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Cornuside alleviates cognitive impairments induced by Aβ1−42 through attenuating NLRP3-mediated neurotoxicity by promoting mitophagy

Alzheimer's Research & Therapy volume 17, Article number: 47 (2025) Cite this article

A Correction to this article was published on 19 March 2025

This article has been updated

Abstract

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder in which mitochondrial dysfunction and neuroinflammation play crucial roles in its progression. Our previous studies found that cornuside from Cornus officinalis Sieb.Et Zucc is an anti-AD candidate, however, its underlying mechanism remains unknown. In the present study, AD mice were established by intracerebroventricular injection of Aβ1−42 and treated with cornuside (3, 10, 30 mg/kg) for 2 weeks. Cornuside significantly ameliorated behavioral deficits, protected synaptic plasticity and relieved neuronal damage in Aβ1−42 induced mice. Importantly, cornuside decreased NLRP3 inflammasome activation, characterized by decreased levels of NLRP3, ASC, Caspase-1, GSDMD, and IL-1β. Furthermore, cornuside promoted mitophagy accompanied by decreasing SQSTM1/p62 and promoting LC3B-I transforming into LC3B-II, via Pink1/Parkin signaling instead of FUNDC1 or BNIP3 pathways. In order to investigate the relationship between NLRP3 inflammasome and mitophagy in the neuroprotective mechanism of cornuside, we established an in-vitro model in BV2 cells exposed to LPS and Aβ1−42. And cornuside inhibited NLRP3 inflammasome activation and subsequent cytokine release, also protected neurons from damaging factors in microenvironment of conditional culture. Cornuside improved mitochondrial function by promoting oxidative phosphorylation and glycolysis, decreasing the production of ROS and mitochondrial membrane potential depolarization. Besides, mitophagy was also facilitated with increased colocalization of MitoTracker with LC3B and Parkin, and Pink1/Parkin, FUNDC1 and BNIP3 pathways were all involved in the mechanism of cornuside. By blocking the formation of autophagosomes by 3-MA, the protective effects on mitochondria, the inhibition on NLRP3 inflammasome as well as neuronal protection in conditional culture were eliminated. There is reason to believe that the promotion of mitophagy plays a key role in the NLRP3 inhibition of cornuside. In conclusion, cornuside re-establishes the mitophagy flux which eliminates damaged mitochondria and recovers mitochondrial function, both of them are in favor of inhibiting NLRP3 inflammasome activation, then alleviating neuronal and synaptic damage, and finally improving cognitive function.

Introduction

Alzheimer’s disease (AD) is the most common form of dementia characterized by amyloid β (Aβ) deposition and neurofibrillary tangles (NFTs) containing hyper-phosphorylated tau protein in the brain [1, 2]. Aβ and tau aggregates have been observed to damage synaptic function and have a profound effect on neuronal loss in the AD brain [3,4,5]. Several hypotheses have been put forward to explain the pathogenesis of AD. Among these, mitochondrial dysfunction, oxidative stress, and excessive neuroinflammation are vital processes mediating neurodegeneration [6–7]. Microglia are the major resident immune cells in the brain and are responsible for constantly surveying the microenvironment and maintaining homeostasis of the central nervous system. Aβ and NFTs are major stimuli that activate microglia, resulting in elevated production of pro-inflammatory cytokines and reactive oxygen species (ROS) [8,9,10]. Therefore, activated microglia play a vital role in the degenerative process of AD.

NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome is a multi-protein heteromeric complex existing in the cytoplasm, composed of NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), and protease precursor Pro-caspase-1 [11]. Upon activation of the NLRP3 inflammasome, Pro-caspase-1 is converted into its active form, Caspase-1, leading to the secretion of mature IL-1β and IL-18, resulting in an inflammatory cell death called pyroptosis [12]. Excessive pyroptosis can lead to widespread neuronal loss, causing neural circuit disruption and neurodegeneration. In the brains of AD patients, microglia surrounding Aβ plaques are significantly activated [13,14,15]. The protein expressions of NLRP3 and Caspase-1 are also increased. Moreover, elevated levels of IL-18 and IL-1β have been detected in the cortex and cerebrospinal fluid of AD patients [16,17,18,19]. These findings imply that NLRP3 inflammasome activation in microglia is involved in the pathogenesis of AD.

Mitochondrial dysfunction induced by Aβ releases damage-associated molecular patterns (DAMPs), such as ROS, mitochondrial DNA (mtDNA), cardiolipin, and ATP, which can trigger the NLRP3 inflammasome [19]. Excessive NLRP3 inflammasome activation further damages mitochondria, leading to mitochondrial depolarization and establishing a positive feedback loop between them. Mitophagy specifically degrades damaged mitochondria and is responsible for maintaining mitochondrial homeostasis and cellular survival [20, 21]. When mitophagy is impaired, damaged mitochondria remain in the cell with increased production of ROS, which activates inflammatory response in microglia via activating NLRP3 inflammasome. Therefore, mitophagy might provide a potential strategy for inhibiting NLRP3 inflammasome mediated neuronal death by removing dysfunctional mitochondria.

Cornuside, an iridoid glycoside derived from Cornus Officinalis Sieb. Et Zucc, has been proved to be a candidate compound for AD [22,23,24,25]. In previous studies, it showed potent activities in inhibiting oxidative stress and neuroinflammation, and RAGE/TXNIP/NF-κB signaling was demonstrated to participate in its activities [23]. NF-κB is a vital inflammatory transcription factor involved in the gene transcription of NLRP3 inflammasome-related proteins, such as NLRP3, pro-IL-1β, and pro-IL-18 [26]. TXNIP is an inhibitor of the ROS scavenger thioredoxin and promotes oxidative stress, as well as being involved in the activation of the NLRP3 inflammasome [27]. Hence, we hypothesized that cornuside may modulate microglial activation and neuroinflammation by inhibiting the NLRP3 inflammasome and subsequent neurotoxicity. In the present study, we established an AD mice model by intracerebroventricular injection of Aβ1−42, as well as a microglia model by LPS and Aβ1−42 stimulation, aiming to investigate the potential mechanism of cornuside against NLRP3 inflammasome activation and elucidate its interplay with mitophagy.

Materials and methods

Reagents and antibodies

Cornuside (C24H30O14) was obtained by our team from the dried ripe fruits of C. officinalis, and the purity was more than 98% determined by HPLC. The isolation process and NMR data of cornuside were detailed in our previously published article [28]. Human Aβ1−42 was obtained from Sangon Biotech (Shanghai, P. R. China). Donepezil was purchased from Yuanye Biotechnology (Shanghai, P. R. China). 3-MA (S2767) was obtained from Selleck (Houston, USA). LPS (L3023) and DMSO (D2650) were obtained from Sigma (St. Louis, USA). Dulbecco’s Modified Eagle’s Medium (C11995500BT) and Fetal Bovine Serum (1600044) were sourced from Gibco (Carlsbad, USA). Protease inhibitor (A8260), phosphatase inhibitor (P1260), BCA Protein Assay Kit (PC0020), and RIPA buffer (R0010) were obtained from Solebao (Beijing, China). Primary antibodies anti-NeuN (ab177487), anti-PSD95 (ab18258), anti-NLRP3 (ab263899), anti-Caspase-1 (ab207802), anti-IL-1β (ab283818), anti-ASC (ab283684), anti-SQSTM1/p62 (ab56416), anti-PINK1 (ab23707), anti-Parkin (ab15954), anti-BNIP3 (ab239976), anti-FUNDC1 (ab224722), anti-LC3B (ab48394), anti-PINK1 (ab23707), anti-Parkin (ab15954), anti-BNIP3 (ab239976), anti-FUNDC1 (ab224722) were from Abcam (Cambridge, UK). Primary antibodies also included anti-GSDMD (G7422, Sigma, St. Louis, MO, USA), anti-β-actin (20536-1-AP, Proteintech, Rosemont, IL, USA). Other antibodies included rabbit IgG secondary antibody (31430 and 31460, Thermo, Waltham, USA), Alexa Fluor 488-conjugated goat anti-rabbit and Alexa Fluor 594 goat anti-mouse IgG antibodies (A11008 and A11005, Invitrogen, Waltham, MA, USA). MitoTracker Deep Red (M22426, Sigma, USA) and DAPI Solution (C0065, Solarbio, Beijing, China) were used in immunofluorescent staining for mitochondria and nuclear.

Aβ1−42 preparation

Aβ1−42 was dissolved in hexafluoroisopropanol (HFIP) and incubated at room temperature for at least 1 h to achieve monomerization and structural randomization. Then, the sample was dried in a speed-vacuum centrifuge to remove HFIP, dissolved in anhydrous DMSO to a concentration of 5 mM, and diluted to 500 µM with PBS. Next, the solution was incubated at 4–8 °C for 48 h and centrifuged at 14,000 g for 10 min. Soluble oligomers were present in the supernatant. The supernatant was diluted and used for the experiment.

Mice surgery and treatment

Male ICR mice (6 weeks old, 20 ± 2 g) were purchased from Vital River Laboratory (Beijing, P. R. China) and housed in an experimental animal room with a constant temperature of 23 ± 2 °C, humidity of 50% ± 10%, and a 12 h light-dark cycle. All mice had free access to food and water. All procedures were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Animal Ethics Committee of China-Japan Friendship Hospital.

