*Article* **Inhibiting NLRP3 Inflammasome Activation by CY-09 Helps to Restore Cerebral Glucose Metabolism in 3***×***Tg-AD Mice**

**Shuangxue Han 1,2, Zhijun He 1,6, Xia Hu 3, Xiaoqian Li 1, Kaixin Zheng 1, Yingying Huang 1, Peng Xiao 3, Qingguo Xie 3,7,\*, Jiazuan Ni 1,4,5 and Qiong Liu 1,4,5,\***


**Abstract:** The reduction of the cerebral glucose metabolism is closely related to the activation of the NOD-like receptor protein 3 (NLRP3) inflammasome in Alzheimer's disease (AD); however, its underlying mechanism remains unclear. In this paper, 18F-flurodeoxyglucose positron emission tomography was used to trace cerebral glucose metabolism in vivo, along with Western blotting and immunofluorescence assays to examine the expression and distribution of associated proteins. Glucose and insulin tolerance tests were carried out to detect insulin resistance, and the Morris water maze was used to test the spatial learning and memory ability of the mice. The results show increased NLRP3 inflammasome activation, elevated insulin resistance, and decreased glucose metabolism in 3×Tg-AD mice. Inhibiting NLRP3 inflammasome activation using CY-09, a specific inhibitor for NLRP3, may restore cerebral glucose metabolism by increasing the expression and distribution of glucose transporters and enzymes and attenuating insulin resistance in AD mice. Moreover, CY-09 helps to improve AD pathology and relieve cognitive impairment in these mice. Although CY-09 has no significant effect on ferroptosis, it can effectively reduce fatty acid synthesis and lipid peroxidation. These findings provide new evidence for NLRP3 inflammasome as a therapeutic target for AD, suggesting that CY-09 may be a potential drug for the treatment of this disease.

**Keywords:** Alzheimer's disease; glucose metabolism; NLRP3 inflammasome; insulin resistance; oxidative stress; CY-09

## **1. Introduction**

Alzheimer's disease (AD) is a common neurodegenerative disease in elder people. Deposits of amyloid-β (Aβ) and hyperphosphorylated tau protein are its main characteristics [1,2]. Due to the failure of anti-amyloid and anti-tau aggregation drugs, neuroinflammation has been considered as a new therapeutic target for AD treatment.

NLRP3 inflammasome, composed of nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 (NLRP3) and apoptosis-associated speck-like proteins of CARD (caspase recruitment domain) (ASC) and pro-caspase-1, plays an important role in neuroinflammation. Activation of NLRP3 inflammasome causes the increase in caspase-1

**Citation:** Han, S.; He, Z.; Hu, X.; Li, X.; Zheng, K.; Huang, Y.; Xiao, P.; Xie, Q.; Ni, J.; Liu, Q. Inhibiting NLRP3 Inflammasome Activation by CY-09 Helps to Restore Cerebral Glucose Metabolism in 3×Tg-AD Mice. *Antioxidants* **2023**, *12*, 722. https:// doi.org/10.3390/antiox12030722

Academic Editor: Yan-Zhong Chang

Received: 31 January 2023 Revised: 6 March 2023 Accepted: 8 March 2023 Published: 15 March 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

and the release of interleukin-1β (IL-1β) [3,4]. Increased activation of NLRP3 inflammasome is closely related with the reduction of cerebral glucose metabolism. Studies show that the translocation of hexokinase (HK), a key enzyme in glucose metabolism, can activate NLRP3 inflammasome, and inhibition of NLRP3 inflammasome can restore the expression and distribution of HK in AD model cells [5,6]. In AD mice, NLRP3 binds to the mitochondria and is then activated by mitochondrial reactive oxygen species (mtROS) [7,8]. HK binding to mitochondria reduces mtROS transport into the cytoplasm and further reduces the activation of NLRP3 inflammasome [9,10]. A positive correlation occurs between glucose metabolism and neuroinflammation in early AD, but disappears as the pathological course progresses [11]. In addition, increased neuroinflammation leads to a shift in energy metabolism from oxidative phosphorylation (OxPhos) to aerobic glycolysis in microglia. Activated microglia compete with neurons for glucose, which limits the energy availability of neurons [12]. Our previous studies have shown that inhibiting the activation of NLRP3 inflammasome can increase the expression and distribution of HK in vitro [5], but determining whether it could restore cerebral glucose metabolism in this process requires further exploration in vivo.

