Professor Soo Young Choi, Department of Biomedical Science, Research Institute of Bioscience and Biotechnology, Hallym University, 1 Hallymdaehak-gil, Chuncheon, Gangwon 24252, Republic of Korea
*Contributed equally
Oxidative stress is strongly implicated in the pathogenesis of Parkinson's disease (PD) through degeneration of dopaminergic neurons. The present study was designed to investigate the underlying mechanisms and therapeutic potential of Brain Factor-7® (BF-7®), a natural compound in silkworm, in a mouse model of PD induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MPTP (20 mg/kg) was intraperitoneally injected into mice to cause symptoms of PD. Mice were orally administered BF-7® (a mixture of silk peptides) before and after MPTP treatment. Rotarod performance test was used to assess motor performance. Fluoro-Jade B staining for neurons undergoing degeneration and immunohistochemistry of tyrosine hydroxylase for dopaminergic neurons, 4-hydroxy-2-nonenal (4HNE) for lipid peroxidation, 8-hydroxy-2'-deoxyguanosine (8OHdG) for DNA damage and superoxide dismutase (SOD) 1 and SOD2 for antioxidative enzymes in the pars compacta of the substantia nigra were performed. Results showed that BF-7® treatment significantly improved MPTP-induced motor deficit and protected MPTP-induced dopaminergic neurodegeneration. Furthermore, BF-7® treatment significantly ameliorated MPTP-induced oxidative stress. Increased 4HNE and 8OHdG immunoreactivities induced by MPTP were significantly reduced by BF-7®, whereas SOD1 and SOD2 immunoreactivities decreased by MPTP were significantly enhanced by BF-7®. In conclusion, BF-7® exerted protective and/or therapeutic effects in a mouse model of PD by decreasing effects of oxidative stress on dopaminergic neurons in the substantia nigra pars compacta.
Parkinson's disease (PD) is a prevalent neurodegenerative disorder with defective motor function, which is characterized by a progressive loss of dopaminergic neurons located in the pars compacta of the substantia nigra (SNpc) with subsequent exhaustion of dopamine in the striatum (
Oxidative stress is the result of many metabolic processes essential to the body. However, it can exert toxic and deleterious roles in the body (
Studies have demonstrated that various kinds of natural resources with significant antioxidant activities are effective against PD (
To the best of the authors' knowledge, neuroprotective effects of cocoon hydrate such as BF-7® against PD has been poorly investigated. Thus, the aim of the present study was to evaluate the neuroprotective potential of BF-7® in a mouse model of MPTP-induced PD. For experimental studies on PD, acute, subacute, or chronic administrations of MPTP in rodents is used to replicate pathological hallmarks shown in human PD patients (
A total of 30 adult male C57BL/6 mice (10 weeks old; 27±2 g) were used for this experiment. The mice were obtained from the Experimental Animal Center of Kangwon National University (Chuncheon, South Korea). They were housed in pathogen-free condition with standard temperature (~23˚C) and humidity (~60%) on 12-h light/dark cycle. Freely accessible pellet feed (DBL Co., Ltd.) and water were provided to the animals.
The protocol of all experiments was approved on 28 January, 2020 by the Ethics Committee of Kangwon National University (approval no., KW-200113-2). All experimental procedures adhered to the guidelines described in the 'Current International Laws and Policies', which is included in the Guide for the Care and Use of Laboratory Animals (
BF-7® used in this experiment was supplied by Famenity Co., Ltd. BF-7® is a mixture of silk peptides obtained from the cocoon shell of silkworm (
A total of 30 mice were randomly assigned to three groups: i) Vehicle (saline) treated group (control group, n=10), ii) vehicle and MPTP treated group (vehicle + MPTP group, n=10) and iii) BF-7® + MPTP group (n=10).
