The experiments were performed on Sprague-Dawley (SD) rats less than 24 h old purchased from the Shanghai Institute of the Chinese Academy of Science. This study was approved by the Fudan University experimental standards and followed the international guidelines on the ethical treatment of experimental animals. Primary cultures of dissociated hypothalamic cells were prepared according to a previously described method (12) with specific modifications. The hypothalamic tissue was dissected from neonatal SD rats, stripped of the meninges and blood vessels, and minced in Hanks’ balanced salt solution (HBSS) without Ca²⁺ and Mg²⁺. The tissue was dissociated using 0.125% trypsin (Beijing Solarbio Science and Technology Co., Ltd., Beijing, China) digestion for 15 min at 37°C and gentle titration through a series of glass pasteur pipettes. The cells were then plated on poly-L-lysine (molecular weight, 30,000–70,000; 0.1mg/ml; Sigma, St. Louis, MO, USA) coated-glass coverslips, 96-well plates, or 100 mm dishes at a density of 1×10⁶ cell/ml and maintained at 37°C in a humidified 5% CO₂ incubator. Neurons were cultured in neurobasal-A medium (Gibco BRL, Grand Island, NY, USA) and supplemented with 2% B27 supplement (Gibco BRL), 10 μl/ml penicillin-streptomycin, 2 mM glutamine (Solarbio), 5 ng/ml bovine fibroblast growth factor 2 (FGF2; R&D Systems, Minneapolis, MN, USA) and 1% fetal bovine serum (FBS). After a 24-h culture period, cultures grown in serum-free neurobasal medium yielded greater than 80% neurons, as estimated by immunocytochemical staining with antibodies against neurofilament proteins. The cultured neurons were used for in vitro studies on day 8 (DIV 8).

CORT (Sigma) was initially dissolved in ethanol as a stock solution and then in culture media (final concentration of ethanol, 0.1%). Icariin (purity >99%) was purchased from the Shanghai Winherb Medical S&T Development Co., Ltd. (Shanghai, China). Eight-day primary hypothalamic neurons were washed twice with Mg²⁺-free, HEPES-buffered saline (HBS; 146 mM NaCl, 10 mM HEPES, 2 mM CaCl₂, 5 mM KCl, 10 mM D-glucose, pH 7.4) and pretreated with different concentrations of icariin with or without 50 μM LY294002 (Sigma) for 2 h at 37°C, and then exposed to CORT for 24 h.

After exposure to various concentrations of CORT, icariin and LY294002, cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl (MTT) assay system (Beyotime Institute of Biotechnology, Shanghai, China) (11). Cell cytotoxicity following various treatments was evaluated by lactate dehydrogenase (LDH) release. This was acheived using a Quantitative Detection kit according to the manufacturer’s instructions (GenMed Scientifics Inc., Arlington, MA, USA) (11).

To evaluate the morphological changes in primary cultures of hypothalamic cells, cultures were observed under a Leica DFIL inverted microscope with a phase-contrast optic lens. Images were captured using Leica QWin plus 3 Image Processing Software (Media Cybernetics, Silver Spring, MD, USA) through a Leica DFC300 FX camera device. We analyzed neurite preference from five images per condition.

Caspase-3 activity in primary cultures of hypothalamic lysate was determined using the Chemicon caspase colorimetric activity assay kit according to the manufacturer’s instructions (11). In brief, treated cells were resuspended in 50 μl of cell lysis buffer and incubated on ice for 30 min. After centrifugation for 5 min at 10,000 × g, the supernatant was transferred to a fresh tube followed by the addition of reaction buffer and caspase-3 substrate for 4 h at 37°C in the dark. The free chromophore p-nitroaniline (pNA) was quantified using a microtiter plate reader at 405 nm. The fold increase in caspase-3 activity was determined by comparing the absorbance from each sample with untreated neurons.

Following treatment, hypothalamic cell cultures grown on 96-well plates were loaded with Rhodamine 123 (Rho 123) (10 μM) (Beyotime Institute of Biotechnology, Shanghai, China) at 37°C in the dark for 15 min and then washed 3 times with phosphate-buffered saline (PBS). The cell fluorescence intensity of Rho 123 was quantified using a fluorescence microplate reader (TECAN Infinite 200 microplate reader; Tecan Trading AG, Männedorf, Switzerland) with excitation at 485 nm and emission at 530 nm. The background fluorescence signal of Rho 123 was determined without cells and subtracted from those obtained in hypothalamic neurons.

Intracellular ROS levels were analyzed by H2DCF-DA assay according to the method previously described (13). Cells (1×10⁶ cells/well) were rinsed with Kreb’s ringer solution and 10 mM H2DCF-DA was added. After 15 min incubation at 37°C, cells were washed with PBS, harvested and pelleted by centrifugation and then resuspended in 0.5 ml PBS. Fluorescence intensity was then monitored using a fluorescence microplate reader. The data was analyzed and expressed as a percentage of the control. DCF labeled cells were observed under fluorescence microscopy.

Following treatment, the cultures were washed twice with PBS, then placed into ice-cold PBS (0.1 M, containing 0.05 mM EDTA) and homogenized. The homogenate was then centrifuged at 4°C at 10,000 × g for 30 min, after which the protein concentration was determined using the Bradford method, using bovine serum albumin (BSA) as a reference standard. Measurement of superoxide dismutase (SOD) activity was performed according to the reagent kit manufacturer’s instructions.

