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Acupuncture stimulation at GB34 suppresses 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced oxidative stress in the striatum of mice

Acupuncture stimulation at GB34 suppresses 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced... J Physiol Sci (2018) 68:455–462 https://doi.org/10.1007/s12576-017-0547-7 OR IGINAL PAPER Acupuncture stimulation at GB34 suppresses 1-methyl-4- phenyl-1,2,3,6-tetrahydropyridine-induced oxidative stress in the striatum of mice 1 1 1 1 2 • • • • • Yukyung Lee Gaeun Choi Hyongjun Jeon Dongsoo Kim Sun Ryu 1,2 2 1,2 • • Sungtae Koo Ki-Tae Ha Seungtae Kim Received: 9 December 2016 / Accepted: 28 May 2017 / Published online: 10 June 2017 The Physiological Society of Japan and Springer Japan 2017 Abstract Recent studies have suggested that increased Keywords Parkinson’s disease  Acupuncture oxidative stress is a potential etiology in Parkinson’s dis- Antioxidant  DJ-1  Superoxide dismutase  Catalase ease (PD). In this study, we investigated whether 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine acupuncture regulates antioxidants in the striatum (ST) of a PD mouse model. Male C57BL/6 mice were administered 30 mg/kg of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine Introduction (MPTP) intraperitoneally once a day for 5 days and given acupuncture stimulation at SI3 or GB34 (Yanglingquan) Parkinson’s disease (PD) is a representative neurodegen- was for 12 consecutive days. Dopaminergic neuronal sur- erative disorder that is accompanied by symptoms such as vival in the nigrostriatal pathway and DJ-1 expression in rigidity, postural instability, akinesia, bradykinesia, and the ST was evaluated by immunostaining, and the activities tremor [1]. From an etiological point of view, PD is gen- of superoxide dismutase (SOD) and catalase (CAT) in the erally caused by dopaminergic neuronal degeneration in ST was by enzyme-linked immunosorbent assay. MPTP the nigrostriatal pathway, and it is well known that it is administration induced dopaminergic neuronal death in the influenced by several factors including environmental nigrostriatal pathway, which was suppressed by acupunc- conditions and genetics. Moreover, recent studies have ture stimulation at GB34. MPTP administration also sup- suggested that increased oxidative stress is a potential eti- pressed DJ-1 expression and SOD and CAT activities in the ology in PD [2]. ST, which were restored by acupuncture stimulation at To induce PD-like symptoms and neuronal death, a few GB34. These results indicate that the neuroprotective effect neurotoxins that restrain complex I of the mitochondrial of acupuncture stimulation is due to regulation of the respiratory chain are used. Among them, 1-methyl-4-phe- antioxidants. nyl-1,2,3,6-tetrahydropyridine (MPTP) is well recognized for causing the traits of sporadic PD [3]. When MPTP is absorbed by an organism, it passes through the brain because it can penetrate the blood–brain-barrier. In the brain, monoamine oxidase B converts MPTP to 1-methyl- 4-phenylpyridinium (MPP?), which is a toxic metabolite Yukyung Lee and Gaeun Choi contributed equally to this work. that subsequently permeates into dopaminergic neurons through dopamine transporters. The MPP? in the & Seungtae Kim [email protected] dopaminergic neurons deters the complex I activation, which induces oxidative stress and apoptosis in sequence, Division of Meridian and Structural Medicine, School of leading to development of PD symptoms [4]. Oxidative Korean Medicine, Pusan National University, Busandaehak- stress also plays a significant role in the onset and progress ro 49, Mulgeum-eup, Yangsan-si, Gyeongsangnam-do 50612, Republic of Korea of PD [5]; therefore, suppression of oxidative stress can alleviate PD symptoms and protect against dopaminergic Korean Medicine Research Center for Healthy Aging, Pusan neuronal death in PD patients and animal models. National University, Yangsan-si, Republic of Korea 123 456 J Physiol Sci (2018) 68:455–462 Acupuncture has been broadly applied to treat various (MPTP), a MPTP-injected plus acupuncture at SI3 (Houxi) diseases [6], and there have been several reports of elec- acupoint group (MPTP ? SI3), and a MPTP-injected plus troacupuncture being able to enhance antioxidants. For acupuncture at GB34 acupoint group (MPTP ? GB34). example, Kim et al. showed that 100-Hz electroacupunc- ture at GB34 (Yanglingquan) and GB39 enhances cyto- MPTP injection chrome c oxidase [7], while Lv et al. reported that 100-Hz electroacupuncture alleviates nuclear factor-E2-related Except for the saline group, all of the mice were injected factor-2 and heme oxygenase-1 [8], and Wang et al. sug- with MPTP-HCl (30 mg/kg of free base; Sigma, St. Louis, gested that electroacupuncture stimulation at ST36 and SP6 MO, USA) in sterilized normal saline (vehicle) intraperi- increases glutathione concentration and superoxide dis- toneally at 24-h intervals for 5 days. The mice in the saline mutase (SOD) activity [9]. Taken together, these studies group were injected with vehicle intraperitoneally follow- indicate that 100-Hz electroacupuncture stimulation ing the same schedule [13]. enhances antioxidant activities in the striatum (ST) of MPTP-administrated mice, but it is still not clear whether Acupuncture treatment manual acupuncture stimulation can also enhance them. The GB34 acupoint has been used to cure a variety of Two hours after MPTP injection, mice in the MPTP ? SI3 diseases in lower limbs, muscles, and the gall bladder, as and MPTP ? GB34 groups received acupuncture stimu- well as paralysis, and recent studies have confirmed the lation at SI3 (MPTP ? SI3 group) or GB34 effects of acupuncture stimulation at GB34 on PD animal (MPTP ? GB34 group). SI3 is located at the ulnar end of models [10]. For example, acupuncture stimulation at the distal palmar crease proximal to the 4th metacarpal GB34 suppressed MPTP-induced behavioral impairment phalangeal joint at the junction of the red and white skin, and dopaminergic neuronal destruction in the substantia and GB34 is located at the point at which lines from the nigra (SN) and ST by regulating the PI3K-Akt pathway anterior borders to the head of the fibula intersect. Mice in [11] and enhancing synaptic dopamine availability [12]. the MPTP ? SI3 group were lightly immobilized, after However, although several studies have shown the effects which