Mice were randomly divided into seven groups: control group, sham group, model group, cornuside-treated groups (3, 10, and 30 mg/kg), and donepezil group (3 mg/kg). Except for mice in the control group, all mice were subjected to surgery. Aβ1−42 (3 µL, 500 µM) was administered via intracerebroventricular injection at a location 1 mm posterior to the bregma, 1.9 mm lateral to the sagittal suture, and 2.4 mm in depth. After the surgery, mice received different interventions. Fifteen days later, behavioral tests were conducted 30 min after drug administration. For mice treatment, cornuside was dissolved in distilled water with the concentration 0.3, 1, 3 mg/mL. Donepezil was also dissolved in distilled water at a concentration of 1 mg/mL. Cornuside and donepezil were administered intragastrically once daily at a volume of 10 mL/kg body weight.

Behavioral testing

Morris water maze test

The Morris water maze test was performed in a round water pool (100 cm in diameter) with a platform (15 cm in diameter) hidden 1 cm below the water surface. The water temperature was maintained at 22 ± 1 °C. The pool was surrounded by a white wall. Dark posters with different shapes (one per wall), provided distant landmarks. During the four-day navigation trials, mice were trained twice daily. During training, mice were placed in the water and allowed to search for the platform for 60 s. If mice found the platform and stayed on it for 5 s, the trial was finished. If mice could not find the platform within 60 s, they were guided to the platform and allowed to stay on it for 15 s. On the fifth day, the platform was removed from the pool, and mice were allowed to swim for 60 s. Video tracking system (Zhongshi Science & Technology, P. R. China) was used to record the time and route of swimming.

Y maze test

The Y-maze test was conducted in a Y-shaped apparatus constructed from black polyvinyl chloride (PVC) plastic, with 40 cm in length, 3 cm in width, and 15 cm in height. The mice were placed into one arm of the maze and allowed to explore freely. The number of entries and the sequence of arm entries were recorded over a period of 10 min. Spontaneous alternation was defined as a series of consecutive entries into three different arms (e.g., ABC, CAB, or BCA), excluding sequences such as BAB and CBC. After each test, the Y maze was meticulously cleaned with 75% alcohol to eliminate any residual odors. The percentage of spontaneous alternation was calculated as: Alternation (%) = (Number of alternations) / (Total arm entries − 2) × 100%.

Novel object recognition test

Novel object recognition test was performed in a rectangular box with two objects. The test involved three phases. In the first phase, mice were placed into the box to adapt for 5 min. In the second phase, two identical red cylinders were put in, and mice were allowed to explore for 5 min. In the third phase, one of red cylinders was replaced with blue cube, and mice were allowed for 5 min exploration with time exploring the old (red cylinder) and new object (blue cube) recorded. The discrimination index (DI) was calculated as follows: DI (%) = (Time exploring new object − Time exploring old object) / (Time exploring new object + Time exploring old object) × 100%.

Nest building test

This test was conducted prior to the nocturnal activity period of mice (from 20:00 to 8:00 the next day). Mice were individually housed in cages with 24 h adaptation, then two stacks of tissues were put in as nesting material. Nests were scored at 2 and 12 h after the test began, according to the criteria below (Table 1).

Table 1 Criteria for nest scoring
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Three-chamber social interaction test

The three-chamber social interaction test (TBSIT) consisted of three chambers separated by doors. In the first stage, test mouse was made to adapt for 5 min in the empty condition. Then stranger mouse 1 was put into one side, test mouse was made to socialize for 10 min. In the third stage, another stranger mouse 2 was put into the other side, and the test mouse was made to free social for 10 min. The processes were recorded and analyzed the time communicating with mouse 1 (familiar mouse) and mouse 2 (novel mouse).

Cell culture and treatment

BV2 cells were obtained from the Cell Resource Center of the Institute of Basic Medical Sciences, Peking Union Medical College and Chinese Academy of Medical Sciences (Beijing, P. R. China). BV2 cells were maintained in DMEM supplemented with 10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37 °C and 5% CO₂ in a humidified incubator (Thermo Scientific, Langenselbold, Germany). In the present study, BV2 cells were seeded in 24-well or 6-well plates and after 24 h, the cells reached 70–80% confluence. Cells were pretreated with or without cornuside for 1 h, then treated with Aβ1−42 (2.5 µM) for 1 h, followed by LPS (1 µg/mL) stimulation. Cells were then incubated for the indicated time periods: 12 h for ROS and MitoSOX assays, 18 h for JC-1 staining, and 24 h for protein expression analysis. To gain further insight into the role of mitophagy in the NLRP3 inflammasome activation, 3-MA was used to inhibit the formation of autophagosomes, which was added to cells before cornuside treatment. In the in-vitro test, cornuside was dissolved in DMSO at a concentration of 10 mM, then further diluted with DMEM, ensuring the final DMSO concentration in the cell culture medium was less than 0.1%. LPS was dissolved in PBS at a concentration of 1 mg/mL.

For the preparation of conditioned medium, BV2 microglial cells were seeded in 6-well plates on the first day and cultured overnight to facilitate cell adhesion. On the second day, the medium was refreshed, and cells were pretreated with cornuside for 1 h. Following this pretreatment, LPS was added, and incubation continued for an additional hour. Subsequently, Aβ₁-₄₂ was added, and cells were co-incubated for 24 h. Concurrently, HT22 hippocampal neurons were seeded in 6-well plates and cultured overnight. On the third day, the culture supernatant from treated BV2 cells was collected and centrifuged at 3,000 rpm for 5 min to remove cell debris. The BV2 cell-conditioned medium was then applied to HT22 cells, replacing their medium, for an additional 24 h. Treated HT22 cells were subsequently used for further analyses, including cell viability, apoptosis, and additional assays.

TUNEL staining

In accordance with the manufacturer’s instructions for the TUNEL (Elabscience Biotechnology, Wuhan, China) in situ cell death detection kit, brain slices were incubated with the TUNEL reaction mixture for 1 h. Then images were captured at 525 nm using Zeiss fluorescence microscope (Jena, Germany).

Transmission electron microscopy (TEM)

Brain sections of 1 mm³ were fixed in 2.5% glutaraldehyde solution at 4 °C for 3 days. Subsequently, the samples were post-fixed in 1% osmium acid with 0.1 M phosphate buffer (pH 7.4) at room temperature for 2 h. The tissues were then dehydrated in graded acetone series (50%, 70%, 90%, 100%), followed by infiltration, embedding, and polymerization in Epon 812 resin. Ultrathin Sect. (70 nm) were cut using an ultramicrotome and stained with uranyl acetate and lead citrate. The sections were observed and imaged using a transmission electron microscope (JEM-1400 Plus, JEOL, Tokyo, Japan).

Immunohistochemistry (IHC)

Brain tissue was transcardially perfused with 4% paraformaldehyde (PFA), then underwent post-fixed, dehydrated, and embedded, and finally cut into 4-µm-thick brain slides. Brain slides were treated with 3% H2O2 to quench the endogenous peroxidase, and were blocked with 10% goat serum. Brain slides were incubated with primary antibodies overnight at 4 °C. On the following day, slides were incubated with HRP-conjugated secondary antibodies for 1 h at 37 °C after washing with PBS. DAB solution was used to develop signals, which were captured by Roche microscope slide scanner (Basel, Switzerland), and further analyzed by ImageJ.

Immunofluorescent staining (IF)

Mice brains were fixed in 4% PFA at 4℃ overnight, dehydrated in 30% sucrose, embedded in OCT medium, cut into 10-µm-thick brain slides, and washed in PBS. Tissues were permeabilized with 0.5% Triton X-100 at room temperature for 10 min. Samples were blocked with 3% BSA at room temperature for 1 h, then incubated with primary antibodies at 4℃ overnight, followed by secondary antibodies at room temperature for 1 h, and finally DAPI staining. Representative fluorescent images were captured using a ZEISS confocal fluorescence microscope and further analyzed using ImageJ.

For cultured cells, BV2 cells were seeded onto a 24-well plate containing coverslips and incubated with cornuside, LPS and Aβ1−42 successively. Afte 6 h later, cells were fixed in 4% PFA at room temperature for 10 min. After washing with PBS, cells were permeabilized with 0.5% Triton X-100 for 10 min and blocked with 3% BSA for 1 h at room temperature. Cells were incubated with primary antibodies at 4℃ overnight, then with secondary antibodies coupled to Alexa Fluor 488 or 594 at room temperature for 1 h. After DAPI staining, images were captured using a ZEISS confocal fluorescence microscope and further analyzed using ImageJ.

Annexin V-FITC/PI staining

Wash the cells twice with cold PBS, then resuspend them in 1× binding buffer at a concentration of 1 × 10⁶ cells/mL. Transfer 100 µL solution (1 × 10⁵ cells) to a 5 mL culture tube, and add 5 µL of FITC Annexin V and PI solution. Incubate in the dark at room temperature for 15 min. Finally, add 400 µL of 1× binding buffer to each tube and analyze the samples using flow cytometry (CytoFLEX LX, Beckman, Indianapolis, USA) at PE and FITC channels.

Detection of intracellular ROS and mitochondrial ROS

Intracellular ROS was detected using the DCFH-DA staining (Nanjing Jiancheng Bioengineering, Nanjing, China). Briefly, BV2 cells were incubated with DMEM containing 10 µM DCFH-DA at 37 °C for 30 min, and then washed twice with PBS. Subsequently, samples were observed and photographed under ZEISS confocal fluorescence microscope or resuspended in cold PBS for flow cytometry analysis at FITC channel.

Mitochondrial ROS were detected using the MitoSOX Red kit (Invitrogen, Carlsbad, USA). BV2 cells were incubated with 2.5 µM MitoSOX at 37 °C for 30 min, followed by two washes with PBS. Afterward, cells were observed and photographed using a ZEISS confocal fluorescence microscope (Jena, Germany) or resuspended in cold PBS for flow cytometry analysis at PE channel.

