Regents

A total cholesterol assay kit (#A111-1-1), triglyceride assay kit (#A110-1-1), low-density lipoprotein cholesterol assay kit (#A113-1-1), high-density lipoprotein cholesterol assay kit (#A112-1-1), acetylcholine assay kit (#A105-1-1), choline acetyltransferase assay kit (#A079-1-1), acetylcholinesterase assay kit (#A024-1-1), dopamine assay kit (#H170), norepinephrine assay kit (#H096), and estradiol 2 assay kit (#H102-1) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Alisol B 23-acetate (#PCS0958) was purchased from Chengdu Index Pure Biotechnology Co., Ltd. (Chendu, China). Estradiol (Lot: 356,564) was purchased from Abbott Healthcare Products (Netherlands). Simvastatin (Lot: 1,870,401) was purchased from Yangtze River Pharmaceutical Group (Taizhou, China). Palmitic acid (#SLBZ9610), MPP (#M70068), and PHTPP (#SML1355) were purchased from Sigma–Aldrich (New Jersey, USA). G-15 (#HY-103,449) was purchased from MedChemExpress (New Jersey, USA). PSD-95 antibody (#20665-1-AP), Tau antibody (#10274-1-AP), Bcl-2 antibody (#26593-1-AP), Bax antibody (#60267-1-Ig), β-actin antibody (#66009-1-Ig), and β-actin antibody (#20536-1-AP) were purchased from Proteintech (Wuhan, China). ERα antibody (#ab32063) was purchased from Abcam (Cambridge, England). ERβ antibody (#A2546) and GPER antibody (#A10217) were purchased from Abclone (Hangzhou, China). A metal enhanced DAB substrate kit (#DA1015), PMSF (100 mM) (#P0100), RIPA buffer (high) (#R0010), MTT cell proliferation and cytotoxicity assay kit (#M1020), and neutral balsam (#G8590) were purchased from Solarbio (Suzhou, China). Sensitive ECL chemiluminescent substrate (#BL520B), fetal bovine serum (#BL201A), high glucose DMEM (#BL301A), and trypsin solution (phenol red, EDTA-free) (#BL527A) were purchased from Biosharp (Hefei, China).

GEO database analysis

The GSE36318 data set and GSE1297 data set were downloaded from the GEO database (https://www.ncbi.nlm.nih.gov/gds). Sample information and sequencing data were obtained by R studio. The groups were defined using GEO2R, and the sequencing data for the samples were standardized. The differentially expressed genes between the defined groups were analyzed by GEO2R, and a table of the results was downloaded and used for gene set enrichment analysis (https://david.ncifcrf.gov). The enrichment analysis results were visualized using a cloud plotting platform (http://vip.sangerbox.com).

Experimental animal and postmenopausal dyslipidemia mouse model

C57BL/6J and LDLR−/− mice (SPF grade, female, 15–20 g) were purchased from Jiangsu Jicuiyaokang Biotechnology Co., Ltd. All animals were raised in the Experimental Animal Center of Nanjing University of Chinese Medicine (Ethic No. 201912A012).

Before the experiment, the mice were adaptively fed an SPF grade experimental diet for 1 week. The groups included the C57BL/6J + normal diet group, C57BL/6J + high-fat diet group, LDLR−/−+ normal diet group, LDLR−/−+ high-fat diet group, LDLR−/− ovariectomy + high-fat diet group, LDLR−/− + ovariectomy + high-fat diet + 17-β estradiol (E2, 0.13 mg/kg) group and LDLR−/− + ovariectomy + high-fat diet + simvastatin (SIM, 4.5 mg/kg) group. N = 5 in each group.