stainless-steel acupuncture needles of acupuncture on dopaminergic neuronal degeneration in (0.18 mm 9 8 mm; Dongbang acupuncture, Boryeng, PD mouse models, it is still not clear if manual acupuncture Republic of Korea) were inserted to a depth of 2 mm at the at GB34 alleviates MPTP-induced oxidative stress in the SI3 acupoint, turned at a rate of two revolutions per second ST. Therefore, this study investigated the potential for for 30 s, and then immediately removed. The acupuncture acupuncture stimulation to improve motor function, pre- stimulation was performed from the left to right acupoint in vent dopaminergic neuronal death, and alleviate the levels sequence (treatment lasting 60 s). Mice in the of anti-oxidants including DJ-1, SOD, and catalase (CAT) MPTP ? GB34 group underwent the same procedure, in the ST of MPTP-treated mice. except they received acupuncture stimulation at GB34. Mice in the saline and the MPTP groups were also lightly immobilized for 60 s without acupuncture, then returned to Materials and methods their cage. This procedure continued at 24-h intervals for 12 consecutive days. Animals Behavioral test (pole test) This study was conducted with the approval of the Pusan National University Institutional Animal Care and Use A modified version of the method established by Abe et al. Committee. We purchased 8-week-old male C57BL/6 mice [14] was applied for the pole test. Mice were positioned weighing 20–23 g from Orientbio Inc. (Seongnam, Korea) head-downwards near the top of a rough-surfaced wood and housed them at room temperature (22 ± 2 C) under a pole (1 cm in diameter and 55 cm in height), after which standard 12-h light/dark cycle (lights on at 07:00 h) with the time taken to arrive at the bottom of the pole was food and water provided ad libitum. measured. The test was repeated three times at 30-s time intervals, and behavioral changes were evaluated based on Group classification the average of the three times. The test was performed 1 day before the first MPTP administration (day 0), 2 h Mice were randomly placed into four groups of 16: a sal- after the fifth acupuncture stimulation on day 5, and 2 h ine-injected group (saline), a MPTP injected group after the last acupuncture stimulation on day 12. 123 J Physiol Sci (2018) 68:455–462 457 Immunofluorescent staining polyacrylamide gels and transferred to nitrocellulose membranes. The membrane was blocked with 5% bovine After the pole test was conducted on day 12, mice (n = 6 serum albumin in Tris-buffered saline containing 0.1% at each group) were anesthetized with isoflurane (JW Tween-20 for 1 h at room temperature and incubated Pharmaceutical, Seoul, Republic of Korea) and perfused overnight with anti-DJ-1 (Abcam), anti-caspase-3, anti- with 4% paraformaldehyde in 0.1 M phosphate buffer. The phosphorylated AKT and anti-AKT (Cell Signaling Tech- brain was then quickly removed and postfixed in 4% nology) antibodies that were diluted 1:1000 in blocking paraformaldehyde solution for 48 h, after which it was solution antibody at 4 C. Then the membrane was washed washed in 0.1 M phosphate buffer, then immersed in 30% and incubated with horseradish peroxidase-conjugated sucrose solution for storage at 4 C until sectioning. The secondary anti-rabbit antibody (1:2000, Santa Cruz brains were cut to a thickness of 30 lm using a Leica Biotechnology) at room temperature for 1 h. After washing CM3050S cryostat (Leica Microsystems, Wetzlar, Ger- the membrane, bands were detected using the enhanced many). The SN sections located between AP -3.08 and chemiluminescence detection kit (Thermo Scientific, -3.28 mm from the bregma and the ST sections located Rockford, IL, USA). Then, these blots were re-probed with between AP ?0.48 and ?0.68 mm from the bregma were rabbit monoclonal anti-b-actin antibody (1:1000; Santa incubated with 1% H O in 0.05 M phosphate-buffered Cruz Biotechnology) as a loading control for all experi- 2 2 saline for 15 min, followed by 0.3% Triton X-100 and 3% ments. Quantification of immunoreactivity corresponding normal blocking serum in PBS at room temperature for 1 h, to the total bands was performed by densitometric analysis then stained overnight at room temperature using a primary using an Image Quant LAS 4000 (Fujifilm, Tokyo, Japan). antibody. An anti-tyrosine hydroxylase (TH, 1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), an anti-cas- Assessment of superoxide dismutase and catalase pase-3 antibody (1:1000; Abcam, Cambridge, UK) and an in mouse brain anti-DJ-1 antibody (1:100; Cell Signaling Technology, Danvers, MA, USA) were used as a primary antibody. On Mice (n = 6 at each group) were sacrificed and their STs the following day, the sections were reacted with anti- were immediately enucleated after completion of the pole rabbit Alexa-488 IgG for 1 h at room temperature. The test on day 12. Collected ST tissues were homogenized and tissues were then mounted on gelatin-coated slides, air- centrifuged, then the supernate was collected and stored at dried, dehydrated, and coverslipped. -80 C until assayed. The activities of SOD and CAT in Histological features were captured using an Axio the ST were detected using enzyme-linked immunosorbent Observer Z1 microscope (Carl ZEISS, Oberkochen, Ger- assay (ELISA) kits (Cusabio Biotech, Wuhan, China) many). The number of TH-positive neuronal cells in the SN according to the manufacturer’s instructions. Briefly, after was counted to evaluate the survival of dopaminergic 100 ll of sample per well was added and incubated for 2 h neurons. An independent observer who was not aware of at 37 C, the liquid of each well was removed. Then 100 ll the expected test results manually counted the TH-positive of biotin-antibody was added to each well and incubated neurons bilaterally in five continuous SN sections. The for 1 h at 37 C. After each well was aspirated and washed, number of cells was counted and confirmed three times to 100 ll of horseradish peroxidase-avidin was added and validate the data. To evaluate the changes in dopaminergic incubated for 1 h at 37 C. After being washed, 90 llof 0 0 neuronal fibers in the ST, the mean values of optical den- 3,3 ,5,5 -tetramethylbenzidine substrate was added, incu- sity (OD) of the TH, DJ-1, and caspase-3 in the ST were bated for 3 min at 37 C with light protection, 50 ll of stop determined using Image-Pro Plus 6.0 (Media Cybernetics, solution was added to each