Determination of mitochondrial membrane potential

Mitochondrial membrane potential (MMP) was assessed using the fluorescent probe JC-1 (Invitrogen, Carlsbad, USA). When the membrane potential is high, JC-1 is present as aggregates and shows red fluorescence. When the membrane potential is low, JC-1 is present as a monomer and shows green fluorescence. Briefly, BV2 cells were incubated with 2.5 µg/mL JC-1 at 37 °C for 30 min and then washed twice with PBS. The samples were then observed and photographed using a ZEISS confocal fluorescence microscope (Jena, Germany) or resuspended in cold PBS for flow cytometry analysis.

Enzyme-linked immunosorbent assay (ELISA)

The concentrations of cytokines TNF-α, IL-1β, IL-18 and IL-6 in culture medium were determined by ELISA. All procedures were according to the manufacturer’s instructions (Elabscience Biotechnology, Wuhan, China). Finally, the absorbance was detected using a microplate reader (BioTek Synergy Multifunctional Microwell Plate Testing System, USA).

Lactate dehydrogenase (LDH) assay

Carefully collect supernatants from conditionally cultured HT22 cells, and detect the LDH level according to the instructions. In a new 96-well plate, 120 µL supernatant and 60 µL working solution were mixed and incubated for 25 min at room temperature in the dark. Then the absorbance was measured at 490 nm and calculated LDH level.

Seahorse assay

The oxygen consumption rate (OCR) and real-time extracellular acidification rate (ECAR) in BV2 cells were measured using a Seahorse XF24 Extracellular Flux Analyzer (Agilent Technologies, California, USA). Cellular glycolysis (ALS22022) and mitochondrial oxidative phosphorylation (OXPHOS) assay kits (ALS22012) were obtained from Alicelligent (Beijing, China). BV2 cells were seeded at 8000 cells/well in Seahorse XF plates and cultured in DMEM for 24 h at 37℃ in a 5% CO2 incubator. After drug treatment as mentioned above, cells were incubated in Seahorse assay medium with substrate supplementation for 40 min at 37℃ in a CO2-free incubator. To assess oxidative phosphorylation, three baseline measurements were recorded, followed by measurements after sequential incubation with oligomycin (1.5 µM), FCCP (1 µM), rotenone (1 µM) and antimycin A (1 µM). To assess glycolysis, three baseline measurements were recorded, followed by measurements after sequential incubation with rotenone (1 µM), antimycin A (1 µM) and 2-DG (50 mM). Data normalization was performed by cell numbers via an automated cell imaging and analysis system (Falcon S300, Alicelligent Technologies).

Western blot assay

Cultured cells and brains were lysed in lysis buffer containing protease inhibitors and a phosphatase inhibitor. Protein concentrations were measured using the Micro BCA Kit. Samples containing 30-µg-protein were separated by 10%, 12.5%, or 15% SDS-PAGE gels and transferred to PVDF membranes (IPVH00010, Millipore, Massachusetts, USA). After blocking with 5% BSA, the PVDF membranes were cropped according to the molecular weight of target proteins. This approach is more efficient to obtain the bands of multiple target proteins in one experiment. Subsequently, the cropped membranes were incubated with the respective primary antibodies overnight at 4 °C. After washing with TBST, membranes were incubated with HRP-conjugated secondary antibodies for 2 h at 37 °C. The ChemiDoc™ imaging system (Bio-Rad, Hercules, CA, USA) was used to capture membrane images via an enhanced chemiluminescence (ECL) detection reagent. ImageJ software was used to analyze the gray value of bands.

Statistical analysis

Data were expressed as mean ± standard error of mean (SEM) and analyzed with one-way ANOVA followed by Dunnett’s multiple comparison test in the GraphPad Prism 8.0.2 software (San Diego, USA). The difference between cornuside group and cornuside + 3-MA group was evaluated with Student’s t-test. The difference was considered significant when p < 0.05.

Results

Cornuside improved the capability of learning and memory in Aβ1−42 induced mice

Morris water maze test

The Morris water maze test is a classical method for evaluating the spatial memory of mice, consisting of a navigation trial conducted in the first four days and a spatial probe trial on the fifth day. Performance on the fourth day served as the final result of the navigation trial, with swimming routes recorded and analyzed (Fig. 1A). The swimming routes of mice in the control and sham groups demonstrated a sense of purpose, whereas those in the model group appeared aimless and confused. Administration of cornuside enhanced the purposefulness of Aβ1−42-induced mice in searching for the hidden platform. Similar to the swimming routes, the escape latency of the model group was significantly longer than that of the control and sham groups (Fig. 1B). Cornuside at the doses of 3, 10, and 30 mg/kg reduced escape latency (Fig. 1B). Additionally, there was no significant difference in average speed among the groups (Fig. 1C), indicating that learning and memory capabilities, rather than motor skills, affected behavioral performance in the Morris water maze test.

Fig. 1
figure 1

The effect of cornuside on the learning and memory of Aβ1−42 intracerebroventricular injected mice in the behavioral tests. Data were expressed as the mean ± SEM (n = 13–15). A. Exploring routes during the navigation trial. B, C. Escape latency and average speed in the navigation trial. D. Exploring routes in the spatial probe trial. E, F. Latency to the platform and crossing times in the spatial probe trial. G, H. Representative nests built by mice, scored at 2 and 12 h in the nest building test. I. Discrimination index in the novel object recognition test. J. Spontaneous alternation rate in the Y-maze. K. Ratio of interaction with a novel mouse in the novel object recognition test. #p < 0.05 and ###p < 0.001 vs. control group; &p < 0.05 and &&p < 0.01 vs. sham group; *p < 0.05, **p < 0.01 and ***p < 0.001 vs. model group

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In the spatial probe trial, the platform was moved out of the water, and mice swam for 60 s to search for the platform according to the previous memory. Swimming routes were recorded (Fig. 1D); mice in the control and sham groups searched purposefully in the first quadrant, while those in the model group lacked purpose in their searching. Mice in the cornuside-administered groups exhibited more purposeful searching routes than those in the model group. Further analysis revealed that mice in the model group had a longer latency to first crossing the original platform location (Fig. 1E), with significantly fewer cross times (Fig. 1F). Administration of cornuside improved spatial memory in mice, resulting in shorter latencies (Fig. 1E) and increased crossing times at the original platform location (Fig. 1F).

Nest building test

The nesting results of Aβ1−42 induced mice were assessed at 2 h and 12 h, respectively. The nesting performance was not significantly different at 2 h, with nesting scores of mice in the model group slightly lower than those of other groups. This implied that mice in the model group started to build nests later than other groups. After 12 h-work, there were significant differences in nest shapes among the groups, as shown in Fig. 1G. Mice in the model group were unable to construct a nest-like structure by utilizing tissues. Mice in the CO-3 group piled the tissues together without nest structure, while mice in control, sham, CO-10, CO-30 and the donepezil groups could establish nests for hiding to varying degrees. The nesting scores at 12 h were notably higher than those at 2 h, the score of model group was obviously lower than that of the control and sham group (Fig. 1G), and the administration of cornuside raised the nesting ability and score of Aβ1−42 induced mice (Fig. 1H).

Novel object recognition test

The novel object recognition test evaluates the memory by recording the time mice spend exploring the old and new objects, and calculating a discrimination index (DI). As shown in Fig. 1I, the DI of the model mice decreased obviously, which was reverted by treatment with cornuside at 3, 10, and 30 mg/kg (Fig. 1I).

Y maze test

The Y-maze is used to evaluate the short-term spatial memory by utilizing the curiosity of mice, and mice were allowed to explore three arms with the spontaneous alternation rate used to estimate the memory of mice. As shown in Fig. 1J, the spontaneous alternation rate of the model mice decreased, cornuside at 30 mg/kg could increase the spontaneous alternation rate of Aβ1−42 induced mice (Fig. 1J), which indicated that each time mice switched to explore a new arm, they could remember the direction they had explored before.

Three-box social interaction test

The three-box social interaction test is widely used to study the social interest and memory to unfamiliar mouse. In this test, the processes of mice interacting with familiar and unfamiliar mouse were tracked and further analyzed. As shown in Fig. 1K, the ratio of model mice interacting with unfamiliar mouse was lower than that of control or sham group, and cornuside at 10 and 30 mg/kg could promote Aβ1−42 induced mice to interact with unfamiliar mouse (Fig. 1K).

Cornuside protected synapses and neurons in the cortex and hippocampus of Aβ1−42 induced mice

PSD95 is a vital marker of synaptic plasticity, which is disrupted by Aβ and induces memory dysfunction. We measured the expression levels of PSD95 in the hippocampus and cortex. The protein level of PSD95 was significantly reduced in Aβ1−42 induced mice by WB and IF (Fig. 2A-D). Administration of cornuside increased the expression of PSD95 in hippocampus and cortex, and the results of IF were similar to those of WB (Fig. 2A, B). The ultrastructure of hippocampal synapses was further investigated using TEM (Fig. 2E). In the hippocampus of Aβ1−42-induced mice, synaptic gaps appeared blurrier, synaptic vesicles were reduced or absent, and the number of synapses decreased significantly (Fig. 2E), suggesting that Aβ1−42 induced synaptic damage and loss in hippocampus of mice. Compared to the model group, cornuside at 30 mg/kg exhibited a protective effect on synapses, characterized by clearer synaptic gaps, intact structures, and an increased number of synapses (Fig. 2E).

