
==== Front
Biomol Ther (Seoul)
Biomol Ther (Seoul)
Biomolecules & Therapeutics
1976-9148
2005-4483
The Korean Society of Applied Pharmacology

39092515
10.4062/biomolther.2024.075
bt-32-5-523
Original Article
Coadministration of 6-Shogaol and Levodopa Alleviates Parkinson’s Disease-Related Pathology in Mice
Kim Jin Hee 1
Kim Jin Se 1
Ju In Gyoung 2
Huh Eugene 2ψ
Choi Yujin 1
Lee Seungmin 1
Cho Jun-Young 3
Park Boyoung Y. 3
Oh Myung Sook 12*
1Department of Biomedical and Pharmaceutical Sciences, Graduate School, Kyung Hee University, Seoul 02447, Republic of Korea
2 Department of Oriental Pharmaceutical Science and Kyung Hee East-West Pharmaceutical Research Institute, College of Pharmacy, Kyung Hee University, Seoul 02447, Republic of Korea
3 Department of Fundamental Pharmaceutical Science, Kyung Hee University, Seoul 02447, Republic of Korea
* Corresponding Author E-mail: msohok@khu.ac.kr, Tel: +82-2-961-9436, Fax: +82-2-963-9436
ψ Present Address Department of Formulae Pharmacology, College of Korean Medicine, Gachon University, Seongnam 13120, Republic of Korea

1 9 2024
2 8 2024
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32 5 523530
10 5 2024
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Copyright © 2024, The Korean Society of Applied Pharmacology
2024
https://creativecommons.org/licenses/by-nc/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Parkinson’s disease (PD) is a neurodegenerative disease caused by the death of dopaminergic neurons in the nigrostriatal pathway, leading to motor and non-motor dysfunctions, such as depression, olfactory dysfunction, and memory impairment. Although levodopa (L-dopa) has been the gold standard PD treatment for decades, it only relieves motor symptoms and has no effect on non-motor symptoms or disease progression. Prior studies have reported that 6-shogaol, the active ingredient in ginger, exerts a protective effect on dopaminergic neurons by suppressing neuroinflammation in PD mice. This study investigated whether co-treatment with 6-shogaol and L-dopa could attenuate both motor and non-motor symptoms and dopaminergic neuronal damage. Both 6-shogaol (20 mg/kg) and L-dopa (80 mg/kg) were orally administered to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine/probenecid-induced PD model mice for 26 days. The experimental results showed that L-dopa alleviated motor symptoms, but had no significant effect on non-motor symptoms, loss of dopaminergic neuron, or neuroinflammation. However, when mice were treated with 6-shogaol alone or in combination with L-dopa, an amelioration in both motor and non-motor symptoms such as depression-like behavior, olfactory dysfunction and memory impairment was observed. Moreover, 6-shogaol-only or co-treatment of 6-shogaol with L-dopa protected dopaminergic neurons in the striatum and reduced neuroinflammation in the striatum and substantia nigra. Overall, these results suggest that 6-shogaol can effectively complement L-dopa by improving non-motor dysfunction and restoring dopaminergic neurons via suppressing neuroinflammation.

6-shogaol
Levodopa
Parkinson’s disease
Non-motor symptom
Dopaminergic neuron
Neuroinflammation
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pmcINTRODUCTION

Parkinson’s disease (PD) is a neurodegenerative disease that is predominantly caused by damage to dopaminergic neurons in the brain (Kalia and Lang, 2015; Park et al., 2019). Although the pathogenesis of PD remains incompletely understood, it is recognized that a multitude of factors, including oxidative stress, impaired mitochondrial function, and neuroinflammation, can contribute to the death of dopaminergic neurons (Ham et al., 2022; Morris et al., 2024). Recent studies have revealed evidence of chronic neuroinflammation in patients with PD, including activation of glial cells and increased pro-inflammatory cytokines (Tansey et al., 2022; Dzamko, 2023). As such, neuroinflammation has emerged as a promising therapeutic target for PD, primarily due to its implication in disease progression.

The primary symptoms of PD encompass motor disorders such as rigidity, tremors, and bradykinesia (Kalia and Lang, 2015). Recent studies have shown that 50-70% of patients with PD suffer from non-motor disorders such as depression, olfactory dysfunction, and memory impairment, in addition to motor symptoms (Pfeiffer, 2016; Schapira et al., 2017; Weintraub et al., 2022). These non-motor symptoms are considered a major cause of decreases in the quality of life and interference with daily life among PD patients (Wu et al., 2022).