Mice accepting ovariectomy were anesthetized by intraperitoneal injection of 0.5% pentobarbital sodium. Then, a small incision was made on both sides of the back, and the ovaries were separated. After that, uterine ligation was performed, the ovaries were removed, and the wound was sutured on both sides of the back. One week after the operation, the mice were subjected to vaginal smears for 7 consecutive days, and the vaginal smears were observed under a microscope to observe whether the mice had an estrous cycle. The absence of the estrous cycle indicated that the mice were in a postmenopausal state and that the model was successfully replicated. Mice were given different diets according to the experimental groups. The administration group was treated once a day by gavage. The other groups were gastric administered 0.5% sodium carboxymethyl cellulose by gavage for 90 days.

Cognitive behavior tests in miceNovel object recognition test

Three objects A, B and C were used in the test; objects A and B were exactly the same, and object C was completely different from objects A and B. A box (0.4 m*0.4 m*0.3 m) was used to place the objects and mice. On the training day, objects A and B were placed on the two corners of one side of the box. The mice were then placed into the box with the back toward the two objects. Then, the video equipment was immediately opened to record the sniffing for the two objects of the mice for 5 min. After 24 h, object B was changed to object C, the mice were then placed into the box with the back toward the two objects again, and the sniffing of the two objects of the mice for 5 min was recorded. The discrimination index = (time to explore new things − time to explore old things)/ (time to explore new things + time to explore old things) *100%.

Y maze task

Before the experiment, the three arms of the Y maze were labeled arm A, arm B and arm C. The mice were placed in the same starting arm A with their heads facing the backplane. Then, the mouse was allowed to move freely for 8 min. A camera was used to record the behavior of the mice. Only when all four legs of the mouse entered one arm was it recorded as an arm entrance; when the mouse entered three different arms in turn, it was recorded as an alternate arm entrance. Maximum arm entrance = Total arm entrance − 2. Alternate behavior score = Total number of alternate arm entrances/maximum arm entrances * 100%.

Morris water maze

Positioning navigation experiments and space exploration experiments were included in the Morris water maze. The positioning navigation experiment was divided into a 4-day training and a one-day test. During the training period, mice were put into the pool from the entry point in each quadrant of the water maze and allowed to swim in the water and find the escape platform. The time limit for each quadrant was 60 s. During the test period, mice were put into each quadrant of the water maze to explore freely according to the training method, and the video software recorded the latency and swimming path of the mice. On the sixth day, a space exploration experiment was performed by removing the platform and putting the mice into the water facing the pool wall from the third quadrant to freely explore for 60 s. The video software recorded the times that cross the quadrant that the platform used to be placed.

Detection of lipids in mice

Orbital blood was taken from all mice at the end of the administration. Centrifugation was performed at 3000 rpm/min for 10 min to collect the serum. TC, TG, HDL-C and LDL-C levels in serum were determined by referring to the instructions of the test kits. A small amount of hippocampal tissue or liver tissue was put into a precooled glass homogenate tube, and anhydrous ethanol was added at a ratio of 1:9 by mass volume and ground until the tissue was homogenized. Centrifugation was performed at 3000 rpm/min for 10 min, and the supernatant was collected. Then, the protein concentration of the sample was detected by using a BCA kit. Then, the TC and TG levels were detected by referring to the instructions of the test kits.

ELISA and enzyme activity detection in mice

Orbital blood was taken from all mice at the end of the administration. Centrifugation was performed at 3000 rpm/min for 10 min before collecting the serum. The serum estradiol levels were determined by referring to the instructions of the estradiol ELISA detection kit. A small amount of hippocampal tissue was placed into a precooled glass homogenate tube, and saline or homogeneous solution provided by the kits was added at a ratio of 1:9 by mass volume and ground until the tissue was homogenized. Centrifugation was performed at 3000 rpm/min for 10 min, and the supernatant was collected. Then, the protein concentration of the sample was detected by using a NanoDrop System. The same total protein of the samples was used to detect Ach, NE, and DA levels according to the instructions of their ELISA detection kit. The activity of AChT and AchE in hippocampal tissues was detected by referring to the instructions of their detection kits.

