Materials
Adult male Wistar rats (4–5 weeks old) were obtained from the Animal Resource Center of Jilin University and housed at 22 ± 2 °C in a controlled environment with a 12-h light and 12-h dark cycle. All rats were treated humanely and in compliance with the recommendations of the animal care committee of Jilin University. We used the minimum number of mice required to draw a conclusion and tried to minimise their suffering as much as possible.
3-(4, 5- Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), and dimethyl sulfoxide (DMSO), L-Glu were purchased from Sigma. Cell culture media, Dulbecco’s modified Eagle’s medium (DMEM), heat-inactivated foetal bovine serum (FBS) and trypsin were supplied by Gibco. Antibodies were purchased from Santa Cruz (CA93446, USA). A Luciferase Reporter Gene Assay Kit was purchased from Beyotime. (RS)-2-Chloro-5-hydroxyphenylglycine (CHPG), a selective mGluR5 agonist and 2-Methyl-6-(phenylethynyl)-pyridine (MPEP), a selective mGluR5 antagonist, were purchased from Selleckchem.
Cell Culture
The SH-SY5Y, HEK293T and 3 T3 cell lines were cultured in DMEM, and the Jurkat cell line was cultured in RPMI1640 medium supplemented with 10 % FBS, 100 units/mL penicillin and 100 units/mL streptomycin (Invitrogen).
Cytotoxicity Assay
In the cytotoxicity assay, cells were seeded at a density of 1 × 104 cells/well in 96-well plates and incubated for 24 h. Cells were treated with pu-erh tea at various concentrations for 24 h. MTT (5 mg/mL, 20 μL) was added to the medium 24 h later. After incubation at 37 °C for 4 h, the supernatant was aspirated and 100 μL of DMSO was added to each well, followed by vibration for 10 min. The absorbance in the experimental wells was measured at 570 nm with a microplate reader.
Affymetrix Microarrays
RNA was extracted with TRI reagent (Sigma) and DNAse I (Invitrogen) from Jurkat in triplicate with or without pu-erh tea treatment for 12 h. RNA quality and concentration were assessed with an Agilent 2100 Bioanalyser and Nanodrop ND-1000. Data from Affymetrix 430 2.0 microarray chips (SCIBLU, Affymetrix) was analysed with Arraystar 3 software (DNA STAR Inc.), which was quantile-normalised and processed by the RMA (Affymetrix) algorithm.
Luciferase Assay
Promoter regions were amplified by PCR amplification and subcloned into the KpnI/HindIII sites of the luciferase reporter vector pGL3-Basic (Promega) upstream of the firefly luciferase gene (Fig. 2). SH-SY5Y and HEK293T cells were transfected with mGluR5 promoter-pGL3 recombinant plasmid by electroporation according to the manufacturer’s instructions. A monolayer of 80 % confluent cells was trypsinised, washed with appropriate media and washed again in ice-cold PBS buffer. Cell suspension for the electroporation was first prepared in 400 μL ice-cold PBS buffer (2.5 × 106 cells/mL) in a 0.4-cm electric rotary cup, followed by adding 10 μg plasmid. The electroporation conditions were adopted from previous work, as follows: U = 225 V, R = ∞, Ω = 1050 μF. After electroporation, cells were seeded in 12-well plates at a density of 4 × 104 cells per well. After 12 h, transfected cells were incubated with pu-erh, black and green teas. Cells were then harvested and firefly luciferase activity was determined using a Sirius luminometer (Berthold Detection System). Experiments were performed in triplicates and repeated at least three times independently.
Western Blot
Cells were prepared in ice-cold lysis buffer (100 mM NaCl, 50 mM Tris-HCl, 1 mM EDTA, 10 mM MgCl2, pH 7.2) containing 1 % Triton X-100, phosphatase and protease inhibitor cocktail (Dinggguo, China) for 10 min. Cell lysates were centrifuged at 12,000g for 10 min at 4 °C, and protein concentrations of supernatants were determined with BCA assay. Lysates were added 5 × SDS containing 100 mM DTT. Equal amounts of protein were loaded onto and separated on 12 % SDS-PAGE gels and transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were incubated with primary antibodies overnight, washed three times in washing buffer (0.1 % Tween-20 in PBS), incubated with HRP-coupled secondary antibodies for 1 h, washed three times in washing buffer and developed using chemiluminescent HRP detection substrate. When proteins were extracted from hippocampus tissue, hippocampus tissues were placed in lysis buffer (as above) and homogenised for 1 min. The homogenate was then centrifuged at 12,000g for 10 min at 4 °C. The final supernatant was collected and processed as above.
