Animals
Female C57/BL6J mice (5 months old) were purchased from Weitong Lihua Experimental Animal Technology (Beijing, China) and carefully reared in the SPF laboratory of the animal experimental center of Shandong University. All mice were housed in a pathogen-free condition at 23°C and 45% humidity with a photoperiod of 12-h light and 12-h dark and fed with sterile fodder and drinking water. After a week of acclimatization, the animals were randomly divided into three experimental groups, i.e., OVX, sham, and OVX + sE2 groups. All animal experiments followed the ARRIVE guidelines and were conducted in accordance with the U.K. Animals (Scientific Procedures) Act, 1986 and related guidelines. Completely randomized design and blinding were adopted in animal experiments.
Ovariectomy
Mice were anesthetized with isoflurane (2.5%) after the lower back was shaved and cleaned with betadine and then an alcohol swab. A small incision was then made into the muscle wall with the uterine horn located and drawn through the muscle layer using blunt forceps. The ovaries were removed after the oviduct was ligated. The muscle layer was then sutured to close, while the skin was stapled. The sham-operated animals were treated with the same procedure without ligating the fallopian tube and the ovary excision. Finally, mice were placed in a heated cage until they recovered from anesthesia. Drug or control solvent injections were performed one week after surgery.
Animal treatments
E2 was purchased from Med Chem Express (Beijing, China) and dissolved in olive oil. The mice in the OVX + sE2 group received a daily intraperitoneal injection of E2 (0.5 mg/kg) for 5 weeks. This dose of E2 was 2–3 times higher than that of the circulating serum levels in the normal maintenance of mice [20–21]. The same volume of solvent was used in both OVX and sham-operated groups of mice. The body weights and food intakes in mice were recorded daily with the E2 dosage adjusted according to the change in body weights.
Open field test
The open field test consisted of 5 min trial in a white opaque arena of 45 cm × 45 cm × 30 cm in size (Maze Engineers, Boston, USA). The test arena was divided equally into nine square quadrants. Each mouse was placed into the central quadrant of the open field and allowed to freely explore the arena. The trajectory, the total distance moved, and the time spent in the central area of each mouse was recorded by the camera and analyzed using ANY-mazeTM video-tracking software (Stoelting Co., Chicago, IL, USA).
Y-maze test
The Y-maze test was carried out based on the method previously described [22]. Briefly, each mouse was placed in the arm designated (A) of the Y-maze and allowed to freely explore the maze for 5 min. The number of entries, excluding the first two, and the number of triads were recorded with the percentage of alternation calculated.
Forced swim test
A forced swim test was performed according to the method previously described [23]. Briefly, the mice were placed into plastic buckets, each 18 cm in diameter and 25 cm in height and filled with water, and maintained at 23°C. After 1 min of habituation, the immobility time (in the unit o sec) of the mice were recorded for a period of 5 min.
Sucrose Preference Test
Mice were habituated to 1% sucrose water for 3 days prior to the test. After 24 h deprivation of water and food, mice were provided with 1% sucrose water and pure water. The consumption levels were measured after 24 h testing. Sucrose preference (%) = (sucrose water intake/(sucrose water intake + pure water intake)) × 100.
Tissue processing
After the behavioral tests, the mice were deeply anesthetized with diethyl ether (10%, Buxco Electronics, Inc., Wilmington, NC, USA) and sacrificed for the preparation of blood extraction and brain tissue collection. One part of the brain tissue of each mouse was fixed in 4% paraformaldehyde for subsequent histopathological analysis. After the brain tissue was dehydrated, embedded in paraffin, and sectioned with 6 µm thickness. The other part of the brain tissue was frozen in liquid nitrogen for subsequent RNA extraction, proteomic analysis and untargeted metabolomic analysis.
Hormone assay
The serum level of E2 was measured with ELISA immunoassay (Estradiol EIA kit, Oxford Biomedical Research, Oxford, MI) according to the manufacturer’s instructions.
Immunostaining
The immunohistochemical experiments were conducted as previously described [24]. The antibodies used in this study included an anti-Iba1 antibody (1:50; ab178846; ab283319; Abcam, MA, USA), an anti-CD86 antibody (1:200; ab220188; Abcam, MA, USA), anti-IL-1β antibody (1:200; ab254360; Abcam, MA, USA), anti-TNF-α antibody (1:150; ab1793; Abcam, MA, USA), anti-IL-6 antibody (1:200; ab290735; Abcam, MA, USA), anti-ERα antibody (1:200; ab32063; Abcam, MA, USA), anti-GPER antibody (1:200; PA5-77396; Invitrogen, Karlsruhe, Germany), anti-ERβ antibody (1:150; PA1-311; Thermo Fisher Scientific, Shanghai, China), anti-NF-κB p65 antibody (1:200; ab32536; Abcam, MA, USA), anti-COX1 antibody (1:500; ab109025; Abcam, MA, USA), anti-CB1 antibody (1:200; ab3558; Abcam, MA, USA), anti-IL-1 (1:200; ab254360; Abcam, MA, USA), Alexa Fluor 488 secondary antibody (1:1,000; Invitrogen, Karlsruhe, Germany), Alexa Fluor 594 secondary antibody (1:1,000; Invitrogen), and IgG H&L (HRP) antibody (1:500; ab97051; Abcam, MA, USA). The nuclei were stained with DAPI (Sigma-Aldrich, Beijing, China) and images were captured on the LSM700 laser scanning confocal microscope (Zeiss, Tokyo, Japan) with the mean fluorescence intensities (MFI) determined using the FlowJo software [25].