Currently, the gold standard therapy for PD is levodopa (L-dopa), a precursor of dopamine which effectively functions to promptly replenish dopamine levels (Fahn, 2008). However, the effects of L-dopa are limited to improvements in motor symptoms, with no significant impact on non-motor symptoms or PD progression (Pantcheva et al., 2015; Hormann et al., 2021). Hence, it is necessary to develop a combination drug that can overcome the limitations of L-dopa while maintaining its efficacy in improving motor symptoms.

The compound 6-Shogaol (6-SHO), the primary pharmacological component of ginger (Zingiber officinale), has been shown to have a strong anti-inflammatory effect (Ballester et al., 2022). For example, one previous study comparing the anti-inflammatory effect of the main ingredients in ginger showed that 6-SHO exhibited the most significant efficacy (Ho and Chang, 2018). The outstanding anti-inflammatory properties of ginger have since been attributed to 6-SHO. Additionally, 6-SHO has been reported to exhibit inhibitory effects on neuroinflammation in mouse models of PD (Park et al., 2013; Huh et al., 2020, 2023). Park et al. (2013) reported that 6-SHO protects dopaminergic neurons by reducing neuroinflammation in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model. Additionally, Huh et al. (2020, 2023) revealed that 6-SHO inhibited inflammation and α-syn aggregation in the brain and gut of MPTP- and Proteus mirabilis-induced PD mice. Therefore, 6-SHO has been proposed as a promising PD treatment, but whether it can complement L-dopa remains unclear.

The aim of this study was to determine whether concurrent treatment with 6-SHO and L-dopa could improve motor and non-motor dysfunction and protect dopaminergic neuronal damage through inhibition of neuroinflammation in MPTP/probenecid (MPTP/p)-induced mice. Further, we investigated the effects of 6-SHO and L-dopa on motor and non-motor dysfunctions and dopaminergic neuron loss using behavioral tests and histological analysis of the brain.

MATERIALS AND METHODS

Materials

Bovine serum albumin (BSA), tribromoethanol, MPTP and probenecid were purchased from Sigma Aldrich (St Louis, MO, USA). Rabbit anti-tyrosine hydroxylase (TH), rat anti-dopamine transporter (DAT), immobilon-P transfer membranes, dimethyl sulfoxide (DMSO) were purchased from Merck Millipore (Burlington, MA, USA). Rabbit anti-ionized calcium-binding adapter molecule-1 (Iba-1) was purchased from Fujifilm Wako (Chuo-Ku, Osaka, Japan). Mouse horseradish peroxidase (HRP)-conjugated β-actin and mouse anti-tumor necrosis factor- α (TNF-α) antibodies were purchased from Santa Cruz Biotechnology (Temecula, CA, USA). Goat anti-Glial fibrillary acidic protein (GFAP) was purchased from Invitrogen (Middlesex County, MA, USA). Sodium dodecyl sulfate, protein assay reagent, Tween 20, ammonium persulfate, acrylamide, enzyme-linked chemiluminescence reagent, and skimmed milk were purchased from Bio-Rad Laboratories (Hercules, CA, USA). Anti-rabbit and anti-rat HRP secondary antibodies were purchased from Enzo Life Science, Inc. (Farmingdale, NY, USA).

Animals and experimental design

All animal studies were performed in accordance with the “Guide for the Care and Use of Laboratory Animals, 8th edition” (National Institutes of Health, 2011) and approved by the “Animal Care and Use Guidelines” of Kyung Hee University, Seoul, Korea (Approval number: KHSASP-22-416). Twenty-nine C57BL/6 mice (8–week–old, male) were obtained from DBL Inc. (Eumseong, Korea) and housed under 12-h light/dark cycle, at a constant temperature (23 ± 1°C) and humidity (60 ± 10%) in standard cages.