Fig. 2
figure 2

The effect of cornuside on synapses of hippocampus and cortex in Aβ1−42 intracerebroventricular injected mice. Data were expressed as the mean ± SEM (n = 3–6). A. Protein levels of PSD95 in the hippocampus and cortex. B-D. Fold changes of PSD95 and representative immunofluorescence images (scale bar: 40 μm) in the hippocampus and cortex. E. Number of synapses in the hippocampus, with representative transmission electron microscopy (TEM) photomicrographs (scale bar: 2 μm). #p < 0.05, ###p < 0.001 vs. control group; *p < 0.05 and ***p < 0.001 vs. model group

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Neuronal injury and loss are further induced by Aβ and constitute the direct basis of cognitive impairment. Tunel staining is used to observe cellular apoptosis, Aβ1−42 induced a significant increase in apoptotic cells, as shown in Fig. 3A-D, and cornuside dose-dependently decreased apoptotic cells in the hippocampus and cortex. Moreover, the expression of NeuN was further explored by WB and IHC which were shown in Fig. 3F-H, compared to the control group, NeuN in the model group was significantly reduced in hippocampus and also showed a decreasing trend in cortex (Fig. 3H). Cornuside could reversed the decline of NeuN in the hippocampus and cortex (Fig. 3H), and the result of IHC were similar (Fig. 3F-G). In addition, the ultrastructure of neurons was also studied by the means of TEM, as shown in Fig. 3E. Neurons in the control group showed a regular nucleus with small nucleolus and clear margin, around by mitochondrion and endoplasmic reticulum. Neurons in the model group had enlarged nucleolar condensation in unregular nucleus with blurry nuclear membrane, expanded endoplasmic reticulum, and swollen mitochondria with reduced or even disappeared spines, implying that neuronal damage was induced by Aβ in the hippocampus. Cornuside at 30 mg/kg could partly rescue Aβ1−42-induced neuronal damage, with the morphology of nuclear and organelles return to normal to some extends.

Fig. 3
figure 3

The effect of cornuside on neuron in the hippocampus and cortex of Aβ1−42 intracerebroventricular injected mice. Data were expressed as the mean ± SEM (n = 3–6). A, C. Representative images of TUNEL staining for the hippocampus and cortex, with a scale bar of 100 μm. B, D. The percentage of apoptotic cells in these regions. E, F. Representative images of IHC staining (scale bar: 100 μm) in CA3 and cortex. G Protein levels of NeuN in the hippocampus and cortex. H. TEM photomicrographs showing neuronal structures from different treatment groups (scale bar: 2 μm). The orange arrow indicates the nucleolus; the yellow arrow indicates the nuclear membrane; the blue arrow indicates mitochondrial ultrastructure; and the green arrow indicates the endoplasmic reticulum. #p < 0.05, ###p < 0.001 vs. control group; *p < 0.05, **p < 0.01 and ***p < 0.001 vs. model group

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Cornuside inhibited NLRP3 inflammasome activation in Aβ1−42 induced mice

NLRP3 inflammasome activation is a key aspect leading to inflammatory damage and neurotoxicity in the pathogenesis of AD. In the activation step, NLRP3 oligomerizes, further recruits ASC and binds with pro-caspase1. NLRP3 and ASC were firstly immunofluorescence co-stained in hippocampus and cortex, which were induced to be a higher fluorescence intensity by Aβ1−42 (Fig. 4A-F). Cornuside significantly lower fluorescence intensity of NLRP3 and ASC in hippocampus and cortex. The protein levels of NLRP3 and ASC were further affirmed by WB, and similar results were found (Fig. 4G-H).

Fig. 4
figure 4

The effect of cornuside on NLRP3 inflammasome in Aβ1−42 intracerebroventricular injected mice. Data are expressed as the mean ± SEM (n = 4–6). A, B. Representative images of NLRP3 and ASC in the hippocampus and cortex (scale bar: 40 μm). C-F. Fold changes of NLRP3 and ASC are displayed. G, H. Protein levels of NLRP3, GSDMD, ASC, Caspase-1, and IL-1β. #p < 0.05, ##p < 0.01, ###p < 0.001 vs. control group; *p < 0.05, **p < 0.01 and ***p < 0.001 vs. model group

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Pro-caspase1 is converted into Caspase-1 (active form) following inflammasome activation. Caspase1 was found to be a higher level in hippocampus and cortex (Fig. 4G and H), while cornuside remarkably inhibited the expression of caspase1 (Fig. 4G and H). Caspase-1 produces the mature of IL-1β from its precursor, and also cleaves Gasdermin D (GSDMD) to promote cytokine release. GSDMD and IL-1β were induced to be increased by Aβ (Fig. 4G and H), which were decreased by cornuside in hippocampus and cortex (Fig. 4G and H).

Cornuside improved mitophagy in Aβ1−42 induced mice

Autophagy is a main pathway to negatively regulate NLRP3 inflammation activation. LC3B and SQSTM1/P62 are vital markers for autophagy, which was first studied to determine the level of autophagy by IF and WB. The total level of LC3B were revealed by IF, no significant difference among groups was found in hippocampus and cortex as shown in Fig. 5A and B, though cornuside at 30 mg/kg had a tendency to increase the fluorescence intensity of LC3B. Then LC3B I and II were respectively investigated in WB, and the ratio of LC3B II to LC3B I was decreased in hippocampus and cortex of model group (Fig. 5C and I), indicating the inhibited autophagy in Aβ-induced mice. Cornuside could promote the transformation of LC3B I to LC3B II in autophagy flux, with increased ratio of LC3B II to LC3B I (Fig. 5C and I). The level of SQSTM1/P62 are the substrate in autophagy flux, which was found to be reduced in cornuside-administrated mice (Fig. 5C and H).

Fig. 5
figure 5

The effect of cornuside on mitophagy of hippocampus and cortex in Aβ1−42 intracerebroventricular injected mice. Data are expressed as the mean ± SEM (n = 4–6). A, B. Representative images of LC3B in the hippocampus and cortex (scale bar: 40 μm). C-I. Protein levels of PINK1, SQSTM1/p62, Parkin, BNIP3, FUNDC1 and LC3B. #p < 0.05, ##p < 0.01 vs. control group, *p < 0.05, **p < 0.01 and ***p < 0.001 vs. model group

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PINK1/Parkin, BNIP3 and FUNDC1 pathways are involved in the regulation of mitophagy, which is both a vital part of autophagy and an important factor regulating the NLRP3 inflammasome activation. In the present study, PINK1/Parkin pathway was significantly inhibited in Aβ1−42 induced mice (Fig. 5D and E). However, there was no significant change in BNIP3 and FUNDC1 (Fig. 5F and G), indicating their weak effect in the Aβ1−42 induced mitophagy.

Cornuside protected neurons from inflammatory injury via inhibiting NLRP3 inflammasome activation in LPS + Aβ1−42 induced BV2 cells

Furthermore, microglia exposed to LPS and Aβ1−42 was used to investigate the neuroprotective effects of cornuside. Pro-inflammatory cytokines in the cell supernatant were detected in Fig. 6A-D, the levels of IL-1β, IL-18, IL-6 and TNF-α were significantly elevated in LPS + Aβ1−42 induced BV2 cells (Fig. 6A-D), which were inhibited by cornuside administration (Fig. 6A-D).

Fig. 6
figure 6

The effect of cornuside on LPS + Aβ1−42 induced NLRP3 inflammasome activation of BV2 cells and conditioned stimulation of HT22 cells. Data are expressed as the mean ± SEM (n = 3–6). A-D. Cytokine levels determined in the supernatant of cultured BV2 cells. E, G. Protein levels of NLRP3, ASC, Pro-caspase-1, Caspase-1, Pro-IL-1β, IL-1β and GSDMD-NT. F. The representative images of NLRP3 in BV2 cells (scale bar: 20 μm). H, I. Neuron survival rate and LDH levels in supernatant. J, K. Flow cytometric representation and statistical analysis of apoptosis in conditioned medium. #p < 0.05, ##p < 0.01, ###p < 0.001 vs. control group; *p < 0.05, **p < 0.01 and ***p < 0.001 vs. model group

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In addition, the components of NLRP3 inflammasome, including NLRP3, ASC and Pro-caspase-1, were inhibited by cornuside (Fig. 6E and G), meanwhile the IF results of NLRP3 supported the WB results mentioned aboved. Then, the active form Caspase-1 was determined to be lower level in cornuside-given group (Fig. 6E and G). IL-1β and its precursor pro-IL-1β in cell lysate were suppressed by cornuside (Fig. 6E and G). GSDMD is cleaved by Caspase-1, with the N-terminal (GSDMD-NT) produced and forming pores in the membrance. The production of GSDMD-NT was remarkably decreased by cornuside at 10 and 30 µM (Fig. 6E and G). Therefore, cornuside could inhibit NLRP3 inflammasome activation and subsequent release of inflammatory cytokines.

Activated NLRP3 inflammasome releases inflammatory cytokines, such as IL-1β and IL-18, from GSDMD-NT pores and alters microenvironment, which might lead to neuronal damage. The culture medium from LPS + Aβ1−42 induced BV2 cells exhibited significant toxicity towards HT22 cells as shown in Fig. 6H-K. The medium from LPS + Aβ1−42 induced BV2 cells co-treated with cornuside recovered the neuronal survival rate (Fig. 6H), reduced the LDH release (Fig. 6I) and cell apoptosis (Fig. 6J and K). The neuronal protection of cornuside was mediated by improving micro-environment via inhibiting NLRP3 inflammasome activation.

