In this study, we screened the CHMs and their active compounds that could improve the symptoms of SCA17.
Three steps were set up:
(1) To study whether CHMs and the active phytochemicals inhibit the neurotoxicity mediated by glutamate by using the SH-SY5Y cell model
(2) To investigate the effects of active phytochemicals of CHMs on the cytotoxicity of nTBP/Q36-EGFP and nTBP/Q79-EGFP by using inducible SCA17 cell method
(3) To study that the phytochemicals improve defective phenotypes in the transgenic mice by SCA17 transgenic mice model
3 Materials and Methods 3.1 Materials
Human neuroblastoma SH-SY5Y cells were from ATCC.
Dulbecco’s Modified Eagle Medium with nutrient mixture F-12 (DMEM/F12), 0.5% Trypsin-EDTA, penicillin/streptomycin (P/S), and Fluo-4 AM were obtained from Invitrogen Corporation. Fetal bovine serum (FBS) was from Falcon.
Primary antibodies against Calpain-2, Bax, cleaved PARP, cleaved caspase-9, and cleaved caspase-3 were obtained from Cell Signaling Technology. Cytochrome C, Bcl-2, TBP (1C2) and TBP (N12) were purchased from Santa Cruz Technology.
Actin and SBDPs was purchased from Millpore Corporation.
Secondary antibodies of Horseradish peroxidase (HRP)-conjugated goat anti-mouse secondary antibody and goat anti-rabbit secondary antibody were obtained from Minipore Corporation.
3- (4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and retinoic acid (RA) was purchased from Sigma-Aldrich. Annexin V-FITC assay Kit was supplied from
BioVision Corporation. MitoProbe™ JC-1 Assay Kit was obtained from Invitrogen Corporation. Protease inhibitors cocktail were obtained from Roche Applied Science. Pure compounds were supplied by Sigma-Aldrich.
3.2 Cell culture
Human neuroblastoma SH-SY5Y cells, nTBP/Q36-EGFP cells and nTBP/Q79-EGFP cells, kindly supplied by Dr.
Guey-Jen Lee-Chen, National Taiwan Normal University (NTNU), were cultured in DMEM/F12 supplemented with 10%
FBS, 100 units/mL penicillin and 100 ug/mL streptomycin at 37 ºC in a 5% CO2 humidified incubator, and cells were passaged until 80% to 90% confluency in a one tenth ratio.
3.3 MTT assay
This assay is based on the ability of succinate dehydrogenase to convert MTT into water-insoluble purple formazan crystals. Human SH-SY5Y neuroblastoma cells were plated in 96-well plates (2 x 104 cells/well). After 24 h, cells were pre-treated with the various concentrations of NH043-1 or
10 uM MK801 (NMDA receptor antagonist) for 1 h, and then challenged with 100 mM glutamate for 24 h.
To investigate the effects of NH043-1 on SCA17 inducible cell, nTBP/Q36-EGFP and nTBP/Q79-EGFP were seeded in 96-well plate (2 x 104 cells/well). After 24 h, cells were pre-treated with the various concentrations of NH043-1 for 1 d, and then challenged with 10 ug/mL doxycycline and retinoic acid (RA) for 1, 3, and 5 d.
After the treatment, 0.5 mg/ml MTT was added to culture media, and cells were incubated for 3 h at 37oC. To dissolve the formazan crystals, 100 uL lysis solution [10% sodium dodecyl sulfate (SDS) and 0.01N HCl] was added and the absorbance was read at OD570 with an ELISA reader (uQuant, bio-tek INK, Vermont, USA). The blank control wells were used for zeroing absorbance. The percentage of cell viability was calculated as follows:
Cell viability (%) = (OD570 of experimental well / OD570 of control well) x 100%
3.4 Flow cytometric measurement of apoptotic cells
The apoptosis induced by glutamate was measured by flow cytometry by using Annexin V-FITC/propidium iodide (PI) double-labeling method. SH-SY5Y cells were seeded (1 x 106 cell/well) in 6 cm dish, and were treated with 100 mM glutamate and 15 uM NH043-1 or 10 uM MK801 for 24 h. Apoptotic cells were then trypsinized and collected by centrifugation at 2,000 rpm for 3 min. After washing with PBS, cells were then double-stained with Annexin V-FITC and PI. According to the manual, cells were resuspended in Annexin V-FITC binding buffer, incubated with Annexin V-FITC for 30 min, and were then incubated with PI. Samples were analyzed with a flow cytometery (FACS Sorter, Becton, Dickinson and Company) by two parameter-dot-plots, and a total of 10,000 cells were recorded in each case.
