Curcumin exerts neuroprotective effects on proliferation of neural stem cells in vitro and APP/PS1 mouse model in vivo

Abstract

Alzheimer’s disease (AD) is characterized by the accumulation of amyloid beta (Aβ) plaques, leading to neuronal death. Notably, there are no therapeutically efficacious treatments for this disorder. This study investigates the neuroprotective mechanisms of curcumin. In vitro experiments demonstrated that curcumin significantly promoted the proliferation of neural stem cells (NSCs) derived from E16 mouse embryos. Additionally, in the APP/PS1 transgenic mouse model, curcumin improved cognitive function, as evidenced by a shorter escape latency and increased platform crossing frequency in the Morris water maze test and enhanced novel object recognition. Histological analysis showed that curcumin reduced Aβ accumulation in the hippocampal and cortical regions. Molecular studies revealed that curcumin upregulated the expression of BDNF and enhanced phosphorylation of CREB. Collectively, these findings indicate that curcumin exerts neuroprotective effects through multiple mechanisms, including promoting NSC proliferation, reducing Aβ accumulation, and the potential modulation of the BDNF-CREB signaling pathway.

Introduction

Alzheimer’s disease (AD), the most prevalent neurodegenerative disorder, is a heterogeneous condition with complex pathobiology. The primary pathological diagnostic feature of AD is senile plaque resulting from the accumulation of anti-beta-amyloid (Aβ) protein outside nerve cells and the aggregation of neurofibrillary tangles formed from the phosphorylation of Tau protein inside nerve cells¹. This abnormal accumulation of protein disrupts the synaptic signal transmission, induces synaptic degeneration, and neuronal apoptosis, leading to cognitive and memory dysfunctions observed in AD patients^2,3. Traditional therapeutic strategies for AD involve the administration of pharmacological agents—including cholinesterase inhibitors, N-methyl-D-aspartate (NMDA) receptor antagonists, Aβ drugs, nerve growth factors, and non-steroidal anti-inflammatory drugs. While these agents help to alleviate the AD-related symptoms, they exhibit limited efficacy in preventing the disease progression⁴. Consequently, the use of stem cells in AD treatment has garnered significant attention, with the results indicating therapeutic potential. However, several key challenges persist in the application of this approach, including the absence of an advanced technological support system, ethical concerns, and immune-related issues⁵.

In the 1990s, Johnasson et al. discovered neural stem cells (NSCs) within the embryonic and adult central nervous system. These cells exhibit key characteristics, including self-renewal, multipotent differentiation, migratory capacity, efficient tissue fusion, and low immunogenicity. Notably, NSCs can differentiate into neurons, astrocytes, and oligodendrocytes⁶. Consequently, NSC therapy has gained significant attention in AD treatment due to the inherent ability of the NSCs to repair and replenish damaged nerve cells, strengthen the connection between neural synapses, establish new neural circuits, and improve cognitive function^7,8. Studies have shown that AD patients can partially induce the activation of endogenous NSCs, facilitating limited compensatory recovery of neurological functions. However, this endogenous activation is insufficient to fully repair the neural damage. Furthermore, as the disease progresses and patient’s age increases, the capacity for endogenous NSC activation progressively diminishes, resulting in impaired regenerative potential. Therefore, the activation of endogenous NSCs, with the capacity to differentiate into new neural cells to replace dead neurons, serves as a therapeutically efficacious approach to treat patients with AD⁹. In recent years, research in the medical field has significantly focused on the activation of endogenous NSCs through exogenous means to treat patients with AD¹⁰.

Research has shown that certain natural compounds exhibit the capacity to activate endogenous NSCs through various pathways. This activation promotes NSC proliferation, self-renewal, and directed differentiation, as well as enhanced migratory activity to the target site, where they undergo further differentiation and integration, thereby contributing to effective treatment of AD^11,12. For instance, turmeric roots contain a polyphenol compound known as curcumin. This compound possesses various pharmacological activities, such as anti-inflammatory, antioxidant, and anti-tumor effects^{13, 14, 15–16}. Additionally, curcumin exhibits neuroprotective effects, as well as the promotion of synaptic axon regeneration and reconstruction^{17, 18–19}.

Consequently, we investigated whether curcumin can promote the proliferation of NSCs, reduce the accumulation of Aβ protein, and improve cognitive dysfunction in APP/PS1 mice using in vivo and in vitro experiments. Additionally, we explored its underlying mechanisms by which it exerts these therapeutic effects.