Mice were randomly divided into 5 groups as follows: (1) normal (NOR) group (10% DMSO p.o. treated+i.p. saline injected group, N=8); (2) MPTP/p group (10% DMSO p.o. treated+i.p. MPTP/p-injected group, N=5); (3) 6-SHO group (6-shogaol p.o. treated+i.p. MPTP/p-injected group, N=5); (4) L-dopa group (L-dopa p.o. treated+i.p. MPTP/p-injected group, N=5); (5) 6-SHO+L-dopa group (6-shogaol and L-dopa p.o. treated+i.p. MPTP/p-injected group, N=6). Mice in MPTP/p or 6-SHO or L-dopa or 6-SHO+L-dopa were injected with MPTP (25 mg/kg in saline) along with probenecid (100 mg/kg in 5% NaHCO3). Probenecid was administered 30 min prior to MPTP injection. These mice received 9 injections of MPTP in combination with probenecid. The 9 injections were administered at an interval of 3.5 days between consecutive doses. After the third MPTP/p injection, 6-SHO was dissolved in 10% DMSO with saline and administered at a dose of 20 mg/kg daily until sacrifice. 6-SHO was synthesized and provided by Professor Boyoung Y. Park at Kyung Hee University.

Behavior tests

Pole test: To evaluate motor deficits, the current study used the pole and rotarod tests. The pole test was performed three days after the last MPTP/p injection. The mice were placed head-up on a pole (diameter=8 mm, height=55 cm, rough surface). The times required for head down (T-turn) and landing (T-LA) were recorded. Each trial had a cut off limit of 1 min.

Rotarod test: The rotarod test was performed four days after the last MPTP/p injection. Each mouse was trained for 3 min on a rotating spindle (30 mm diameter, JD-A-07-TSM, Jeung Do Bio & Plant Co., Ltd., Seoul, Korea) before the rotarod test. The time spent on the rotating spindle until the first drop (latency) was recorded.

Sucrose splash test (SST): To evaluate depression-like behaviors, the current study used the SST and tail suspension test (TST). The SST and TST were performed two days after the last MPTP/p injection. A 10% sucrose solution was sprayed on the back coat of animals placed individually in a cylinder (8.5 cm diameter, 12 cm height) and recorded for 6 min. The time taken for the animals to start grooming was measured for 4 min, excluding the first 2 min, by a highly trained observer who was unaware of the group.

TST: Mice were suspended in a visually isolated area with the tip of their tail firmly clamped to a metal bar. After 6 min of video recording, immobility time was measured. Measurements were taken for 4 min, excluding the first 2 min, by a highly trained observer who was unaware of the group.

Buried food test: To assess olfactory impairment, the buried food test was performed as previously described (Choi et al., 2018). In brief, the experimental setup comprised a clean mouse cage filled with clean sawdust to a depth of 3-4 cm, in which pieces of cheese were buried to a depth of 1 cm in different locations for each mouse. Mice were fasted without food or water 24 h before the experiment. During the experiment, individual mice were placed in any corner of the cage. The time until the mouse discovered the hidden piece of cheese (when the mouse touched the pellet) was measured.

Y-maze: To evaluate memory deficits, the current study used Y-maze. The Y-maze was performed one day after the last MPTP/p injection. The test apparatus consisted of three arms (3×40×12 cm) at a 120° angle from each other. Mice were located in the middle of the Y-maze and allowed to explore the three arms for 8 min. The number and sequence of arm entries were recorded. The percentage alternation, a measure of spatial working memory, was calculated as follows: (the total number of alternations/total number of arm entries−2)×100. An alternation behavior was defined as consecutive entries into all three arms (i.e., ABC, BCA or CAB but not BAB).

Preparation of tissues

On day 5 of last MPTP/p injection, mice were anesthetized with tribromoethanol (300 mg/kg, i.p.) 1 h after drugs treatment. The mice were decapitated, and the striatum (ST) and substantia nigra (SN) was isolated and stored at –80°C until use.

Western blot

Tissues were lysed in RIPA buffer containing a protease/phosphatase inhibitor cocktail. Proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. The membranes were blocked with 5% BSA or skim milk for 30 min, then incubated overnight at 4°C with primary antibodies as follows: TH (1:1000), DAT (1:1000), Iba-1 (1:1000), GFAP (1:1000), TNF- α (1:500) and β-actin-HRP (1:3000). After washing with Tris-buffered saline (10 mM Tris-HCl, 150 mM NaCl, pH 7.5) containing 0.1% Tween 20, the membrane was incubated with secondary antibodies (1:3000) at room temperature for 1 h. Visualization and quantification of bands were performed using Image Lab Software (Bio-Rad Laboratories).

