Figure 1. Effects of CHMs on MSG-induced cell death in SH-SY5Y cells
(A) 3.0 x 104 SH-SY5Y cells were treated with various concentrations of MSG for 24hr and cell viability was determined by MTT assay. The IC50
of MSG is found to be 80 mmole/L. (B) 3.0 x 104 SH-SY5Y cells were co-treated with nine CHMs at indicated concentrations (IC50 listed in Table 1), or 10 μmole/L MK801 (antagonist of NMDA), or 80 mmole/L MSG. Cell viability was measured by MTT assay at 24hr treatment. The results were expressed as mean±SEM, n=6. *P < 0.05, ** P < 0.01, comparing to that of MSG treatment.
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Figure 2.NH018-1 decreased the glutamate-induced cell death in SH-SY5Y cells.
3.0 x 104 SH-SY5Y cells were treated with 80 mmole/L MSG and various concentrations of NH018-1 or 10 μmole/L MK801 for 24 hr. Cell
viability was measured by MTT assay. The results were expressed as mean±SEM, n=3. *P < 0.05, comparing to MSG treatment.
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Figure 3. NH018-1 inhibited the LDH release from SH-SY5Y cells induced by MSG.
1.0 x 105 SH-SY5Y cells were co-treated with 80 mmole/L MSG and various concentrations of NH018-1, or 10 μmole/L MK801 for 24 hr. The cell damage was determined by measuring the amount of LDH release.
The results were expressed as mean±SEM, n=3. *P < 0.05, comparing to MSG treatment.
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Figure 4.Effects of NH018-1 on Bcl-2 and Bax expression induced by MSG in SH-SY5Y cells
(A) 1.2 x 106 SH-SY5Y cells were co-treated with 80 mmole/L MSG and 10 μmole/L NH018-1, or 10 μmole/L MK801 for 6 hr. The Bcl-2 and Bax expression were measured by immunoblotting. (B) and (C) Quantification of the intensities of Bcl-2 and Bax protein bands was determined by
Image J and expressed as the ratio versus actin (internal control). The results were expressed as mean±SEM, n=3. *P < 0.05, comparing to MSG treatment.
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Figure 5. NH018-1 suppressed cytochrome C release from mitochondria to cytosol mediated MSG in SH-SY5Y cells.
(A) 1.2 x 106 SH-SY5Y cells were co-treated with 80 mmole/L MSG and 10 μmole/L NH018-1, or 10 μmole/L MK801 for 24 hr. The level of cytochrome C in cytosol fraction was assayed by immunoblotting. (B) Quantification of the intensities of cytochrome C protein bands was measured by Image J and expressed as the ratio versus actin. The results were expressed in mean±SEM, n=3. *P < 0.05, comparing to MSG treatment.
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Figure 6. NH018-1 decreased the cleaved-Caspase-9,
cleaved-Caspase-3, and cleaved-PARP expression induced by MSG in SH-SY5Y cells.
(A) 1.2 x 106 SH-SY5Y cells were co-treated with 80 mmole/L MSG and 10 μmole/L NH018-1, or 10 μmole/L MK801 for 12 hr. The expression of cleaved-caspase-9, cleaved-caspase-3, and cleaved-PARP, was measured by immunoblotting. (B), (C), and (D). Quantification of the intensities of cleaved-caspase-9, cleaved-caspase-3, and cleaved-PARP protein bands was carried out by Image J and expressed as the ratio versus actin. The results were expressed in mean±SEM, n=3. *P < 0.05, comparing to MSG treatment.
B
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Figure 7. NH018-1 suppressed Calpain-2 and SBDP expression induced by MSG in SH-SY5Y cells.
(A) 1.2 x 106 SH-SY5Y cells were co-treated with 80 mmole/L MSG and 10 μmole/L NH018-1, or 10 μmole/L MK801 for 12 hr. The expression of Calpain-2 and SBDP, as assessed by immunoblotting. (B), (C)
Quantification of the intensities of Calpain-2 and SBDP protein bands was performed by Image J and expressed as the ratio versus actin. The results were expressed as mean±SEM, n=3. *P < 0.05, comparing to MSG treatment.
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Figure 8. NH018-1 reduced MSG-induced apoptosis in SH-SY5Y cells.
(A) 1.2 x 106 SH-SY5Y cells were co-treated with 80 mmole/L MSG and 10 μmole/L NH018-1, or 10 μmole/L MK801 for 24 hr. PI/Annexin V-FITC double staining was performed to detect cell death/apoptosis after MSG treatment and measured with a flow cytometer. (B)
Quantification of cell apoptosis percentage by measuring the lower right region. The results were expressed as mean±SEM, n=3.
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Figure 9. NH018-1 suppressed MSG-induced ROS production.