Results

NSCs in primary culture can proliferate and differentiate into neural cells

During 1 week of primary NSC culturing, gradual formation of standard neuroglobular-like cell aggregations was observed (Fig. 1A) beginning on day 6. These aggregates expressed the nestin of NSC marker (Fig. 1B,D). These NSCs co-expressed EdU and nestin, indicating their proliferative ability (Fig. 1E,G). The culture medium was discarded and replaced with a differentiation medium. Subsequently, the neuron-specific marker β-tubulin III and the astrocyte-specific marker GFAP staining were expressed in the cultured NSCs, indicating the neurospheres differentiated into neurons and astrocytes (Fig. 1H,K).

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Fig. 1

Cultured NSCs proliferate and differentiate into neural cells. (A) Representative neurospheres in culture medium. Scale bar = 100 μm (B–D). The neurospheres express nestin + labeling. Scale bar = 50 μm. (E, F) Immunostaining of the neurospheres shows EdU + and nestin + labeling. Scale bar = 50 μm. (H–K) NSCs differentiated into neurons (Tuj1+) and astrocytes (GFAP+). Scale bar = 25 μm.

Curcumin promotes the proliferation of NSCs

CCK-8 assay results indicated that the 0.5 µM curcumin group exhibited optimal proliferative effects in neural stem cells, with values significantly higher than the control group (p < 0.05) (Fig. 2B). Furthermore, after twelve days of treatment with varying concentrations of curcumin, we randomly selected three microscopic fields to measure and statistically analyze neurosphere diameter and quantity (representative fields shown in Fig. 2A). The 0.5 µM curcumin-treated group demonstrated the largest neurosphere diameter (p < 0.05), indicating the strongest proliferative capacity of cells within neurospheres at this concentration. Notably, the 2.5 µM curcumin group yielded the highest number of neurospheres (p < 0.05) (Fig. 2D), suggesting a potential enhancement of neural stem cells’ (NSCs) clonal formation capacity. In contrast, the 62.5 µM curcumin group displayed evident cytotoxic effects, showing significant inhibition of neurosphere proliferation (p < 0.001).

To further verify that curcumin promoted the proliferation of NSCs, we treated NSCs with varying concentrations of curcumin: 0.5 µM, 2.5 µM, 12.5 µM, and 62.5µM. Subsequently, we assessed the NSC marker, nestin, along with cell proliferation using EdU for a co-staining assay. Compared to the control group, there was a significant increase in the proliferation of NSCs after treatment with curcumin at 0.5 µM concentration(p<0.05) (Fig. 2E,F). To explore the potential molecular mechanism by which curcumin promotes NSC proliferation, we examined the expression of BDNF and p-CREB/CREB ratio in NSCs following curcumin treatment (Fig. 2G). Results from Western blot assays showed that curcumin treatment at a 0.5 µM concentration significantly upregulated BDNF expression(p<0.001) (Fig. 2H) and increased the ratio of p-CREB to CREB (Fig. 2I) (p<0.0001). These findings indicate that the BDNF-CREB signaling pathway is potentially involved in curcumin-induced proliferation of NSCs.

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Fig. 2

Effects of curcumin on neural stem cell (NSC) proliferation, neurosphere formation, and associated molecular mechanisms. (A) Representative images of neurospheres and their population following curcumin intervention. Scale bar = 100 m. (B) Proliferation rate indexes of NSCs after curcumin treatment. Fold changes in proliferation were assessed using the CCK-8 assay with absorbance measured at 450 nm. (C) Quantification of neurosphere diameters. (D) Quantification of neurosphere numbers. (E) Dissociated NSCs were treated with the indicated concentrations of curcumin in the reduced growth medium for 72 h. Then, cells were subjected to EdU assay (red), counterstained with nestin (green) or Hoechst (blue), and the images obtained through fluorescent microscopy. (F) Quantification of newly proliferated NSCs. (G) Western blot was performed to assess the effect of curcumin on CREB, p-CREB, and BDNF levels in NSCs. (H) Quantification of BDNF protein expression. (I) Quantification of p-CREB/CREB protein expression. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Scale bar = 50 μm.