Mouse brain tissue stainingNissl staining

The brain was fixed with 4% paraformaldehyde. After the brain was dehydrated, cleared and waxed, the wax blocks were obtained by paraffin embedding. A paraffin slicer was used for slicing, and sections with a thickness of 5 microns were obtained. The sections were dewaxed with xylene and then rehydrated, soaked and stained with Nissl’s dye A solution according to a Nissl staining kit. After differentiation with Nissl’s dye B solution, the sections were stained with hematoxylin. After that, the sections were dehydrated, cleared and sealed. A microscope was used to observe and take photographs of the brain.

Immunohistochemistry staining

Brain sections of 5 microns thickness were obtained. After the sections were dewaxed, rehydrated, and soaked in 3% hydrogen peroxide, they were washed with PBS 3 times. Then, the sections were soaked in citrate buffer at 95 °C for 20 min. After cooling, the slides were sealed with 5% BSA for 20 min. Primary antibodies against ERα, ERβ, and GPER were incubated with the brain overnight at 4 °C. After washing with PBS 3 times, the brain was incubated with the secondary antibody for 2 h at room temperature. A DAB chromogenic kit was used to incubate the brain for color development. After washing with PBS 3 times, the sections were stained with hematoxylin. After that, the sections were dehydrated, cleared and sealed. A microscope was used to observe and take photographs of the brain.

Immunofluorescence staining

Brain sections of 5 microns thickness were prepared. Following dewaxing and rehydration, the sections were treated with 3% hydrogen peroxide, then washed with PBS three times. Subsequently, the sections were immersed in citrate buffer at 95 °C for 20 min. After cooling, the slides were blocked with 5% BSA for 20 min. Primary antibodies against PSD-95 and TAU were applied to the sections and incubated overnight at 4 °C. After washing with PBS three times, the sections were incubated with the fluorescently-labeled secondary antibody for 2 h at room temperature. The sections were then covered with an anti-fade mounting medium containing DAPI. Photographs were taken using a fluorescence microscope.

TUNEL staining

Brain sections of 5 microns thickness were processed. After dewaxing, rehydrating, and treating with 3% hydrogen peroxide, the sections were washed three times with PBS. The sections were then immersed in citrate buffer at 95 °C for 20 min before cooling. The TUNEL reaction mixture was then applied to the sections according to the manufacturer’s instructions and incubated in a humidified atmosphere. Following this, the sections were washed thoroughly with PBS. They were then incubated with a FITC-conjugated secondary antibody specific to the TUNEL reaction. To counterstain the nuclei, the sections were covered with an anti-fade mounting medium containing DAPI. Observations and photographs of the fluorescently-labeled apoptotic cells were taken using a fluorescence microscope.

Western blot of the brains of mice

The hippocampus of the mice was collected, and the lysate was prepared according to the ratio of RIPA:PMSF = 100:1. The lysate was added according to the ratio of weight: volume = 50 mg: 1 mL. Two steel balls with a diameter of 3 mm were added, the tissue homogenizer was set at 50 Hz, and the brains were crushed for 30 s. Centrifugation was performed at 12,000 rpm/min for 10 min to collect the supernatant. The protein concentration of the sample was detected by using a NanoDrop System. Then, 5× sample loading buffer was added and cooked at 100 °C for 15 min on a dry thermostat. After SDS–PAGE and membrane transfer, primary antibodies against ERα (55 kDa), ERβ (65 kDa), GPER (55 kDa), PSD-95 (80 kDa), Tau (79 kDa), Bax (21 kDa), and Bcl-2 (26 kDa) were diluted according to the manufacturer’s instructions. As for loading control, a separate piece of membrane loaded with the same amount of protein of the samples was incubated with β-actin (43 kDa). The PVDF membrane was then incubated overnight with the diluted primary antibodies in a refrigerator at 4 °C. The membrane was washed the next day, and the second antibody was used to incubate the membrane at room temperature for 2 h. After washing again, a chemiluminescence kit and gel imaging system were used to expose the blots and analyze the data using relatively quantitative methods.