Following treatment, cells in each of the 6-well plates were rinsed twice with cold PBS, followed by the addition of cell lysis buffer containing 150 mM NaCl, 50 mM Tris-HCl, 5 mM EDTA, 1% Nonidet P-40, 0.5% deoxycholate, 1% SDS with proteinase inhibitor cocktail (Sigma) on ice for 15 min, and then centrifuged for 20 min at 12,000 × g. The supernatant was collected and the protein concentration was measured using the Bradford method. Fifty milligrams of total protein was dissolved in sample buffer and heated for 5 min prior to loading onto polyacrylamide gels. Proteins were then transferred to poly(vinylidine difluoride) filter membranes, and blocked with 5% non-fat dry milk in Tris-buffered saline/0.05% Tween-20. The membrane was incubated with a monoclonal antibody against phospho-Akt (Ser473), Akt (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), followed by incubation with horseradish peroxidase-conjugated (HRP) secondary antibodies and visualized using an enhanced chemiluminescence kit.

All data are expressed as the mean ± SD. Differences between groups without particular comments were generally examined for statistical significance using one-way ANOVA analysis with a post-hoc Dunnett’s test. P≤0.05 indicated a statistically significant difference.

Cultured hypothalamic neurons were exposed to an increasing concentration of CORT (0.01–5 μM) for 24 h and cell viability was assessed using an MTT reduction assay. CORT induced cell death in a concentration-dependent manner (Fig. 1A). Exposure to CORT (1 μM) for 24 h was used for inducing excitotoxic neuronal injury in the subsequent experiments.

To investigate the neuroprotective effects of icariin on CORT-induced neuronal damage, hypothalamic neurons were pretreated with icariin at 0.1 μM and 1 μM for 2 h, followed by challenge with CORT (1 μM) for 24 h. Icariin pretreatment at 0.1 μM and 1 μM enhanced cell viability compared with the CORT-treated control (Fig. 1B). The protective effect of icariin on CORT-induced cytotoxicity was also analyzed using the LDH test. Pretreatment with 0.1 μM and 1 μM icariin caused a marked decrease in LDH release (Fig. 1C).

Morphological analysis at the subcellular level remains the most conclusive method for distinguishing apoptosis from necrosis. The protective effect of icariin on CORT-induced cytotoxicity was also analyzed by microscopic examination. Cultured hypothalamic neurons were pretreated with or without 1 μM icariin for 2 h, followed by challenge with CORT (1 μM) for 24 h. Cells treated with vehicle were healthy with networks of neurites and vacuole-free cell bodies. The synapse connections between neurons were clearly observed. Neurite fragmentation, shrinkage of cell bodies and evident cell loss were observed when the neuronal culture was exposed to 1 μM CORT for 24 h. Pretreatment of icariin with 1 μM was able to protect cortical neurons against CORT toxicity as demonstrated by the fine morphology (Fig. 2A).

Biochemical markers for caspase-3 activation following treatment with CORT were consistent with apoptotic cell death. Following 24-h CORT treatment, caspase-3 activity increased in the hypothalamic neurons. In the cultured hypothalamic neurons exposed to 1 μM CORT plus 1 μM icariin, caspase-3 activity was similar to that the of the control (Fig. 2B)

Mitochondria are recognised to be involved in apoptosis. Permeability changes can lead to caspase-dependent cytotoxicity and downstream apoptotic signaling, while a loss of mitochondrial transmembrane potential, denoted as mitochondrial dysfunction, leads to cytochrome c release from the mitochondria and triggers other apoptotic factors. In the present study, we evaluated mitochondrial transmembrane potential using Rho 123 as fluorescent dye. Dye distribution was examined under inverted microscope (Leica DFIL). In the control cultures, Rho 123 was markedly aggregated in hypothalamic neurons, reflecting the baseline for healthy mitochondria in cells. CORT-treated neurons demonstrated a decrease in Rho 123 of 37%. Pretreatment of 1 μM, but not 0.1 μM icariin, significantly inhibited these changes (Fig. 2C and D).

To determine whether icariin attenuates cell death by blocking ROS generation, we detected the intracellular level of ROS using H2DCF-DA fluorescent dye. Treatment of hypothalamic neurons with 1 μM CORT for 24 h resulted in a significantly increased DCF signal compared with the control group, but this was significantly reduced by icariin pretreatment (Fig. 3A and B).

The level of SOD provided further evidence of the protective effects of icariin. Incubation with 1 μM CORT for 24 h resulted in a significantly decreased activity of SOD (42.5%) in the hypothalamic neurons compared to the controls. However, pretreatment with 1 μM icariin resulted in a significant increase in the activity of SOD (37.5%) when compared to cells treated with CORT. Together, these results suggest that pretreatment with icariin prevents ROS generation and attenuates changes in SOD activity induced by treatment with CORT (Fig. 3C).

Activation of the PI3-K/Akt signal pathway by CORT and icariin in cultured hypothalamic neurons was determined by measuring phospho-Akt levels. Neurons were pretreated with icariin or control media for 30 min followed by stimulation with CORT for 1, 2 and 3 h. Western blot analysis revealed a significant increase in phospho-Akt at 2 h following treatment with CORT in cultured hypothalamic neurons, similar levels were also recorded in the cells treated for 3 h. In the icariin-treated groups, Akt phosphorylation was increased compared with the CORT-treated control group. As expected, the cells treated with LY294002 demonstrated a significant decrease in the amount of phosphorylated Akt at 2 h (Fig. 4).