Glucose metabolism provides over 70% of energy for the brain. Glucose is transported into cells via glucose transporters (GLUTs), is phosphorylated by HK, and then catalyzed by a series of enzymes to produce energy. GLUT1 and GLUT3 are expressed in the brain and are responsible for the entry of glucose from the blood to the intracellular environment [13–15]. In AD, decreased expression levels of GLUT1 and GLUT3 lead to reduced glucose transport. GLUT4 is also expressed in the brain. Unlike GLUT1 and GLUT3, it is sensitive to insulin, and when insulin levels rise, it is transferred to cell membranes for the transport of glucose, [16,17]. The transfer of GLUT4 is regulated by the insulin-PI3K-AKT pathway [18]. Several studies have shown that insulin resistance is a key event leading to AD pathology [17,19,20]. Insulin receptors (IR) recognize insulin and then self-phosphorylate to recruit insulin receptor substrate (IRS) and activate the IRS-AKT-AS160 pathway [21,22]. AS160 is a guanosine triphosphate enzyme activating protein, the phosphorylation of which promotes translocation of GLUT4 [18,23]. In AD, lower insulin levels in cerebrospinal fluid result in decreased expression of p-IR, p-AKT, and p-AS160, a s well as increased IR [24]. A positron emission tomography (PET) study confirmed that the expression of IR was increased in the lower glucose metabolism region.

Oxidative stress also contributes to the pathology of AD. The generation of an excessive amount of ROS results in oxidative stress and the activation of NLRP3 inflammasome. Moreover, oxidative stress causes iron metabolism disorders and leads to ferroptosis [25,26]. Ferroptosis manifests as increased cellular Fe2+, decreased glutathione peroxidase 4 (GPX4), and increased lipid peroxidation [27]. Elevated Fe2+ results from increased transferrin transport and ferritinophagy [28]. Transferrin (TF), transferrin receptor (TFR), and ferroportin (FPN) are responsible for the transport of Fe, and nuclear receptor coactivator 4 (NCOA4) participates in ferritinophagy. Long-chain acyl-CoA synthetase 4 (ACSL4) is a key protein linking ferroptosis and lipid peroxidation. Increased ACSL4 is consistent with increased ferroptosis and lipid peroxidation in AD [29–31]. Decreased GPX4 and Solute Carrier Family 7, Member 11 (SLC7A11) are also related to lipid peroxidation.

In the study, we injected CY-09, a specific inhibitor of NLRP3, into non-transgenic (NTg) and triple transgenic AD (3×Tg-AD) mice daily, with a dose of 2.5 mg/kg for six weeks, according to the reference [32]. Then, we explored the effect of NLRP3 inflammasome inactivation on glucose transport, insulin resistance, and glucose metabolic enzymes in vivo. Simultaneously, we investigated whether NLRP3 inflammasome inactivation by CY-09 could reduce AD classical pathology and oxidative stress and improve cognitive deficits. Overall, this study aimed to determine the effect of NLRP3 inflammasome activation on glucose metabolism and to investigate the potentiality of CY-09 as a therapeutic drug for AD treatment.

