AC-186 did not affect the viability of LPS-stimulated BV-2 microglia

BV-2 microglia were treated with AC-186 for 30 min, followed by stimulation with LPS (100 ng/mL) for a further 24 h. Results of MTT assay showed that at concentrations 0.625 µM, 1.25 µM, 2.5 µM and 5 µM, the compound did not reduce cell viability, when compared with cells incubated with 0.2% DMSO (Fig. 2).

Fig. 2

AC-186 (0.625, 1.25, 2.5 and 5 µM) did not reduce viability of BV-2 microglia stimulated with LPS (100 ng/mL)

AC-186 decreased levels of TNFα and IL-6 in LPS-stimulated BV-2 microglia

Stimulation of BV-2 cells with LPS (100 ng/mL) for 24 h resulted in significant (p < 0.0001) increase in the release of TNFα, when compared to unstimulated BV-2 cells (Fig. 3a). However, pre-treatment of cells with 0.625 µM, 1.25 µM, 2.5 µM and 5 µM of AC-186 prior to LPS stimulation resulted in significant (p < 0.05) reduction in TNFα production, when compared to stimulation of cells with LPS alone (Fig. 3a). Similarly, incubation of BV-2 microglia with LPS for 24 h resulted in an elevated production of IL-6 in comparison to unstimulated cells. This increase in IL-6 production was however significantly reduced (p < 0.05) when cells were treated with AC-186 (0.625 µM, 1.25 µM, 2.5 µM and 5 µM) prior to activation with LPS (Fig. 3b).

Fig. 3

AC-186 suppressed TNFα and IL-6 production in BV-2 microglia cells. Microglia cells were treated with LPS (100 ng/mL) in the absence or presence of indicated concentrations of AC-186 for 24 h. TNFα (3A) and IL-6 (3B)  levels were measured using ELISA. Data are expressed as the mean ± SEM (n = 3). **p < 0.01, ***p < 0.001, ****p < 0.0001 versus LPS stimulation alone. AC-186 suppressed NO production and iNOS protein expression in BV-2 microglia cells. Microglia cells were treated with LPS (100 ng/mL) in the absence or presence of indicated concentrations of AC-186 for 24 h. Nitrite levels were measured using the Griess reaction (3C). BV-2 microglia cell lysates were subjected to Western blot for iNOS (3D). Levels of iNOS were normalised actin levels and expressed as a relative change in comparison with the LPS stimulation, which was set at 100% (3E). Data are expressed as the mean ± SEM (n = 3). *p < 0.05, ***p < 0.001, ****p < 0.0001 versus LPS stimulation alone. AC-186 suppressed PGE2 production and COX-2 protein expression in BV-2 microglia cells. Microglia cells were treated with LPS (100 ng/mL) in the absence or presence of indicated concentrations of AC-186 for 24 h. PGE2 levels were measured using enzyme immunoassay (3F). BV-2 microglia cell lysates were subjected to Western blot for COX-2 (3G). Levels of COX-2 were normalised actin levels and expressed as a relative change in comparison with the LPS stimulation, which was set at 100% (3H). Data are expressed as the mean ± SEM (n = 3). **p < 0.01, ***p < 0.001, ****p < 0.0001 versus LPS stimulation alone

AC-186 reduced LPS-induced NO production/iNOS protein expression

Nitrite production in cells is taken as a surrogate marker for nitric oxide production. Analyses of culture supernatants revealed that stimulation of BV-2 microglia with LPS resulted in significant (p < 0.0001) increase in the production of nitrite, in comparison with unstimulated cells. In the presence of 0.625 µM, 1.25 µM, 2.5 µM and 5 µM of AC-186, nitrite concentration was reduced from ~ 12.7 μM to ~ 9.4 μM, ~ 8.2 μM, ~ 7.3 μM and ~ 5.1 μM, respectively (Fig. 3c).