Curcumin improves cognitive dysfunction in APP/PS1 mice

Before euthanization, the cognitive abilities of the mice were evaluated following curcumin intervention. The schematic diagram of animal experiments is shown in Fig. 3A. The MWM tests were used to evaluate the learning and memory ability of AD mice treated with curcumin (Fig. 3B). On day 3 of training, the escape latency between the groups was assessed, with the results indicating that the escape latency of AD mice in the curcumin-treated group (100 mg/kg) was significantly shorter compared to that in the AD model group (p < 0.05, p = 0.0117 ). Additionally, the number of crossing within the 60-second window was significantly increased in mice treated with Cur100mg/kg, compared to that in the AD mouse group (p < 0.01, p = 0.004 Fig. 3C). Furthermore, the trajectory of the mice to the platform was shortened in curcumin-treated mice group compared to the other groups (Fig. 3E). These results indicated that the short-term memory ability of APP/PS1 mice were significantly enhanced following curcumin treatment. Notably, the preference index for novel objects was higher in the Cur100mg/kg group (p < 0.05, p = 0.0304), suggesting curcumin increased the curiosity and interest of the mice in the new object (Fig. 3D). These results indicate that treatment with curcumin improved the spatial exploration and short-term memory in AD mice.

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Fig. 3

The effect of curcumin on the memory of the APP/PS1 mice in the behavioral tests. (A) Flow chart illustrating the process and methodology applied in the animal experiments. (B) Effects of curcumin on the escape latency of mice in the place navigation task of the MWM test. (C) On day 5 of the MWM experiment, after removal of the hidden platform, the number of times mice in each group passed through the platform area within the 60-second window. (D) The NOR experiment recorded the preference index of each group of mice for new objects. (E) Representative images of the path that the mice swam along to find the platform. Data are expressed as mean ± SD, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, *p < 0.05 was considered statistically significant, with n = 6.

Curcumin treatment increases neural regeneration in hippocampal regions in APP/PS1 mice

To determine the number of newly generated neurons in the dentate gyrus of the hippocampus of APP/PS1 mice following curcumin treatment, the new neurons, doubly stained with EdU and NeuN proteins, were quantified. In each group, most of the EdU-labeled new neurons migrated and dispersed into the dentate granular cell layer and were co-localized with NeuN (Fig. 4A). This observation revealed that curcumin significantly increased neuronal regeneration in the hippocampal region of the APP/PS1 mice (Fig. 4B).

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Fig. 4

Curcumin enhances neurogenesis in transgenic AD mice. (A, Upper panel) Fluorescence microscope image showing double staining of EdU (red) and NeuN (green) neurons in the dentate gyrus of the hippocampus. Scale = 100 μm. (B, Lower panel) Data analysis shows that curcumin treatment significantly increased the number of EdU/NeuN double-stained cells in the dentate gyrus. Statistical significance was considered for ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001, with n = 3.

Curcumin reduces Aβ accumulation in both the hippocampal and cortical regions of the APP/PS1 mice

The abnormal accumulation of Aβ plaque is considered a primary pathological feature of AD (Fig. 5A). To explore how curcumin treatment impacts Aβ accumulation in APP/PS1 mouse models, we utilized immunohistochemical analysis to evaluate Aβ plaque levels in the cortical and hippocampal regions of the brain. Our results indicated the presence of Aβ plaques in the hippocampus of APP/PS1 mice, whereas no Aβ accumulation was observed in the WT group (Fig. 5B). Notably, treatment with curcumin in the AD model group significantly diminished Aβ plaque accumulation in both the hippocampal and cortical regions (Fig. 5C,D).

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Fig. 5

Effect of curcumin on amyloid deposits in transgenic AD mice. (A) The underlying mechanism of curcumin in AD. Senile plaques formed by Aβ deposition interfere with intercellular signaling and result in neuronal death. (B) Immunohistochemical analysis of the effect of curcumin on the expression of Aβ in the hippocampus and cortex of the APP/PS1 mice. (C, D) Quantitative analysis of Aβ deposition in the cortex and hippocampus of mice in each group. Data are expressed as mean ± SD. Statistical significance was considered for ^*p < 0.05, ^***p < 0.001, ^****p < 0.0001. Scale bar = 100 μm and n = 3.

