4.1. CHM NH018 rescues SH-SY5Y cell viability after MSG treatment
MSG was used to induce excitotoxicity in neuroblastoma SH-SY5Y cell model to screen the effective CHMs. At first, cells were treated with 40, 80, 120, or 160 mM MSG for 24 hr to determine the IC50.As shown in figure 1 (A), the IC50 of MSG is 80 mmole/L after 24 hr treatment.
Then, the IC50 of various CHM water extracts on SH-SY5Y cells was measured, and half or one-fifth of CHM IC50 (1/2 X or 1/5 X IC50) (IC50
listed in Table 1) was used to screen effective CHMs against MSG by using NMDA receptor antagonist, MK801, as a positive control. As shown in figure 1 (B), Compared to MSG (reduced relative cell viability to about 60%), NH018, NH021, and NH027 exhibited 75%, 70%, and 80% increased of cell viability, respectively. Therefore, NH018 was chosen for further studies.
4.2. Active compound, NH018-1 of NH018 increased
SH-SY5Y cell viability after MSG treatment
The IC50 of various compounds isolated from CHMs against SH-SY5Y cells were determined, and the results were summarized in Table 2. Then 1/2 and 1/5 IC50 of the compounds were used to study their effects on the cell excitotoxicity induced by MSG, and the results showed19
that NH018-1 was found to be the most effective compound. As shown in figure 2 (A), 10 μmole/L NH018-1 was sufficient to significantly rescue cell death induced by MSG (increased relative cell viability from
65%±1.2 (SEM) to 75%±0.5, p < 0.05). Because NH018-1 is the major active component of NH018, the above mentioned results imply that NH018-1 could be the effective compound that exerts protective effect against glutamatergic cytotoxicity.
4.3. Effects of NH018-1 on the release of lactate
dehydrogenase (LDH) from SH-SY5Y cells treated with MSG
To verify the membrane integrity, the passage of substances that are normally detained inside cells to the outside was assessed by monitoring the release of LDH into the culture medium 20. As shown in figure 3, NH018-1 at 2.5-40 μmole/L effectively inhibited the release of LDH induced by MSG (suppressed relative LDH release from ~215% to
~125%). It suggests that NH018-1 exhibits protective effects against MSG-induced cell membrane damage.
4.4. Effects of NH018-1 on Bcl-2, Bax and cytochrome C release for SH-SY5Y cells treated with MSG
To investigate whether mitochondrial apoptotic pathway is involved
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in NH018-1-mediated protection against glutamatergic excitotoxicity, the cell-survival protein Bcl-2 and pro-apoptotic protein Bax, which mediate the release of pro-apoptotic cytochrome C from mitochondria 3 were investigated. Treatment of 80 mmole/L MSG resulted in the 12%
reduction of Bcl-2 and the 60% increment of Bax. When cells were co-treated with MSG and NH018-1, the levels of Bcl-2 and Bax were increased 30% and suppressed 50%, respectively (Figure 4 (A), (B) and(C)). It indicates that NH018-1 is able to reverse the decline of Bcl-2/Bax ratio.
To further study the effect of NH018-1 on the cytochrome C release from mitochondria to the cytosol, cytoplasm and mitochondria were fractionated and analyzed. As shown in figure 5 (A), by Western analyze, NH018-1 significantly decreased the release of cytochrome C from
mitochondria to cytosol induced by MSG (relative fold change 2.8 versus 1.2 , p < 0.05)
4.5. NH018-1 attenuated MSG-induced the activation of Caspase-9, Caspase-3, and PARP expression in
SH-SY5Y cells
Previous study showed that the overactivation of glutamate receptors by NMDA receptor triggered neuronal cell death, associated with
caspase-family downstream of Bcl-2/Bax and cytochrome C such as
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caspase-9, caspase-3 as well as nuclear enzyme PARP activated 2, 8. As shown in figure 6 (A), (B), (C), and (D). MSG remarkably increased the expression of cleaved-Caspase-9, cleaved-Caspase-3, and cleaved-PARP by a relative fold changes, 1.3, 4.3, and 8.1, respectively. Co-treatment of NH018-1 with MSG for 24 hr, it decreased the expression of
cleaved-Caspase-9, cleaved-Caspase-3, and cleaved-PARP by a relative fold changes, 1.0, 3.2, and 5.0, respectively. Based on the
above-mentioned results, it suggests that NH018-1 inhibits the
excitotoxicity via the suppression of the mitochondria apoptotic pathway.