Cornuside protected mitochondrial function and promoted mitophagy in LPS + Aβ1−42 induced BV2 cells

Mitochondrial dysfunction could release several damages associated molecular patterns (DAMPs), such as reactive oxygen species (ROS), mitochondrial DNA (mtDNA) and ATP, which are also vital triggers of NLRP3 inflammasome. Mitochondrial membrane potential (MMP) is detected by JC-1 staining, as shown in Fig. 7A and B, cornuside significantly recovered MMP characterized by increased ratio of JC-1 aggregates (red) to monomers (green) (Fig. 7A). Then cellular ROS and mtROS were explored via flow cytometer as shown in Fig. 7B-C, cornuside notably inhibited the level of ROS in cytoplasm and mitochondrion (Fig. 7C and D).

Fig. 7
figure 7

The effect of cornuside on mitochondrial function in LPS + Aβ1−42 induced d BV2 cells. Data were expressed as the mean ± SEM (n = 4–6). A. JC-1 flow cytometry representation and statistics. B-D. Levels of ROS and mtROS measured by flow cytometry, and the representative pictures with scale bar of 40 μm. E, F. The OCR and ECAR were measured using a Seahorse XF24 Extracellular Flux Analyzer instrument. #p < 0.05, ##p < 0.01, ###p < 0.001 vs. control group; *p < 0.05, **p < 0.01 and ***p < 0.001 vs. model group

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Oxidative phosphorylation (OXPHOS) and glycolysis are of importance to maintain mitochondrial homeostasis in microglia, which were further investigated by analyzing oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in live cells, respectively. In comparison with control group, LPS + Aβ1−42 stimulation resulted in a significant decrease in ECAR, with basal glycolysis, basal proton efflux rate (PER), and compensatory glycolysis reduced, as shown in Fig. 7E. Furthermore, OCR levels also diminished by LPS + Aβ1−42 stimulation, characterized by a marked reduction in basal respiration, maximal respiration, ATP production and spare respiratory capacity as shown in Fig. 7F. Cornuside treatment not only significantly enhanced the ECAR of BV2 cells, including basal glycolysis (Fig. 7E), basal PER (Fig. 7E), and compensatory glycolysis (Fig. 7E); it also significantly promoted the OCR of BV2 cells, including basal respiration (Fig. 7F), maximal respiration (Fig. 7F), ATP production (Fig. 7F), and spare respiration capacity (Fig. 7F). Therefore, mitochondrion function is elevated by cornuside.

Mitophagy is a vital process maintaining the mitochondrion quality and function, which were investigated further in-vitro. Cornuside remarkably decreased SQSTM1/p62 and elevated the transformation of LC3B I to LC3B II (Fig. 8A and B), implying that cornuside facilitated the mitophagy in LPS + Aβ1−42 induced BV2 cells. Co-staining of MitoTracker and LC3B was performed to analyze mitophagy level, expressed as the Pearson correlation coefficient. In LPS + Aβ1−42 induced BV2 cells, the co-localization of mitotraker and LC3B was reduced, and cornuside could revert their co-localization with increased pearson correlation coefficient (Fig. 8C and D). Elevated mitophagy is indicated in the mitochondrial protection of cornuside.

Fig. 8
figure 8

The effect of cornuside on mitophagy in LPS + Aβ1−42 induced d BV2 cells. Data are expressed as the mean ± SEM (n = 4–6). A, B. Protein levels of PINK1, Parkin, FUNDC1, BNIP3, SQSTM1/p62 and LC3B. C, D IF staining of MitoTracker and LC3B colocalization, with Pearson colocalization coefficient. E, F. IF staining of MitoTracker and Parkin colocalization, with Pearson colocalization coefficient. (Scale bar: 20 μm). #p < 0.05, ##p < 0.01, ###p < 0.001 vs. control group; *p < 0.05, **p < 0.01 and ***p < 0.001 vs. model group

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Consistent with the in-vivo experiments, cornuside elevated PINK1 and Parkin levels in LPS + Aβ1−42-induced BV2 cells (Fig. 8A and B). The co-localization of MitoTracker and Parkin was also increased by cornuside, as indicated by an elevated Pearson correlation coefficient (Fig. 8E and F). However, it is worth noting that FUNDC1 and BNIP3 were markedly decreased in LPS + Aβ1−42 induced BV2 cells (Fig. 8A and B), which were returned to normal after cornuside treatment (Fig. 8A and B). PINK1/ Parkin, FUNDC1 and BNIP3 pathways jointly participated in cornuside’s contribution to mitophagy.

Blocking mitophagy hampered the effect of cornuside in mitochondrial protection, NLRP3 inflammasome inhibition

To better understand the interplay between the inhibitory NLRP3 inflammasome and stimulative mitophagy in the neuroprotection of cornuside, autophagy inhibitor 3-MA was applied in further research of LPS + Aβ1−42 induced BV2 cells. Firstly, we detected mitophagy signaling related proteins, as shown in Fig. 9A, compared with CO-10 group, 3-MA alleviated the promotion of mitophagy, characterized by increased SQSTM1/p62 and decreased ratio of LC3B II to LC3B I (Fig. 9A). The effect of cornuside on PINK1/Parkin, FUNDC1 and BNIP3 pathways were also hampered by 3-MA (Fig. 9A). It demonstrated that cornuside’s promotion on mitophagy was disturbed by 3-MA.

Fig. 9
figure 9

The effect of 3-MA on mitophagy, anti-inflammatory and neuroprotective effects of cornuside. Data are expressed as the mean ± SEM (n = 4–6). A Protein levels of PINK1, SQSTM1/p62, Parkin, BNIP3, FUNDC1 and LC3B. B. The red/green ratio of JC-1 was measured by flow cytometry. C. mtROS levels were measured by flow cytometry. D-G. cytokines levels in the supernatant. H, I. Protein levels of NLRP3, Pro-caspase-1, GSDMD-NT, Pro-IL-1β, ASC, Caspase-1 and IL-1β. J. LDH leakage in supernatant. K, L. Flow cytometric representation and statistical analysis of apoptosis in conditioned medium. *p < 0.05, **p < 0.01 and ***p < 0.001

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Furthermore, obvious reduced ratio of JC-1 aggregate (red) to JC-1 monomer (green) (Fig. 9B) and increased mitochondrion ROS (Fig. 9B) were found when cornuside co-tread with 3-MA. Therefore, 3-MA almost eliminated the mitochondrial protection of cornuside.

Additionally, we analyzed the activation of NLRP3 inflammasome in the presence of 3-MA. Compared with CO-10 group, 3-MA weakened the inhibitory effect of cornuside on inflammatory cytokines secretion, including IL-1β, IL-18, IL-6 and TNF-α (Fig. 9D-G). Similar to cytokines secretion, the pro-IL-1β and IL-1β in cell lysate were also upregulated by 3-MA (Fig. 9H-I). The inhibitory effect of cornuside on NLRP3 inflammasome components, such as NLRP3, ASC, and Pro-caspase-1, was reversed in the presence of 3-MA (Fig. 9H and I). Co-treatment with 3-MA also mitigated the downregulation of NLRP3 inflammasome activation by cornuside, resulting in upregulated Caspase-1 and GSDMD-NT (Fig. 9H and I), which further hindered cytokines release. Hence, 3-MA abolished the inhibition of cornuside on NLRP3 inflammasome activation.

To determine whether cornuside’s protective effects on HT22 neurons under conditioned culture were disrupted by 3-MA, we utilized LDH leakage assays and Annexin V-FITC/PI staining. Both assays indicated that the conditioned medium from co-treatment with 3-MA and cornuside significantly diminished the protective effects of cornuside on HT22 cells, as evidenced by increased LDH in the supernatant and a higher ratio of apoptosis rate (Fig. 9J-L). So, 3-MA also canceled the neuronal protection of cornuside on HT22 neurons (Fig. 10).

Fig. 10
figure 10

Proposed mechanism of cornuside alleviating cognitive disorder in Aβ1−42 intracerebroventricular injected mice. Cornuside promoted mitophagy via PINK1/Parkin, BNIP3 and FUNDC1 pathways, leading to the elimination of damaged mitochondria. Following the improvement of mitochondrial quality and function, ROS production was significantly inhibited, which decreased the activation of NLRP3 inflammasome. Both mitochondrial improvement and NLRP3 inhibition could protect the neuron and synapse from damage, indicating the importance of mitophagy in cornuside alleviating neuro-inflammation and neuro-degeneration in Aβ1−42 intracerebroventricular injected mice

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Discussion

Aβ (monomers, oligomers and fibrils) is AD specific pathological hallmarks, initiates series of cell injury processes, which converge on neuroinflammation and eventually resulting in synaptic destruction and neuron loss [29]. Neuroinflammation is induced by innate immune cells, such as microglia and astrocytes, as a self-defense mechanism against cellular injury. However, chronic or excessive inflammation can exacerbate brain damage by increasing the levels of inflammatory cytokines in the micro-environment. The NLRP3 inflammasome has emerged as a critical multi-protein complex widely implicated in chronic inflammation. The NLRP3, Caspase-1 and IL-1β are highly expressed in the brains of AD patients and APP/PS1 mice. Pharmacological inhibition of NLRP3 has been shown to protect APP/PS1 mice from memory loss, suggesting that the NLRP3 inflammasome may be a promising target for AD treatment [30]. Mitophagy is responsible for the removal of the damaged mitochondria, and then negatively regulates NLRP3 inflammasome activation. Considering the anti-AD effect and anti-inflammatory activity of cornuside, we aimed to illuminate its mechanism in relation to NLRP3 inflammasome activation and mitophagy.