Curcumin increases the expression of p-CREB/CREB and BDNF proteins in APP/PS1 mice

To explore whether the improvement in memory and cognitive function in APP/PS1 mice treated with curcumin was associated with alterations in the expression levels of memory-related proteins, we quantified detected BDNF and CREB—these are key functional proteins in memory formation and consolidation (Fig. 6A). Western blot analysis revealed that the expressions of p-CREB and BDNF in the hippocampus of mice in the AD model were significantly lower compared to those in the WT control mice (p < 0.001; Fig. 6B,C). After curcumin intervention, the expressions of P-CREB/CREB and BDNF in the hippocampus of AD mice were significantly increased (p < 0.05; P-CREB/CREB: AD vs. Cur of 100 mg/kg p = 0.0484; AD vs. Cur of 300 mg/kg p = 0.0178; Fig. 6B,C). Additionally, there was a significant increase in the expression of P-CREB/CREB in the cortex of AD mice treated with curcumin (p < 0.05; AD vs. Cur of 100 mg/kg p = 0.0482; AD vs. Cur of 300 mg/kg p = 0.0354; Fig. 6E). Furthermore, there was significant increase in the expression of BDNF following curcumin treatment (AD vs. Cur of 100 mg/kg p = 0.015; AD vs. Cur of 300 mg/kg p = 0.0011; Fig. 6D). These findings indicate that curcumin enhances the expression of BDNF and p-CREB, which are key functional proteins in memory formation.

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Fig. 6

Western blot quantitative analysis of the effect of curcumin on the BDNF-CREB signaling pathway in various groups of mice. (A) Western blot analysis was performed to assess the expression levels of CREB, p-CREB, and BDNF in the hippocampus and cortex across control, disease model, and treatment groups. (B, C) Quantification of BDNF protein expression (B) and the p-CREB/CREB ratio (C) in the hippocampus. (D, E) Quantification of BDNF protein expression (D) and the p-CREB/CREB ratio (E) in the cortex. Data are presented as mean ± standard error of the mean (SEM), ^*p < 0.05, ^**p < 0.01 ^***p < 0.001 vs. AD group. Data are expressed as mean ± SD. n = 3.

Discussion

In this study, the effect of curcumin on the proliferation of NSCs and its potential therapeutic mechanisms were examined both in vitro and in vivo. The results revealed that treatment with 0.5 µM curcumin significantly enhances the proliferation of NSCs. In vivo experiments revealed that following curcumin intervention, the escape latency of AD mice was shortened, with the number of cross-platform within the 60-second window increased. Additionally, the trajectory time of the AD mice to the platform was shorter, and the identifying index of new objects in the AD mice was improved. These findings revealed that curcumin exhibited a beneficial effect on enhancing the spatial exploration and short-term memory capabilities of mice with AD. Histological examinations revealed that curcumin diminished the accumulation of Aβ protein in both the hippocampus and cortex, while promoting the proliferation of endogenous NSCs. These findings reinforce the prospective significance of curcumin in neuroprotection. Subsequently, we further explored the underlying mechanisms by which curcumin exerts its neuroprotective properties in AD by assessing the expression levels of crucial proteins associated with memory function, including p-CREB, CREB, and BDNF. The results showed that curcumin upregulates the expression of BDNF and p-CREB/CREB, indicating that curcumin may play a neuroprotective role by activating the BDNF-CREB signaling pathway²⁰. However, the direct molecular interaction between curcumin and the activation of this signaling cascade remains underexplored. Further investigations employing specific pathway inhibitors or gene knockdown approaches will be necessary to establish causality and delineate the precise underlying mechanisms. This represents a key limitation of the current study and a critical direction for future research.

In this study, curcumin was found to be a potential therapeutic agent for AD due to its significant pleiotropic and excellent safety profile²¹. Previous studies have shown that curcumin, as a multi-target compound, can efficiently bind to Aβ plaques, reduce their neurotoxicity, and promote their degradation, thereby effectively improving cognitive deficits in AD mice^{22, 23–24}. Consequently, curcumin was identified as a potential and potent therapeutic agent in improving the cognitive symptoms of AD²⁵. This observation is corroborated by findings from previous studies that have similarly revealed curcumin as a potential candidate in the treatment of AD^{26, 27, 28–29}. Despite the therapeutic potential of curcumin being well-established, concerns about its safety and appropriate dosage persist, thereby warranting further investigations into its potential side effects. Specifically, off-target effects have not yet been adequately investigated and cannot be ruled out. Further pharmacodynamic and toxicological examination is warranted to fully assess these risks. Additionally, the mode of administration significantly affects the efficacy of curcumin³⁰. Administration of curcumin through intravenous (iv) or intraperitoneal (ip) injections potentially results in the formation of highly stable water-soluble derivatives—thereby increasing its bioavailability—compared to when the administration is conducted orally¹⁷. In this study, APP/PS1 mice were selected as the experimental model mice, with ip injection of curcumin applied. Subsequently, alterations in the body weight of the mice were monitored throughout the experiment.