well. Immediately the SOD and Silver Spring, MD, USA). the CAT activities were measured spectrofluorometrically using a multi-well plate reader, and the value of subtracted Western blot readings at 540 nm from the readings at 450 nm was used. To confirm immunofluorescent data, we performed Wes- Statistical analysis tern-blot analysis. Mice (n = 4 at each group) were sacri- ficed with CO gas on day 12, then ST tissues were All of data were expressed as the means ± the standard errors of mean. All data were analyzed by one-way analysis dissected rapidly and kept at -80 C until use. The tissues were homogenized with protease inhibitor and RIPA buf- of variance (ANOVA) followed by Tukey’s post hoc test fer, centrifuged for 20 min at 4 C at 12,000 rpm, and the using Prism 5 for Windows (GraphPad Software Inc., La supernatants were separated. Equal amounts of protein Jolla, CA, USA). A p \ 0.05 was considered to indicate (30 lg) from each sample were separated on 12% SDS– significance. 123 458 J Physiol Sci (2018) 68:455–462 the OD in the ST. An ANOVA revealed a significant effect Results on the TH-positive neurons in the SN [F(3,23) = 7.953, Effects of acupuncture on MPTP-induced p \ 0.001] and the ST [F(3,23) = 8.131, p\ 0.001] among the groups. The numbers in the MPTP behavioral impairment (72.12 ± 3.15%) and the MPTP ? SI3 (72.39 ± 5.14%) groups were significantly lower than in the saline group The pole test was conducted to verify the efficacy of acupuncture on behavioral impairment by MPTP [14]. No (100 ± 6.47%, p \ 0.01 at each group). However, the number in the MPTP ? GB34 group (89.57 ± 4.63%) was significant differences were observed in the descending time of the mice before MPTP administration (day 0). On significantly higher than in the MPTP and the MPTP ? SI3 groups (p \ 0.05 at each group), and there was no signif- day 5, an ANOVA revealed a significant difference on the icant difference between the saline and the MPTP ? GB34 descending times among the groups [F(3,23) = 6.446, groups. p \ 0.01]. The descending times in all of MPTP-adminis- The OD in the ST of mice was significantly lower in the trated groups were significantly increased relative to the vehicle-administrated (saline) group (p \ 0.05). On day MPTP (53.13 ± 4.96%, p \ 0.001) and the MPTP ? SI3 (62.64 ± 9.48%, p \ 0.01) groups compared with the 12, an ANOVA revealed a significant difference on the descending times among the groups [F(3,23) = 6.929, saline group (100.00 ± 5.05%). Conversely, that in the MPTP ? GB34 group was significantly higher than in the p \ 0.01]. The descending times in the MPTP (12.25 ± 1.32 s) and the MPTP ? SI3 (11.75 ± 1.25 s) MPTP group (79.06 ± 8.33%, p \ 0.05), and there was no significant difference between the saline and the groups were still significantly higher than in the saline MPTP ? GB34 groups (Fig. 2). group (7.60 ± 0.75 s, p\ 0.05 at each group), but that in the MPTP ? GB34 group (7.25 ± 0.63 s) was signifi- Change in caspase-3 expression in the ST cantly decreased compared to those in the MPTP and MPTP ? SI3 groups (p\ 0.05 at each group). There was To confirm the level of MPTP-induced apoptosis, we no significant difference between the saline and the MPTP ? GB34 groups (Fig. 1). investigated the caspase-3 expression in the ST because the caspase-3 is an indicator of apoptosis [16]. ANOVA Neuroprotective effects of acupuncture on MPTP- revealed a significant difference on the caspase-3 expres- sion among the groups [F(3,23) = 9.053, p\ 0.001]. induced neuronal death in the SN and the ST When compared with caspase-3 expression in the ST of mice in the saline group (100.00 ± 6.95%), the expression TH is a standard marker to identify dopaminergic neurons [15]. The neuroprotective effects of acupuncture on MPTP- levels in the MPTP (129.00 ± 5.48%) and the MPTP ? SI3 (117.00 ± 5.25%) groups were significantly induced neuronal death were evaluated by counting the increased (p \ 0.01 and p \ 0.05, respectively). However, number of TH-positive neurons in the SN and measuring the expression in the MPTP ? GB34 group (93.58 ± 4.21%) was significantly decreased compared to the levels in the MPTP (p \ 0.001) and the MPTP ? SI3 (p\ 0.05) groups, and there was no significant difference between the saline and the MPTP ? GB34 groups (Fig. 3). Change in DJ-1 expression in the ST We investigated DJ-1 expression in the ST because it is a PD-related protein [17] as well as an anti-oxidant [18]. ANOVA revealed a significant difference on the DJ-1 expression among the groups [F(3,23) = 5.420, p\ 0.01]. When compared with the DJ-1 expression in the ST of Fig. 1 Pole test results. Acupuncture stimulation at GB34 alleviated mice in the saline group (100.00 ± 4.70%), significant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced reductions were found in the MPTP (82.02 ± 6.20%, behavioral impairment on day 12. Saline saline injected group, MPTP MPTP injected group, MPTP ? SI3 MPTP injected plus p \ 0.05). Conversely, that in MPTP ? GB34 group was acupuncture at SI3 (Houxi) acupoint group, MPTP ? GB34 MPTP significantly higher than that in the MPTP group injected plus acupuncture at GB34 (Yanglingquan) acupoint group. (107.80 ± 2.51%, p \ 0.01), and there was no significant Data shown are the means ± the standard errors of means. *p \ 0.05 difference between the saline and the MPTP ? GB34 versus the saline group, p \ 0.05 versus the MPTP group and groups (Fig. 3). p \ 0.05 versus the MPTP ? SI3 group 123 J Physiol Sci (2018) 68:455–462 459 Fig. 2 Effects of acupuncture stimulation on MPTP-induced bar 200 lm. Data are shown as the means ± the standard errors of dopaminergic neuronal death in the substantia nigra (SN) and the means. **p \ 0.01 and ***p \ 0.001 versus the saline group and striatum (ST). MPTP administration induced dopaminergic neuronal p \ 0.05 versus the MPTP group and p \ 0.05 versus the death, whereas acupuncture stimulation at GB34 prevented it. Scale MPTP ? SI3 group Confirmation of caspase-3 and DJ-1 expressions of mice in the saline group (100.00 ± 7.20%), the by Western-blot analysis activities in the MPTP (65.94 ± 6.36%) and the MPTP ? SI3 (73.66 ± 7.47%) groups were significantly To verify the reliability of the immunofluorescent data, decreased (p \ 0.05 at each group). However, that in the Western-blot analysis was performed. The results of MPTP ? GB34 group (92.98 ± 4.25%) was significantly triplicate Western blots for caspase-3 