#### **2. Materials and Methods**

#### *2.1. Reagents and Antibodies*

CY-09 (Cat#S5774) was purchased from Selleck Chemicals LLC, Houston, TX, USA. Polyoxyl 15 hydroxystearate (Cat#HY-136349) and DMSO (Cat#HY-Y0320) were purchased from MedChemExpress LLC, Deer Park Dr, Suite Q, Monmouth Junction, NJ, USA. Saline (Cat#R22172) was purchased from Shanghai yuanye Bio-Technology Co., Ltd., Shanghai, China. 18F-FDG was provided by Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China. Antibodies against NLRP3 (Cat#AG-20B-0014-C100) and caspase-1 (P20) (Cat#AG-20B-0042-C100) were purchased from Adipogen Corporation, San Diego, CA, USA. Antibodies against IL-1β (Cat#16806-1-AP), GLUT1 (Cat#21829-1-AP), GLUT4 (Cat#66846-1-Ig), GLUT3 (Cat#20403-1-AP), HK2 (Cat#22029- 1-AP), voltage-dependent anion-selective channel protein 1 (VDAC1) (Cat#10866-1-AP), and SLC7A11 (Cat#26864-1-AP) were purchased from Proteintech Group, Inc., Chicago, IL, USA. Antibodies against IRS (Cat#3407), p-IRS-Ser1101 (Cat#2385), AKT (Cat#9272), p-AKT-Ser473 (Cat#4060), GSK3β (Cat#12456), p-GSK3β-Ser9 (Cat#5558), FAS (Cat#3180), ACC (Cat#3676), p-ACC-Ser79 (Cat#11818), and LRP1 (Cat#64099) were purchased from Cell Signaling Technology, Inc., Boston, MA, USA. Antibodies against IR (Cat#sc-57342), p-IR-Tyr1150 (Cat#sc-81500), and ACSL4 (Cat#sc-271800) were purchased from Santa Cruz Biotechnology, Inc., Dallas, TX, USA. The antibodies against AS160 (Cat#ab189890), p-AS160-T642 (Cat#ab131214), HK1 (Cat#ab150423), PDHE1α (Cat#ab168379), COX IV (Cat#ab16056), APP (Cat#ab32136), BACE1 (Cat#ab108394), PSD95 (Cat#ab18258), synaptophysin (Cat#ab32127), tau5 (Cat#ab80579), p-tau-Ser404 (Cat#ab92676), MDA (Cat#ab27642), GPX4 (Cat#ab125066), and HMGCS1 (Cat#ab155787) were purchased from Abcam plc., Cambridge, UK. Antibodies against 6E10 (Cat#803002), sAPPα (Cat#813501), and sAPPβ (Cat#813401) were purchased from BioLegend, Inc., San Diego, CA, USA. The antibody against Aβ1-42 (Cat#AB5078P) was purchased from Millipore Corporation, Boston, MA, USA. Antibodies against HT7 (Cat#MN1000), TF (Cat#PA5-27306), TFR (Cat#13-6800), FPN (Cat#PA5-22993), NOCA4 (Cat#PA5-96398), and SREBP2 (Cat#PA1-338) were purchased from Thermo Fisher Scientific, Waltham, MA, USA. The antibody against β-actin (Cat#AB0033) was purchased from Abways Technology, Inc., Shanghai, China. The antibody against HMGCR (Cat#T56640S) was purchased from Abmart Shanghai Co., Ltd., Shanghai, China. Goat Anti-Rabbit IgG H&L (HRP) secondary antibody (Cat#ab6721) and Goat Anti-Mouse IgG H&L (HRP) secondary antibody (Cat#ab6789) were purchased from Abcam plc., Cambridge, UK. Alexa Fluor® 488 AffiniPure Goat Anti-Rabbit IgG (H+L) secondary antibody (Cat#111-545-003) and Alexa Fluor® 594 AffiniPure Goat Anti-Mouse IgG (H+L) secondary antibody (Cat#111-585-003) were purchased from Jackson ImmunoResearch Inc., West Grove, PA, USA. DAPI Staining Solution (Cat#C1002) was purchased from Beyotime Biotech Inc., Shanghai, China.

#### *2.2. Animals and Treatment*

The impact of sex on AD pathology has been reported in many references. Senior females are more likely to develop AD due to their lower estrogen levels. Ovarian hormone loss causes a bioenergetic deficit and a shift in metabolic fuel availability in AD model mice [33,34]. Thus, we select 9-month-old female C57BL/6J mice (NTg mice) and 3×Tg-AD mice for this study. NTg mice were purchased from Guangdong Medical Laboratory Animal Center and 3×Tg-AD mice which harbor the mutated human genes amyloid precursor protein (APP) (SWE), PS1 (M146V), and Tau (P301L) were purchased from the Jackson Laboratory. Mice were housed in conditions under 22 ◦C in a 12:12 h light/dark cycle, with food and water ad libitum. All animal experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee of Tongji Medical College of Huazhong University of Science and Technology (IACUC Number: 2390; Approved Date: 27 February 2018).