To produce experimental PD in mice, as previously described (
Rotarod test is a well-established way to assess motor coordination and balance for screening side effects of insults in preclinical tests (
F-J B was used as a fluorescent marker for degeneration of neurons. In this experiment, F-J B staining was used to detect loss/death of neurons in the substantia nigra. For the preparation of brain sections containing the substantia nigra, mice were deeply anesthetized with 200 mg/kg pentobarbital sodium obtained from JW pharm. Co., Ltd. and fixed transcardially with 4% paraformaldehyde. Obtained brains were cut into 20-µm coronal sections using an SM2020 R sliding microtome (Leica Microsystems GmbH) equipped with a freezing stage (BFS-40MP; Physitemp Instruments Inc.). F-J B staining was performed according to a published method (
For the analysis for neuronal loss/death in the substantia nigra, seven sections per mouse were selected at corresponding levels. F-J B-stained cells were examined and images captured using a fluorescence microscope (Carl Zeiss AG) equipped with blue (450-490 nm) excitation light. F-J B-stained cells were counted in an area of 200 µm2 using an image analyzing software (Optimas 6.5; CyberMetrics Corporation).
Changes in dopaminergic neurons, lipid peroxidation, DNA oxidation and antioxidant proteins were examined by immunohistochemistry using rabbit anti-TH (diluted 1:200; Abcam; cat. no. ab6211), mouse anti-4HNE (diluted 1:1,200; Abcam; cat. no. ab48506), goat anti-8OHdG (diluted 1:600; MilliporeSigma; cat. no. AB5830), sheep anti-SOD1 (diluted 1:500; Calbiochem; cat. no. 574597) and sheep anti-SOD2 (diluted 1:1,000; Calbiochem; cat. no. 574596). According to a published method (
To analyze numbers of TH-immunostained cells, seven sections per mouse were selected at corresponding levels of the substantia nigra. Images of the TH-immunostained cells were captured using a BX53 upright light microscope (Olympus Corporation). Then two blinded experimenters counted mean numbers of TH-immunostained cells at corresponding areas using an image analyzing system (Optimas 6.5; CyberMetrics Corporation).
To analyze immunoreactivity of SOD1, SOD2, 4HNE and 8OHdG, the choice of the sections and the capture of the images were performed in the same way as described above. Change in each immunoreactivity was presented as a relative optical density (ROD) using Adobe Photoshop 8.0 (Adobe Systems, Inc.) and ImageJ software 1.59 (National Institutes of Health). ROD was calibrated as % compared with the control group (100%).
Regarding sample size, ≤7 mice per group were used at an α error of 0.05 and a power of >80%. The sample size was calculated with power calculator. Using GraphPad Prism software (version 5.0; GraphPad Software, Inc.), a multiple-sample comparison was performed to test the differences between the groups (two-way analysis of variance and Tukey's multiple comparison with a
In the MPTP-induced mice, motor deficit was evaluated to investigate the relationship between DA neuron degeneration using rotarod test and the effect of BF-7® administration in the MPTP-induced mice was assessed (
After noting that BF-7® administration alleviated MPTP-induced motor deficit, the present study continued to examine if there were neuroprotective effects of BF-7® against MPTP-induced dopaminergic cell loss using TH immunohistochemistry and/or F-J B staining in the substantia nigra.
Treatment with 10 mg/kg BF-7® protected dopaminergic neurons in the SNpc from MPTP-induced neuronal death.
In the control group, TH-stained dopaminergic cells (neurons) were distributed in SNpc (
Treatment with 10 mg/kg BF-7® ameliorated MPTP-induced decrease of TH immunoreactivity in the striatum.
The striatum receives dopamine from dopaminergic cells located in the SNpc. In the control group, strong TH immunoreactivity, which is dopaminergic neuropil, was shown in the striatum (
Treatment with 10 mg/kg BF-7® reduced MPTP-induced oxidative stresses in the SNpc.