and DJ-1 were higher than those in the MPTP and the MPTP ? SI3 consistent with those of immunofluorescence staining groups (p \ 0.05 at each group), and there was no sig- (Fig. 4). nificant difference between the saline and the MPTP ? GB34 groups (Fig. 5). Changes in SOD and CAT activities in the ST Similar results were observed for CAT activity. When compared with the saline group (100.00 ± 7.44%), sig- Decreased endogenous antioxidants are risk factors caus- nificant reductions in CAT activity were found in the ing early degeneration of dopaminergic neurons [8]. To MPTP (72.11 ± 7.13%, p \ 0.05) and the MPTP ? SI3 confirm whether acupuncture regulates anti-oxidants in (73.00 ± 6.54%, p \ 0.05) groups. On the other hand, the the ST, the activities of SOD and CAT were measured by activity in the MPTP ? GB34 group was significantly ELISA. ANOVA revealed a significant difference on the higher than in the MPTP and the MPTP ? SI3 groups SOD [F(3,23) = 6.156, p \ 0.01] and the CAT (95.51 ± 3.97%, p\ 0.05 at each group). There was no [F(3,23) = 5.229, p \ 0.01] expressions among the significant difference between the saline and the groups. When compared with the SOD activity in the ST MPTP ? GB34 groups (Fig. 5). 123 460 J Physiol Sci (2018) 68:455–462 Fig. 3 Changes in caspase-3 and DJ-1 expressions in the striatum in 200 lm. Data are shown as the means ± the standard errors of response to MPTP and acupuncture stimulations. MPTP administra- means. *p \ 0.05 and **p \ 0.01 versus the saline group, p \ 0.05, ## ### tion increased caspase-3 expression and suppressed DJ-1 expression; p \ 0.01 and p \ 0.001 versus the MPTP group, and p \ 0.05 however, acupuncture stimulation at GB34 alleviated them. Scale bar versus the MPTP ? SI3 group present study also demonstrated that acupuncture stimu- lation improved MPTP-induced behavioral impairment, protected against dopaminergic neuronal death and sup- pressed caspase-3 expression. In addition, it could increase the level of DJ-1 and the activities of SOD and CAT in the ST. DJ-1, also known as PD protein 7, has been recognized as a major protein in the pathogenesis of PD. Previous studies have shown that DJ-1 inhibits the aggregation of a- synuclein by regulating chaperone activity under oxidative conditions [17], while it protects dopaminergic neurons in Fig. 4 Confirmation of caspase-3 and DJ-1 expressions in the striatum by Western-blot analysis. The results of triplicate Western the nigrostriatal pathway against MPTP-induced oxidative blots for caspase-3 and DJ-1 were consistent with those of stress [18], and that the inhibition of DJ-1 causes immunofluorescence staining dopaminergic neuronal degeneration via increases in reactive oxygen species [19]. Based on these results, DJ-1 plays a crucial role in the pathogenesis of PD by regulating Discussion oxidative stress [20]. In the present study, MPTP admin- istration suppressed DJ-1 expression in the ST, which was Previous studies have shown that acupuncture stimulation alleviated by acupuncture stimulation at GB34. These at GB34 suppresses behavioral impairment and dopaminergic neuronal death from MPTP toxicity. This results indicate that the enhancement of DJ-1 expression by 123 J Physiol Sci (2018) 68:455–462 461 Fig. 5 Changes in superoxide dismutase and catalase activities in the **p \ 0.01 versus the saline group, p \ 0.05 versus the MPTP striatum. MPTP administration suppressed the activities, which were group, and p \ 0.05 versus the MPTP ? SI3 group restored by acupuncture stimulation at GB34. *p \ 0.05 and acupuncture stimulation at GB34 influences the neuropro- by acupuncture stimulation at GB34 may influence the tective effect of the acupuncture stimulation. enhancement of SOD and CAT activities in the ST of A recent study showed that modulation of the Akt MPTP-treated mice. However, additional study is needed pathway is essential for DJ-1 activity, which protects to confirm this. dopaminergic neurons in the nigrostriatal pathway against MPTP toxicity [18]. Interestingly, previous studies have Conclusions shown that the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway plays a central role in the mechanism MPTP administration caused behavioral impairment, underlying acupuncture-induced benefits. For example, dopaminergic neuronal death in the nigrostriatal pathway, acupuncture increased Akt phosphorylation in the brain reduction of DJ-1 expression, and suppression of SOD and after traumatic brain injury [21] and electroacupuncture CAT activities in the ST. These changes were alleviated by ameliorated acute renal injury in lipopolysaccharide-stim- acupuncture stimulation at GB34, indicating that the neu- ulated rabbits [22] and exerted an anti-apoptotic effect on roprotective effect of acupuncture stimulation is due to cerebral ischemia–reperfusion injury [23] through modu- regulation of the antioxidants. Taken together, these find- lating PI3K/Akt pathway. Moreover, Kim et al. reported ings indicate that acupuncture stimulation at GB34 can be that PI3K-Akt signaling pathway mediates acupuncture- an alternative therapy for PD. induced dopaminergic neuronal protection in the brains of MPTP-treated mice [11]. Therefore, the increase of DJ-1 Acknowledgements This work was supported by a National observed in this study may have been caused by the Research Foundation of Korea (NRF) grant funded by the Korean increase of Akt phosphorylation induced by acupuncture government (MSIP) (No. NRF-2014R1A5A2009936). stimulation at GB34. Compliance with ethical standards MPTP is widely used for PD research. Following administration, it is metabolized into 1-methyl-4- Conflict of interest The authors declare that they have no conflicts of phenylpyridinum by monoamine oxidase B, which leads to interest. increased oxidative stress that causes neuronal death [24], as well as interruption of antioxidant activities including References those of SOD and CAT [25]. The combination of increased oxidative stress and decreased endogenous antioxidants is a 1. 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Plant Signal hydroxylase to define dopaminergic neurons for use in cell Behav 5:1034–1036 replacement therapies for Parkinson’s disease. CNS Neurol Dis- ord Drug Targets 11:340–349 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Physiological Sciences Springer Journals

Acupuncture stimulation at GB34 suppresses 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced oxidative stress in the striatum of mice

