Geniposide protects human neuroblastoma SH-SY5Y cells against corticosterone-induced injury＊☆

Liping Chen, Fawei Wang, Miao Geng, Hongyan Chen, Dongmei Duan

1Department of Traditional Chinese Medicine, General Hospital of PLA, Beijing 100853, China

2Institute of Gerontology, General Hospital of PLA, Beijing 100853, China

lNTRODUCTlON

Chronic stress can induce hypothalamicpituitary-adrenal axis hyperfunction and increases in adrenal cortical hormones,resulting in hippocampal hormone receptor overstimulation neuronal injury and dysfunction[1-3].

Cape jasmine is the dried fruit of Gardenia jasminoides Ellis. The main active component of the Chinese herb Fructus Gardeniae is geniposide and genipin[4].

Interestingly, the metabolite of geniposide can suppress inflammation and oxidative stress[5]. Genipin can inhibit xylene- and carrageenan-induced external ear swelling, formaldehyde-induced toe swelling and hydroxyl radical-induced lipid peroxidation in mice[6-7]. Crude extracts of cape jasmine significantly improved the depressive behavior of mice and promoted neurogenesis in the hippocampus[8]. Chinese medicinal formulae containing cape jasmine have been shown to improve clinical symptoms of depression in patients[9]. Therefore, we hypothesized that geniposide may influence growth of in vitro cultured SH-SY5Y cells exposed to high-doses of corticosterone.

This study established a high-dose corticosterone injury model using SH-SY5Y cellsl[10-11]to investigate the protective effect of geniposide on SH-SY5Y cells.

RESULTS

lnfluence of geniposide on cell viability of corticosterone-injured SH-SY5Y cells

The effect of geniposide on the survival rate of SH-SY5Y cells exposed to corticosterone was tested using the chi square test. After exposure to corticosterone for 48 hours(model group), the methyl thiazolyl tetrazolium (MTT) assay showed that corticosterone significantly reduced SH-SY5Y cell survival when compared to untreated cultures (P ＜ 0.01). However, pretreatment with geniposide (50, 5 μg/mL) significantly increased the survival rate of SH-SY5Y cells when compared to non- pretreated cells(96% vs. 41%, P ＜ 0.05), indicating the protective effect of geniposide against corticosterone-induced injury (Figure 1).

Figure 1 Effect of geniposide on the cell viability of SH-SY5Y cells injured by a high-dose of corticosterone using the methyl thiazolyl tetrazolium assay. Geniposide G1: 5 μg/mL geniposide; Geniposide G2: 50 μg/mL geniposide. aP ＜ 0.01, vs. normal group; bP ＜0.05, vs. model group. Data are expressed as mean. Survival rate= Absorbanceexperimental group/Absorbancecontrol group×100%.

Effect of geniposide on morphology of SH-SY5Y cells following corticosterone injury

Following Hoechst 33258 fluorescence staining, nuclei in control cultures appeared normal and evenly stained,and only 0.7 ± 0.3% of cells had apoptotic nuclei. After treatment with a high-dose of corticosterone for 48 hours,nuclei of SH-SY5Y cells changed and cell body area reduced, with 2.3 ± 0.3% cells exhibiting apoptotic nuclei.

After low-dose geniposide pretreatment, the number of apoptotic cells was similar to the model group, while a high-dose of geniposide resulted in a significant decrease in the number of apoptotic cells when compared to the model group (2.3 ± 0.3% vs. 13.67± 1.20%,respectively, P ＜ 0.05), indicating geniposide can attenuate high-dose corticosterone-induced cell injury(Figure 2).

Figure 2 Effect of geniposide on morphology of SH-SY5Y cells injured by a high-dose of corticosterone using Hoechst 33258 and fluorescence microscopy. Scale bar: 100 μm. Nuclei of normal cells were stained blue,while nuclei of apoptotic cells were dense and darkly stained, which were accompanied by pyknosis and white cell bodies.

Effect of geniposide on the cell cycle of corticosterone-injured SH-SY5Y cells

P53 protein is a phosphorylated protein in the nuclei that regulates the cell cycle and apoptosis[12]. P21 is a cell cycle-associated protein[13]. Flow cytometry results showed that the number of SH-SY5Y cells in the G1 and S phase of the cell cycle significantly increased (P ＜0.05), but there was a significant reduction in the number of cells in the G2 phase after high-dose corticosterone injury (P ＜ 0.05). After geniposide treatment, the number of SH-SY5Y cells in the G2 phase significantly increased (P ＜ 0.05). However, the number of cells in the S phase was significantly decreased (P ＜ 0.05; Figure 3,Table 1).

Figure 3 Effect of geniposide on the rate of apoptosis and the cell cycle of SH-SY5Y cells injured by a high-dose of corticosterone. Grey area: apoptosis, first red peak: G1 phase; second red peak: G2 phase; striped area: S phase.