Our results showed that curcumin administered at a dose of 300 mg/kg exhibits no effect on the body weight of mice in a short time, indicating its safety. However, due to the poor solubility of curcumin, higher concentrations of curcumin partially resulted in condensation in the cortex of mice. Wang et al. investigated how curcumin influences cognitive abilities using an APP/PS1 mouse model, and revealed that after six months, curcumin significantly enhanced the cognitive performance of the mice, demonstrating a long-lasting effect that was dosage-dependent. Additional studies have validated that curcumin can effectively impede the aggregation of Aβ, thereby positioning it as the most promising anti-amyloid compound³¹. Immunohistochemical examinations indicated that curcumin notably decreased the quantity of Aβ plaques in the hippocampal and cortical regions of AD mouse models. Furthermore, the behavioral tests showed that the AD model mice exhibited significant improvement in memory and spatial exploration ability after curcumin intervention. This observation further validates that curcumin can improve memory and cognitive impairment in AD patients, mitigating the aggregation of amyloid.

The proliferative ability of endogenous NSCs in AD mice was decreased compared to that of the normal mice³². Treatment with curcumin increased the number of newly formed hippocampal neurons in APP/PS1 mice. Additionally, the newly generated cells within the dentate gyrus of the hippocampus were able to persist for several weeks while consistently integrating into the granular cell layer³³. Notably, EdU—a thymine nucleoside analogue—is one of the raw materials for DNA synthesis. During the proliferative period (S phase) of NSCs, the synthesis of DNA from newborn cells is required. EdU can integrate into DNA molecules that are replicating by replacing thymine³⁴. The EdU positive expression can be detected in the new NSCs. The migration, survival, and differentiation of endogenous NSCs after proliferation can be monitored using EdU. NeuN, a neuron-specific molecule, is not only a stable and reliable marker of mature neurons, but also plays an important role in determining neuronal phenotype; moreover, it may be involved in regulating the transcription process of neuronal development and maturation. Thus, positive expression of EdU/NeuN can be used to identify endogenous NSCs that undergoing differentiation into neuronal cells^35,36. Immunofluorescence staining results showed that curcumin can promote the proliferation of endogenous NSCs in the hippocampal region of the AD model mice. Notably, we primarily observed newly generated neurons in the hippocampus, as indicated by EdU/NeuN double-positive cells. However, evidence of gliogenesis was not observed. This can be attributed to the fact that we specifically focused on neuronal differentiation, using NeuN as a marker of mature neurons, while failing to perform co-staining with markers of glial cells, such as GFAP (for astrocytes) and Olig2 (for oligodendrocytes). Therefore, the absence of gliogenesis is attributed to the lack of application of the corresponding markers within this study. In future experiments, we plan to include glial cell markers to comprehensively assess the differentiation potential of NSCs under curcumin treatment. Moreover, curcumin may preferentially enhance neurogenesis rather than gliogenesis within the hippocampus, a hypothesis that requires further mechanistic investigation to elucidate the underlying pathways and cellular specificity.

Research has shown that BDNF is crucial for the survival of neurons, efficient synaptic plasticity, and memory formation³⁷. The gene for BDNF is a primary target of CREB, with its signaling dependent on phosphorylation of CREB. Serum levels of BDNF were altered in patients with AD, with the results indicating that advanced age affects the secretion of BDNF from neural cells in the brain. Research has indicated that dietary curcumin facilitates the restoration of BDNF and CREB levels in cases of traumatic brain injury induced by oxidative stress, as well as mitigating its associated cognitive deficits²⁰. We discovered that curcumin elevates BDNF and CREB levels within the hippocampal and cortical regions of AD mice. This finding elucidates the potential underlying mechanisms by which curcumin therapeutically improves clinical outcomes in AD and also establishes a robust experimental foundation for developing optimally efficacious therapeutic strategies for this disorder.

Despite the significant contributions revealed through this study, several limitations need to be acknowledged. First, curcumin exhibits limited bioavailability, which may affect its therapeutic efficacy in vivo. Therefore, improving the bioavailability of curcumin is an important direction for future studies³⁸. Second, the underlying mechanisms of action of curcumin have to be deeply investigated to identify its specific key targets. Third, the potential off-target effects of curcumin, particularly at higher concentrations, warrant thorough investigation in future studies to ensure safety and specificity of action. Finally, due to the multifactorial pathogenesis of AD, a single pharmacological agent approach may be insufficient to achieve optimal therapeutic outcomes. Therefore, combination therapies targeting multiple pathways offer a more effective strategy for treating AD.

