Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 Feb 1;17(1):45.
doi: 10.1186/s12974-020-1715-x.

G-protein-coupled estrogen receptor activation upregulates interleukin-1 receptor antagonist in the hippocampus after global cerebral ischemia: implications for neuronal self-defense

Ning Bai  1 , Quanguang Zhang  2 , Wenli Zhang  1 , Bin Liu  3 , Fang Yang  1 , Darrell Brann  4 , Ruimin Wang  5   6
Affiliations

G-protein-coupled estrogen receptor activation upregulates interleukin-1 receptor antagonist in the hippocampus after global cerebral ischemia: implications for neuronal self-defense

Ning Bai et al. J Neuroinflammation. .

Abstract

Background: G-protein-coupled estrogen receptor (GPER/GPR30) is a novel membrane-associated estrogen receptor that can induce rapid kinase signaling in various cells. Activation of GPER can prevent hippocampal neuronal cell death following transient global cerebral ischemia (GCI), although the mechanisms remain unclear. In the current study, we sought to address whether GPER activation exerts potent anti-inflammatory effects in the rat hippocampus after GCI as a potential mechanism to limit neuronal cell death.

Methods: GCI was induced by four-vessel occlusion in ovariectomized female SD rats. Specific agonist G1 or antagonist G36 of GPER was administrated using minipump, and antisense oligonucleotide (AS) of interleukin-1β receptor antagonist (IL1RA) was administrated using brain infusion kit. Protein expression of IL1RA, NF-κB-P65, phosphorylation of CREB (p-CREB), Bcl2, cleaved caspase 3, and microglial markers Iba1, CD11b, as well as inflammasome components NLRP3, ASC, cleaved caspase 1, and Cle-IL1β in the hippocampal CA1 region were investigated by immunofluorescent staining and Western blot analysis. The Duolink II in situ proximity ligation assay (PLA) was performed to detect the interaction between NLRP3 and ASC. Immunofluorescent staining for NeuN and TUNEL analysis were used to analyze neuronal survival and apoptosis, respectively. We performed Barnes maze and Novel object tests to compare the cognitive function of the rats.

Results: The results showed that G1 attenuated GCI-induced elevation of Iba1 and CD11b in the hippocampal CA1 region at 14 days of reperfusion, and this effect was blocked by G36. G1 treatment also markedly decreased expression of the NLRP3-ASC-caspase 1 inflammasome and IL1β activation, as well as downstream NF-κB signaling, the effects reversed by G36 administration. Intriguingly, G1 caused a robust elevation in neurons of a well-known endogenous anti-inflammatory factor IL1RA, which was reversed by G36 treatment. G1 also enhanced p-CREB level in the hippocampus, a transcription factor known to enhance expression of IL1RA. Finally, in vivo IL1RA-AS abolished the anti-inflammatory, neuroprotective, and anti-apoptotic effects of G1 after GCI and reversed the cognitive-enhancing effects of G1 at 14 days after GCI.

Conclusions: Taken together, the current results suggest that GPER preserves cognitive function following GCI in part by exerting anti-inflammatory effects and enhancing the defense mechanism of neurons by upregulating IL1RA.

Keywords: G-protein-coupled estrogen receptor 1 (GPER/GPR30); Global cerebral ischemia; Inflammasome; Interleukin-1 receptor antagonist (IL1RA); NACHT-, LRR- and PYD-containing protein 3 (NLRP3).