The immunoreactivity of 4HNE (a marker for lipid peroxidation) in the control group was very weak in the SNpc (
In the control group, 8OHdG immunoreactivity was observed in the SNpc (
Treatment with 10 mg/kg BF-7® ameliorated MPTP-induced decrease of endogenous antioxidant enzymes.
SOD1 immunoreactivity was shown in the SNpc of the control group (
In the control group, SOD2 immunoreactivity was also found in the SNpc (
PD is a neurodegenerative disease accompanied by gradual loss of dopaminergic neurons in SNpc of the midbrain (
It has been reported that MPTP treatment to mice for PD model can result in significant movement disorders, including increased climbing time and reduced swimming time (
In clinical reports, motor symptoms in PD patients are firstly shown at 50-60% loss (death) of dopaminergic cells in the SNpc and 70-80% degradation of dopamine level in the striatum (
Lipids, as major components of the central nervous system serve crucial roles in neural health and pathology (
DNA integrity is a prerequisite for cell survival (
Dopaminergic neurons in PD are thought to be affected by high oxidative stress from enzymatic and non-enzymatic metabolism and dopamine autoxidation (
The present study examined the neuroprotective effect of BF-7® in a mouse model of Parkinson's disease. However, the current study has a limitation in not investigating the pathways that inhibit and/or alleviate oxidative stresses. Therefore, it is proposed that a follow-up study using
In conclusion, the present study provided evidence that BF-7® can exert a neuroprotective effect against MPTP-induced PD in mice, showing that BF-7® treatment can improve motor deficit, alleviate loss or decrease in dopaminergic cells and nerve terminals and reduce oxidative stress (such as lipid peroxidation, DNA damage and decrease in SODs) in a mouse model of MPTP-induced PD. Therefore, the results strongly suggested that BF-7® has potential as a candidate for treating PD.
The authors would like to thank Mr. Seung Uk Lee and Ms. Hyun Sook Kim (Department of Biomedical Science, Research Institute for Bioscience and Biotechnology, Hallym University) for their technical help in this study.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
TKL, JCL, DWK, JWL, HIK and MCS performed the experiments and measurements. TKL, JCL, SSK and JHC analyzed and interpreted data. TKL, JCL and MHW confirm the authenticity of all the raw data. and MHW and SYC made substantial contributions to conception and design and were involved in drafting, revising the manuscript and interpreting all data. All authors have read and approved for the final manuscript.
The protocol of all experiments was approved on January 28, 2020 by the Ethics Committee of Kangwon National University (approval no. KW-200113-2). All experimental procedures adhered to the guidelines described in the 'Current International Laws and Policies', which is included in the Guide for the Care and Use of Laboratory Animals. Every effort was made to reduce the pain of mice and minimize the number of mice used.
Not applicable.
The authors have declared that there is no conflicting interest. Note that J-WL is employed by Famenity Co., Ltd., who produced the drug BF-7® used in this study.
Experimental timeline. At day 0, MPTP (20 mg/kg) and vehicle was intraperitoneally injected four times with two hours of interval. BF-7® and vehicle (saline) was administrated once a day for seven days. Behavioral test was performed at one and two days before MPTP treatment and one and two days after MPTP treatment. At three days after MPTP treatment, mice were sacrificed for analyses. MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; BF-7®, Brain Factor-7®.
Effects of BF-7® on MPTP-induced motor impairment in mice. Latency time on the rotarod is impaired in the vehicle + MPTP group from day 1 to 2 following MPTP treatment. However, the impairment is ameliorated in the BF-7® + MPTP group (n=10, respectively; *P<0.05 vs. control group and #P<0.05 vs. vehicle + MPTP group). The bars indicate the means ± standard error of mean. BF-7®, Brain Factor-7®; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.