Nuclear Staining with Hoechst 33342
SH-SY5Y cells were seeded in 6-well plates (1.5 × 105 cells/well) for 24 h. Cells in a 6-well plate were incubated with pu-erh, black or green teas for 12 h before treatment with L-Glu for 24 h. Cells were washed with PBS and stained with 500 μL of Hoechst 33342 solution for 15 min at room temperature in the dark. The nuclear morphology of the cells was examined under a fluorescent microscope.
Flow Cytometry Analysis of Apoptosis in SH-SY5Y
SH-SY5Y cells were seeded in 6-well plates (1.5 × 105 cells/well) for 24 h. Cells in each 6-well plate were incubated with pu-erh, black or green teas for 12 h before treatment with L-Glu for 24 h. Cells treated with medium alone were used negative control. Apoptosis of cells was evaluated by measurement of the exposure of phosphatidylserine on the cell membranes using Apoptosis Detection Kits. Cell pellets were resuspended in a staining solution containing propidium iodide (PI) for 5 min and Annexin V-FITC for 15 min at room temperature in the dark. The cells were assessed by fluorescence-activated cell sorting (FACS) using the Cell Quest software (BD, Pharmingen).
Calcium Imaging
SH-SY5Y cells were seeded in 6-well plates (1.5 × 105 cells/well) for 24 h. Cells in a 6-well plate were incubated with pu-erh, black, or green teas for 12 h before treatment with L-Glu for 24 h. Cells treated with medium alone were used as negative control. Cells were loaded with 2 μM Fluo-3 AM at 37 °C for 1 h. Cells were suspended in PBS after being washed three times and analysed by a flow cytometry device equipped with the Cell Quest software at 488 nm.
Epileptic Rat Model
Status epilepticus (SE) was induced in adult Wistar rats by using Licl-pilocarpin [21]. Rats were randomly divided into 6 groups with 6 rats in each group. Three groups were administered intragastricly with pu-erh tea at a dose of 0.5 g/Kg/day, the other three groups were treated with saline. After 20 days, lithium chloride was intraperitoneally injected into the four groups rats that two of them were dministered intragastricly with pu-erh tea, the other two were treated with saline at a 127 mg/kg dosage. After 18 h, the rats were treated with a 40 mg/kg dose of pilocarpine, administered intraperitoneally. Two of the groups that one was treated with pu-erh tea and Licl-pilocarpin, the other was treated with saline and Licl-pilocarpin were treated with 4 mg/kg diazepam, before pilocaroine administered intragastricly. Saline was administered to the control group at the same dosage.
Behavioural Study
Rats were video monitored from day 0 to day 15 post status epilepsy (SE) and were scored by an individual blind to the experimental design. Behavioural seizure was recorded according to a modification of the classification of Racine [22]: 0, exploring; 1, immobility; 2, rigid posture; 3, head nodding; 4, bilateral forelimb clonus and falling; 5, continued clonus and falling; and 6, generalised tonus. Three behavioural patterns of SE could be recognised: I, initial (class 1–2), M, middle (class 3) and C, critical (class 4–6). Seizure number and type were recorded within 1 h of the seizure in day 0. Then, seizure number and type were recorded 30 min every 24 h from day 1 to day 15.
Measurements of IP3 and DAG
The measurement of the contents of IP3 and DAG was conducted using IP3 and DAG ELISA kits (RD, USA). Hippocampus tissues were placed in ice-cold PBS and homogenised for 1 min. The homogenate was then centrifuged at 12,000g for 10 min at 4 °C. The final supernatant was collected.
Haematoxylin and Eosin (HE) Staining
Brains were prepared from control and post-SE rats. Animals were anaesthetised using 20 % urethane at a dose of 0.5 mL/kg. Once deeply anesthetised, animals underwent heart perfusion with ice-cold modified PBS followed by 4 % poly-formaldehyde. The brain was removed and dehydrated in 20 and 30 % sucrose. The brains were then embedded in optimal cutting temperature compound (OCT). Six-micrometre-thick slices were made with a tissue slicer and stained in HE solutions to examine morphological changes.
Immunofluorescence
To detect the expression of mGluR5 in rat brains, 6-μm paraffin sections were subjected to antigen retrieval and blocking of endogenous peroxidase activity, followed by incubation with mGluR5 monoclonal antibody (diluted at 1:200) overnight at 4 °C. The sections were incubated with FITC-conjugated anti-mouse IgG (1:250) for 2 h at room temperature. All images were acquired with a fluorescence microscope.
Statistical Analysis
All values were expressed as mean ± S.D. A One-way analysis of variance (ANOVA) was used to detect statistical significance followed by post hoc multiple comparisons (Dunn’s test) by GraphPad Prism 6.0. A value of P < 0.05 was considered to be significant [23, 24].