Statistical analysis

Differences among the groups were analyzed statistically by one-way analysis of variance (ANOVA) followed by Dunnett’s post-hoc test or student’s t test using GraphPad Prism 8.0.1 software (GraphPad Software Inc., CA, USA). All values are presented as mean ± standard error of the mean (SEM). The differences were considered statistically significant at p<0.05 and are expressed in each figure.

RESULTS

Co-administration of 6-SHO and L-dopa improves motor symptoms in MPTP/p-injected mice

We performed the pole and rotarod tests to evaluate the effects of 6-SHO or L-dopa monotherapy, as well as combined treatment with both 6-SHO and L-dopa, on behavioral disorders. In both tests, the MPTP/p group showed significant motor dysfunctions compared to the NOR group. In the pole test, both T-turn and T-LA times decreased in the 6-SHO- or L-dopa-only treatment groups compared to the MPTP/p group. Additionally, in the 6-SHO+L-dopa group, T-turn and T-LA times decreased similarly to the L-dopa-only group (Fig. 1A, 1B). The rotarod test indicated a tendency for latency time to increase in the 6-SHO-only group, with a significant increase observed in both the L-dopa-only group and the 6-SHO+L-dopa group compared to the MPTP/p group (Fig. 1C).

Co-administration of 6-SHO and L-dopa improves depression-like behaviors, olfactory dysfunction and memory impairment in MPTP/p-injected mice

Non-motor symptoms such as depression, olfactory dysfunction, and memory impairment occur in more than half of patients with PD and are directly related to quality of life (Tibar et al., 2018). Therefore, in the present study, we evaluated depression-like behavior using the SST and TST, olfactory function with the buried food test, and memory function with the Y-maze test. In both the SST and TST, we observed depression-like behavior in the MPTP/p group, with no significant change in the L-dopa-only group. However, the 6-SHO-only group and the 6-SHO+L-dopa group both showed improvements in depression-like behavior compared to the MPTP/p group (Fig. 2A, 2B). In the buried food test, the MPTP/p group showed an increased latency time, while no significant change was observed in the L-dopa-only group. However, latency time was significantly decreased in the 6-SHO-only group and the 6-SHO+L-dopa group (Fig. 2C). Finally, in the Y-maze test, the MPTP/p group showed a tendency towards a decrease in the percentage of spontaneous alternation compared to the NOR group. In addition, there was no change in the L-dopa-only group compared to the MPTP/p group. However, a significant increase in the % of spontaneous alternation was observed in both the 6-SHO-only and the 6-SHO+L-dopa groups (Fig. 2D).

Co-administration of 6-SHO and L-dopa protects against the loss of dopaminergic neurons in MPTP/p-injected mice

In previously study, we revealed that the administration of L-dopa-only in normal mice had no significant effect on dopaminergic neurons (Huh et al., 2018). In addition, our pilot study found that 6-SHO administration did not change the protein level of TH in normal mice (Supplementary Data 1). To investigate whether co-administration of 6-SHO and L-dopa could protect against MPTP/p-induced dopaminergic neuron death in PD condition, the protein levels of TH and DAT were assessed in the ST. The MPTP/p group showed significantly reduced protein levels of TH and DAT in the ST compared to the NOR group. Both the 6-SHO-only group and the 6-SHO+L-dopa group had elevated protein level of TH in the ST compared to the MPTP/p group. The protein level of DAT significantly increased in the 6-SHO-only group, and showed an increasing trend in the 6-SHO+L-dopa group compared to the MPTP/p group. However, the L-dopa-only group showed no significant difference compared to the MPTP/p group (Fig. 3).

Co-administration of 6-SHO and L-dopa inhibits microglia activation in MPTP/p-injected mice

Iba-1 is a biomarker indicating microglia activation associated with inflammatory responses in PD. Therefore, we assessed the protein expression levels of Iba-1 in ST and SN to investigate whether co-administration of 6-SHO and L-dopa could inhibit MPTP/p-induced activation of microglia. These analyses showed that MPTP/p injection increased the protein level of Iba-1 in both the ST and SN. The protein level of Iba-1 in the ST and SN was decreased in the 6-SHO-only and 6-SHO+L-dopa groups compared to the MPTP/p group, but was not significantly reduced in the L-dopa-only group (Fig. 4).