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References (28)

Publisher
Springer Journals
Copyright
Copyright © 2017 by The Physiological Society of Japan and Springer Japan
Subject
Biomedicine; Human Physiology; Neurosciences; Animal Biochemistry; Animal Physiology; Cell Physiology; Neurobiology
ISSN
1880-6546
eISSN
1880-6562
DOI
10.1007/s12576-017-0547-7
pmid
28601951
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Abstract

J Physiol Sci (2018) 68:455–462 https://doi.org/10.1007/s12576-017-0547-7 OR IGINAL PAPER Acupuncture stimulation at GB34 suppresses 1-methyl-4- phenyl-1,2,3,6-tetrahydropyridine-induced oxidative stress in the striatum of mice 1 1 1 1 2 • • • • • Yukyung Lee Gaeun Choi Hyongjun Jeon Dongsoo Kim Sun Ryu 1,2 2 1,2 • • Sungtae Koo Ki-Tae Ha Seungtae Kim Received: 9 December 2016 / Accepted: 28 May 2017 / Published online: 10 June 2017 The Physiological Society of Japan and Springer Japan 2017 Abstract Recent studies have suggested that increased Keywords Parkinson’s disease  Acupuncture oxidative stress is a potential etiology in Parkinson’s dis- Antioxidant  DJ-1  Superoxide dismutase  Catalase ease (PD). In this study, we investigated whether 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine acupuncture regulates antioxidants in the striatum (ST) of a PD mouse model. Male C57BL/6 mice were administered 30 mg/kg of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine Introduction (MPTP) intraperitoneally once a day for 5 days and given acupuncture stimulation at SI3 or GB34 (Yanglingquan) Parkinson’s disease (PD) is a representative neurodegen- was for 12 consecutive days. Dopaminergic neuronal sur- erative disorder that is accompanied by symptoms such as vival in the nigrostriatal pathway and DJ-1 expression in rigidity, postural instability, akinesia, bradykinesia, and the ST was evaluated by immunostaining, and the activities tremor [1]. From an etiological point of view, PD is gen- of superoxide dismutase (SOD) and catalase (CAT) in the erally caused by dopaminergic neuronal degeneration in ST was by enzyme-linked immunosorbent assay. MPTP the nigrostriatal pathway, and it is well known that it is administration induced dopaminergic neuronal death in the influenced by several factors including environmental nigrostriatal pathway, which was suppressed by acupunc- conditions and genetics. Moreover, recent studies have ture stimulation at GB34. MPTP administration also sup- suggested that increased oxidative stress is a potential eti- pressed DJ-1 expression and SOD and CAT activities in the ology in PD [2]. ST, which were restored by acupuncture stimulation at To induce PD-like symptoms and neuronal death, a few GB34. These results indicate that the neuroprotective effect neurotoxins that restrain complex I of the mitochondrial of acupuncture stimulation is due to regulation of the respiratory chain are used. Among them, 1-methyl-4-phe- antioxidants. nyl-1,2,3,6-tetrahydropyridine (MPTP) is well recognized for causing the traits of sporadic PD [3]. When MPTP is absorbed by an organism, it passes through the brain because it can penetrate the blood–brain-barrier. In the brain, monoamine oxidase B converts MPTP to 1-methyl- 4-phenylpyridinium (MPP?), which is a toxic metabolite Yukyung Lee and Gaeun Choi contributed equally to this work. that subsequently permeates into dopaminergic neurons through dopamine transporters. The MPP? in the & Seungtae Kim [email protected] dopaminergic neurons deters the complex I activation, which induces oxidative stress and apoptosis in sequence, Division of Meridian and Structural Medicine, School of leading to development of PD symptoms [4]. Oxidative Korean Medicine, Pusan National University, Busandaehak- stress also plays a significant role in the onset and progress ro 49, Mulgeum-eup, Yangsan-si, Gyeongsangnam-do 50612, Republic of Korea of PD [5]; therefore, suppression of oxidative stress can alleviate PD symptoms and protect against dopaminergic Korean Medicine Research Center for Healthy Aging, Pusan neuronal death in PD patients and animal models. National University, Yangsan-si, Republic of Korea 123 456 J Physiol Sci (2018) 68:455–462 Acupuncture has been broadly applied to treat various (MPTP), a MPTP-injected plus acupuncture at SI3 (Houxi) diseases [6], and there have been several reports of elec- acupoint group (MPTP ? SI3), and a MPTP-injected plus troacupuncture being able to enhance antioxidants. For acupuncture at GB34 acupoint group (MPTP ? GB34). example, Kim et al. showed that 100-Hz electroacupunc- ture at GB34 (Yanglingquan) and GB39 enhances cyto- MPTP injection chrome c oxidase [7], while Lv et al. reported that 100-Hz electroacupuncture alleviates nuclear factor-E2-related Except for the saline group, all of the mice were injected factor-2 and heme oxygenase-1 [8], and Wang et al. sug- with MPTP-HCl (30 mg/kg of free base; Sigma, St. Louis, gested that electroacupuncture stimulation at ST36 and SP6 MO, USA) in sterilized normal saline (vehicle) intraperi- increases glutathione concentration and superoxide dis- toneally at 24-h intervals for 5 days. The mice in the saline mutase (SOD) activity [9]. Taken together, these studies group were injected with vehicle intraperitoneally follow- indicate that 100-Hz electroacupuncture stimulation ing the same schedule [13]. enhances antioxidant activities in the striatum (ST) of MPTP-administrated mice, but it is still not clear whether Acupuncture treatment manual acupuncture stimulation can also enhance them. The GB34 acupoint has been used to cure a variety of Two hours after MPTP injection, mice in the MPTP ? SI3 diseases in lower limbs, muscles, and the gall bladder, as and MPTP ? GB34 groups received acupuncture stimu- well as paralysis, and recent studies have confirmed the lation at SI3 (MPTP ? SI3 group) or GB34 effects of acupuncture stimulation at GB34 on PD animal (MPTP ? GB34 group). SI3 is located at the ulnar end of models [10]. For example, acupuncture stimulation at the distal palmar crease proximal to the 4th metacarpal GB34 suppressed MPTP-induced behavioral impairment phalangeal joint at the junction of the red and white skin, and dopaminergic neuronal destruction in the substantia and GB34 is located at the point at which lines from the nigra (SN) and ST by regulating the PI3K-Akt pathway anterior borders to the