To observe the impact of NLRP3 inflammasome activation on cerebral glucose metabolism in AD, we used CY-09 to inhibit NLRP3 inflammasome activation. Here, 24 female mice were randomly divided into four groups: NTg mice, CY-09-treated NTg mice (NTg + CY-09 mice), 3×Tg-AD mice, and CY-09-treated 3×Tg-AD mice (3×Tg-AD + CY-09 mice). CY-09 was dissolved in a vehicle containing 10% DMSO, 10% Polyoxyl 15 hydroxystearate, and 80% saline. It was then injected into the mice daily, with a dose of 2.5 mg/kg for six weeks, according to the reference [32].

## *2.3. 18F-FDG PET*

18F-FDG PET experiments were applied to examine the glucose metabolism in the mouse brain [35]. All the mice were fasted for 12–16 h and then injected by vein with 7.4 MBq 18F-FDG before the PET scan. For the static PET scan, the mice were scanned for 10 min after 60 min of free metabolism. However, in the dynamic scan, the mice were scanned for 60 min immediately after injection. PET data were acquired with Trans-PET Discoverist 180 (Raycan Technology Co., Ltd., Suzhou, China) and reconstructed with a 3D OSEM algorithm. The time segmentary schemes for dynamic data reconstruction were as follows: 5 s × 6, 10 s × 3, 30 s × 4, 60 s × 2, 120 s × 5, 300 s × 3, 600 s × 1, 300 s × 1, 900 s × 1. To calculate the cerebral metabolic rate of glucose (CMRglu), blood glucose was measured from the second drop of tail blood using a Roche blood glucose meter (Roche Pharma (Schweiz) Ltd., Basel, Switzerland). Standard uptake values (SUVs) of the whole brain, cortex, and hippocampus, as well as the CMRglu of the whole brain, were quantified by Amide 1.0.4 software (Crump Institute for Molecular Imaging, UCLA School of Medicine, CA, USA), Crimas 2.9 (Turku PET center, Turku, Finland), MATLAB 2019b (MathWorks Inc., Natick, MA, USA), and SPM12 (The Wellcome Centre for Human Neuroimaging, UCL Queen Square Institute of Neurology, London, UK).

#### *2.4. Morris Water Maze Test*

The Morris water maze test is one of the behavioral experiments used to detect the spatial learning and memory ability of mice. In the study, we mainly refer to the previous protocol, with slight modifications [36,37]. None of the mice were trained on the test before experimentation. However, an additional process was carried out to familiarize the mice with the environment before the formal experiment started. The Morris water maze test includes place navigation tests and spatial probe tests. In the place navigation test, the mice were first placed on the platform for 1 min to familiarize themselves with the environment. Subsequently, the mice were placed into the water from the four quadrants. The time (escape latency) from entering the water to finding the platform was recorded within 60 s. The place navigation test lasted for five days. In the spatial probe test, the platform was removed and the mice were placed into the diagonal quadrant. Then, the time spent by the mice in the target quadrant in 24 h and 72 h was recorded. The Morris water maze WMT-200A (Chengdu Techman Software Co., Ltd., Chengdu, China) and the Animal Behavior Analysis System BAS-100 (Chengdu Techman Software Co., Ltd., Chengdu, China) were used for the recording of data.

#### *2.5. Glucose Tolerance Test (GTT) and Insulin Tolerance Test (ITT)*

GTT and ITT were performed to respectively detect the ability to regulate blood glucose and insulin sensitivity [32,38]. In GTT, the mice were fasted for 12 h and then injected with glucose in the dose of 2 g glucose per Kg mice (glucose was purchased from Beyotime Biotech Inc., Shanghai, China, Cat#ST1228). In ITT, mice were fasted for 4 h and then injected with insulin in the dose of 0.75 U insulin per Kg mice (insulin was purchased from Beyotime Biotech Inc., Shanghai, China, Cat#P3376). All experiments were required to detect and record the blood glucose in time points of 0, 30, 60, 90, 120, and 150 min after the injection. Blood glucose was measured from the second drop of tail blood using a Roche blood glucose meter (Roche Pharma (Schweiz) Ltd., Basel, Switzerland).

#### *2.6. Brain Tissues Extraction and Preservation*

After a week of recovery, the mice were euthanized after isoflurane anesthesia to extract the brain and blood. The hemibrain of each mouse was used for immunofluorescence experiments and stored with 4% paraformaldehyde. The total proteins of the rest of the hippocampus were extracted using the Phosphorylated Protein Extraction Kit (Cat#KGP950, Jiangsu Keygen Biotech Corp., Ltd., Nanjing, China) for Western blot experiments. The of the rest cortex was used for LC-MS/MS analysis, ROS measurement, and mitochondrion isolation. All tissues were stored at −80 ◦C before the experiments. Blood was tested for insulin directly after the extraction.

