD neuro2a cells;. N2a cells viabilities shown as % of control (Group 1) were evaluated by MTT assay. Data were presented as mean ± SD % of control (n=3). * p<0.05 compared to control. # p<0.05. Ctrl, control; Veh, vehicle.

ELISAs were performed to detect the concentrations of the proinflammatory cytokine TNF-α and the anti-inflammatory cytokine IL-10 in the conditioned medium from N9 cells and to further investigate the mechanisms by which the conditioned medium from N9 cells treated with 4R modulated the viability of Neuro2a cells cultured under OGD conditions. Treatment with 1 μM 4R alone did not affect TNF-α release from N9 cells cultured under normal conditions compared to the vehicle treatment (Figure 7a). However, 1 h of OGD stimulation significantly increased TNF-α release from N9 cells to 203.4% ± 30.6 of the control group, and treatment with 1 μM 4R attenuated the increased level of TNF-α released from N9 cells subjected to 1 h of OGD to 156.1% ± 5.8 of the control group (Figure 7a). Vehicle treatment did not affect TNF-α release from N9 cells after OGD. In addition, we also detected the IL-10 level in the medium of N9 cells. One hour of OGD did not significantly change the IL-10 level in the medium of N9 cells, and vehicle did not exert a significant effect on IL-10 release (117.0% ± 14.9 of the control) in N9 cells cultured under normal conditions (Figure 7b). Notably, 1 μM 4R treatment significantly increased IL-10 release from N9 cells cultured under both normal conditions and OGD conditions. The level of IL-10 in N9 cell medium was increased to 223.8% ± 13.3 of the control after 1 μM 4R treatment under normal conditions and 329.4% ± 30.4 of the control under OGD conditions (Figure 7b).

Fig. 7. 4R inhibited OGD-induced pro-inflammatory cytokine release, while increased anti-inflammatory cytokine release in N9 cells.

Fig. 7.

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N9 cells were stimulated with OGD for 1 hour then treated with vehicle (DMSO) or 1 μM 4R for 24 hrs. The cytokine release of a) TNF- α concentration and b) IL-10 concentration in medium were determined by ELLISA assay. Data are represented as mean ± SD % of control (n=3), * p<0.05 compared to control. # p<0.05 compared to OGD with vehicle. Ctrl, control; Veh, vehicle.

DISCUSSION

M1 microglia exacerbate postischemic injury by inducing inflammatory responses and cell injury, but M2 microglia exert a beneficial effect on enhancing tissue repair and neuronal survival through their anti-inflammatory and debris-clearing functions. In response to ischemia, the M2 microglial population increases at the beginning of injury and then gradually decreases to the basal level, but M1 microglial population expansion begins in the later period of ischemia (Peng et al. 2017). A higher ratio of M1 to M2 microglia is associated with the severity of postischemic and inflammatory injury (Hu et al. 2012). Therefore, therapeutic approaches designed to promote M2 and/or inhibit M1 activation will be effective against ischemic stroke or neuroinflammatory diseases. Previously, we reported neuroprotective effects of 4R on brain ischemia-, NMDA-, 6-OHDA- or organophosphorus insecticide-induced injury (Martins et al. 2015; Ferchmin et al. 2005; Hu et al. 2017; Ferchmin et al. 2015; Ferchmin et al. 2014; Eterovic et al. 2013; Eterovic et al. 2011). In the present study, we used a microglial cell line, N9 cells, to study the effects of 4R on microglial activation in an LPS-inflammatory model or OGD conditions and to evaluate the effect of conditioned medium from 4R-treated N9 cells on the viability of Neuro2a cells subjected to OGD conditions, which mimicked brain ischemia-induced cell injury in vitro. Although LPS treatment is different from OGD conditions, both models trigger similar inflammatory responses. We used both conditions to investigate the mechanisms underlying the neuroprotective and anti-inflammatory effects of 4R. The concentrations (50 nM, 1 μM or 8 μM) of 4R selected in the present study were based on our previous studies examining the neuroprotection induced by 4R (Hu et al. 2017; Martins et al. 2015; Ferchmin et al. 2005).