head of the fibula intersect. Mice in [11] and enhancing synaptic dopamine availability [12]. the MPTP ? SI3 group were lightly immobilized, after However, although several studies have shown the effects which stainless-steel acupuncture needles of acupuncture on dopaminergic neuronal degeneration in (0.18 mm 9 8 mm; Dongbang acupuncture, Boryeng, PD mouse models, it is still not clear if manual acupuncture Republic of Korea) were inserted to a depth of 2 mm at the at GB34 alleviates MPTP-induced oxidative stress in the SI3 acupoint, turned at a rate of two revolutions per second ST. Therefore, this study investigated the potential for for 30 s, and then immediately removed. The acupuncture acupuncture stimulation to improve motor function, pre- stimulation was performed from the left to right acupoint in vent dopaminergic neuronal death, and alleviate the levels sequence (treatment lasting 60 s). Mice in the of anti-oxidants including DJ-1, SOD, and catalase (CAT) MPTP ? GB34 group underwent the same procedure, in the ST of MPTP-treated mice. except they received acupuncture stimulation at GB34. Mice in the saline and the MPTP groups were also lightly immobilized for 60 s without acupuncture, then returned to Materials and methods their cage. This procedure continued at 24-h intervals for 12 consecutive days. Animals Behavioral test (pole test) This study was conducted with the approval of the Pusan National University Institutional Animal Care and Use A modified version of the method established by Abe et al. Committee. We purchased 8-week-old male C57BL/6 mice [14] was applied for the pole test. Mice were positioned weighing 20–23 g from Orientbio Inc. (Seongnam, Korea) head-downwards near the top of a rough-surfaced wood and housed them at room temperature (22 ± 2 C) under a pole (1 cm in diameter and 55 cm in height), after which standard 12-h light/dark cycle (lights on at 07:00 h) with the time taken to arrive at the bottom of the pole was food and water provided ad libitum. measured. The test was repeated three times at 30-s time intervals, and behavioral changes were evaluated based on Group classification the average of the three times. The test was performed 1 day before the first MPTP administration (day 0), 2 h Mice were randomly placed into four groups of 16: a sal- after the fifth acupuncture stimulation on day 5, and 2 h ine-injected group (saline), a MPTP injected group after the last acupuncture stimulation on day 12. 123 J Physiol Sci (2018) 68:455–462 457 Immunofluorescent staining polyacrylamide gels and transferred to nitrocellulose membranes. The membrane was blocked with 5% bovine After the pole test was conducted on day 12, mice (n = 6 serum albumin in Tris-buffered saline containing 0.1% at each group) were anesthetized with isoflurane (JW Tween-20 for 1 h at room temperature and incubated Pharmaceutical, Seoul, Republic of Korea) and perfused overnight with anti-DJ-1 (Abcam), anti-caspase-3, anti- with 4% paraformaldehyde in 0.1 M phosphate buffer. The phosphorylated AKT and anti-AKT (Cell Signaling Tech- brain was then quickly removed and postfixed in 4% nology) antibodies that were diluted 1:1000 in blocking paraformaldehyde solution for 48 h, after which it was solution antibody at 4 C. Then the membrane was washed washed in 0.1 M phosphate buffer, then immersed in 30% and incubated with horseradish peroxidase-conjugated sucrose solution for storage at 4 C until sectioning. The secondary anti-rabbit antibody (1:2000, Santa Cruz brains were cut to a thickness of 30 lm using a Leica Biotechnology) at room temperature for 1 h. After washing CM3050S cryostat (Leica Microsystems, Wetzlar, Ger- the membrane, bands were detected using the enhanced many). The SN sections located between AP -3.08 and chemiluminescence detection kit (Thermo Scientific, -3.28 mm from the bregma and the ST sections located Rockford, IL, USA). Then, these blots were re-probed with between AP ?0.48 and ?0.68 mm from the bregma were rabbit monoclonal anti-b-actin antibody (1:1000; Santa incubated with 1% H O in 0.05 M phosphate-buffered Cruz Biotechnology) as a loading control for all experi- 2 2 saline for 15 min, followed by 0.3% Triton X-100 and 3% ments. Quantification of immunoreactivity corresponding normal blocking serum in PBS at room temperature for 1 h, to the total bands was performed by densitometric analysis then stained overnight at room temperature using a primary using an Image Quant LAS 4000 (Fujifilm, Tokyo, Japan). antibody. An anti-tyrosine hydroxylase (TH, 1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), an anti-cas- Assessment of superoxide dismutase and catalase pase-3 antibody (1:1000; Abcam, Cambridge, UK) and an in mouse brain anti-DJ-1 antibody (1:100; Cell Signaling Technology, Danvers, MA, USA) were used as a primary antibody. On Mice (n = 6 at each group) were sacrificed and their STs the following day, the sections were reacted with anti- were immediately enucleated after completion of the pole rabbit Alexa-488 IgG for 1 h at room temperature. The test on day 12. Collected ST tissues were homogenized and tissues were then mounted on gelatin-coated slides, air- centrifuged, then the supernate was collected and stored at dried, dehydrated, and coverslipped. -80 C until assayed. The activities of SOD and CAT in Histological features were captured using an Axio the ST were detected using enzyme-linked immunosorbent Observer Z1 microscope (Carl ZEISS, Oberkochen, Ger- assay (ELISA) kits (Cusabio Biotech, Wuhan, China) many). The number of TH-positive neuronal cells in the SN according to the manufacturer’s instructions. Briefly, after was counted to evaluate the survival of dopaminergic 100 ll of sample per well was added and incubated for 2 h neurons. An independent observer who was not aware of at 37 C, the liquid of each well was removed. Then 100 ll the expected test results manually counted the TH-positive of biotin-antibody was added to each well and incubated neurons bilaterally in five continuous SN sections. The for 1 h at 37 C. After each well was aspirated and washed, number of cells was counted and confirmed three times to 100 ll of horseradish peroxidase-avidin was added and validate the data. To evaluate the changes in dopaminergic incubated for 1 h at 37 C. After being washed, 90 llof 0 0 neuronal fibers in the ST, the mean values of optical den- 3,3 ,5,5 -tetramethylbenzidine substrate was