#### *2.7. ELISA Assay and Reactive Oxygen Species Measurement*

All experiments were conducted following the instructions of each kit. Fasting blood insulin was detected using a Highly Sensitive Mouse Insulin Immunoassay Kit (Cat#HMS200, EZassay Ltd., Shenzhen, China). The secretion of IL-1β was detected using a Mouse IL-1β ELISA Kit (Cat#EMC001b, Neobioscience Technology Company, Shenzhen, China). ROS levels were measured using the Reactive Oxygen Species Assay Kit (Cat#E004- 1-1, Nanjing Jiancheng Bioengineering Institute, Nanjing, China). These data were detected by a SpectraMax® L Microplate Reader (Molecular Devices, LLC, San Jose, CA, USA).

#### *2.8. LC-MS/MS*

LC-MS/MS was performed by Triple TOF 6500 (AB Sciex LLC, Framingham, MA, USA) to determine whether CY-09 crosses the BBB and enters the brains of the mice. In this experiment, metabolites were extracted from brain tissues using a 300 μL methanol acetonitrile mixture (methanol: acetonitrile = 2:1), as previously described [39]. After vortexing for 1 min, sonicating for 10 min at 0 ◦C, and centrifuging for 15 min at 13,000× *g*, the samples were held at −20 ◦C until further detection. The conditions for the chromatographic separation of metabolites were as follows—flow rate: 0.3 mL/min; injection volume: 6 μL; mobile phase: phase A, water (containing 0.1% formic acid); phase B, acetonitrile (containing 0.1% formic acid). The ion source parameters of mass spectrometry were as follows—curtain gas: 35 psi; ion spray voltage: −4500 V; source temperature: 550 ◦C; ion source gas1: 55psi; ion source gas2: 55psi. MultiQuant 3.02 (AB Sciex LLC, Framingham, MA, USA) and ProteoWizard 1.3.5.0 (ProteoWizard, Palo Alto, CA, USA) were used to process the data.

#### *2.9. Mitochondrion Isolation and Hexokinase Activity*

The brain mitochondria were isolated following the product instructions for the mitochondrial isolation kit (Cat#C3606, Beyotime Biotech Inc, Shanghai, China). Isolated mitochondria and cytoplasm were collected and stored at −80 ◦C. A NanoDrop 2000c (Thermo Fisher Scientific, Waltham, MA, USA) was used to detect the mitochondria concentration. The hexokinase activity was measured by Micro Hexokinase Assay Kit (Cat#BC0745, Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). All experiments were carried out according to the product instructions.

#### *2.10. Western Blot Analysis*

The total proteins of the brain tissues were extracted using the Phosphorylated Protein Extraction Kit (Cat#KGP950, Jiangsu Keygen Biotech Corp., Ltd., Nanjing, China). The PierceTM BCA Protein Assay Kit (Cat#23225, Thermo Fisher Scientific, Waltham, MA, USA) was used for the detection of protein concentrations. Experimental protocols were the same as previously described [5]. In short, 10% and 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels were used in the experiments. Proteins larger than 100 kD were separated using a 10% SDS-PAGE gel, while proteins smaller than 100 kD were separated using a 12% SDS-PAGE gel. Equal amounts of total protein lysates (20 μg per well) of each sample were loaded for the electrophoresis. Then, the proteins were transferred to a 0.45 μm polyvinylidene difluoride (PVDF) membrane (Cat#ISEQ00010, Millipore Corporation, Boston, MA, USA). After the electrotransfer, the membranes were

blocked in 5% non-fat milk for 2 h and washed four times (10 min each time) with TBST buffer (150 mM NaCl, 10 mM Tris, 0.1% Tween-20, pH 7.4). Afterwards, the membranes were incubated with primary antibodies overnight at 4 ◦C. The next day, the membranes were incubated with secondary antibodies at room temperature for 2 h after the washing of the primary antibody with TBST buffer. After the washing of the secondary antibodies, immunoreactive bands were visualized by the Tanon 5200 Series Image Analysis System (Tanon Science & Technology Co., Ltd., Shanghai, China). Grayscale analysis was used to quantify the protein expression. Grayscale values of the target protein were normalized to the grayscale values of β-actin or VDAC1. ImageJ 1.53C (NIH, Bethesda, MD, USA) was used to quantify the grayscale values of each immunoreactive band.