Increasing evidence demonstrated that the over-activation of NLRP3 inflammasome in microglia drives Aβ-induced synaptic damage and neuronal loss in the progress of AD [30, 31]. NLRP3 inflammasome can be activated by a diverse range of stimuli, including lipopolysaccharides (LPS), ATP, ROS, various crystals, Aβ peptides and neurofibrillary tangles [32,33,34,35,36,37]. Canonical activation of the NLRP3 inflammasome occurs in two steps: priming and activation. During the priming step, NLRP3 expression is enhanced by Toll-like receptors (TLRs), such as TLR2 and TLR4, and several cytokine receptors, including the IL-1β receptor and TNF-α receptor [38,39,40,41]. In the activation step, on sensing agonists, NLRP3 oligomerizes via homotypic NACHT domain interactions, then recruits ASC via PYD domain and induces ASC specks that enable signals amplification, which further recruits Pro-caspase-1 via CARD domain to form the NLRP3-ASC-Pro-caspase-1 protein complex. Subsequently, active Caspase-1 is generated, leading to the maturation of IL-1β and IL-18 from their precursors. Caspase-1 also cleaves GSDMD to produce the N-terminal fragment (GSDMD-NT), which oligomerizes into membrane pores, facilitating the release of IL-1β and IL-18, and inducing pyroptosis [42, 43]. In Aβ1−42 intracerebroventricular injected mice, cornuside treatment could significantly improve mice performance in series of behavioral tests, including Morris water maze test, nest building test, novel object recognition test, Y maze and three-box social interaction test. Synapses and neurons are the structural basis for learning and memory, both of which are impaired by exogenous Aβ1−42. Cornuside can restore down-regulated levels of PSD95, NeuN, and synapse numbers, reduce the percentage of apoptotic cells, and enhance the ultrastructure of neuronal organelles. In addition, exogenous Aβ1−42 could stimulate the NLRP3 inflammasome activation, while cornuside could inhibit activated NLRP3 inflammasome. We next adopted a cellular model to simulate canonical NLRP3 inflammasome activation, in which microglia were stimulated with LPS and Aβ1−42. In LPS + Aβ1−42 stimulated BV2 cells, cornuside suppressed the expression of NLRP3 inflammasome components (NLRP3, ASC and Pro-caspase-1), reduced the activated Caspase-1, as well as inhibited the cleavage and release of IL-1β, IL-18 and GSDMD. Thus, inhibiting NLRP3 inflammasome activation is a crucial molecular mechanism underlying the neuroprotective effects of cornuside, warranting further investigation.

The brain requires a continuous supply of ATP, primarily produced through oxidative phosphorylation in mitochondria, supplemented by aerobic glycolysis in the cytoplasm [44]. Mitochondrial dysfunction has been widely confirmed in both clinical and experimental studies of AD [45,46,47]. Soluble Aβ oligomers adversely impact on mitochondrial oxidative phosphorylation through disrupting functions of respiratory chain [48–49], and result in abundant release of ROS and mtDNA, which function as assembly activators to promote and amplify NLRP3 inflammasome activation [50]. Mitophagy is a defense mechanism in eukaryotic cells that selectively removes excess or damaged mitochondria and maintains cellular homeostasis [51]. Several pathways mediate mitophagy, including PINK1-Parkin signaling and other Parkin-independent pathways such as BINP3 and FUNDC1 [52,53,54,55]. As dysfunctional mitochondria accumulate, PINK1 recruits and activates Parkin on the outer mitochondrial membrane (OMM), mediating the poly-ubiquitination of OMM proteins along with autophagic adaptor molecules such as OPTN and the LC3 family [56, 57]. Parkin, an E3 ubiquitin ligase, mediates the interaction between ubiquitinated mitochondria and LC3 protein through the adaptor protein SQSTM/p62 [58]. PINK1 and Parkin deficiency results in the accumulation of dysfunctional mitochondria in AD patients and rodent models [30, 32]. Restoring mitophagy through the PINK1-Parkin pathway improves memory deficits in overexpressed cells and 5×FAD mice [29, 59]. In fact, FUNDC1 and BINP3 are mainly associated with hypoxia-induced mitophagy. The tolerance of mice to hypoxia may be higher than that of humans [60,61,62], which alleviate FUNDC1 or BINP3-mediated mitophagy in mouse brains. In addition, mitophagy has been reported to negatively regulate NLRP3 inflammasome activation through promoting degradation of damaged mitochondria [19, 63]. Here, in LPS + Aβ1−42 stimulated BV2 cells, oxidative phosphorylation and glycolysis were obvious impaired, with MMP depolarization and ROS over-production, and cornuside provided mitochondrial protection and maintained cellular homeostasis by promoting glycolysis and oxidative phosphorylation. In Aβ1−42 induced mice and LPS + Aβ1−42 stimulated BV2 cells, cornuside promoted mitophagy-related proteins, including LC3B II/LC3B I, SQSTM1/p62, PINK1, and Parkin. However, the changes in FUNDC1 and BNIP3 were inconsistent. Furthermore, specific inhibition of mitophagy by 3-MA blocked the process of mitophagy, blunted the protective effect of cornuside on mitochondrion, and hampered the inhibitory effect of cornuside on NLRP3 inflammasome activation and IL-1β secretion. It suggested that cornuside inhibited NLRP3 inflammasome in microglia in a mitophagy-denpendent manner.

The crosstalk between microglia and neuron amplifies nerve injury of excessive NLRP3 activation, resulting in the neuronal and synaptic disorder. In the present study, we collected the culture media from LPS + Aβ1−42 stimulated BV2 cells, which was further used for the conditional culture on HT22 cells. Interestingly, culture media from LPS + Aβ1−42 stimulated BV2 cells exhibited significant toxicity towards HT22 cells, characterized by reduced viability of neurons, increased LDH leakage and apoptosis. When cornuside was present in the LPS + Aβ1−42 stimulated BV2 cells, the cytotoxicity of conditional media barely exhibited, with neuronal survival, LDH leakage and apoptosis returning to the baseline level. Thus, it is the micro-environment mediated by microglial excessive NLRP3 activation that impact neurons fates. Furthermore, in the presence of 3-MA and cornuside, the neuronal damage of culture media from LPS + Aβ1−42 stimulated BV2 cells was still obvious, suggesting that blocking mitophagy was accountable for the reduced neuro-protection of cornuside via NLRP3 inflammasome activation.

Cornuside, a natural product in C. Officinalis, has been demonstrated to be effective on the AD-related mice. Based on the previous anti-inflammatory effect, we explored its mechanism in-depth from the aspects of NLRP3 inflammasome and mitophagy in the present study. Cornuside could maintain oxidative respiratory chain and mitochondria function via promoting mitophagy, further reduced NLRP3 inflammasome activation, protect neurons and synaptic from damage, and ultimately ameliorate cognitive function. PINK1/Parkin signaling showed obvious participation in facilitating mitophagy of cornuside in-vivo and in-vitro, while the roles of BNIP3 and FUNDC1 signalings remained to be elucidated.

Some limitations in this study should be acknowledged. Regarding experimental design, cornuside’s inhibition on NLRP3 activation and promotion of mitophagy are primarily based on cell experiments. Pharmacological inhibition mitophagy in mice should be included to strengthen the causal link between cornuside, mitophagy, NLRP3 inflammasome and cognitive improvement. Regarding the technical aspect, the experimental techniques used in this study were traditional; advanced technical methods should be adopted to improve the efficiency and accuracy of the study, as well as generate perfect images.

Conclusion

Cornuside could alleviate Aβ-induced cognitive impairment in mice, as well as promote the recovery of synapse and neuron from Aβ-induced damage. PINK1/Parkin dependent mitophagy was found to be a key mechanism of cornuside in inhibit NLRP3 inflammasome activation and subsequent neuronal damage.

Data availability

No datasets were generated or analysed during the current study.

Change history

References

  1. Lane CA, Hardy J, Schott JM. Alzheimer’s disease. Eur J Neurol. 2018;25:59–70.

    Article  CAS  PubMed  Google Scholar 

  2. Hardy J, Selkoe DJ. The amyloid hypothesis of Alzheimer’s disease: progress and problems on the road to therapeutics. Science. 2002;297:353–6.

    Article  CAS  PubMed  Google Scholar 

  3. Reddy PH, Manczak M, Yin X, Grady MC, Mitchell A, Tonk S, Kuruva CS, Bhatti JS, Kandimalla R, Vijayan M, et al. Protective effects of Indian Spice Curcumin against Amyloid-β in Alzheimer’s Disease. J Alzheimers Dis. 2018;61:843–66.

    Article  PubMed  PubMed Central  Google Scholar 

  4. Oddo S, Caccamo A, Shepherd JD, Murphy MP, Golde TE, Kayed R, Metherate R, Mattson MP, Akbari Y, LaFerla FM. Triple-transgenic model of Alzheimer’s disease with plaques and tangles: intracellular abeta and synaptic dysfunction. Neuron. 2003;39:409–21.