3.5 Western blotting
3.5.1 Preparation of cell lysates
Human SH-SY5Y neuroblastoma cells were treated with NH043-1 at indicated concentrations or 10 uM MK801 for 1 h, then challenged with 100 mM MSG. The nTBP/Q36-EGFP and nTBP/Q79-EGFP cells were treated with indicated concentrations of NH043-1, and then treated with 10 ug/mL doxycycline and RA for 5 d, washed with phosphate buffered saline (PBS), and then lysed with radioimmounoprecipitation buffer (RIPA) (150 mM NaCl, 50 mM Tris-HCl, 6 mM deoxycholate, and 1% Nonidet P-40 at pH 7.4) on ice for 5 min, followed by sonication and centrifugation at 13,000 rpm for 5 min at 4oC.
3.5.2 Quantification of protein concentration
The protein concentrations were determined with bicinchoninic acid (BCA) protein assay kit (Thermo scientific). A standard curve was generated by serial dilution of bovine serum albumin (BSA) solutions, 800,
400, 200, 100, and 50 ug/mL in ddH2O in a 96-well plate. One uL protein sample was mixed with 19 uL ddH2O in well, and the BCA reagent was prepared by mixing 20 uL of solution A and 1 mL of solution B, then 100 uL of mixed solution was added to each well.
The plate was incubated at 37oC for 30 min, and absorbance was read at OD562 with an ELISA reader.
3.5.3 Preparation of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)
The separating solution was loaded to the glass sandwish, and 0.2% SDS was added slowly into the glass sandwish. After the separating gel became solid, upper liquid was removed, and the stacking solution was added into the glass sandwish. The gel was prepared after the gel was polymerized (Table 1).
Table 1. The formula of SDS-PAGE
Separation gel Stacking
gel
Percentage 7.5% 10% 12.5%
A solution 1.50 mL 2.00 mL 2.50 mL 0.45 mL B solution 1.50 mL 1.50 mL 1.50 mL
C solution 0.75 mL
ddH2O 3.00 mL 2.50 mL 2.00 mL 1.80 mL
TEMED 9.0 uL 4.5 uL
10% APS 60.0 uL 13.0 uL
A solution: 30% acrylamide/bis-acrylamide.
B solution: 1.5M Tris-HCl and 0.4% SDS at pH 8.8.
C solution: 0.5M Tris-HCl and 0.4% SDS at pH 6.8.
TEMED: Tetramethylethylenediamine APS: Ammonium persulfate.
3.5.4 Protein sample preparation
Protein samples were mixed with 4X protein loading dye (125 mM Tris-HCl, 4% SDS, 30% glycerol, and 0.2% bromophenol blue at pH 6.8), and heated at 95oC for 5 minutes.
3.5.5 Electrophoresis
Equal amount of protein samples (40 mg) and the standard protein molecular weight marker were resolved on 7.5% or 12.5% SDS polyacrylamide gel electrophoresis. The electrophoresis apparatus was linked to an electric power provider with 70 V of the voltage and 20 mA of the current.
3.5.6 Semi-dry blotting
To perform semi-dry blotting, a polyvinylidene fluoride (PVDF) membrane and filter papers were cut and soaked with methanol, anode I buffer (0.3 M Tris-HCl and 10% methanol, pH 10.4), anode II buffer
(25 mM Tris-HCl and 10% methanol, pH 10.4), and cathode buffer (25 mM Tris-HCl, 40 mM glycine, and 10% methanol, pH 9.4). The transfer board was prepared on the experimental table, then one piece of paper with anode I buffer was plated on transfer board.