4.6. NH018-1 reduced MSG-induced the activation of Calpain-2 and Calpain specific-SBDP expression in SH-SY5Y cells
It was reported that excessive glutamate leads to intracellular calcium inflow, and then to activation of calpain. The calpain-specific α-spectrin cleaved by the activation of calpain produces the fragments of 150 kD and 145 kD [also known α-spectrin breakdown product (SBDP)].
Accordingly, SBDP could be used for the indicator of calpain activity. As shown in figure 7 (A), 7 (B), and figure 7 (C), compared to MSG
(increased relative fold changes of Calpain-2 expression to about 1.2 and SBDP expression to about 1.3), NH018-1 significantly reduced the levels of Calpain-2 (0.9 fold) and SBDP (1.0 fold). It suggests that NH018-1 indeed alleviates glutamatergic excitotoxicty.
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4.7. Effects of NH018-1 on MSG-induced
phosphatidylserine (PS) externalization and apoptotic induction in SH-SY5Y cells
In order to study effects of NH018-1 on cell apoptosis, Annexin-V was used to stain externalization of PS, which is an apoptotic marker and measured by flow cytometer method. As shown in figure 8 (A) and (B), MSG significantly increased cell apoptosis, and the co-treatment of NH018-1 with MSG decreased the percentage of cell apoptosis (11%
versus 17%).
4.8. NH018-1 inhibited the ROS production induced by MSG treatment in SH-SY5Y cells
To investigate whether NH018-1 could block the ROS production induced by MSG treatment in SH-SY5Y cells, luminol-dependent chemiluminescence was used. Luminol is activated with an oxidant to exhibit its luminescence, the emission of energy as a photon, indicating the change of electrons from excited state to ground state. The emission produces is blue glow and detected with a chemiluminescence detector.
As shown in figure 9 (A) and (B), the degree of luminescence intensity increased to about 2000 after MSG treatment, but decreased to about 1500 after co-treating with NH018-1. It demonstrated that NH018-1 reduced MSG-induced ROS production.
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4.9. Effects of CHMs and NH018-1 on cell viability of DOX induced SCA17 (79Q) cell model
The abovementioned results imply that NH018-1 possesses
neuroprotective effects on glutamatergic excitotxicity. To further study effects of NH018-1 on SCA17, SCA17 inducible cell model (SCA17 nTBP/Q79-EGFP cells) was used. At first, cells were treated with 5, 10, or 20 μg/mL DOX to induce the expression of nTBP-EGFP(79Q) for 24, 48, and 72 hr to determine the IC50 of DOX. As shown in figure 10 (A), the IC50 of DOX is about 10 μg/mL after 48 hr treatment. From Result 4.1., NH018, NH021, or NH027 significantly increased SH-SY5Y cell
viability after MSG treatment. 1/5X IC50 dose of NH018, NH021, or NH027 increased cell viability significantly after nTBP-EGFP(79Q) induction for 48 hr. As shown in figure 10 (B), comparing to DOX (reduced relative cell viability to about 75%), NH018, NH021, and NH027 increased the relative cell viability to 85%, 90%, and 100%, respectively which indicates the CHMs exhibited protective effects against nTBP-EGFP(79Q)-induced cell death. Furthermore, NH018-1 of NH018 also suppressed nTBP-EGFP(79Q)-induced cytotoxicity
[increased relative cell viability to about 70%, figure 10 (C)].
4.10. NH018-1 attenuated nTBP-EGFP(79Q)-induced
activated Caspase-9, Caspase-3, and PARP expression
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in SCA17 cells
To study the effects of NH018-1 on nTBP-EGFP(79Q) induction of activated Caspase-9, Caspase-3, and PARP expression in SCA17 cells, the cells were treated with 10 μg/mL DOX for 5 days. As shown in figure 11 (A), (B), (C), and (D). nTBP-EGFP(79Q) induction significantly increased the expression of cleaved-Caspase-9, cleaved-Caspase-3, and cleaved-PARP by relative fold changes, 1.8, 3.2, and 7.2, respectively.