Proteomic analysis
Proteomic analysis (OVX and OVX + sE2 groups, each n = 5) was performed using mass spectrometry in data-dependent acquisition mode. To designate significant changes in protein expression, a fold-change of > 1.2 or < 0.83 and a P-value of < 0.05 using Student’s t-test were set as cut-off values. The differential proteins were mapped to the KEGG database (https://www.kegg.jp/kegg/pathway.html) to enrich KEGG pathways.
Untargeted metabolomic analysis
Untargeted metabolomics analysis of both OVX and OVX + sE2 groups (each n = 5) was performed by LC-MS/MS as previously described [26]. The LC-MS/MS analysis was conducted on a quadrupole electrostatic field orbitrap high-resolution mass spectrometer (Thermo Fisher Scientific, USA). Assignment of metabolites was determined based on the published data and publicly available databases such as HMDB (http://redpoll.pharmacy.ualberta.ca/hmdb/HMDB/), KEGG (http://www.genome.jp/kegg), PubChem compound database (http://pubchem.ncbi.nlm.nih.gov), and SMPDB (http://smpdb.ca).
Hematoxylin and eosin (HE) staining
HE staining was conducted according to the routing protocols. Briefly, the brain sections of mice were deparaffinized, rehydrated, stained with hematoxylin (Biyuntian, Beijing, China), differentiated with acidic ethanol, stained with eosin (Biyuntian, Beijing, China), dehydrated, and mounted with Permount (Fisher Scientific, Shanghai, China).
Nissl staining
Nissl staining was performed to detect neuronal injury. The brain sections of mice were stained with 1% Cresyl Violet (C5042, Sigma-Aldrich, St Louis, MO, USA) and covered with 50% glycerin. The stained sections were photographed by a Keyence microscope (BZ9000, Osaka, Japan).
Cell lines and treatments
The microglial cell line BV-2 was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and was cultured in a matched dedicated complete medium (CM-0493; Procell, Wuhan, China) at 37°C with 5% CO2. The hippocampal cell line HT22 was purchased from the Procell Cell Bank (Wuhan, China) and was cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (Pan-Biotech, Aidenbach, Germany).
Primary microglia and neurons were extracted from the brains of neonatal female C57/BL6J mice (n = 32; 1–2 days old) as previously described [27]. The microglia were cultured in Neurobasal-A medium containing 2% B27, 2-mM L-glutamine, 50-U/ml penicillin, and 50-U/ml streptomycin (Gibco, USA). The culture medium for the neuron was DMEM/F12 containing 10% fetal bovine serum, 1 mM sodium pyruvate, 2 mM l-glutamine, 100 mM nonessential amino acids, 50 U/ml penicillin, and 50 mg/ml streptomycin (Gibco, USA).
BV-2 cells or primary microglia were seeded in the 24-well culture plates with cell-climbing slices. Then, the cells were exposed to E2 of different concentrations (0 to 3200 nM) for 24 h. To investigate the effect of E2 on the activated microglia, the cells were exposed to lipopolysaccharide (LPS; 100 ng/ml, Sigma) for 1 h and then co-treated with both LPS and E2 at either a high (800 nM) or a low concentration (200 nM). Inhibition of NF-κB was performed using the NF-κB inhibitor QNZ (EVP4593, Med Chem Express, Beijing, China) at a concentration of 30 nM for 24 h.
Both BV-2 and HT22 cells or primary microglia and neurons were co-cultured in a 2-layered co-culture chamber with the HT22 cells or primary neurons (2 × 105 cells) cultured in the upper layer and BV2 cells or primary microglia (2 × 105 cells) cultured in the lower layer. The intermediate membrane allowed the passage of culture fluid but not cells. Following the establishment of the co-culture system, cells were incubated with E2 (0 or 800 nM) for 0 to 72 h, respectively. Then, the viability of HT22 cells or primary neurons was determined using the CCK-8 assay.
CCK-8 assay
The cell counting kit-8 (CCK-8) assay kit (Dojindo, Shanghai, China) was used to evaluate the effects of E2 at different concentrations and treatment durations on cell viability according to the manufacturer’s protocol.
RNA extraction and qPCR
Total RNA was extracted from the brain tissues of mice or cells after the treatments using RNA Extraction Kit (Qiagen Sciences, Maryland, USA) according to the manufacturer's protocol. RNA was reverse transcribed via the Super Script II Reverse Transcriptase Kit (Invitrogen, Beijing, China) followed by quantitative real-time PCR (qRT-PCR) using Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, USA) based on primers given in supplementary Table S1.
Suppression of estrogen receptors using siRNA
ERα siRNA, ERβ siRNA, GPER siRNA, and control siRNA were purchased from Thermo Fisher Scientific (Shanghai, China). The transfections were performed as previously described [28]. The siRNA sequences were given in supplementary Table S2.
Western blot analysis
Western blot analysis
Western blot analysis was performed as previously described [29]. The antibodies used in this study included an anti-CD86 antibody (1:200; ab220188; Abcam, MA, USA), anti-NF-κB p65 antibody (1:200; ab32536; Abcam, MA, USA), anti-GADPH antibody (Santa Cruz Biotechnology, CA, USA), anti-Histone H3(1:1000; ab1791; Abcam, MA, USA), and goat anti-Rabbit IgG H&L (HRP; 1:2000; ab6721; Abcam, MA, USA). The original image of the WB stain is given in Supplementary Material.
Data analysis and statistics
Statistical analyses were performed with the SPSS software (SPSS Standard version 24.0, SPSS). The statistical analysis among groups was performed using one-way ANOVA followed by Tukey’s post hoc test. The significance of differences between the two groups was determined by Student’s t-test (unpaired, two-tailed). The qRT-PCR quantification was conducted using the 2−ΔΔCt method. A value of P < 0.05 was considered statistically significant.