Conclusion

Curcumin can effectively promote the proliferation of endogenous NSCs in the dentate gyrus of the hippocampus and reduce Aβ accumulation in the hippocampal and cortical regions in AD model mice. Additionally, curcumin upregulates the expression of BDNF and p-CREB/CREB, suggesting that the BDNF-CREB signaling pathway may be involved in its neuroprotective effects. However, whether this pathway directly mediates the reduction of Aβ levels remains to be experimentally validated through targeted mechanistic studies. Collectively, these highlight the multi-targeted therapeutic potential of curcumin in the treatment of AD.

Materials and methods

Primary NSC culture and expansion

Neural stem cells (NSCs) were cultured to generate neurospheres according to the protocols established by Reynolds and Weiss³⁹. Telencephalic tissue was isolated from embryonic day 16 (E16) mice and subjected to mechanical dissociation (repeated trituration using a pipette) to obtain a single-cell suspension. All animal procedures were approved by Shandong First Medical University Affiliated Liaocheng Hospital Ethics Committee (approval number: D2022033). The cells were then resuspended in a growth medium composed of a 1:1 mixture of DMEM/F12 (Gibco, 11320033), supplemented with 20 ng/ml epidermal growth factor (EGF; Thermo Fisher, E3477), 20 ng/ml basic fibroblast growth factor (bFGF; Life technologies, PHG0264), and 2% B27 supplement (Gibco, 17504044). The cultures were then incubated at 37 °C in a humidified atmosphere with 5% CO2, with half of the culture medium being replaced every other day. After 7 days of culture when standard neurospheres had formed, they were dissociated with Accutase (Sigma, A6964) for 10 min, followed by mechanical trituration to generate a single-cell suspension, which was then passaged at a 1:3 ratio. Secondary or tertiary neurospheres were collected for further analysis. For differentiation, the neurospheres were transferred to a differentiation medium composed of DMEM/F12 (1:1) supplemented with 1% penicillin streptomycin (Gibco, 15140122), 2% fetal bovine serum (Gibco, 15140122), and 2% B27 supplement without growth factors. The cells were cultured under these conditions for 4 days to induce differentiation.

Curcumin intervention

To investigate the effects of varying concentrations of curcumin (Aladdin, C140600-5 g) on NSCs proliferation, we employed two experimental approaches: (1) Neurospheres Suspension Culture: Single-cell suspensions were prepared by digesting NSCs with Accutase for 6 min, followed by mixing using mechanical pipetting. Cells were then seeded at a density of 5 × 10³ cells per well in 24-well plates containing 500 µL of culture medium supplemented with varying concentrations of curcumin (0 µM, 0.5 µM, 2.5 µM, 12.5 µM, and 62.5 µM). The medium containing different concentrations of curcumin was replaced every 3 days, and the cells were cultured until day 12. To prevent neurosphere fusion during the culture process, the state of neurospheres was observed daily under a microscope. Once fusion was detected, a Pasteur pipette was gently used to pipette up and down to separate the neurospheres. (2) EdU/Nestin Immunofluorescence Staining (Adherent Culture): The NSCs were seeded as single-cell suspensions at a density of 5 × 10³ cells per well into 24-well plates pre-coated with poly-L-lysine (Sigma, P3513). The cells were then cultured for 48 h in a culture medium supplemented with varying concentrations of curcumin (0 µM, 0.5 µM, 2.5 µM, 12.5 µM, and 62.5 µM). Subsequently, 10 µM EdU (BeyoClick™ EdU-555, C0075S) was added, and the cells were incubated for an additional 24 h. The cells were then fixed and subjected to immunofluorescence co-staining with EdU and the NSC marker Nestin (Abcam, ab22035) to analyze NSC proliferation under varying concentrations of curcumin.

CCK-8 assay

The effects of varying concentrations of curcumin on NSC proliferation were further assessed using CCK-8 cell counting kit-8 (Beyotime; China, C0037) following the manufacturer’s instructions. A 100 µL of monolayer cell suspension was inoculated into a 96-well plate at a density of 5 × 10³ cells/mL and then cultured in 100 µL of base medium. After 24 h of culturing, curcumin at varying concentrations (0 µM, 0.5 µM, 2.5 µM, 12.5 µM, 62.5 µM) was added. After an additional 48 h of treatment, 10 µL of CCK-8 solution was added to each well, followed by incubation at 37 °C for 40 min. Subsequently, absorbance was measured at 450 nm.