Two pieces of paper with anode II buffer were placed on top of paper. The PVDF membrane was put on top of it, then, the gel was putted on the PVDF membrane, and three pieces of paper with cathode buffer were set on the top. The transfer apparatus was linked to electric power provider with 30 V of the voltage and 70 mA of the current for 80 min.
3.5.7 Immunoblotting
After semi-dry blotting, the PVDF membrane was stained with Ponceau S to verify the protein transferred to membrane. The PVDF was washed with TBST buffer (1M Tris-HCl, 5M NaCl, and 0.1% Tween-20, pH 7.4) to de-stain. The PVDF membrane was blocked with 5% non-fat milk solution in TBST at room temperature for 1 h or overnight at 4 oC. The PVDF
membrane was washed thrice with TBST for 5 min, then, incubated with primary antibody in dilute buffer (1% BSA, 0.05% Tween-20, and 0.02% NaN3) at room temperature for 3 h or overnight at 4oC. After three quick washes in TBST buffer for 5 min, the membrane was treated with HRP-conjugated secondary antibodies for 1 h, and the proteins were visualized by using an enhanced chemiluminesence (ECL) detection reagent (Millipore) and detected with an ImageQuestTM LAS-4000 (Fujifilm Co., Tokyo, Japan). The expressions of protein were quantified by ImageJ (National Institute of Health, USA).
The percentage of neuroprotection was estimated as following: percentage of neuroprotection = 100%-[(X-Z)/(X-Y) x100%], X, the intensity of protein in control sample, Y, the intensity of protein in negative control, Z, the intensity of protein in CHMs-pretreated sample.
3.6 Measuring reactive oxygen species (ROS) activity in vitro by Chemiluminescence (CL)
The effects of NH043-1 on the ROS generations by glutamate-induced excitotoxicity in SH-SY5Y cells were measured by using CL analysis system. SH-SY5Y cells were plated in 6 cm dish (1 x 106 cells/dish) for 24 h, and the cells were pre-treated with 15 uM NH043-1 or 10 uM MK-801 for 1 h, then challenged with 100 mM MSG for 24 h. Then the cells were washed with PBS and lysed with 60 uL RIPA on ice for 5 min, and sonicated and centrifuged at 13,000 rpm for 20 min at 4oC. The 200 uL protein samples (20 ug) were mixed with 0.5 mL of 0.2 mM luminal (5-amino-2, 3-dihydro-1, 4-phthalazinedione, Sigma). After 5 min, CL was measured by the amounts of chemiluminescence with a CL analysis system (CLD-110, Tohoku Electronic Inc. Co., Sendai, Japan).
3.7 Measuring mitochondrial membrane potential in vitro by flow cytometery
The effects of NH043-1 on the mitochondrial membrane potential by glutamate-induced excitotoxicity in SH-SY5Y cells
were measured by using flow cytometry with JC-1 dye staining.
SH-SY5Y cells were plated in 6 cm dish (1 x 106 cells/dish) for 24 h, and then the cells were pre-treated with 15 uM NH043-1 or 10 uM MK-801 for 1 h, followed by being challenged with 100 mM MSG for 12 h. The cells were washed and collected by centrifugation at 2000 rpm for 3 min, and after washing with PBS, the cells were stained using JC-1 dye for 30 min. For positive control group, 5 uM carbonyl cyanide 3-chlorophenyl hydrazine (CCCP) was added to induce mitochondrial membrane depolarization. According to the kit’s protocol, the cells were washed and re-suspended in PBS, and analyzed with a flow cytometer by two parameter dot-plots. A total of 10,000 cells were recorded in each case.
3.8 Dot-blot filter retardation assay
The effects of NH043-1 on the nTBP/Q36-EGFP and nTBP/Q79-EGFP aggregation in nTBP/Q36-EGFP and nTBP/Q79-EGFP SH-SY5Y cells, respectively, induced by doxycycline were measured by using BRL dot-blot instrument (Bio-Rad). The nTBP/Q36 and nTBP/Q79 cells were plated in 6 cm dish (1 x 106 cells/dish) for 24 h, and the cells were
pre-treated with 15 uM NH043-1 for 1 h, then treated with 10 uM doxycycline and 10 uM RA for 5 d. The cells were washed and collected by centrifugation at 2,000 rpm for 3 min at 4 oC, and then removed PBS. The cells were incubated on ice for 30 min in the buffer (100 mM NaCl, 5 mM MgCl2, 50 mM Tris-HCl pH 8.8, 0.5% NP-40, 100 mM EDTA, and cocktail).