Co-administration of 6-SHO and L-dopa inhibits astrocyte activation in MPTP/p-injected mice

GFAP is a well-known biomarker of astrocyte activation, which is further associated with inflammatory responses in PD, alongside microglia. Therefore, we measured the protein levels of GFAP in the ST and SN to assess the effect of co-administration of 6-SHO and L-dopa on neuroinflammation. In the MPTP/p group, the protein level of GFAP was increased in both ST and SN compared to the NOR group. However, the protein level of GFAP tended to be decreased in the 6-SHO-only group and was significantly decreased in the 6-SHO+L-dopa group compared to the MPTP/p group. Conversely, there was no significant reduction in the L-dopa-only group (Fig. 5).

Co-administration of 6-SHO and L-dopa decreases protein level of TNF-α in MPTP/p-injected mice

Activated microglia and astrocytes release proinflammatory cytokines, which can induce neuronal death (Shin et al., 2014). Therefore, we evaluated the effect of co-administration of 6-SHO and L-dopa on the expression of TNF-α, a representative proinflammatory cytokine, in the ST and SN. In the MPTP/p group, protein levels of TNF-α were increased in both the ST and SN compared to the NOR group. However, the protein level of TNF-α was significantly decreased in the 6-SHO-only and 6-SHO+L-dopa groups compared to the MPTP/p group. However, there was no significant decrease in the L-dopa-only group (Fig. 6).

DISCUSSION

In the current study, we investigated whether 6-SHO and L-dopa, when administered concurrently, could improve motor and non-motor dysfunction and could further protect against dopaminergic neuronal damage through inhibition of neuroinflammation in MPTP/p-induced mice. Our results revealed that co-administration with 6-SHO and L-dopa exerted significant effects not only on motor symptoms but also on non-motor symptoms, dopaminergic neuron damage, and neuroinflammation in MPTP/p-induced PD mice. Conversely, the L-dopa-only group showed only significant alleviation of motor impairment, with no other observed effects. Overall, these results indicate that 6-SHO, when administered together with L-dopa, can overcome the limitations of L-dopa.

L-dopa, as a precursor of dopamine, replenishes the deficient levels of dopamine in PD patients (Choi et al., 2023). L-dopa has been widely utilized as the gold standard therapy for decades due to its immediate effect in improving behavioral disorders (Hauser, 2009; Tambasco et al., 2018). However, L-dopa therapy has several limitations, including that it does not alleviate non-motor symptoms or prevent PD progression (Pantcheva et al., 2015; Hormann et al., 2021). In addition, previous studies have reported that long-term use of L-dopa can trigger an oxidative stress response termed auto-oxidation, which can accelerate PD progression (You et al., 2018). Despite decades of development, no drug has surpassed the significant efficacy of L-dopa in alleviating motor symptoms. Therefore, the development of combination therapies that maintain the superior behavioral improvement effects of L-dopa while addressing its limitations is emerging as a key therapeutic strategy.

Neuroinflammation can directly affect the death of dopaminergic neurons in PD (Kim et al., 2021). Cells involved in neuroinflammation, such as microglia and astrocytes, can secrete pro-inflammatory cytokines including TNF-α upon activation (Prasad et al., 2015). It is widely known that pro-inflammatory cytokines induce death of dopaminergic neurons (Shin et al., 2014). Therefore, inhibiting the activation of microglia and astrocyte can protect dopaminergic neuron death and alleviate symptoms of PD. Previous studies have reported that 6-SHO protected the death of dopaminergic neurons by suppressing neuroinflammation in multiple PD models. Therefore, we predicted that 6-SHO could be an excellent combination agent that can compensate for the limitations of L-dopa in preventing disease progression, and we proved this through research.

In this study, we chose MPTP/p mice as a model to reflect clinical symptoms and timing of drug administration. Among the various causes of PD, MPTP stands out as a representative toxin that specifically targets the DAT, leading to a loss of dopaminergic neurons (Langston, 2017). However, MPTP is rapidly eliminated by the brain and kidneys, making it unsuitable for chronic administration (Petroske et al., 2001). Therefore, to address this issue, a method involving concurrent injection of probenecid during chronic administration was previously devised. The widely-used chronic MPTP/p model is primarily induced by injections once every 3.5 days, with mice up to the third injection classified as in the early stages of PD (Choi et al., 2018). Therefore, considering that in clinical situations, PD diagnosis and L-dopa prescription generally occur after the early disease stage, L-dopa and 6-SHO were administered following 3 injections of MPTP/p. This suggests that the therapeutic effect of co-administration of 6-SHO and L-dopa shown in this study has the potential to be applied in clinical practice.

