To explore the neuroprotective effects of osteocalcin (OCN) on an Alzheimer’s disease (AD) cell model and its potential mechanisms, providing a scientific basis for new therapeutic targets for AD.

Human neuroblastoma cell line SH-SY5Y was treated with 40 nmol/L okadaic acid (OA) for 24 h to establish an AD cell model. The cells were divided into a normal group (untreated SH-SY5Y cells), a model group (40 nmol/L OA intervention), and an OCN intervention group (intervention with various concentrations of OCN in the AD cell model), and AKT knockout/overexpression groups (AKT-KO group and AKT-OE group), and AKT-KO OCN group and AKT-OE OCN group. CCK-8 assay was used to detect the changes in cell viability. Wright’s staining was employed to observe the morphological changes of AD cells. Western blotting was utilized to detect the protein levels of Tau, p-Tau, Bax, Bcl-2, Caspase-3 and their lytic types, as well as the expression of Tau, p-Tau, mTOR, AKT and p-AKT in each group after AKT knockout/overexpression. TUNEL staining and flow cytometry were applied to detect the changes in early and late apoptotic cells and the apoptotic rate in the OCN-treated AD cell model.

① Compared to the normal group, the model group exhibited a significant decrease in cell viability, noticeable morphological and structural damage, upregulation of p-Tau and Caspase-3, increased early and late apoptosis, and a significantly higher apoptotic rate (P<0.05). ② After treatment of different concentrations of OCN for 24 h, cell viability was increased to varying degrees compared to the AD model group, with the 100 pg/mL OCN group showing a significant increase in cell viability (P<0.01) and marked improvement in cell number and morphology (P<0.01). ③ Compared to the AD cell model group, the p-Tau/Tau ratio was decreased in all OCN treatment groups, particularly in the 100 pg/mL OCN intervention group, where the p-Tau/Tau ratio was significantly lower than that of the model group (P<0.01). ④ Compared to the model group, a significant concentration-dependent decrease in the Cleaved Caspase-3/Caspase-3 ratio was observed when OCN concentrations ranged from 1 to 100 pg/mL, with a significant reduction in the Bax/Bcl-2 ratio in the 100 pg/mL group (P<0.0001). ⑤ The results of TUNEL staining and flow cytometry showed that, compared to the model group, all concentrations of OCN effectively inhibited the apoptosis in the AD model cells, with a significant reduction in early and late apoptotic cells and apoptotic rate in the 100 pg/mL OCN group. ⑥ Compared with the control group and the model group, the P-AKT was significantly increased in the AKT-OE group after AKT overexpression (P<0.05). The expression level of AKT protein was decreased in the AKT-KO group after AKT knockout (P<0.05). When the AKT pathway was inhibited, the expression level of p-Tau was higher in the AKT-KO group than the control group (P<0.05), and when the AKT was overexpressed, the expression level was significantly inhibited (P<0.05).

OCN may inhibit cell apoptosis and reduce p-tau protein level by regulating the ratio of Caspase-3/Caspase-3 and Bax/Bcl-2, and thereby improve the morphology of AD model cells and effectively protect nerve cells, which may be related to the regulation of the AKT/mTOR pathway.

To investigate whether icariin (ICA) ameliorates cognitive impairment and modulates relevant endocrine hormone alterations in Alzheimer's disease (AD) model mice by regulating osteocalcin (OCN) secretion.

An AD mouse model was established by intraventricular injection of okadaic acid (OA). Using a stratified design, 4-week-old male SPF mice (weighing 18 to 22 g) were randomly divided into 8 groups (n=6): wild-type (WT) control group (intragastric administration of 60 mg/kg normal saline); WT-AD group (WT mice with left intracerebroventricular injection of 1 μL OA); WT+ICA group (60 mg/kg ICA daily); WT-AD+ICA group (WT-AD mice with daily 60 mg/kg ICA); OCN^-/- control group (OCN^-/- mice with 60 mg/kg normal saline); OCN^-/--AD group (OCN^-/- mice with left intracerebroventricular injection of 1 μL OA); OCN^-/-+ICA group (OCN^-/- mice with 60 mg/kg ICA daily); and OCN^-/--AD+ICA group (OCN^-/--AD mice with 60 mg/kg ICA daily). The Morris water maze test was conducted to assess learning and memory abilities, while open field test was performed to evaluate anxiety levels. Hippocampal histopathological changes were observed with HE staining and silver impregnation staining; Nissl bodies in hippocampal neurons were detected with Nissl staining. Immunohistochemistry assay was employed to determine the expression of amyloid β-protein (Aβ) and OCN, and Western blotting was used to measure the protein levels of Aβ and OCN. ELISA was applied to detect the expression levels of corticotropin-releasing hormone (CRH), glucose (GLU), glucocorticoid (GC), and cortisol (COR).

Morris water maze test demonstrated that compared with the WT-AD group, the WT-AD+ICA group exhibited significantly shortened escape latency, prolonged time spent in the target quadrant, and increased platform crossing frequency (all P<0.05); similarly, the OCN^-/--AD+ICA group showed improvements than the OCN-/--AD group (all P<0.05). Open field test results indicated that compared with the WT-AD group, the WT-AD+ICA group displayed significantly increased total exploration distance, distance in the center, and time in the center, enhanced activity willingness, and reduced time in peripheral zone and distance in peripheral zone (all P<0.05); similar improvements were observed in the OCN^-/--AD+ICA group compared with the OCN^-/--AD group (all P<0.05). Staining results revealed that ICA intervention attenuated hippocampal pathology, restored hippocampal morphological architecture, reduced neurofibrillary tangles (NFTs), and promoted gradual recovery of Nissl bodies; however, the OCN^-/- mice failed to achieve complete normalization. Immunohistochemistry assay showed that compared with the WT-AD group, the WT-AD+ICA group exhibited decreased Aβ levels and increased OCN levels (all P<0.05); similar trends were observed in the OCN^-/--AD+ICA group relative to the OCN^-/--AD group (all P<0.05). Western blotting demonstrated that compared with the WT-AD group, the WTAD+ICA group showed reduced Aβ expression and elevated OCN expression (both P<0.05); comparable changes were found in the OCN^-/--AD+ICA group compared with the OCN^-/--AD group (both P<0.05). ELISA indicated that compared with the WT-AD group, the WT-AD+ICA group exhibited significantly decreased levels of CRH, GLU, GC, and COR (all P<0.05); similar reductions were observed in the OCN^-/--AD+ICA group compared with the OCN^-/--AD group (all P<0.05).

ICA ameliorates cognitive impairment and reduces levels of related hyperactive endocrine hormones in AD model mice through regulating OCN secretion.