Table 1 Changes in apoptosis and cell cycle of corticosterone-injued SH-SY5Y cells after geniposide treatment

Geniposide inhibited P53 and P21 expression in corticosterone-injured SH-SY5Y cells

Western blot results showed that P53 and P21 expression was increased in corticosterone-injured SH-SY5Y cells when compared with untreated control cultures (P ＜ 0.05). After geniposide treatment, P53 and P21 expression decreased (P ＜ 0.05), indicating geniposide can downregulate P53 and P21 expression in corticosterone-injured SH-SY5Y cells (Figure 4, Table 2).

Figure 4 Effect of geniposide on P53 and P21 protein expression in SH-SY5Y cells injured by a high-dose of corticosterone. 1: Normal group; 2: model group; 3:low-dose geniposide group; 4: high-dose geniposide group.

Table 2 Effect of geniposide on P53 and P21 protein expression in corticosterone-injured SH-SY5Y cells (Gray value)

DlSCUSSlON

The major active component of cape jasmine is ridoids,and geniposide is the most active constituent.

Geniposide can be acid hydrolyzed to form genipin[14],which has been shown to mitigate stress, act as an anti-inflammatory, protect against liver dysfunction, have antioxidant properties, and act as a neuroprotectant[7-9,15-16].

The hippocampus is an important constituent of the limbic system, which can regulate emotion, learning,memory and behavior[17-18]. Hippocampal morphology and molecular changes have been observed after chronic stress reaction[19]. The hippocampus contains abundant neurotransmitters and receptors and is fragile to stress injury[20]. Long-term chronic stress can induce abnormal hormone production and stimulate hippocampal hormone receptors, resulting in neuroendocrine disturbance and monoamine neurotransmitter imbalance, which manifests as a lack of interest in the surrounding environment, reduced appetite, and reduced learning and memory capacity[21-22].Hyperfunction of the hypothalamic-pituitary-adrenal axis is gradually restored with ameliorated depression in patients[23-24].

This study is the first to report the protective effects of geniposide on high-dose corticosterone-induced SH-SY5Y cell injury. That is, geniposide can significantly improve survival rate and reduce apoptosis of SH-SY5Y cells. Fluorescence staining showed that SH-SY5Y cells became sparse and cell body area was reduced after high-dose corticosterone treatment for 48 hours, but these morphological changes were restored after high-dose geniposide treatment. Flow cytometry results showed that the number of SH-SY5Y cells undergoing G1 and S phase of the cell cycle increased after high-dose corticosterone exposure. However, the number of cells at the G2 phase decreased. In contrast, after geniposide pretreatment, the number of SH-SY5Y cells at the G2 phase increased, and the number of cells in the S phase decreased. These results demonstrate that geniposide can strongly inhibit SH-SY5Y cell apoptosis possibly by regulating P53 and P21 protein expression.

External stimuli such as chemical pollution, ion radiation-induced DNA injury can increase P53 protein levels[25], which can activate downstream target gene transcription. P53 protein controls the transition of cells from the growth arrest stage, i.e. G0 phase, to the G1 phase,and regulates cell proliferation after cells enter the cell cycle. When cells at G1 phase are injured, P53 can trigger a cell cycle restriction point to arrest cell growth[26],but if injured cells have entered the S phase, P53 can trigger apoptosis[27]. Studies have demonstrated that P21 is also involved in DNA injury repair and apoptosis[28-29].

Western blot results revealed that a high-dose corticosterone significantly increased P53 and P21 protein expression in SH-SY5Y cells, which increased apoptosis.

High-dose geniposide decreased P53 and P21 protein expression, indicating geniposide inhibited corticosterone-induced SH-SY5Y cell apoptosis by regulating P53 and P21 protein expression.

In conclusion, geniposide can significantly inhibit SH-SY5Y cell apoptosis induced by high-dose corticosterone, and regulate P53 and P21 protein expression.

However, apoptosis and the cell cycle are multifactorially regulated, further studies are needed to verify our results.

MATERlALS AND METHODS

Design

In vitro cell culture experiment.

Time and setting

The experiment was performed in the laboratory of the Institute of Gerontology, General Hospital of PLA, China from March 2010 to March 2011.

Materials

Cells

The human dopaminergic neuroblastoma cell strain(SH-SY5Y) was graciously provided by the Academy of Military Medical Sciences.

老道也看出王祥有跑路的想法，不过王祥许诺把剩下的玉器也留给自己当做补偿，也就没有要王祥为难，痛痛快快地和他立了字据。

Drugs

Geniposide, C17H24O11, was purchased from the National Institutes for Food and Drug Control (No.110749-200814).

Methods

SH-SY5Y cell culture

SH-SY5Y cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% (v/v) fetal bovine serum (FBS; Hyclone, Logan, Utah, USA), 100 U/mL penicillin and 100 U/L streptomycin in 5% CO2at 37°C. The culture solution was replaced every week.Cells reaching 90% confluency were trypsinized (0.25%(w/v); Gibco, Carlsbad, CA, USA) to detach and passage cells. Cell density was adjusted to 1×105/mL and seeded in 96-well culture plates with 80 μL per well.