Immunohistochemistry for TH and F-J B histofluorescence in the SNpc. Representative images of (Aa-Af) TH immunohistochemistry and (Ag-Ai) F-J B staining in the SNpc of the (a, d and g) control group, (b, e and h) vehicle + MPTP group and (c, f and i) BF-7® + MPTP group three days after MPTP treatment. In the vehicle + MPTP group, TH-stained dopaminergic cells are significantly reduced compared with the control group and F-J B-stained cells are abundantly found; however, in the BF-7® + MPTP group, the number of TH-stained dopaminergic cells is high as compared with the vehicle + MPTP group and the number of F-J B-stained cells is significantly low as compared with the vehicle + MPTP group. Scale bars (a-c) 400 and (d-i) 50 µm. Mean number of (B) TH-stained and (C) F-J B-stained cells (n=10, respectively; *P<0.05 vs. control and #P<0.05 vs. vehicle + MPTP group). The bars indicate the means ± standard error of mean. TH, tyrosine hydroxylase; F-J B, Fluoro-Jade B; SNpr, substantia nigra pars reticulata.
Immunohistochemistry for TH in the striatum. (A) TH immunoreactivity in the striatum of the (a) control group, (b) vehicle + MPTP group and (c) BF-7® + MPTP group three days after MPTP treatment. TH immunoreactivity in the control group is strong. In the vehicle + MPTP group, TH immunoreactivity is significantly decreased three days after MPTP treatment; however, the TH immunoreactivity is ameliorated in the BF-7® + MPTP group compared with the vehicle + MPTP group. Scale bar=200 µm. (B) ROD of TH immunoreactivity in the striatum (n=10, respectively; *P<0.05 vs. control group). The bars indicate the means ± standard error of mean. TH, tyrosine hydroxylase; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; BF-7®, Brain Factor-7®; ROD, relative optical density.
Immunohistochemistry for 4HNE and 8OHdG in the SNpc. Immunohistochemical images of (Aa-Ac) 4HNE and (Ad-Af) 8OHdG in the SNpc of the (a and d) control group, (b and e) vehicle + MPTP group and (c and f) BF-7® + MPTP group three days after MPTP treatment. 4HNE and 8OHdG immunoreactivity (arrows) are shown in the SNpc of the control group. In the vehicle + MPTP group, 4HNE and 8OHdG immunoreactivity are increased three days following MPTP treatment; however, the two increased immunoreactivities are apparently reduced in the BF-7® + MPTP group as compared with the vehicle + MPTP group. Scale bar=50 µm. ROD of (B) 4HNE and (C) 8OHdG immunoreactivity in the SNpc three days following MPTP treatment (n=10, respectively; *P<0.05 vs. control group and #P<0.05 vs. vehicle + MPTP group). The bars indicate the means ± standard error of mean. 4HNE, 4-hydroxy-2-nonenal; 8OHdG, 8-hydroxydeoxyguanosine; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; SNpc, pars compacta of substantia nigra; ROD, relative optical density.
Immunohistochemistry for SOD1 and SOD2 in the SNpc. Representative images of (Aa-Ac) SOD1 and (Ad-Af) SOD2 immunoreactivity in the SNpc of the (a and d) control group, (b and e) vehicle + MPTP group and (c and f) BF-7® + MPTP group three days after MPTP treatment. SOD1 and SOD2 immunoreactivity (arrows) are shown in the SNpc of the control group. In the vehicle + MPTP group, SOD1 and SOD2 immunoreactivity are apparently decreased three days after MPTP treatment. However, the two decreased immunoreactivities are higher in the BF-7® + MPTP group than the vehicle + MPTP group. Scale bar=50 µm. Scale bar=50 µm. ROD of (B) SOD1 and (C) SOD2 immunoreactivities in the SNpc three days following MPTP treatment (n=10, respectively; *P<0.05 vs. control group and #P<0.05 vs. vehicle + MPTP group). The bars indicate the means ± standard error of mean. SOD, superoxide dismutase; SNpc, pars compacta of substantia nigra; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; ROD, relative optical density.