After centrifugation at 14,000 rpm for 5 min at 4 oC, the precipitates were dissolved with the buffer (15 mM MgCl2, 20 mM Tris-HCl pH 8.0, and 0.5 mg/mL DNase I) at 37 oC for 60 min. The protein concentrations were determined with bicinchoninic acid (BCA) protein assay kit (Thermo scientific).
Twenty mg protein was mixed with 2% SDS solution to 0.2 mL and loaded to dot-blot machine through a cellulose acetate membrane. Membrane was washed with 0.1% SDS solution, blocked with 5% non-fat milk in TBST solution at room temperature for 1 h, and washed thrice with TBST for 5 min, then, incubated with primary TBP (N-12) antibody (1:1000) in dilute buffer overnight at 4oC. After three quick washes in TBST buffer for 5 min, the membranes were treated with HRP-conjugated secondary anti-rabbit antibodies for 1 h, and the proteins were visualized by using an enhanced chemiluminesence (ECL) detection reagent (Millipore), and
detected with an ImageQuestTM LAS-4000 (Fujifilm Co., Tokyo, Japan). The expressions of protein were quantified by ImageJ (National Institute of Health, USA)
3.9 Animal model
SCA17 transgenic mice bearing expanded TBP (Q109) were kindly supplied by Dr. Hsiu-Mei Hsieh, NTNU. Mice were housed in individually ventilated cages with a 12 h light/dark cycle. All mice were bred and maintained in the animal facility at NTNU under specific pathogen-free conditions in accordance with institutional guidelines of The Animal Care and Use Committee at NTNU. The animals were used for motor behavioral assessments, footprint test, and western blotting analysis of aggregated TBP and cleaved-caspase-3 of the mouse cerebellum. The body weights of mice for rotarod test, and footprint test were measured twice a month.
3.9.1 Motor behavioral assessments
The mice were trained on the rotarod before drug treatment to establish the baseline of behavior activity
by using a linear acceleration from 2 rpm to 20 rpm over 5 min, and then maintained at 20 rpm for another 5 min. For experimental period, mice were placed with a linear acceleration from 4 to 30 rpm with over 5 min.
The rotarod analysis was performed every two-week until 20th week. On every test, mice were subjected to three trials, each with a maximum duration of 600 sec and latency of fall was recorded. Mice were tested between 12:00 and 18:00.
3.9.2 Footprint patterns analysis
The test is widely used to determinate motor skill, coordination and balance. Hind- and forefeet of mice were coated with red and blue nontoxic paint, and the animals were allowed to walk along a runway over a fresh sheet of white paper. The distances between the center of the hind and preceding front footprint, length of steps and distance of parallel fore-paw/hind-paw were recorded over a sequence of six consecutive steps, excluding footprints made at the beginning and end of the run. The footprint patterns were determined
quantitatively by the measurements of stride length and fore-paw/hind-paw overlap. The tests were measured by every half of month.
3.9.3 Western blot analysis of aggregated TBP and cleaved-caspase-3 protein in the cerebellum of tested mice
All mice were anesthetized using urethane (1.5 mg/kg, i.p), and after 10 min, cerebella of mice (n=6, for each group) were collected in cold RIPA buffer, homogenized by 22G and 26G (32 mm and 13 mm) needles (TERUMO® Needle, NEOLUS) on ice, and stored at -80oC. The protein concentrations of homogenates of cerebella were measured with bicinchoninic acid (BCA) protein assay kit (Thermo scientific). Then, 40 mg protein was resolved on 12.5%
SDS-PAGE, and after electrophoresis and semi-dry blotting, the PVDF membrane was blocked with 5%
non-fat milk solution in TBST at room temperature for 1 h or overnight at 4oC. The PVDF was washed thrice with TBST for 5 min, then, incubated with primary
antibody in dilute buffer overnight at 4oC. After three quick washes in TBST buffer for 5 min, the membrane was treated with HRP-conjugated secondary antibodies for 1 h, and the proteins were visualized by using an enhanced chemiluminesence (ECL) detection reagent (Millipore) and detected with an ImageQuestTM LAS-4000 (Fujifilm Co., Tokyo, Japan). The expression of proteins was quantified by ImageJ (National Institute of Health, USA).