Immunoblotting of cell lysates further showed that pre-treatment with AC-186 (0.625 µM, 1.25 µM, 2.5 µM and 5 µM) resulted in reduction of iNOS protein expression to 46.5%, 19.3%, 9.7% and 2.4%, respectively when compared to LPS stimulation (100%) (Fig. 3d and e).

AC-186 reduced PGE2 release/COX-2 protein expression

The outcome of enzyme immunoassays on culture supernatants showed that stimulation of BV-2 microglia with LPS (100 ng/mL) resulted in elevated (p < 0.001) production of PGE2, when compared to unstimulated cells (Fig. 3f). However, pre-treating cells with AC-186 (0.625–5 µM) prior to LPS resulted in significant (p < 0.05) reduction in increased secretion of PGE2, in comparison with LPS stimulation alone.

Results of immunoblotting in Fig. 3g and h show that significant increase in COX-2 protein expression induced by LPS was reduced in a -concentration dependent fashion by AC-186 (0.625–5 µM).

AC-186 targets NF-κB  signalling to produce anti-inflammatory activity

Based on results showing that AC-186 could reduce the production of pro-inflammatory mediators in LPS-activated BV-2 cells, experiments were conducted to evaluate the effects of the compound on mechanisms and molecular targets involved in the activation of the NF-κB transcription factor.

One of the upstream mechanisms involved in the activation of NF-κB is the phosphorylation of the p65 subunit and IκB complex in the cytoplasm. Immunoblotting analyses of BV-2 microglia stimulated with LPS (100 ng/mL) showed a significant (p < 0.001) increase in protein expression of the phospho-p65 subunit (Fig. 4a and b). When cells were treated with AC-186 (0.625–5 µM) prior to LPS stimulation, a concentration-dependent and significant (p < 0.001) reduction in phospho-p65 protein expression was observed. Furthermore, in the presence 1.25 µM, 2.5 µM, and 5 µM of AC-186, there was a concentration-dependent and significant (p < 0.0001) reduction in LPS-induced increased expression of phospho-IκBα protein (Fig. 4c and d). Interestingly significant (p < 0.05) reduction in LPS-induced increased phospho-IκBα protein levels was not observed with the lowest concentration of the compound (0.625 µM) investigated (Fig. 4c and d).

Fig. 4

Inhibition of LPS-induced NF-κB activation by AC-186 in BV-2 cells. Cells were activated with LPS (100 ng/mL) with or without AC-186 (0.625–5 µM). Lysates of treated cells were used to detect phospho-p65 (4A, 4B) and phospho-IκBα (4C, 4D) expression. Actin was used as an internal loading control. Data are expressed as the mean ± SEM (n = 3). ns-not significant at p < 0.05, ****p < 0.0001 versus LPS stimulation alone. (4E). AC-186 inhibited LPS-induced NF-κB-dependent gene expression in BV-2 cells. Transfected cells were treated with AC186 (0.625–5 µM) and stimulated with LPS (100 ng/mL) for 6 h, followed by luciferase reporter gene assay. (4F) Reduction in LPS-induced increased binding of p65 NF-κB to consensus sites by AC-186. BV-2 cells were treated with AC186 (0.625–5 µM) and stimulated with LPS (100 ng/mL) followed by NF-κB transcription factor assay on nuclear extracts. Data are expressed as the mean ± SEM (n = 3). ns-not significant at p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 versus LPS stimulation alone. (4G and 4H) AC-186 interferes with acetylation of p65 sub-unit following activation with LPS. AC-186-treated BV-2 microglia were stimulated with LPS (100 ng/mL). Western blotting on nuclear extracts showed reduction in protein expression of acetyl-p65. Lamin B was used as an internal loading control. Data are expressed as the mean ± SEM (n = 3). ****p < 0.0001 versus LPS stimulation alone

LPS-mediated phosphorylation of p65 subunit and IκBα result in the degradation of the latter and translocation of the former into the nucleus where it regulates the expression of pro-inflammatory genes including iNOS, COX-2 and the pro-inflammatory cytokines. Based on results showing inhibitory effects of AC-186 on the production of pro-inflammatory mediators, as well as phosphorylation of p65 in the cytoplasm, its effect on nuclear transactivation of NF-κB p65 was investigated.