In the present study, iNOS and p65 expression were upregulated under both OGD conditions and LPS stimulation conditions. Furthermore, 4R treatment mitigated the increases in iNOS expression (Figure 3a, 4a) and NF-κB phosphorylation (p-p65, Figure 5) induced by LPS or OGD in N9 cells, while 4R treatment alone did not exert a significant effect on the levels of these proteins. Furthermore, we also evaluated the levels of the M2 markers Arg-1 and IL-10 in the same samples. 4R treatment upregulated Arg-1 and IL-10 expression in N9 cells under normal, LPS, and OGD conditions (Figures 2, 3b, 4b, 7b). In addition, LPS also suppressed M2 marker Arg-1 expression, but 1 h of OGD did not exert a significant effect on M2 microglial polarization in N9 cells (Figures 3b, 4b). Although 1 h of OGD did not reduce M2 polarization, it induced M1 polarization, and the overall ratio of M1/M2 N9 cells detected after 1 h of OGD was higher than that under normal conditions, consistent with previous reports (Weber et al. 2007; Burger et al. 2009; Liu et al. 2013). Importantly, conditioned medium from 1 μM 4R-treated OGD-stimulated N9 cells protected Neuro2a cells from OGD-induced injury and increased their viability compared to the vehicle treatment group (Figure 6). We performed ELISAs to detect the levels of TNF-α and IL-10 in the conditioned medium and elucidate the mechanism underlying the protective effect of conditioned medium from OGD- and 4R-treated N9 cells on Neuro2a cells. TNF-α concentrations were increased in the conditioned medium from N9 cells subjected to 1 h of OGD, and 4R treatment reduced TNF-α concentrations in the conditioned medium of OGD-stimulated N9 cells (Figure 7a). The level of IL-10 in the conditioned medium from N9 cells subjected to 1 h OGD did not show significant changes, but 1 μM 4R treatment increased the IL-10 level in the conditioned medium of N9 cells cultured under OGD conditions for 1 h (Figure 7b). Based on these results, 4R not only inhibits microglial M1 activation but also promotes M2 activation. Meanwhile, 4R-induced anti-inflammatory cytokines released in the conditioned medium protected Neuro2a cells from OGD-induced injury. The 1 h OGD condition applied to the N9 cells in our present study was shorter than the OGD conditions in some previously reported studies, ranging from 6 h to 30 h OGD to induce microglial polarization (del Zoppo et al. 2012; Hatakeyama et al. 2019). The difference in the OGD duration between our present study and the aforementioned studies may be due to the different methods of OGD induction used. We induced OGD conditions using a Tri-gas cell culture incubator with 1% oxygen during the OGD period and incubated the cells with glucose/serum-free medium. The OGD conditions in the abovementioned studies were induced in a hypoxia chamber (Billups-Rothenburg, Del Mar, CA, USA), which was flushed with a mixture of 95% N2 and 5% CO2 for 1 h and then closed for the duration of the experiment. Furthermore, the cells were incubated in low-glucose medium instead of glucose/serum-free medium. In fact, we performed experiments with longer OGD durations, such as 3 h and 6 h OGD, using N9 cells but failed to induce N9 cell polarization. In our study, microglial polarization observed after 1 h of OGD might result from the use of oxygen deprivation and glucose/serum-free medium, which provided stronger stimuli to the cells. Notably, microglial M1/M2 activation is not an all-or-none phenomenon. Instead, the two states may overlap and occur in a continuum. This heterogeneity of microglial states might be another reason for the inconsistency of the OGD duration used under different experimental conditions.