Culture and intervention of SH-SY5Y cells

SH-SY5Y cells were cultured with DMEM complete medium with 10% FBS in a 25T cell flask in an incubator at 37 °C and 5% CO2. When the density of SH-SY5Y cells reached 80%, conventional subculture was performed. Palmitic acid (PA, 0.0307 g) was added to 3 mL of sodium hydroxide solution (0.1 mM) to prepare the PA storage solution. The mixture was placed at 75 °C for 30 min. A 40% BSA solution was mixed with the PA storage solution in a 1:1 ratio to obtain a 20 nM PA working solution. The PA working solution was then diluted to different concentrations as required and used for intervention in SH-SY5Y cells. AB23A (40 µM), MPP (10 µM), PHTPP (10 µM), and G15 (100 nM) were also used to treat SH-SY5Y cells according to different requirements.

Cell viability detection of SH-SY5Y cells

When the density of SH-SY5Y cells reached 80%, the medium was discarded, and 2 mL trypsin solution was added to collect the cells. Then, SH-SY5Y cells were counted by the cell counter and diluted with medium to seed into a 96-well plate with 1 × 104 cells per well. The cells were cultured in the incubator until the density of SH-SY5Y cells reached 80%. Different concentrations of PA were added as needed. After incubation, 200 µL MTT solution was added to each well. The 96-well plate was placed in a 37 °C incubator and incubated for 4 h. Finally, the supernatant was discarded, and 200 µL DMSO was added to each well. The absorbance (OD) value was measured at 490 nm by using a microplate reader.

Immunofluorescence staining of SH-SY5Y cells

SH-SY5Y cells were grown on 24-well plates. Before cell seeding, a circular sterile glass sheet of 24 mm was added to each well. Cells were then cultured and treated with different reagents. After a specific time, the culture medium was discarded, and the wells were washed twice with PBS. Then, SH-SY5Y cells were fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton for 20 min. SH-SY5Y cells were incubated with 5% BSA for 20 min. Primary antibodies against ERα, ERβ, GPER, PSD-95 and Tau, according to the instructions, were diluted and added to incubate the cells overnight at 4 °C. On the second day, after washing with PBS three times, the secondary antibody was added, and the cells were incubated at room temperature for 2 h. After washing with PBS three times, the cells were incubated with DAPI for 5 min to stain the nucleus. After washing with PBS three times, the sterile glass sheets were removed and sealed with glycerin-gelatin sealing liquid. A fluorescence microscope was used to observe and take photographs of the SH-SY5Y cells.

Western blot of SH-SY5Y cells

The medium was discarded from the petri dish, and the SH-SY5Y cells were rinsed with 500 µL PBS solution. Then, 250 µL of mixed lysate was added to each dish, and the SH-SY5Y cells were lysed for 0.5 h. Centrifugation was performed at 12,000 rpm/min for 10 min to collect the supernatant. The protein concentration of the samples was detected by using a Nano Drop System.

The following operations were the same as “Western blot of brain in mice”.

Statistical analysis

The sample information of the GSE36318 data set and GSE1297 data set was compared by unpaired parametric t test. ImageJ was used to count Nissl bodies in the hippocampus of mice and exported measurement data. Image-Pro Plus 6.0 was used to quantify the mean optical density of immunostaining in in vivo and in vitro experiments and exported measurement data. Quantity One 4.5.2 was used to perform the relative quantification of protein expression in in vivo and in vitro experiments and exported measurement data. All measurement data are shown as the mean ± standard deviation. An unpaired parametric t test was used to analyze the difference between two groups. Ordinary one-way ANOVA with multiple comparisons was used to analyze the difference between more than two groups. When P < 0.05, the difference was considered significant. All measurement data were visualized and analyzed using GraphPad 9.0.