Results of these experiments revealed that stimulation of BV-2 microglia with LPS (100 ng/mL) significantly enhanced (p < 0.001) NF-κB luciferase activity, and thus its transcriptional activity when compared with unstimulated cells (Fig. 4e). LPS-induced increased NF-κB luciferase activity was however reduced from 100% to 51.6%, 44.9%, 37% and 22% by 0.625, 1.25, 2.5 and 5 µM concentrations of AC-186, respectively (Fig. 4e).

NF-κB regulates gene transcription by binding to specific kappa B (κB) sites in the DNA. Results showing that AC-186 could reduce NF-κB transcriptional activity prompted investigations to determine whether the compound could interfere with ability of NF-κB to bind to DNA consensus sites. ELISA-based DNA binding assays revealed that the binding activity of nuclear NF-κB-p65 was significantly (p < 0.001) increased following stimulation of BV-2 cells with LPS (100 ng/mL) (Fig. 4f). These experiments also revealed that pre-treatment with AC-186 (1.25, 2.5 and 5 µM) prior to LPS stimulation resulted in decreased NF-κB-p65 DNA binding activity, whereas at 0.625 µM the compound did not produce significant (p < 0.05) reduction in DNA binding activity (Fig. 4f).

AC-186 promotes deacetylation of p65 sub-unit of NF-κB in LPS-stimulated BV-2 microglia

Results in Fig. 4g and h indicate that protein levels of acetyl-NF-κB p65 were significantly (p < 0.001) elevated in BV-2 cells stimulated with LPS (100 ng/mL), in comparison to unstimulated BV-2 microglial cells. However, AC-186 treatment prior to LPS stimulation resulted in significant (p < 0.001) deacetylation of NF-κB p65 with protein levels falling to 42.4%, 34.1%, 28.1% and 20.9% in the presence of 0.625, 1.25, 2.5 and 5 µM of the compound, respectively (Fig. 4g and h).

LPS-indued repression of SIRT-1 protein expression was reversed by AC-186

SIRT-1 is a class III histone deacetylase (HDAC), which is involved in the deacetylation and regulation of the transcriptional activity of NF-κB p65. Encouraged by results showing that AC-186 prevented LPS-induced acetylation of NF-κB p65, experiments were conducted to evaluate effects of the compound on SIRT-1 protein expression in LPS-activated BV-2 microglia. Results in Fig. 5a and b depict significant (p < 0.001) suppression of nuclear SIRT-1 protein levels in cells stimulated with LPS, when compared with unstimulated cells. Significant (p < 0.001) reversal of LPS-induced suppression of nuclear SIRT-1 protein was reversed in the presence of AC-186, with ~ 3.3, ~ 3.6, ~ 4.5, and ~ 5.7-fold increase in expression produced by 0.625, 1.25, 2.5 and 5 µM concentrations of the compound, respectively (Fig. 5a and b).

Fig. 5

AC-186 increases SIRT-1 protein in BV-2 microglia. Microglia cells were treated with LPS (100 ng/mL) in the absence or presence of indicated concentrations of AC-186 followed by immunoblotting of nuclear extracts for SIRT-1. Lamin B was used as an internal loading control. Data are expressed as the mean ± SEM (n = 3). ****p < 0.0001 versus LPS stimulation alone

AC-186 increased oestrogen receptor beta (ERβ) expression and transcriptional activity of oestrogen-response element (ERE) in BV-2 cells

Based on reports indicating that AC-186 is a selective non-steroidal oestrogen receptor β agonist [10], and studies suggesting that oestrogen modulates neuroinflammation by interacting with ERβ [16], experiments were conducted to determine whether AC-186 could affect protein expression of ERβ in BV-2 microglia. Figure 6a and b show that ERβ is expressed in BV-2 microglial cells and treating these cells with 0.625, 1.25, 2.5 and 5 µM of AC-186 resulted in ~ 2.2, ~ 3.2-fold, ~ 4.6-fold and ~ 5.1-fold increase in expression of ERβ protein, respectively when compared to control cells. Interestingly, results of reporter gene assays revealed that significant (p < 0.05) increase in ERE transcriptional activity was only produced in the presence of 5 µM of the compound while treatment of BV-2 microglia with lower concentrations of the compound resulted in insignificant (p < 0.05) increase (Fig. 6c).