added, incu- sity (OD) of the TH, DJ-1, and caspase-3 in the ST were bated for 3 min at 37 C with light protection, 50 ll of stop determined using Image-Pro Plus 6.0 (Media Cybernetics, solution was added to each well. Immediately the SOD and Silver Spring, MD, USA). the CAT activities were measured spectrofluorometrically using a multi-well plate reader, and the value of subtracted Western blot readings at 540 nm from the readings at 450 nm was used. To confirm immunofluorescent data, we performed Wes- Statistical analysis tern-blot analysis. Mice (n = 4 at each group) were sacri- ficed with CO gas on day 12, then ST tissues were All of data were expressed as the means ± the standard errors of mean. All data were analyzed by one-way analysis dissected rapidly and kept at -80 C until use. The tissues were homogenized with protease inhibitor and RIPA buf- of variance (ANOVA) followed by Tukey’s post hoc test fer, centrifuged for 20 min at 4 C at 12,000 rpm, and the using Prism 5 for Windows (GraphPad Software Inc., La supernatants were separated. Equal amounts of protein Jolla, CA, USA). A p \ 0.05 was considered to indicate (30 lg) from each sample were separated on 12% SDS– significance. 123 458 J Physiol Sci (2018) 68:455–462 the OD in the ST. An ANOVA revealed a significant effect Results on the TH-positive neurons in the SN [F(3,23) = 7.953, Effects of acupuncture on MPTP-induced p \ 0.001] and the ST [F(3,23) = 8.131, p\ 0.001] among the groups. The numbers in the MPTP behavioral impairment (72.12 ± 3.15%) and the MPTP ? SI3 (72.39 ± 5.14%) groups were significantly lower than in the saline group The pole test was conducted to verify the efficacy of acupuncture on behavioral impairment by MPTP [14]. No (100 ± 6.47%, p \ 0.01 at each group). However, the number in the MPTP ? GB34 group (89.57 ± 4.63%) was significant differences were observed in the descending time of the mice before MPTP administration (day 0). On significantly higher than in the MPTP and the MPTP ? SI3 groups (p \ 0.05 at each group), and there was no signif- day 5, an ANOVA revealed a significant difference on the icant difference between the saline and the MPTP ? GB34 descending times among the groups [F(3,23) = 6.446, groups. p \ 0.01]. The descending times in all of MPTP-adminis- The OD in the ST of mice was significantly lower in the trated groups were significantly increased relative to the vehicle-administrated (saline) group (p \ 0.05). On day MPTP (53.13 ± 4.96%, p \ 0.001) and the MPTP ? SI3 (62.64 ± 9.48%, p \ 0.01) groups compared with the 12, an ANOVA revealed a significant difference on the descending times among the groups [F(3,23) = 6.929, saline group (100.00 ± 5.05%). Conversely, that in the MPTP ? GB34 group was significantly higher than in the p \ 0.01]. The descending times in the MPTP (12.25 ± 1.32 s) and the MPTP ? SI3 (11.75 ± 1.25 s) MPTP group (79.06 ± 8.33%, p \ 0.05), and there was no significant difference between the saline and the groups were still significantly higher than in the saline MPTP ? GB34 groups (Fig. 2). group (7.60 ± 0.75 s, p\ 0.05 at each group), but that in the MPTP ? GB34 group (7.25 ± 0.63 s) was signifi- Change in caspase-3 expression in the ST cantly decreased compared to those in the MPTP and MPTP ? SI3 groups (p\ 0.05 at each group). There was To confirm the level of MPTP-induced apoptosis, we no significant difference between the saline and the MPTP ? GB34 groups (Fig. 1). investigated the caspase-3 expression in the ST because the caspase-3 is an indicator of apoptosis [16]. ANOVA Neuroprotective effects of acupuncture on MPTP- revealed a significant difference on the caspase-3 expres- sion among the groups [F(3,23) = 9.053, p\ 0.001]. induced neuronal death in the SN and the ST When compared with caspase-3 expression in the ST of mice in the saline group (100.00 ± 6.95%), the expression TH is a standard marker to identify dopaminergic neurons [15]. The neuroprotective effects of acupuncture on MPTP- levels in the MPTP (129.00 ± 5.48%) and the MPTP ? SI3 (117.00 ± 5.25%) groups were significantly induced neuronal death were evaluated by counting the increased (p \ 0.01 and p \ 0.05, respectively). However, number of TH-positive neurons in the SN and measuring the expression in the MPTP ? GB34 group (93.58 ± 4.21%) was significantly decreased compared to the levels in the MPTP (p \ 0.001) and the MPTP ? SI3 (p\ 0.05) groups, and there was no significant difference between the saline and the MPTP ? GB34 groups (Fig. 3). Change in DJ-1 expression in the ST We investigated DJ-1 expression in the ST because it is a PD-related protein [17] as well as an anti-oxidant [18]. ANOVA revealed a significant difference on the DJ-1 expression among the groups [F(3,23) = 5.420, p\ 0.01]. When compared with the DJ-1 expression in the ST of Fig. 1 Pole test results. Acupuncture stimulation at GB34 alleviated mice in the saline group (100.00 ± 4.70%), significant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced reductions were found in the MPTP (82.02 ± 6.20%, behavioral impairment on day 12. Saline saline injected group, MPTP MPTP injected group, MPTP ? SI3 MPTP injected plus p \ 0.05). Conversely, that in MPTP ? GB34 group was acupuncture at SI3 (Houxi) acupoint group, MPTP ? GB34 MPTP significantly higher than that in the MPTP group injected plus acupuncture at GB34 (Yanglingquan) acupoint group. (107.80 ± 2.51%, p \ 0.01), and there was no significant Data shown are the means ± the standard errors of means. *p \ 0.05 difference between the saline and the MPTP ? GB34 versus the saline group, p \ 0.05 versus the MPTP group and groups (Fig. 3). p \ 0.05 versus the MPTP ? SI3 group 123 J Physiol Sci (2018) 68:455–462 459 Fig. 2 Effects of acupuncture stimulation on MPTP-induced bar 200 lm. Data are shown as the means ± the standard errors of dopaminergic neuronal death in the substantia nigra (SN) and the means. **p \ 0.01 and ***p \ 0.001 versus the saline group and striatum (ST). MPTP administration induced dopaminergic neuronal p \ 0.05 versus the MPTP group and p \ 0.05 versus the death, whereas acupuncture stimulation at GB34 prevented it. Scale MPTP ? SI3 group Confirmation of caspase-3 and DJ-1 expressions of mice in the saline group (100.00 ± 7.20%), the by Western-blot analysis activities in the MPTP (65.94 ± 6.36%) and the MPTP ? SI3 (73.66 ± 7.47%) groups were significantly To verify the reliability of the