#### *2.11. Immunofluorescence Assay*

An immunofluorescence assay was performed for the brain slices following the previously established protocol [40]. Briefly, the brain slices were dewaxed and the xylene removed with dimethylbenzene and different concentrations of alcohol (100%, 95%, 85%, 70%, 50%, 30%, and 0%), respectively. Next, the brain slices were incubated with primary antibodies overnight after antigen retrieval, membrane rupture, and blocking. The next day, brain slices were washed using PBST for 30 min and then incubated with a second antibody for 2 h in the dark. After the washing of the second antibody, the brain slices were placed on slides and observed using an Olympus BX53 microscope (Olympus Corporation, Tokyo, Japan).

#### *2.12. Statistical Analysis*

In this study, semi-quantitative analysis and absolute quantification analysis were used to quantify the SUVs and the CMRglu, respectively [41]. The equation for *SUV* is as follows:

$$\text{SLIV} = \frac{\mathcal{C}\_T}{D\_{\text{Inj}}} \cdot \mathcal{W}\_s \tag{1}$$

*CT*, *DInj*, and *WS* represent the radioactivity in the region of interest, the injected dose of radioactivity, and the weight of the mouse, respectively.

The simplified equation for *CMRglu* is as follows:

$$\text{CMR}\_{\text{glu}} = \frac{\text{C}\_{\text{g}}}{\text{LC}} \cdot K \tag{2}$$

*Cg* is the concentration of blood glucose. *LC* is the lumped constant, and it reflects the difference between the metabolism of 18F-FDG and glucose. *K* is the uptake rate of 18F-FDG.

All data were presented as mean ± SD and analyzed with GraphPad Prism 9 (Graph-Pad Software Inc., La Jolla, CA, USA). The normality of distribution of the results was checked by Shapiro–Wilk test. A normal distribution of the data was indicated by the test; thus, a one-way ANOVA, followed by Bonferroni's multiple comparisons test, were performed in the study. Every possible comparison was explored, and significant differences (*p* < 0.05) between each group were shown in the figures.

#### **3. Results**

#### *3.1. CY-09 Could Cross the Blood-Brain Barrier In Vivo*

CY-09 inhibits the assembly and activation of the NLRP3 inflammasome by its combination with the ATP-binding motif of the NLRP3 NACHT domain to inhibit the ATPase activity. To investigate whether it is possible for CY-09 to cross the blood–brain barrier (BBB) in mice, initially, we adopted LC-MS/MS to detect the content of CY-09 in the brain of each group, i.e., the NTg, NTg + CY-09, 3×Tg-AD, and 3×Tg-AD + CY-09 mice. The results were shown in Figures 1a and S1; CY-09 was found in the brain tissues of NTg + CY-09 and 3×Tg-AD + CY-09 mice, which indicated that CY-09 could cross the BBB in vivo.

**Figure 1.** CY-09 attenuated activation of NOD-like receptor protein 3 (NLRP3) inflammasome in triple transgenic AD (3×Tg-AD) mice. (**a**) Content of CY-09 in 3×Tg-AD and 3×Tg-AD + CY-09 mice brains; (**b**–**g**) Western blot analysis of NLRP3, pro-caspase-1, caspase-1 (P20), pro-interleukin-1β (pro-IL-1β), and IL-1β in non-transgenic (NTg), NTg + CY-09, 3×Tg-AD, and 3×Tg-AD + CY-09 mice; (**h**) detection of IL-1β by ELISA in the four groups of mice. (n = 6, mean ± SD, one-way ANOVA and Bonferroni post hoc test; \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001 vs. NTg mice, & *p* < 0.05, && *p* < 0.01, &&& *<sup>p</sup>* < 0.001 vs. NTg + CY-09 mice, # *<sup>p</sup>* < 0.05, ## *<sup>p</sup>* < 0.01 vs. 3×Tg-AD mice).