4 Results
4.1 NH043-1 effectively protects SH-SY5Y cells against MSG-induced excitotoxicity.
MTT assay, a colorimetric assay, was performed for measuring the activity of cellular enzymes representing the cell viability. MSG was used to induce excitotoxicity in neuroblastoma SH-SY5Y cell. First, cells were treated with 20, 40, 60, 80, and 100 mM MSG for 24 h to determine the IC50
toxicity of MSG. The IC50 toxicity of MSG was determined to be 100 mM for 24 h (Figure 1A), and 100 mM MSG was used with following experiments. Then, the IC50 of active compounds of CHMs on SH-SY5Y cells were measured, and half or one-fifth of compounds IC50 (1/2X or 1/5X IC50) were used to screen the most effective compound against MSG. As shown in Table 1, NH043-1 exhibited 85% increased of cell viability, compared to MSG. Therefore, NH043-1 was chosen for further studies.
The treatment of SH-SY5Y cells with 5, 10, 15, 20, and 25 uM of NH043-1 against MSG for 24 h showed that 15 uM of NH043-1 significantly protected the cells against 100 mM MSG-induced excitotoxicity, exhibiting 85% cell viability, as 10
uM MK801 did. MK801 is an antagonist of NMDA receptor and was used as positive control in this study (Figure 1B).
Collectively, our results indicate that NH043-1 showed a remarkably inhibitory effect on MSG-induced excitotoxicity.
4.2 NH043-1 attenuates the apoptosis of SH-SY5Y cells induced by MSG.
To examine whether NH043-1 inhibits apoptosis of SH-SY5Y cells mediated by MSG, flow cytometry analysis using Annexin-V staining, which is an apoptosis marker, was carried out. The results demonstrated that the treatment of 100 mM MSG for 24 h induced 54.13% apoptotic cells, however, NH043-1 at 15 uM and MK801 at 10 uM caused 9.04% and 9.73% apoptosis in the presence of MSG, respectively (Figure 2).
It indicates that NH043-1 effectively reduced apoptosis of SH-SY5Y cells induced by MSG.
4.3 NH043-1 decreases the expressions of calpain-2 and SBDPs in SH-SY5Y cells treated with MSG.
To study whether NH043-1 protects SH-SY5Y cell apoptosis against MSG-induced excitotoxicity via calcium-dependent apoptosis, Western blot analysis was performed. Calpain-2, a thiol proteinase, is activated by the increases of intracellular free calcium ions and the reduction of Bcl-2 level, and SBDPs was elevated by calcium-induced calpain-2 in glutamate-induced cell death (Anderton et al. 2011, Miao et al. 2012). The calpain-2 and SBDPs were investigated.
The 20% increments of both calpain-2 and SBDPs respectively were found by treating the cells with 100 mM MSG for 6 h. By pretreatment with 15 uM NH043-1 and MSG for 6 h, the expression of calpain-2 and SBDPs were decreased 200% and 250% (Figure 3). It indicates that NH043-1 significantly protected cell apoptotic death through calcium-induced apoptosis in MSG-treated SH-SY5Y cells.
4.4 NH043-1 decreases the expressions of Bax, but increases that of Bcl-2 in SH-SY5Y cells treated with MSG.
To study whether NH043-1 protects SH-SY5Y cell apoptosis against MSG-induced excitotoxicity via mitochondria-dependent apoptosis, Western blot analysis was performed. The increases of intracellular free calcium ions were related to the reduction of Bcl-2 level and the increment of Bax level (Anderton et al. 2011, Miao et al. 2012). The cell-survival protein, Bcl-2, and pro-apoptotic protein, Bax, which both mediate the release of cytochrome C from mitochondria, were investigated. The 50% increments of Bax, and the 40%
decrement of Bcl-2, respectively were found by treating the cells with 100 mM MSG for 6 h. By pretreatment with 15 uM NH043-1 and MSG for 6 h, the expression of Bax were decreased 100%, while that of Bcl-2 was increased 150%
(Figure 4). It indicates that NH043-1 significantly protected cell apoptotic death through mitochondria-dependent apoptosis in MSG-treated SH-SY5Y cells.