The NF-κB signaling cascade regulates the production of proinflammatory mediators, including IL-1, IL-2, IL-6, TNF-α, iNOS, and COX-2 (Ridder and Schwaninger 2009). Therefore, inhibiting NF-κB activation may decrease the release of proinflammatory mediators to reduce neuronal injury. Previous studies have indicated that NF-κB activation occurs during M1 polarization but inhibited during M2 polarization (Wang, Liang, and Zen 2014). Consistent with previous studies, our results showed that the modulation of N9 cell polarization by 4R was associated with regulating NF-κB phosphorylation (Figure 5). Our ELISA results revealed that the inhibition of NF-κB phosphorylation led to a decrease in downstream cytokine production. Moreover, 4R treatment reduced the OGD-induced the release of the proinflammatory cytokine TNF-α (Figure 7a) but increased the secretion of the anti-inflammatory cytokine IL-10 in N9 cells cultured under both normal and OGD conditions, consistent with the high level of the microglial M2 marker Arg-1 detected in the cells after 4R treatment (Figures 2, 3b, 4b, 7b). TNF-α is considered to cause neuronal death by increasing glutamate production (Ramesh, MacLean, and Philipp 2013), and IL-10 has been shown to reduce rat brain injury following ischemic stroke (Spera et al. 1998). Therefore, inhibiting M1 microglial activation and enhancing M2 microglial activation will reduce proinflammatory cytokine release but increase anti-inflammatory cytokine release to contribute to the protective function of 4R. This finding might explain why conditioned medium from OGD-stimulated N9 cells treated with 4R protected Neuro2a cells from OGD-induced injury by reducing proinflammatory cytokine release and increasing anti-inflammatory cytokine release. Interestingly, although 4R treatment increased microglial anti-inflammatory cytokine release under normal conditions, the conditioned medium from 4R-treated normal microglia did not increase neuronal cell survival after OGD compared to the control conditioned medium (Figure 6). The assumption that microglia are skewed toward a protective and anti-inflammatory phenotype under normal conditions might explain this phenomenon, as resting microglia also secrete some neurosupportive cytokines, such as IGF-1 and BDNF. Thus, the conditioned media from “resting” and 4R-treated microglia should both exhibit neuroprotective properties. Notably, only IL-10 and iNOS levels were evaluated in the cell culture medium in the present study, and more details of the cytokine release profile may provide more insights into 4R regulation of microglia.

CONCLUSIONS

According to previous studies, 4R-mediated neuroprotection is associated with the activation of the PI3-kinase/Akt cascade in neurons and inhibition of ICAM-1, VCAM-1 and NF-κB activities in brain endothelial cells (Martins et al. 2015; Ferchmin et al. 2005; Hu et al. 2017). Our present study further suggests that 4R inhibits microglial M1 activation induced by LPS or OGD and promotes microglial M2 activation. In addition, modulation of microglial activation by 4R may contribute to its protective effect on Neuro2a cells under OGD conditions by decreasing proinflammatory cytokine release and increasing anti-inflammatory cytokine release. This report is the first to show that 4R exerts an anti-inflammatory effect on microglia, leading to neuroprotection. However, further studies, such as a transcriptome analysis, are still needed to fully characterize 4R functions in CNS inflammation and diseases. In summary, building on previous knowledge of 4R, the present study has provided more insights and information on the therapeutic effects of 4R on ischemic stroke and Parkinson’s disease at the molecular level. The modulation of microglial activation is one of the mechanisms that contributes to the neuroprotective effects of 4R. Further animal studies are necessary to elucidate the effects of 4R-mediated modulation of microglial activation in vivo and to further investigate the mechanism of action underlying 4R-induced neuroprotection.

Supplementary Material

1

Highlights.

  • M1 microglia is associated with pro-inflammatory and pro-killing functions;

  • M2 microglia is protective and associated with anti-inflammation, tissue healing/repair.

  • 4R inhibits M1 activation and promotes M2 activation of microglia.

  • Protective effect of 4R on neuro2a cells against OGD-induced injury.

  • 4R reduces pro-inflammatory cytokines release, but increases anti-inflammatory cytokines release.

ACKNOWLEDGEMENTS

This work was supported by grants R01NS105787 (J.H.) and R01NS100947 (J.J.) and R21NS109687 (J.J.) from the National Institutes of Health (NIH)/National Institute of Neurological Disorders and Stroke (NINDS), and the Michael J. Fox Foundation for Parkinson’s Research, RAPID RESPONSE INNOVATION AWARDS, (J.H.).

Footnotes

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CRediT authorship contribution statement

Hefei Fu: Planned and performed the experiments, Analysis data. Jie Wang: Experimental design and performed the experiments. Jiapeng Wang: Analysis data and performed the experiments. Langni Liu: Analysis data and Writing- Reviewing and Editing. Jianxiong Jiang: Supervision, Writing, Reviewing and Editing. Jiukuan Hao: formulated the working hypothesis, planned the experiments and Supervision, Writing manuscript. All authors critically proof-read the manuscript and approved for publication

Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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