Fig. 6

Implications of treating BV-2 microglia with AC-186 on ERβ. a, b ERβ protein expression was significantly elevated when cells were treated with AC-186. c AC-186 (5 μM) increases ER-dependent luciferase activity in BV-2 microglia. Data are expressed as the mean ± SEM (n = 3). ns-not significant at p < 0.05, *p < 0.05, ****p < 0.0001 versus control alone

Anti-inflammatory effects of AC-186 in BV-2 microglia are dependent on ERβ

Studies have provided evidence linking anti-inflammatory action of oestrogen to its interaction with ERβ. Results of anti-inflammatory effects of AC-186, coupled with its reported ERβ agonist activity prompted investigations to determine whether its anti-inflammatory effect was dependent on ERβ. Results in Fig. 7 show that there were significant decreases in both TNFα (Fig. 7a) and IL-6 (Fig. 7b) levels in control siRNA-transfected BV-2 microglia which were treated with AC-186 (5 µM) and stimulated with LPS (100 ng/mL). In ERβ siRNA-transfected cells however, AC-186 (5 µM) failed to reduce TNFα and IL-6 production following stimulation with LPS.

Fig. 7

Effects of ERβ knockdown on anti-inflammatory effects of AC-186. Control siRNA- and ERβ siRNA-transfected BV-2 cells were treated with AC-186 (5 μM) prior to stimulation with LPS (100 ng/mL) for 24 h. Culture supernatants were analysed for TNFα a and IL-6 b. Data are expressed as the mean ± SEM (n = 3). ns-not significant at p < 0.05, **p < 0.05, ****p < 0.0001

Incubation of LPS-activated BV-2 microglia with AC-186 prevented neuroinflammation-mediated neuronal damage

Excessive production of pro-inflammatory mediators from the microglia has been proposed as one of the principal mechanisms involved in neuronal damage. Exposing BV-2 microglia co-cultured with HT-22 hippocampal neurons to a high concentration (1 μg/mL) of LPS resulted in significant (p < 0.001) increase in LDH release by the neurons when compared with unstimulated cells (Fig. 8a), suggesting neuronal death. Interestingly, supernatants collected from the BV-2 layer showed significant (p < 0.0001) increase in levels of both TNFα and IL-6 (Fig. 8b). However, pre-treating the microglial layer with AC-186 (5 μM) resulted in significant (p < 0.01) reduction in LDH release by the neurons, while levels of TNFα and IL-6 in microglial culture supernatants were also reduced (Figs. 8a and b). MTT assay to assess viability of HT-22 neurons showed that activation of BV-2 microglia with LPS (1 µg/mL) resulted in a significant (p < 0.01) reduction in the number of viable adjacent neurons, when compared to neurons co-cultured with unstimulated cells (Fig. 8c). However, neuronal viability was improved (p < 0.05) when BV-2 cells were treated with AC-186 (5 μM) prior to LPS stimulation (Fig. 8c).

Fig. 8

Neuroprotection by AC-186 in microglia-neuron co-culture. BV-2 microglia-HT-22 neurons were co-cultured in a transwell system and treated with AC-186 (5 μM) prior to stimulation with LPS (1 μg/mL) for 48 h. a. Neuronal viability was determined using LDH assay. b. Reduction in the levels of TNFα and IL-6 were measured using mouse ELISA kits. c. Viability of neurons was assessed using MTT assay. Data are expressed as the mean ± SEM (n = 3). **p < 0.01, ***p < 0.001, ****p < 0.0001 versus LPS stimulation alone