    Article  CAS  PubMed  Google Scholar 

  5. Goel P, Chakrabarti S, Goel K, Bhutani K, Chopra T, Bali S. Neuronal cell death mechanisms in Alzheimer’s disease. An insight. Front Mol Neurosci. 2022;15:937133.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Su B, Wang X, Nunomura A, Moreira PI, Lee HG, Perry G, Smith MA, Zhu X. Oxidative stress signaling in Alzheimer’s disease. Curr Alzheimer Res. 2008;5:525–32.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Fang EF, Hou Y, Palikaras K, Adriaanse BA, Kerr JS, Yang B, Lautrup S, Hasan-Olive MM, Caponio D, Dan X, et al. Mitophagy inhibits amyloid-β and tau pathology and reverses cognitive deficits in models of Alzheimer’s disease. Nat Neurosci. 2019;22:401–12.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Tan MS, Yu JT, Jiang T, Zhu XC, Tan L. The NLRP3 inflammasome in Alzheimer’s disease. Mol Neurobiol. 2013;48:875–82.

    Article  CAS  PubMed  Google Scholar 

  9. Prinz M, Priller J, Sisodia SS, Ransohoff RM. Heterogeneity of CNS myeloid cells and their roles in neurodegeneration. Nat Neurosci. 2011;14:1227–35.

    Article  CAS  PubMed  Google Scholar 

  10. Lucin KM, Wyss-Coray T. Immune activation in brain aging and neurodegeneration: too much or too little? Neuron. 2009;64:110–22.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Barczuk J, Siwecka N, Lusa W, Rozpedek-Kaminska W, Kucharska E, Majsterek I. Targeting NLRP3-Mediated Neuroinflammation in Alzheimer’s Disease Treatment. Int J Mol Sci. 2022; 23.

  12. Tang T, Lang X, Xu C, Wang X, Gong T, Yang Y, Cui J, Bai L, Wang J, Jiang W, Zhou R. CLICs-dependent chloride efflux is an essential and proximal upstream event for NLRP3 inflammasome activation. Nat Commun. 2017;8:202.

    Article  PubMed  PubMed Central  Google Scholar 

  13. Zhang Y, Zhao Y, Zhang J, Yang G. Mechanisms of NLRP3 inflammasome activation. Its role in the treatment of Alzheimer’s Disease. Neurochem Res. 2020;45:2560–72.

    Article  CAS  PubMed  Google Scholar 

  14. Freeman LC, Ting JP. The pathogenic role of the inflammasome in neurodegenerative diseases. J Neurochem. 2016;136(Suppl 1):29–38.

    Article  CAS  PubMed  Google Scholar 

  15. Feng YS, Tan ZX, Wu LY, Dong F, Zhang F. The involvement of NLRP3 inflammasome in the treatment of Alzheimer’s disease. Ageing Res Rev. 2020;64:101192.

    Article  CAS  PubMed  Google Scholar 

  16. Blum-Degen D, Müller T, Kuhn W, Gerlach M, Przuntek H, Riederer P. Interleukin-1 beta and interleukin-6 are elevated in the cerebrospinal fluid of Alzheimer’s and de novo Parkinson’s disease patients. Neurosci Lett. 1995;202:17–20.

    Article  CAS  PubMed  Google Scholar 

  17. Li L, Ismael S, Nasoohi S, Sakata K, Liao FF, McDonald MP, Ishrat T. Thioredoxin-interacting protein (TXNIP) Associated NLRP3 Inflammasome activation in human Alzheimer’s Disease Brain. J Alzheimers Dis. 2019;68:255–65.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Ojala J, Alafuzoff I, Herukka SK, van Groen T, Tanila H, Pirttilä T. Expression of interleukin-18 is increased in the brains of Alzheimer’s disease patients. Neurobiol Aging. 2009;30:198–209.

    Article  CAS  PubMed  Google Scholar 

  19. Zhong Z, Umemura A, Sanchez-Lopez E, Liang S, Shalapour S, Wong J, He F, Boassa D, Perkins G, Ali SR, et al. NF-κB restricts Inflammasome Activation via Elimination of Damaged Mitochondria. Cell. 2016;164:896–910.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Cao S, Shrestha S, Li J, Yu X, Chen J, Yan F, Ying G, Gu C, Wang L, Chen G. Melatonin-mediated mitophagy protects against early brain injury after subarachnoid hemorrhage through inhibition of NLRP3 inflammasome activation. Sci Rep. 2017;7:2417.

    Article  PubMed  PubMed Central  Google Scholar 

  21. Youle RJ, Narendra DP. Mechanisms of mitophagy. Nat Rev Mol Cell Biol. 2011;12:9–14.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Yu L, Che RM, Zhang WK, Xu JK, Lian WW, He J, Tu SX, Bai X, He XL. Cornuside, by regulating the AGEs-RAGE-IκBα-ERK1/2 signaling pathway, ameliorates cognitive impairment associated with brain aging. Phytother Res. 2023;37:2419–36.

    Article  CAS  PubMed  Google Scholar 

  23. Lian WW, Wang ZX, Zhou FL, Yuan XT, Xia CY, Wang WP, Yan Y, Cheng YC, Yang H, Xu JK, et al. Cornuside ameliorates cognitive impairments via RAGE/TXNIP/NF-κB signaling in Aβ(1–42) induced Alzheimer’s disease mice. J Neuroimmune Pharmacol. 2024;19:24.

    Article  PubMed  Google Scholar 

  24. Zhang WK, Xu JK, He XL, He J, Lian WW, Wang ZX. Application of cornuside in the preparation of drugs for the treatment of Alzheimer’s disease. CN110433168B.

  25. Xu JK, Zhang WK, He J, Lian WW, Peng ZC, Zhang J, Wang ZX, Pan XG. A kind of preparation method of cornuside raw material drug. CN113480585B.

  26. Liu T, Zhang L, Joo D, Sun SC. NF-κB signaling in inflammation. Signal Transduct Target Ther. 2017;2:17023.

    Article  PubMed  PubMed Central  Google Scholar 

  27. Sbai O, Djelloul M, Auletta A, Ieraci A, Vascotto C, Perrone L. Correction to. RAGE-TXNIP axis drives inflammation in Alzheimer’s by targeting Aβ to mitochondria in microglia. Cell Death Dis. 2022;13:368.

    Article  PubMed  PubMed Central  Google Scholar 

  28. Wang ZX, Lian WW, He J, He XL, Wang YM, Pan CH, Li M, Zhang WK, Liu LQ, Xu JK. Cornuside ameliorates cognitive impairments in scopolamine induced AD mice: involvement of neurotransmitter and oxidative stress. J Ethnopharmacol. 2022;293:115252.

    Article  CAS  PubMed  Google Scholar 

  29. Wang N, Yang J, Chen R, Liu Y, Liu S, Pan Y, Lei Q, Wang Y, He L, Song Y, Li Z. Ginsenoside Rg1 ameliorates Alzheimer’s disease pathology via restoring mitophagy. J Ginseng Res. 2023;47:448–57.

    Article  PubMed  Google Scholar 

  30. Heneka MT, Kummer MP, Stutz A, Delekate A, Schwartz S, Vieira-Saecker A, Griep A, Axt D, Remus A, Tzeng TC, et al. NLRP3 is activated in Alzheimer’s disease and contributes to pathology in APP/PS1 mice. Nature. 2013;493:674–8.

    Article  CAS  PubMed  Google Scholar 

  31. Ising C, Venegas C, Zhang S, Scheiblich H, Schmidt SV, Vieira-Saecker A, Schwartz S, Albasset S, McManus RM, Tejera D, et al. NLRP3 inflammasome activation drives tau pathology. Nature. 2019;575:669–73.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Lee HJ, Park JH, Hoe HS. Idebenone regulates Aβ and LPS-Induced neurogliosis and cognitive function through inhibition of NLRP3 Inflammasome/IL-1β Axis Activation. Front Immunol. 2022;13:749336.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Bai H, Yang B, Yu W, Xiao Y, Yu D, Zhang Q. Cathepsin B links oxidative stress to the activation of NLRP3 inflammasome. Exp Cell Res. 2018;362:180–7.

    Article  CAS  PubMed  Google Scholar 

  34. Aminzadeh M, Roghani M, Sarfallah A, Riazi GH. TRPM2 dependence of ROS-induced NLRP3 activation in Alzheimer’s disease. Int Immunopharmacol. 2018;54:78–85.

    Article  CAS  PubMed  Google Scholar 

  35. Martinon F, Pétrilli V, Mayor A, Tardivel A, Tschopp J. Gout-associated uric acid crystals activate the NALP3 inflammasome. Nature. 2006;440:237–41.

    Article  CAS  PubMed  Google Scholar 

  36. Duewell P, Kono H, Rayner KJ, Sirois CM, Vladimer G, Bauernfeind FG, Abela GS, Franchi L, Nuñez G, Schnurr M, et al. NLRP3 inflammasomes are required for atherogenesis and activated by cholesterol crystals. Nature. 2010;464:1357–61.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Stancu IC, Cremers N, Vanrusselt H, Couturier J, Vanoosthuyse A, Kessels S, Lodder C, Brône B, Huaux F, Octave JN, et al. Aggregated tau activates NLRP3-ASC inflammasome exacerbating exogenously seeded and non-exogenously seeded tau pathology in vivo. Acta Neuropathol. 2019;137:599–617.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Bauernfeind FG, Horvath G, Stutz A, Alnemri ES, MacDonald K, Speert D, Fernandes-Alnemri T, Wu J, Monks BG, Fitzgerald KA, et al. Cutting edge: NF-kappaB activating pattern recognition and cytokine receptors license NLRP3 inflammasome activation by regulating NLRP3 expression. J Immunol. 2009;183:787–91.

    Article  CAS  PubMed  Google Scholar 

  39. Franchi L, Eigenbrod T, Núñez G. Cutting edge: TNF-alpha mediates sensitization to ATP and silica via the NLRP3 inflammasome in the absence of microbial stimulation. J Immunol. 2009;183:792–6.

