Contributed equally
Excessive corticosterone (CORT) is acknowledged to induce neuronal damage in a number of regions of the brain, particularly the hippocampus, the main area implicated in depression. However, little research has been conducted on alterations to hypothalamic neurons in depression and the cellular and molecular basis for these changes. In the present study, we aimed to determine whether CORT causes apoptosis in primary cultured hypothalamic neurons, and to investigate the protective effects of icariin, an active natural ingredient from the Chinese plant,
Depression is one of the most common types of mood disorder and is associated with significant disability and mortality. It is projected to become the second leading cause of burden of disease in 2030 (
Considerable evidence suggests that dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis is associated with depression (
Recently, we reported that icariin, a biologically active purified compound from the Chinese herbal plant
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 (
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) (
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 (
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 (
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 (
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 (
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 (
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 (
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 (
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 (
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 (
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 (
The present study demonstrates that icariin prevents CORT-induced apoptosis in primary cultured hypothalamic neurons. Exposure of hypothalamic neurons to CORT resulted in a significant loss of viability and apoptosis of the cells. In parallel, CORT significantly increased the intracellular ROS elevation and decreased SOD activity. However, pretreatment of the cells with icariin prior to CORT exposure noticeably suppressed these CORT-induced cellular events. Furthermore, icariin is able to prevent CORT-induced hypothalamic cell death via activation of the PI3-K/Akt pathway.
Patients with major depression and other neurological afflictions frequently display hyperactivity of the HPA axis (
Several studies have demonstrated that ROS may lead to neuronal apoptosis in neurodegenerative disorders (
The involvement of the PI3-K/Akt signal pathway in neuronal survival involves different mechanisms. To identify the intracellular signaling pathways that mediate the neuroprotective actions of icariin, changes in the phosphorylation of key signaling proteins were analyzed by immunoblots with phospho-specific antibodies. In the present study, 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 an increase in phospho-Akt at 2 h following treatment with CORT in cultured hypothalamic neurons and these levels of phospho-Akt were sustained for at least 3 h. This is consistent with previous studies that revealed CORT increases the phosphorylation of PI3-K/Akt in neurons grown in neurobasal medium supplemented with B27 and 500 μm L-glutamine (NBM+) (
In conclusion, icariin was identified to be able to prevent CORT-induced hypothalamic cell death via activation of the PI3-K/Akt pathway. This study not only demonstrates the potential pharmacological uses of icariin, but also reveals the key neuroprotective role of the PI3-K/Akt pathway.
This study was supported by a grant from the National Basic Science Program of China (No. 2009CB523000) and the National Natural Science Foundation of China (No. 81102562).
corticosterone
phosphoinositide 3-kinase/protein kinase B
3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide
lactate dehydrogenase
reactive oxygen species
hypothalamic-pituitary-adrenal
icarrin
glucocorticoid receptor
mitogen-activated protein kinases
rhodamine 123
mitochondrial membrane potential
superoxide dismutase
Icariin protected primary cultured neuronal cells from CORT-induced neurotoxicity. (A) CORT-induced reduction viability of primary cultured neuronal cells in a dose-dependent manner. Eight-day-old primary hypothalamic neurons were treated with icariin for 2 h prior to a 24-h exposure to 1 μM CORT. Following treatment, (B) cell viability and (C) LDH release were measured. Data represents the mean ± SD of three independent experiments, each was performed in triplicate. Statistical significance using a t-test was set as ##P<0.05, #P<0.01 vs. vehicle control, **P<0.05, *P<0.01 vs. model,⋆P<0.01 vs. icariin group. CORT, corticosterone; ICA, icariin; LDH, lactate dehydrogenase.
Icariin inhibited CORT-induced apoptosis in cultured hypothalamic neurons. (A) Morphological characteristics of cultured cortical neurons were visualized under microscopic analysis. Scale bar = 50 μM. (B) Activation of caspase-3. (C) Mitochondrial function was determined by Rho 123 fluorescence intensity. Scale bar = 1 mm for all images. (D) Rho 123 fluorescence intensity was quantitatively analyzed. Data are expressed as fold over the control group in five to six coverslips from separate cultures. Statistical significance using a t-test was set as ##P<0.05, #P<0.01 vs. vehicle control, **P<0.05, *P<0.01 vs. model, ⋆⋆P<0.01 vs. icariin group. CORT, corticosterone; ICA, icariin; LY, LY294002.
Icariin inhibited CORT-induced intracellular accumulation of ROS and rescued loss of SOD. (A) Intracellular ROS were imaged using the H2DCF-DA fluorescent dye. (B) Quantitative analysis of ROS in cortical neurons was determined by fluorescence microplate reader. (C) Measurement of the SOD. Statistical significance using a t-test was set as ##P<0.05, #P<0.01 vs. vehicle control, **P<0.05, *P<0.01 vs. model, ⋆⋆P<0.01 vs. icariin group. CORT, corticosterone; ICA, icariin; ROS, reactive oxygen species; SOD, superoxide dismutase; LY, LY294002.
Effects of corticosterone and ICA on the levels of phosphorylated Akt and total Akt in cultured hypothalamic neurons. The intensity of the bands for the phosphorylation of Akt and total Akt was analyzed by western blot analysis. The experiments were repeated three times independently, one representative result is shown. ICA, icariin.