Collectively, the present study showed that co-administration of 6-SHO and L-dopa exerted a similar behavioral improvement effect to L-dopa-alone in MPTP/p-injected mice. Moreover, the co-administration of 6-SHO and L-dopa demonstrated effects in suppressing neuroinflammation, preserving dopaminergic neurons, and mitigating non-motor symptoms such as depression-like behavior, olfactory impairment, and memory decline, which were not observed when L-dopa was administered alone (Fig. 7). Therefore, we suggest that 6-SHO may be a promising candidate for combination treatment with L-dopa in PD patients.

ACKNOWLEDGMENTS

This study was supported by the National Research Foundation of Korea Grant and Commercialization Promotion Agency for R&D Outcomes (COMPA) (2021M3A9G1015618). This research was also supported by grants from the National Research Foundation of Korea, funded by the Korean government (grant number 2022M3A9B6017813).

Fig. 1 Effect of co-administering 6-SHO and L-dopa on motor dysfunction in MPTP/p-injected mice. To evaluate motor function, T-turn (A) and T-LA (B) in the pole test and latency to fall (C) in the rotarod test and were measured. Values are given as the mean ± SEM (N=5-8). Data were analyzed by One-way ANOVA followed by Dunnett’s multiple comparison test. *p<0.05, **p<0.01 and ***p<0.001 vs. MPTP/p group.

Fig. 2 Effect of co-administering 6-SHO and L-dopa on depression-like behaviors, olfactory and memory dysfunctions in MPTP/p-injected mice. SST (A) and TST (B) were performed to evaluate depression-like behavior. Buried food test was performed to evaluate olfactory function (C). Y-maze was performed to evaluate memory function (D). Values are given as the mean ± SEM (N=5-8). Data were analyzed by one-way ANOVA followed by Dunnett’s multiple comparison test or Student’s t-test. *p<0.05 and **p<0.01 vs. MPTP/p group (One-way ANOVA); $p<0.05 vs. MPTP/p group (Student’s t-test).

Fig. 3 Effect of co-administering 6-SHO and L-dopa on dopaminergic neurons in ST of MPTP/p-injected mice. Western blot was performed to measure protein level of TH (A) and DAT (B) in the ST. Values are given as the mean ± SEM (N=5-8). Data were analyzed by one-way ANOVA followed by Dunnett’s multiple comparison test or Student’s t-test. *p<0.05 and ***p<0.001 vs. MPTP/p group; $$p<0.01 vs. MPTP/p group (Student’s t-test).

Fig. 4 Effect of co-administering 6-SHO and L-dopa on expression of Iba-1 in ST and SN of MPTP/p-injected mice. Western blot was performed to measure protein level of Iba-1 in the ST (A) and SN (B). Values are given as the mean ± SEM (N=5-8). Data were analyzed by one-way ANOVA followed by Dunnett’s multiple comparison test or Student’s t-test. *p<0.05 and **p<0.01 vs. MPTP/p group; $$p<0.01 vs. MPTP/p group (Student’s t-test).

Fig. 5 Effect of co-administering 6-SHO and L-dopa on expression of GFAP in ST and SN of MPTP/p-injected mice. Western blot was performed to measure protein level of GFAP in the ST (A) and SN (B). Values are given as the mean ± SEM (N=5-8). Data were analyzed by one-way ANOVA followed by Dunnett’s multiple comparison test. *p<0.05 and **p<0.01 vs. MPTP/p group.

Fig. 6 Effect of co-administering 6-SHO and L-dopa on expression of TNF-α in ST and SN of MPTP/p-injected mice. Western blot was performed to measure protein level of TNF-α in the ST (A) and SN (B). Values are given as the mean ± SEM (N=5-8). Data were analyzed by one-way ANOVA followed by Dunnett’s multiple comparison test or Student’s t-test. *p<0.05 vs. MPTP/p group; $p<0.05 vs. MPTP/p group (Student’s t-test).

Fig. 7 Summary of the mechanism of action with co-administration of 6-SHO and L-dopa. Created with BioRender.com.
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