Co-treatment of NH018-1 with DOX for 5 day, it decreased the
expression of cleaved-Caspase-9, cleaved-Caspase-3, and cleaved-PARP by relative fold changes, 1.6, 2.6, and 4.0, respectively. Take together the results, nTBP-EGFP(79Q)-induced activation of caspase-family, and PARP were suppressed significantly by the NH018-1 in SCA17 inducible cell model.
4.11. Effects of NH018-1 on body weight changes and rotarod performance in SCA17 mice model
To study the effects of NH018-1 on behavioral aspects in SCA17 mice, the rotarod performance was used to evaluate its efficacy 11. After 1.5 mg/kg NH018-1 administration for 12 weeks, there was no significant difference in body weight change between wild-type and transgenic mice (figure 12 (A)), implying that the NH018-1 dosage used here was not toxic to mice. As shown in figure 12 (B), the SCA17 transgenic mice that received saline vehicle performed poorly on an accelerating rotarod (from
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4 to 30 rpm in 5 min) and the latency to fall kept declining from 15- to 21-week. On the contrary, the motor coordination SCA17 transgenic mice were significantly improved after receiving 1.5 mg/kg NH018-1 from 17- to 21-week.
4.12. Effects of NH018-1 on SCA17 mice footprinting
The efficacy of NH018-1 on SCA17 transgenic mice was further investigated by assessing gait abnormalities at 8- and 21-week (figure 13 (A)). The front/hind footprint overlap was significantly increased in the SCA17 transgenic mice that received saline (figure 13 (B) and (C)), but the stride length and parallel length were not altered (figure 13 (D) to (I)) even at 8 and 21 week. NH018-1 significantly suppressed gaitabnormalities of SCA17 transgenic mice by reducing front/hind footprint overlap (figure 13 (B) and (C)), and intensifying the stride and parallel lengths (figure 13 (D) and (I)).
4.13. NH018-1 attenuated SCA17 mice-induced activated TBP (N12), Calapin-2, and cleaved-Caspase-3
expression
To study the effects of NH018-1 on aggregation (TBP (N12)), and the expression of Calpain-2, and cleaved-Caspase-3 was measured in cerebellum of SCA17 mice, mice were sacrificed after 21 weeks. As
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shown in figure 14 (A), (B), (C), and (D). The expression of TBP (N12), Calpain-2, and cleaved-Caspase-3 were increased by a relative fold changes, 1.8, 1.3, and 1.7, respectively in SCA17 mice. The treatment of NH018-1 from 10- to 21-week in SCA17 mice, it decreased the
expression of TBP (N12), Calpain-2, and cleaved-Caspase-3 by a relative fold changes, 1.3, 0.9, and 0.6, respectively. Based on the
above-mentioned results, it suggests that NH018-1 inhibits the aggregation and excitotoxicity-induced cell death.
4.14. Effects of NH018-1 on acetylcholinesterase (AChE) activity of cerebrum and cerebellum in SCA17 mice
The significant reduction of AChE activity in the cerebral and cerebellar cortex with inherited olivopontocerebellar atrophy (OPCA) was observed in the postmortem 35. It was also reported that AChE activity was decreased in SCA3 and SCA6 47, but the AChE activity in the cerebellum in SCA17 disorder is remained to be studied. To
investigate whether NH018-1 improves the SCA17 disorder via
inhibition of AChE activity in the cerebellar, mice were sacrificed, and the AChE activity in cerebellar was measured. As shown in figure 15 (A), cerebral AChE activities were similar within control and
NH018-1 treated groups. The cerebellar AchE activity, however, was remarkably increased in SCA17 transgenic mice (figure 15 (B)).
Treatment of NH018-1 did not inhibit cerebellar AChE activity in SCA17 transgenic mice. It suggests that amelioration of SCA17 by
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NH018-1 is not via inhibiting of AChE activity.
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