4.5 NH043-1 inhibits the expressions of caspase family proteins mediated by MSG.
Caspases, a family of cysteine proteases implicated in classical apoptotic death, were initially shown to play a role in delayed excitotoxic injury, which is associated with caspase-9, caspase-3 and nuclear enzyme PARP activation. To study whether NH043-1 protects SH-SY5Y cells death by MSG-induced excitotoxicity through caspase-dependent apoptosis, Western blot analysis was carried out. Treating the cells with 100 mM MSG for 24 h resulted in the 40%, 60%, and 190% increases of cleaved-caspase-9, cleaved-caspase-3, and cleaved-PARP respectively. By treating 15 uM NH043-1 against MSG for 24 h, the expressions of cleaved-caspase-9, cleaved-caspase-3, and cleaved-PARP, were significantly decreased 125%, 87%, and 52% inhibition, respectively, that of MSG treated cells, suggesting that NH043-1 increased cell viability by inhibiting the expressions of caspase family proteins in MSG-treated SH-SY5Y cells (Figure 5).
4.6 NH043-1 inhibits the intracellular ROS induced by MSG.
The glutamate-induced cytotoxicity was associated with mitochondrial dysfunction and increased ROS production in neuronal cells (Votyakova and Reynolds 2005). To investigate that NH043-1 would inhibit the accumulation of ROS implicated by MSG-induced excitotoxicity, the ROS level was measured by CL assay. Luminol is activated with an oxidant to exhibit its chemiluminescence. The emission spectrum is blue glow and can be detected with a chemiluminescence detector. We found that the intracellular ROS was 80% increment by 100 mM MSG-induced excitoxtoicity, but extracellular ROS was not affected. By treating with 10 uM NH043-1 or 10 uM MK801 for 24 h after MSG treatment, the intracellular ROS was 80%
inhibition and the extracellular ROS did not alter (Figure 6). The results suggested that NH043-1 reduced the intracellular ROS production induced by MSG.
4.7 NH043-1 blocks the decrease of mitochondrial membrane potential (MMP) mediated by MSG.
To study whether NH043-1 blocks the decrease of MMP in SH-SY5Y cells mediated by MSG, flow cytometry analysis was performed. JC-1 dye could be used as an indicator of mitochondrial membrane potential in a variety of cell types. The results demonstrated that the treatment of 100 mM MSG exhibited 62±10% MMP in relative to that of the control after 24 h MSG treatment. However, NH043-1 at 15 uM showed 85±13% MMP by MSG and the treatment of 10 uM MK801 showed 90±5% MMP. Five uM CCCP, the disruptor of electron transport chain, showed 18±10% MMP (Figure 7). It suggested that NH043-1 effectively reduced the loss of MMP in SH-SY5Y cells arose by MSG.
4.8 NH043-1 effectively increases cell viability against Dox-induced nTBP/Q
79-EGFP cells.
To further study the effect of NH043-1 on SCA17, inducible nTBP/Q36-EGFP and nTBP/Q79-EGFP cells were used.
The cells were induced with 10 ug/mL Dox to express
nTBP/Q36-EGFP and nTBP/Q79-EGFP with or without 15 uM of NH043-1 for 5 d. The cell viability was not changed at each group in nTBP/Q36-EGFP. The treatment of 15 uM NH043-1 protected the cells against nTBP/Q79-EGFP-induced cytotoxicity for 5 d, and the increase of 20% cell viability was observed. It
nTBP/Q36-EGFP and nTBP/Q79-EGFP with or without 15 uM of NH043-1 for 5 d. The cell viability was not changed at each group in nTBP/Q36-EGFP. The treatment of 15 uM NH043-1 protected the cells against nTBP/Q79-EGFP-induced cytotoxicity for 5 d, and the increase of 20% cell viability was observed. It