Animals and drug administration

A total of 30 SPF-grade five-month-old APP/PS1 double transgenic mice were used for the in vivo experiments. All animals were purchased from Beijing HFK Bioscience Co., Ltd., China. All animal treatment protocols were approved by the Shandong First Medical University Affiliated Liaocheng Hospital Ethics Committee (approval number: D2022033) and conducted in accordance with the laboratory animal care guidelines set forth by the National Institute of Health (NIH). Significant efforts were made to minimize animal suffering and to reduce the number of animals used in the study. Additionally, the experiments adhered to the ARRIVE guidelines (https://arriveguidelines.org) to ensure complete and transparent reporting of the methods and results. The mice were randomly assigned into five groups (n = 6 for each group): the WT control group, the AD model group, a low concentration curcumin group (100 mg/kg), a high concentration curcumin group (300 mg/kg), and a positive control group treated with 0.3 mg/kg of Rivastigmine (sigma, SML0881-50MG). The selection of curcumin doses (100 mg/kg and 300 mg/kg) was based on previous studies that demonstrated their neuroprotective and anti-inflammatory effects in vivo without observable toxicity when administered intraperitoneally^{40, 41–42}. These concentrations were also chosen to address curcumin’s known limitations in bioavailability and to explore potential dose-dependent effects. Rivastigmine was selected as a reference dose based on its proven efficacy in ameliorating cognitive dysfunction in AD mouse models⁴³. The WT control and AD model groups received an equal dose of DMSO. The drugs were administered intraperitoneally into experimental mice for 7 days. Additionally, from days 4 to 7, the mice were injected with EdU (50 mg/kg) once a day to evaluate neurogenesis.

Morris water maze (MWM) test

The MWM test, a classic experiment used to assess the memory and cognitive ability of mice, was classified into two domains: (1) positioning navigation, which assesses memory ability; and (2) space exploration, which tests space exploration ability. The MWM test spanned 6 days. During days 1 to 5, the mice were sequentially placed in the first, second, and third quadrants of the maze. The time taken to reach the hidden platform within a 60-second window was recorded as the escape latency. A shorter escape latency reflects better spatial learning and memory performance in the mice. On day 6, the platform was removed, and the frequency of the mice crossing the platform was recorded, with a higher frequency indicating a greater spatial exploration capability.

Novel object recognition test

Additionally, a novel object recognition (NOR) test was conducted to evaluate the short-term recognition memory of the mice. This test examines the memory capabilities of mice by leveraging their inherent tendency to explore unfamiliar objects. The exploration process is divided into three stages lasting for three days: adaptation period, familiarity period, and test period. Adaptation period: On day 1, the mice were sequentially introduced into the test box and allowed to explore freely for 10 min. This acclimatized the mice to the environment, thereby eliminating any anxiety and other behaviors that may potentially arise from the subsequent experiment. Familiarization period: On day 2, two identical objects were placed in the designated positions of the blank test box. Subsequently, the mice were sequentially introduced into the test box at the same location where they were previously positioned. Test period: On day 3, one of the objects was replaced with a new object, and then the mice were reintroduced into the test box to explore freely, with the time taken to explore both the old and new objects recorded. The NOR memory was determined using the following formula: Novel object recognition index (NOI) = [(exploration time of the novel object) / (exploration time of familiar and novel objects)] × 100%.

Immunofluorescence

The animals underwent perfusion, after which the brain tissues were dissected and fixed in PFA overnight for preservation. The tissue was dehydrated using 30% Sucrose (Sigma, V900116-X500G), sectioned into 15 μm-thick slices using a freezing microtome, followed by rinsing of the hippocampal sections using PBS. To prevent nonspecific binding, the sections were incubated for 1 h with a solution supplemented with 10% normal goat serum (Sigma, NS02L) combined with 0.3% Triton X-100 (China, Sangon Biotech, A417820-0100). Appropriate concentrations of the primary antibody, rabbit anti-NeuN (1:500, Abcam, ab177487), were applied to the slides and then incubated at 4 °C overnight. After washing with PBS, the brain sections were treated with the secondary antibody, anti-rabbit IgG conjugated with Alexa 488 (1:1000, Abcam, Cambridge, UK) for 1 h at room temperature. The EdU color reaction system (BeyoClick™ EdU Cell Proliferation Kit equipped with Alexa Fluor 555) was prepared according to the kit manufacturer’s instructions, with the room temperature maintained and protected from light for 30 min. After washing with PBS, nuclear staining was conducted on the sections using Hoechst 33,258 (1:10000, Sigma, 94403).