PubMed Disclaimer

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
GPER activation attenuates neuroinflammation in the hippocampal CA1 region following GCI and enhances neuronal survival. a Representative images of double-immunofluorescent staining for GPER (green) and Iba1 (red) in indicated groups (yellow), indicating co-localization of GPER with Iba1, and DAPI (blue) counter stains nuclei. Quantitative analysis of Iba1 intensity (b) and co-localization of GPER with Iba1 intensity (ratio to sham) of hippocampal CA1 region (c). d Representative images of double-immunofluorescent staining for NeuN (red) and GPER (green) in indicated groups, which shows that G1 administration enhanced GPER immunoreactive levels in hippocampal neurons. e Representative photographs of NeuN staining (green) and TUNEL (red) in the indicated groups (reperfusion at 14 days). Quantification was performed by counting the number of NeuN-positive neurons (f) or TUNEL-positive (g) per 250 μm length in the medial CA1 pyramidal cell layer. Magnifications are zooms of the boxed areas. Scale bar, 50 μm. n = 7–8, *P < 0.05 vs. IR group, #P < 0.05 vs. G1-treated group. IR: ischemia reperfusion
Fig. 2
Fig. 2
The GPER agonist, G1 attenuates protein expression of the NLRP3 inflammasome components in the hippocampal CA1 region following GCI. a Immunofluorescent staining of NLRP3 (green) and Iba1 (red), b CD11b (red), and ASC (green), showing the increased intensity of NLRP3 and ASC in the 14-day IR group, as compared to the G1-treated group, and G36 reversed the effect. Co-expression of NLRP3 with Iba1 or ASC with CD11b is indicated as yellow in the merged photomicrographs. c Western blot analysis of NLRP3 and ASC. Quantification was carried out according to the density of blot bands ratio to GAPDH. Data was expressed as means ± SE. GAPDH was used as a loading control. d Duolink analysis showed the interaction (red particles) of NLRP3 with ASC in both the 14-day IR and G36-treated groups compared to the sham or G1 group, DAPI (blue) counterstaining nucleus. #P < 0.05 vs. sham group, ##P < 0.05 vs. 14-day IR group, ###P < 0.05 vs. G1-treated group. n = 4–5, scale bar 50 μm
Fig. 3
Fig. 3
The effects of the GPER agonist, G1 upon caspase 1 activation and IL1β production in the hippocampal CA1 region following GCI. a–d Western blot analysis for CD11b, a marker of activated microglia, Cle-cas1 (cleaved caspase 1) and cleaved IL1β. Semi-quantitative analysis was carried out according to the band density of target protein ratio to that of loading control (GAPDH). Data was expressed as mean ± SE. #P < 0.05 vs. 14-day IR group, ##P < 0.05 vs. G1-treated group, e Double-immunofluorescent staining for CD11b (red) and cleaved IL1b (green), showing a strong increase in CD11b and cleaved IL1b intensity in the 14-day IR animals compared to the G1-treated group, while G36 reversed the increase. Scale bar 50 μm, magnification 40× n = 4–5
Fig. 4
Fig. 4
The effects of the GPER agonist, G1 upon IL1RA immunoreactive protein levels in the hippocampal CA1 region following GCI. a Representative images of double-immunofluorescent staining for a IL1RA (green) and CD11b (red), b IL1RA (green) and NeuN (red). Co-localization of IL1RA immunoreactive protein with the microglia marker, CD11b, and neuronal survival marker, NeuN, is shown in magnification images. DAPI (blue) counter stain for the cell nucleus. c Western blot analysis for IL1RA. #P < 0.05 vs. 14-day IR group, ##P < 0.05 vs. G1-treated group. n = 4 in sham and G1 groups and n = 5 in IR and G36 + G1 groups. d, e The specificity of IL1RA antibody was addressed using Western blot and immunofluorescent staining of IL1RA, and DAPI (red) counter stain for cell nucleus. f–h Effect of G1 on IL1RA protein expression in primary hippocampal neurons. Representative photographs of double-immunofluorescence staining for IL1RA (green) and MAP 2 (red) at 6 h after G1 or G1 + G36-administration, DAPI (blue) counter stain for cell nucleus (f). Western blot analysis showed the IL1RA protein expression in indicated groups (g, h). × 40 magnification, scale 50 μm
Fig. 5
Fig. 5
IL1RA knockdown abolishes the anti-inflammatory effects of GPER activation at 3 days of reperfusion in the hippocampal CA1 region following GCI. a Western blot analysis showed that IL1RA antisense oligodeoxynucleotide (AS) knockdown significantly decreased IL1RA protein expression (n = 4 in each group). Samples for Western blot were from sham, IR, G1 plus IL1RA missense oligodeoxynucleotide (MS), and G1 plus AS. Antibodies of CD11b (n = 4 in sham, IR, G1 groups, and n = 5 in G36 + G1-treated group), NLRP3 (b) (n = 4 in each group), cleaved-IL1β (c) (n = 4 in sham, IR, G1 groups, and n = 5 in G36 + G1-treated group), and loading control GAPDH. #P < 0.05 vs. 3-day IR group, ##P < 0.05 vs. G1 + MS group. Double staining for NLRP3 (green) and CD11b (red) (d) or cleaved-IL1b (green) and CD11b (red) (e) in the indicated groups. × 40 magnification, scale bar 50 μm