(A) 1.2 x 106 SH-SY5Y cells were co-treated with 80 mmole/L MSG and 10 μmole/L NH018-1, or 10 μmole/L MK801 for 24 hr. The amounts of ROS was measured by luminol-dependent chemiluminescence patterns detected with a chemiluminescence detector. (B) Quantification of the degree of ROS production. The results were expressed as mean±SEM, n=3.
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Figure 10. Effects of three CHMs and NH018-1 on DOX-induced SCA17 (79Q) cell model
(A) 1.5 x 104 SCA17 cells were exposed to DOX at 5-20 μg/mL for 24-72 hr and cell viability was determined by MTT assay. The IC50 of DOX is about 10 μg/mL for 48 hr. (B) 1.5 x 104 SCA17 cells were co-treated with CHMs at 1/5 X IC50 concentrations, 10 μmole/L MK801, 10 μmole/L D-AP5 (antagonist of NMDA), and 10 μg/mL DOX. Cell viability was measured by MTT assay at 48 hr treatment. (C) 1.5 x 104 SCA17 cells were co-treated with 10 μmole/L NH018-1, 10 μmole/L MK801, and 10 μg/mL DOX for 48 hr. The results were expressed as mean±SEM, n=3.
*P < 0.05, ** P < 0.01, comparing to DOX treatment.
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Figure 11. Effects of NH018-1 on cleaved-Caspase-9,
cleaved-Caspase-3, and cleaved-PARP expression after MSG exposure in SCA17 cells
(A) 1.0 x 106 SCA17 (36 & 79Q) cells were co-treated with 10 μg/mL DOX and 10 μmole/L NH018-1, or 10 μmole/L MK801 for 5 days. The expression of cleaved-caspase-9, cleaved-caspase-3, and cleaved-PARP, was measured by immunoblotting. (B), (C), and (D) Quantification of the intensities of cleaved-caspase-9, cleaved-caspase-3, and cleaved-PARP protein bands determined by Image J and expressed as the ratio versus actin from SCA17 (79Q) cells.
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Figure 12. Effects of NH018-1 on body weight and motor performance in SCA17 transgenic (TG) mice
Mice were grouped into WT+Saline, TG+Saline, and TG+NH018-1 groups. (A) Mice body weight was measured from 8- to 20-week-old. (B) The performance of motor coordination was determined by rotarod test.
The results were expressed as mean±SEM, n=6. *P < 0.05, comparing to TG+saline treatment. Mice were intraperitoneal-injected with saline (0.1 ml) or NH018-1 (1.5 mg/kg) at ten-week after the birth by three times a week.
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Figure 13. NH018-1 improved gait abnormalities
Gait analysis of WT and TG mice at 8-week and 21-week, mice were divided into three groups, WT+saline, TG+saline, and TG+1.5 mg/kg NH018-1. Saline or 1.5 mg/kg NH018-1 three times a week was i.p. after ten- week. (A) The footprint patterns of WT and TG for control and NH018-1 groups. (B), (C) The front/hind footprint overlaps. (D) - (G) The front/hind stride length. (H), (I) The front/hind parallel length of WT and TG for control and Hup A groups. The results were expressed as mean±SEM, n=6. *P < 0.05, comparing to TG+NH018-1 (8 week) treatment.
B C
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Figure 14. Effects of NH018-1 on TBP (N12), Calpain-2, and cleaved-Caspase-3 expression in SCA17 transgenic mice
(A) The expression of TBP (N12), Calpain-2, and cleaved-caspase-3 in the cerebellum was measured by immunoblotting at 21-week. (B), (C), and (D). Quantification of the intensities of TBP (N12), Calpain-2, and cleaved-caspase-3 protein bands was carried out by Image J and
expressed as the ratio versus actin.
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Figure 15. Effects of NH018-1 on cerebrum and cerebellum AChE activity in SCA17 mice
Mice were sacrificed at 21-week and the AChE activity of cerebrum and cerebellum were measured with AChE activity kit. (A) AChE activity of cerebrum in WT+saline, TG+saline, and TG+1.5 mg/kg NH018-1 mice.
(B) AChE activity of cerebellum in WT+saline, TG+saline, and TG+1.5 mg/kg NH018-1 mice. The results were expressed as mean±SEM, n=3.
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8. Tables
Table 1. IC50- of nine CHMs studied.
Table 2. IC50-cytotoxicity of nine compounds studied.
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9. Supplementary figure
Supplementary figure 1. Effects of nine compounds on MSG-induced cell death in SH-SY5Y cells
3.0 x 104 SH-SY5Y cells were co-treated with 80 mmole/L MSG and IC50
of nine compounds as listed in Table, or 10 μmole/L MK801, cell
viability was measured by MTT assay at 24hr treatment. The results were expressed as mean±SEM, n=4. *P < 0.05, compared to MSG treatment.