    Article  CAS  PubMed  Google Scholar 

  40. Lin KM, Hu W, Troutman TD, Jennings M, Brewer T, Li X, Nanda S, Cohen P, Thomas JA, Pasare C. IRAK-1 bypasses priming and directly links TLRs to rapid NLRP3 inflammasome activation. Proc Natl Acad Sci U S A. 2014;111:775–80.

    Article  CAS  PubMed  Google Scholar 

  41. Xing Y, Yao X, Li H, Xue G, Guo Q, Yang G, An L, Zhang Y, Meng G. Cutting Edge: TRAF6 mediates TLR/IL-1R Signaling-Induced Nontranscriptional Priming of the NLRP3 inflammasome. J Immunol. 2017;199:1561–6.

    Article  CAS  PubMed  Google Scholar 

  42. Christgen S, Place DE, Kanneganti TD. Toward targeting inflammasomes: insights into their regulation and activation. Cell Res. 2020;30:315–27.

    Article  PubMed  PubMed Central  Google Scholar 

  43. Broz P, Pelegrín P, Shao F. The gasdermins, a protein family executing cell death and inflammation. Nat Rev Immunol. 2020;20:143–57.

    Article  CAS  PubMed  Google Scholar 

  44. Cunnane SC, Trushina E, Morland C, Prigione A, Casadesus G, Andrews ZB, Beal MF, Bergersen LH, Brinton RD, de la Monte S, et al. Brain energy rescue: an emerging therapeutic concept for neurodegenerative disorders of ageing. Nat Rev Drug Discov. 2020;19:609–33.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Beal MF. Mitochondria take center stage in aging and neurodegeneration. Ann Neurol. 2005;58:495–505.

    Article  CAS  PubMed  Google Scholar 

  46. Bubber P, Haroutunian V, Fisch G, Blass JP, Gibson GE. Mitochondrial abnormalities in Alzheimer brain: mechanistic implications. Ann Neurol. 2005;57:695–703.

    Article  CAS  PubMed  Google Scholar 

  47. Lustbader JW, Cirilli M, Lin C, Xu HW, Takuma K, Wang N, Caspersen C, Chen X, Pollak S, Chaney M, et al. ABAD directly links Abeta to mitochondrial toxicity in Alzheimer’s disease. Science. 2004;304:448–52.

    Article  CAS  PubMed  Google Scholar 

  48. Cardoso SM, Santos S, Swerdlow RH, Oliveira CR. Functional mitochondria are required for amyloid beta-mediated neurotoxicity. Faseb j. 2001;15:1439–41.

    Article  CAS  PubMed  Google Scholar 

  49. Askanas V, McFerrin J, Baqué S, Alvarez RB, Sarkozi E, Engel WK. Transfer of beta-amyloid precursor protein gene using adenovirus vector causes mitochondrial abnormalities in cultured normal human muscle. Proc Natl Acad Sci U S A. 1996;93:1314–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Elliott EI, Sutterwala FS. Initiation and perpetuation of NLRP3 inflammasome activation and assembly. Immunol Rev. 2015;265:35–52.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Hara T, Nakamura K, Matsui M, Yamamoto A, Nakahara Y, Suzuki-Migishima R, Yokoyama M, Mishima K, Saito I, Okano H, Mizushima N. Suppression of basal autophagy in neural cells causes neurodegenerative disease in mice. Nature. 2006;441:885–9.

    Article  CAS  PubMed  Google Scholar 

  52. Sita G, Graziosi A, Hrelia P, Morroni F. NLRP3 and infections: beta-amyloid in Inflammasome beyond Neurodegeneration. Int J Mol Sci. 2021;22(13):6984.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Milner MT, Maddugoda M, Gotz J, Burgener SS, Schroder K. The NLRP3 inflammasome triggers sterile neuroinflammation and Alzheimer’s disease. Curr Opin Immunol. 2021;68:116–24.

    Article  CAS  PubMed  Google Scholar 

  54. Kelley N, Jeltema D, Duan Y, He Y. The NLRP3 inflammasome. An overview of mechanisms of activation and regulation. Int J Mol Sci. 2019;20(13):3328.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Lou G, Palikaras K, Lautrup S, Scheibye-Knudsen M, Tavernarakis N, Fang EF. Mitophagy and Neuroprotection. Trends Mol Med. 2020;26:8–20.

    Article  CAS  PubMed  Google Scholar 

  56. Zhang CW, Hang L, Yao TP, Lim KL. Parkin regulation and neurodegenerative disorders. Front Aging Neurosci. 2015;7:248.

    PubMed  Google Scholar 

  57. Gkikas I, Palikaras K, Tavernarakis N. The role of Mitophagy in Innate Immunity. Front Immunol. 2018;9:1283.

    Article  PubMed  PubMed Central  Google Scholar 

  58. Harper JW, Ordureau A, Heo JM. Building and decoding ubiquitin chains for mitophagy. Nat Rev Mol Cell Biol. 2018;19:93–108.

    Article  CAS  PubMed  Google Scholar 

  59. Zheng W, Li K, Zhong M, Wu K, Zhou L, Huang J, Liu L, Chen Z. Mitophagy activation by rapamycin enhances mitochondrial function and cognition in 5×FAD mice. Behav Brain Res. 2024;463:114889.

    Article  CAS  PubMed  Google Scholar 

  60. Zhang J, Ney PA. Role of BNIP3 and NIX in cell death, autophagy, and mitophagy. Cell Death Differ. 2009;16:939–46.

    Article  CAS  PubMed  Google Scholar 

  61. Hou X, Watzlawik JO, Cook C, Liu CC, Kang SS, Lin WL, DeTure M, Heckman MG, Diehl NN, Al-Shaikh FSH, et al. Mitophagy alterations in Alzheimer’s disease are associated with granulovacuolar degeneration and early tau pathology. Alzheimers Dement. 2020;17:417–30.

    Article  PubMed  Google Scholar 

  62. Kim J, Lee HJ, Park SK, Park JH, Jeong HR, Lee S, Lee H, Seol E, Hoe HS. Donepezil regulates LPS and Aβ-Stimulated neuroinflammation through MAPK/NLRP3 Inflammasome/STAT3 signaling. Int J Mol Sci. 2021;22(19):10637.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. de Rivero Vaccari JP, Dietrich WD, Keane RW. Activation and regulation of cellular inflammasomes: gaps in our knowledge for central nervous system injury. J Cereb Blood Flow Metab. 2014;34:369–75.

    Article  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank all lab members for their valuable comments and discussions on this study.

Funding

This work was supported by National Natural Science Foundation of China (82404883; 82273809; 82273815; 82073731; 82474100; 82204676 ), National High Level Hospital Clinical Research Funding & Elite Medical Professionals Project of China-Japan Friendship Hospital (2024-NHLHCRF-JBGS-WZ-07; 2023-NHLHCRF-CXYW-01; ZRJY2024-BJ01; ZRJY2023-QM10; ZRJY2023-QM28; ZRJY2023-QM22), and Central Universities Fundamental for Basic Scientific Research of Peking Union Medical College (3332023096).

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Author notes

  1. Fulin Zhou and Wenwen Lian contributed equally to this work.

Authors and Affiliations

  1. Institute of Clinical Medical Sciences, Department of Pharmacy, China-Japan, Friendship Hospital 2nd, Yinghua Dongjie, Chaoyang District, Beijing, 100029, People’s Republic of China

    Fulin Zhou, Wenwen Lian, Congyuan Xia, Yu Yan, Wenping Wang, Jun He & Weiku Zhang

  2. School of Life Science, Beijing University of Chinese Medicine, No. 11, Bei San Huan Dong Lu, Chaoyang District, Beijing, 100029, People’s Republic of China

    Xiaotang Yuan, Zexing Wang, Zhuohang Tong & Jiekun Xu

  3. Department of Pharmacology, School of Medicine, Yale University, Connecticut, New Haven, USA

    Yungchi Cheng

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  1. Fulin Zhou
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Contributions

F.Z. contributed to Conceptualization, Methodology, Formal analysis, Data curation, and Writing original draft. W.L. contributed to Conceptualization, Project administration, Funding acquisition, Data curation, Visualization, Writing - original draft, and Writing - review & editing. X.Y. contributed to Methodology, Formal analysis, and Data curation. Z.W. contributed to Methodology, Formal analysis, and Data curation. C.X. contributed to Project administration, Data curation, and Visualization. Y.Y. contributed to Data curation and Visualization. W.W. contributed to Methodology and Visualization. Z.T. contributed to Writing - review & editing. J.X. contributed to Project administration, Funding acquisition, and Writing - review & editing. J.H. contributed to Supervision, Funding acquisition, and Writing - review & editing. W.Z. contributed to Resources, Project administration, Supervision, Funding acquisition, and Writing - review & editing.

Corresponding authors

Correspondence to Jiekun Xu, Jun He or Weiku Zhang.

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All animal procedures were approved by the Animal Care and Use Committee of China-Japan Friendship Hospital.

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The original version of this article was revised: the author’s name Yungchi Cheng was incorrectly written as Yunchi Cheng.

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Zhou, F., Lian, W., Yuan, X. et al. Cornuside alleviates cognitive impairments induced by Aβ1−42 through attenuating NLRP3-mediated neurotoxicity by promoting mitophagy. Alz Res Therapy 17, 47 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13195-025-01695-w

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Keywords

Alzheimer's Research & Therapy

ISSN: 1758-9193