immunofluorescent data, decreased (p \ 0.05 at each group). However, that in the Western-blot analysis was performed. The results of MPTP ? GB34 group (92.98 ± 4.25%) was significantly triplicate Western blots for caspase-3 and DJ-1 were higher than those in the MPTP and the MPTP ? SI3 consistent with those of immunofluorescence staining groups (p \ 0.05 at each group), and there was no sig- (Fig. 4). nificant difference between the saline and the MPTP ? GB34 groups (Fig. 5). Changes in SOD and CAT activities in the ST Similar results were observed for CAT activity. When compared with the saline group (100.00 ± 7.44%), sig- Decreased endogenous antioxidants are risk factors caus- nificant reductions in CAT activity were found in the ing early degeneration of dopaminergic neurons [8]. To MPTP (72.11 ± 7.13%, p \ 0.05) and the MPTP ? SI3 confirm whether acupuncture regulates anti-oxidants in (73.00 ± 6.54%, p \ 0.05) groups. On the other hand, the the ST, the activities of SOD and CAT were measured by activity in the MPTP ? GB34 group was significantly ELISA. ANOVA revealed a significant difference on the higher than in the MPTP and the MPTP ? SI3 groups SOD [F(3,23) = 6.156, p \ 0.01] and the CAT (95.51 ± 3.97%, p\ 0.05 at each group). There was no [F(3,23) = 5.229, p \ 0.01] expressions among the significant difference between the saline and the groups. When compared with the SOD activity in the ST MPTP ? GB34 groups (Fig. 5). 123 460 J Physiol Sci (2018) 68:455–462 Fig. 3 Changes in caspase-3 and DJ-1 expressions in the striatum in 200 lm. Data are shown as the means ± the standard errors of response to MPTP and acupuncture stimulations. MPTP administra- means. *p \ 0.05 and **p \ 0.01 versus the saline group, p \ 0.05, ## ### tion increased caspase-3 expression and suppressed DJ-1 expression; p \ 0.01 and p \ 0.001 versus the MPTP group, and p \ 0.05 however, acupuncture stimulation at GB34 alleviated them. Scale bar versus the MPTP ? SI3 group present study also demonstrated that acupuncture stimu- lation improved MPTP-induced behavioral impairment, protected against dopaminergic neuronal death and sup- pressed caspase-3 expression. In addition, it could increase the level of DJ-1 and the activities of SOD and CAT in the ST. DJ-1, also known as PD protein 7, has been recognized as a major protein in the pathogenesis of PD. Previous studies have shown that DJ-1 inhibits the aggregation of a- synuclein by regulating chaperone activity under oxidative conditions [17], while it protects dopaminergic neurons in Fig. 4 Confirmation of caspase-3 and DJ-1 expressions in the striatum by Western-blot analysis. The results of triplicate Western the nigrostriatal pathway against MPTP-induced oxidative blots for caspase-3 and DJ-1 were consistent with those of stress [18], and that the inhibition of DJ-1 causes immunofluorescence staining dopaminergic neuronal degeneration via increases in reactive oxygen species [19]. Based on these results, DJ-1 plays a crucial role in the pathogenesis of PD by regulating Discussion oxidative stress [20]. In the present study, MPTP admin- istration suppressed DJ-1 expression in the ST, which was Previous studies have shown that acupuncture stimulation alleviated by acupuncture stimulation at GB34. These at GB34 suppresses behavioral impairment and dopaminergic neuronal death from MPTP toxicity. This results indicate that the enhancement of DJ-1 expression by 123 J Physiol Sci (2018) 68:455–462 461 Fig. 5 Changes in superoxide dismutase and catalase activities in the **p \ 0.01 versus the saline group, p \ 0.05 versus the MPTP striatum. MPTP administration suppressed the activities, which were group, and p \ 0.05 versus the MPTP ? SI3 group restored by acupuncture stimulation at GB34. *p \ 0.05 and acupuncture stimulation at GB34 influences the neuropro- by acupuncture stimulation at GB34 may influence the tective effect of the acupuncture stimulation. enhancement of SOD and CAT activities in the ST of A recent study showed that modulation of the Akt MPTP-treated mice. However, additional study is needed pathway is essential for DJ-1 activity, which protects to confirm this. dopaminergic neurons in the nigrostriatal pathway against MPTP toxicity [18]. Interestingly, previous studies have Conclusions shown that the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway plays a central role in the mechanism MPTP administration caused behavioral impairment, underlying acupuncture-induced benefits. For example, dopaminergic neuronal death in the nigrostriatal pathway, acupuncture increased Akt phosphorylation in the brain reduction of DJ-1 expression, and suppression of SOD and after traumatic brain injury [21] and electroacupuncture CAT activities in the ST. These changes were alleviated by ameliorated acute renal injury in lipopolysaccharide-stim- acupuncture stimulation at GB34, indicating that the neu- ulated rabbits [22] and exerted an anti-apoptotic effect on roprotective effect of acupuncture stimulation is due to cerebral ischemia–reperfusion injury [23] through modu- regulation of the antioxidants. Taken together, these find- lating PI3K/Akt pathway. Moreover, Kim et al. reported ings indicate that acupuncture stimulation at GB34 can be that PI3K-Akt signaling pathway mediates acupuncture- an alternative therapy for PD. induced dopaminergic neuronal protection in the brains of MPTP-treated mice [11]. Therefore, the increase of DJ-1 Acknowledgements This work was supported by a National observed in this study may have been caused by the Research Foundation of Korea (NRF) grant funded by the Korean increase of Akt phosphorylation induced by acupuncture government (MSIP) (No. NRF-2014R1A5A2009936). stimulation at GB34. Compliance with ethical standards MPTP is widely used for PD research. Following administration, it is metabolized into 1-methyl-4- Conflict of interest The authors declare that they have no conflicts of phenylpyridinum by monoamine oxidase B, which leads to interest. increased oxidative stress that causes neuronal death [24], as well as interruption of antioxidant activities including References those of SOD and CAT [25]. The combination of increased oxidative stress and decreased endogenous antioxidants is a 1. 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Journal

The Journal of Physiological SciencesSpringer Journals

Published: Jun 10, 2017

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