Fig. 6
Fig. 6
IL1RA knockdown reverses G1 regulation of NF-κB signaling in the hippocampal CA1 region following GCI. a-b Western blot analysis for NF-κB p65 in cytoplasm and nucleus fractions, and GAPDH, H2A as the loading controls of cytoplasm and fraction, respectively. #P < 0.05 vs. sham group, ##P < 0.05 vs. 3-day IR group, and ###P < 0.05 vs. G1 + MS group. c Double straining of NF-κB-p65 (red) and Iba1 (green). d Colocalization analysis of NFkB-P65 and Iba1 by Pearson Correlation Coefficient (PCC). #P < 0.05 vs. 3-day IR group, and ##P < 0.05 vs. G1 + MS group. × 40 magnification, scale bar 50 μm, n = 4–5
Fig. 7
Fig. 7
IL1RA knockdown abolishes the neuroprotective effect of G1 following GCI in the hippocampal CA1 region. a Western blot analysis of anti-apoptotic protein Bcl2, CREB activation, and an executioner of apoptosis, cle-cas3 (cleaved caspase 3). b Semi-quantitative analysis was carried out according to the band density of Bcl2 or cle-cas3 and p-CREB (c) ratio to that of loading control (tubulin). d Representative images of double-immunofluoscence staining for NeuN (red) and p-CREB (green). Data was expressed as mean ± SE. #P < 0.05 vs. 3-day IR group, ##P < 0.05 vs. G1 + MS-treated group. n = 4–5. e–g Representative photomicrographs of NeuN staining (green) and TUNEL analysis (red) in the indicated groups. n = 5, scale bar 50 μm
Fig. 8
Fig. 8
G1 enhancement of spatial learning and memory after GCI are abolished by G36 treatment or IL1RA knockdown. a Barnes maze was performed to examine spatial learning and memory. a The time (s) spent finding target hole (TH) at days 7, 8, and 9 after ischemia insult. b Exploration time spent in the target quadrant (TQ) that initially contained the TH at day 10 following reperfusion. c Moving speed of the rats in the probe trail on the fourth day of the test. d, e Representative traces indicating the sample paths of the rats from the maze latency trials and probe trials. Data are expressed as mean ± SE from 5 different animals. P < 0.05 considered as statistic difference between the groups. 1 sham, 2 IR, 3 G1 + G36, 4 G1, 5 G1 + IL1RA-AS
Fig. 9
Fig. 9
G1 enhancement of reference memory after GCI is abolished by G36 treatment or IL1RA knockdown. The novel object recognition (NOR) test was performed following GCI. Five-minute NOR tests at 13 and 14 days after 12-min ischemia were performed to monitor the long-term memory (a). b Time spent exploring the two familiar objectives on the training day. c The time spent exploring each object (familiar object and novel object) and d the discrimination index percentage (the time spent exploring novel object divided by total time spent). e Representative traces of the indicated groups on the testing day. All the data are expressed as means standard error from 6 to 7 animals in each group. *P < 0.05 considered as a statistic difference between the groups. n.s., no significant change; Ob, object; Fa-ob, familiar object; Nov-ob, novel object
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Neumann JT, Cohan CH, Dave KR, Wright CB, Perez-Pinzon MA. Global cerebral ischemia: synaptic and cognitive dysfunction. Curr Drug Targets. 2013;14:20–35. doi: 10.2174/138945013804806514. - DOI - PMC - PubMed
    1. Sekhon MS, Ainslie PN, Griesdale DE. Clinical pathophysiology of hypoxic ischemic brain injury after cardiac arrest: a “two-hit” model. Crit Care. 2017;21:90. doi: 10.1186/s13054-017-1670-9. - DOI - PMC - PubMed
    1. Pulsinelli WA, Brierley JB, Plum F. Temporal profile of neuronal damage in a model of transient forebrain ischemia. Ann Neurol. 1982;11:491–498. doi: 10.1002/ana.410110509. - DOI - PubMed
    1. Kim YM, Yim HW, Jeong SH, Klem ML, Callaway CW. Does therapeutic hypothermia benefit adult cardiac arrest patients presenting with non-shockable initial rhythms?: a systematic review and meta-analysis of randomized and non-randomized studies. Resuscitation. 2012;83:188–196. doi: 10.1016/j.resuscitation.2011.07.031. - DOI - PubMed
    1. Tang H, Zhang Q, Yang L, Dong Y, Khan M, Yang F, Brann DW, Wang R. GPR30 mediates estrogen rapid signaling and neuroprotection. Mol Cell Endocrinol. 2014;387:52–58. doi: 10.1016/j.mce.2014.01.024. - DOI - PMC - PubMed

MeSH terms

Substances