Corticosterone-induced injury and geniposide treatment

SH-SY5Y cells at a density of 1×105/mL were assigned to the control, model, high-, and low-dose geniposide groups. After culture for 12 hours[30-31], cells from the high- and low-dose geniposide groups were treated with 50, and 5 μg/mL geniposide, respectively, for 12 hours.Cells from the two geniposide groups and the model group were than cultured with 10 μL corticosterone(400 μmol/L; Sigma, St. Louis, MO, USA) for 48 hours[32].

At 48 hours after corticosterone treatment, cells at the logarithmic phase were harvested and their density was adjusted to 1×104/mL. The cells were cultured in 5% CO2at 37°C with MTT (5 g/L; 20 μL per well; Sigma) for 4 hours. When ianthinus crystals formed, the supernatant was discarded. Dimethyl sulphoxide (Sigma; 150 μL)was added to each well, shaken for 10 minutes to dissolve the crystals, and the absorbance was measured at 570 nm using a Polar star Galaxy microplate reader(BMG, Offenburg, Germany). The mean absorbance of six wells was calculated. Survival rate= Absorbanceex-perimentalgroup/Absorbancecontrolgroup×100%. The procedures were repeated in triplicate.

Detection of SH-SY5Y cell morphology after corticosterone and geniposide treatment using Hoechst 33258 fluorescence staining

SH-SY5Y cells (1×104/mL) from each group were incubated for 30 minutes at 37°C in freshly prepared 4% (w/v)paraformaldehyde (pH 7.14), washed with PBS twice,mixed with 5 mg/L Hoechst 33258 staining solution(Sigma) for 30 minutes, washed with PBS, mounted with mounting solution (20 mmol/L citric acid, 50 mmol/L sodium phosphate, 50% (v/v) glycerol; pH 5.15) and observed by fluorescence microscopy (Olympus, Hatagaya,Japan). The number of normal nuclei and apoptotic nuclei in five fields of view were recorded to calculate the percent of apoptotic nuclei out of the total number of nuclei counted.

Detection of P21 and P53 protein expression in SH-SY5Y cells after corticosterone and geniposide treatment by western blot

SH-SY5Y cells at 80% confluency were collected, and mixed with 0.5 mL pre-cooled cell lysis solution (Pierce,Rockford, IL, USA) at 4°C for 30 minutes, followed by incubation in a water bath at 37°C for 10 minutes. Protein was quantified according to the BCA protein quantitation kit (Pierce). Equal amounts of protein were denatured at 98°C for 10 minutes, cooled at room temperature and stored at 4°C. Protein samples were separated by 12%SDS polyacrylamide gel electrophoresis, and GAPDH protein was used as an internal reference. The target and internal reference proteins were transferred to two nitrocellulose membranes, blocked with 5% (w/v) skim milk, incubated with 2 mL rabbit anti-human p21, p53 and GAPDH monoclonal antibodies (1: 100; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 37°C for 2 hours, washed with Tris buffered saline, followed by incubation in horseradish peroxidase-labeled goat anti-rabbit IgG (1: 500; Santa Cruz Biotechnology) at 37°C for 1 hour. The protein was colorized with diaminobenzidine. The protein bands were scanned and gray value was analyzed using Gel-ProAnalyzer (Version 3.0;Media Cybernetics, State of Washington, USA).

Detection of SH-SY5Y cell apoptosis and cell cycle analysis after corticosterone and geniposide treatment using propidium iodide staining

After SH-SY5Y cells were treated with drugs for 48 hours,they were centrifuged at 1 000 r/min for 10 minutes,washed with PBS twice, fixed with 70% (v/v) cold ethanol at -20°C for 24 hours, centrifuged at 1 000 r/min for 10 minutes and washed with PBS twice. Cell density was adjusted to 1×106/mL. Cells were mixed with RNase(Sigma) at a final concentration of 100 μg/mL and placed in a water bath at 37°C for 30 minutes. Propidium iodide(Sigma) was added at a final concentration of 50 μg/mL,left at 4°C for 30 minutes in the dark, and filtrated through a 350-mesh nylon web. DNA content at each phase was determined using an Epics XL flow cytometer (Coulter,CA, USA) with an excitation wavelength of 488 nm and an emission wavelength of 610 nm. The sub-G1 peak that was lower than the DNA content at the G1 phase in the DNA distribution histogram was regarded as the number of apoptotic cells. The procedures were performed in triplicate.

Statistical analysis

Enumeration data were expressed as the mean ± SD.

One-way analysis of variance was performed using SPSS 13.5 (SPSS, Chicago, IL, USA). Intergroup differences were compared determined by the F-test using CHISS software (Beijing Yuanyitong Technology, Beijing,China). The size of the test was α =0.05.

Author contributions:Liping Chen conceived and designed this study, and wrote the draft of the manuscript. Fawei Wang was in charge of funds. Miao Geng and Hongyan Chen provided technical and data support. Dongmei Duan contributed to the evaluation and interpretation of the study.

Conflicts of interest:None declared.

Funding:This study was supported by the Capital Specific Clinical Medical Subject of Beijing Science and Technology Commission (Clinical study of Shuyu Ningxin treatment for heart and liver disorder in depression), No.Z090507017709030.

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