Immunohistochemistry

The immunohistochemical assay was performed using the SP reagent kit (Mouse Streptavidin-Biotin Detection System, China, ZSGB-BIO, SP002). This assay is based on the principle that the biotin-labeled goat anti-mouse IgG binds to the primary antibody on the tissue to create an immune complex, which is subsequently bound to Horseradish peroxidase (HRP)-conjugated streptavidin to form a polymer. HRP in the polymer catalyzes a reaction between the substrate hydrogen peroxide and DAB, generating an insoluble brown chromogen, that facilitates visualization of the specific antigen site in the tissue sections observed under a microscope. Briefly, the tissues were treated with the antigen repair solution (China, ZSGB-BIO, ZLI-9072) to achieve antigen retrieval by incubation at 100 °C for 10 min. The tissue sections were delineated with an immunohistochemistry barrier pen, and incubated with an endogenous peroxidase blocker at room temperature for 10 min. Sections were washed with PBS buffer 3 times for 3 min, followed by incubation with 100 µL of blocking solution containing normal goat serum for 10–15 min. Next, the samples were treated with 100 µL of primary antibody, Anti-Beta (β)-Amyloid antibody (Sigma, MAB348, 1:500) at 37 °C for 60 min, washed with PBS buffer 3 times for 3 min. Subsequently, 100 µL of biotin-conjugated goat anti-mouse IgG was applied to the tissue section and incubated at room temperature for 10 min. As the primary antibody used is a mouse-derived monoclonal IgG, the secondary antibody, goat anti-mouse IgG, binds specifically to the Fc region of the mouse immunoglobulin, allowing effective signal amplification through the streptavidin–biotin detection system, thereby improving sensitivity of antigen visualization. The sections were washed with PBS buffer 3 times for 3 min, and then incubated with the HRP-conjugated streptavidin working solution at room temperature for 10 min, then washed with PBS washes 3 times for 3 min. Freshly prepared DAB chromogenic solution (China, ZSGB-BIO, ZLI-9017) was added for incubation for 5 min at room temperature. The sections were rinsed with tap water and counterstained with hematoxylin for 20 s, differentiated, rinsed, and stained blue. Finally, the sections were dehydrated with ethanol and xylene, then mounted using neutral resin.

Western blot

Proteins were extracted from the samples and rinsed with ice-cold PBS and then lysed using RIPA lysis buffer (China, Beyotime, P0013B) containing 1% PMSF (Beyotime, ST505). The concentration of proteins was measured using the BCA assay (Beyotime, P0012). The protein samples were analyzed using 10% SDS-polyacrylamide gel electrophoresis (Beyotime, P0012AC), and then transferred to a membrane through the wet transfer method. Primary antibodies, including p-CREB (1:1000, Cell Signaling, 9198), BDNF (1:1000, Cell Signaling, 47808), CREB (1:1000, Cell Signaling, 9197), and β-actin (1:1000, Beyotime, A1978), were incubated with the membranes overnight, which had been blocked with BSA. The membranes were then treated with a secondary antibody for two hours and the formed bands were developed using the enhanced chemiluminescence technique, and the signals were quantified using Image Lab software from Bio-Rad.

Statistical analysis

All quantitative data were analyzed using GraphPad Prism 5.0 software (GraphPad Software, USA), and results were expressed as mean ± standard deviation. Two-way ANOVA was performed to analyze escape latency during the MWM training task, and one-way ANOVA was used for the remaining experiments. Tukey’s post hoc test was used for multiple comparisons following ANOVA. A significance level of P < 0.05 was considered statistically significant.

Author contributions

J.C. and C.W. analyzed the data, Completed histological experiments and manuscript writing, Completed protein detection. C.W. and Q.C. performed the animal experiments; Y.Z. performed the cell experiments. X.L. X.Z. W.Z. and F.H. designed the experiments, Guided the experiment, revised the manuscript. All authors reviewed the manuscript.

Funding

This study was supported by the key basic research fund from the Natural Science Foundation of Shandong Province, China (ZR2019ZD39); Shandong Second Medical University Affiliated Hospital Scientific Research Development Fund Project (2024FYQ063); Weifang Science and Technology Development Plan Project (2021Y111); High-talent Startup fund of Yidu Central Hospital (2024); The Opening Project of Hubei Key Laboratory of Cognitive and Affective Disorders (HBCAD2024-13).

Data availability

All the data generated or analyzed during this study are included in this published article.

Declarations

Competing interests

The authors declare no competing interests.

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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