1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine를 이용한 파킨슨병 생쥐 모델에서의 laminin 발현에 대한 양릉천
자침의 조절효과
김연정1⋅김범식2⋅박히준3,4
1경희대학교 간호과학대학, 동서간호학연구소, 2경희대학교 의과대학, 고황의학연구소
3경희대학교 한의과대학 경혈학교실, 4경희대학교 침구경락과학연구센터
Acupuncture at GB34 modulates laminin expression in
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induced PD mouse model
Youn-Jung Kim1, Bum-Shik Kim2, Hi-Joon Park3,4
1East-west Nursing Research Institute, College of Nursing Science, Kyung Hee University,
2Kohwang Medical Research Institute, College of Medicine, Kyung Hee University,
3Department of Meridian &Acupoint, College of Korean Medicine, Kyung Hee University,
4Acupuncture &Meridian Science Research Center, Kyung Hee University Abstract
목 적 : 본 연구의 목적은 양릉천 침 처치 시 C57BL/6 생쥐의 중뇌 흑질에 위치한 도파민성 신경세포 사멸 억 제 효과를 조직화학 염색법을 이용하여 Tyrosine hydroxylase(TH)와 laminin의 발현으로 관찰하고자 한다.
실험방법 : 실험에 이용한 동물은 C57BL/6 생쥐로, 매일 25mg/kg의 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)를 5일간 주사하였고, 매일 MPTP 주사한 뒤 2시간 후에 양릉천에 침치료를 시행하였으며 MPTP 주 사를 종료한 뒤 침치료는 7일동안 계속 시행하였다. 마지막 MPTP 주사 7일 후에 동물을 희생하여 뇌를 적출하 고 고정하였다. 침효과를 확인하기 위해 Thyrosine hydroxylase(TH), laminin의 발현 변화 정도를 조직염 색화학법으로 이용하여 확인하였다. 각 그룹간의 유의성 검증은 one-way ANOVA를 이용하였다.
결 과 : 도파민성 신경세포 선택적인 신경독소인 MPTP에 대한 양릉천 침처치에 의한 신경보호 효과를 도파민 신경세포의 표지자인 TH 발현을 면역화학조직염색법으로 관찰하였다. 대조군에 비해 MPTP 처치 군의 신경세 포 사멸이 유의적으로 감소하였고(P <0.05), MPTP + 침처치 군에서 증가되는 양상을 확인하였다 (P <0.05).
또한 도파민성 신경세포내에 존재하는 laminin의 발현정도 역시 대조군보다 MPTP 처치 군에서 유의적으로 감 소하였고, MPTP + 침처치 군에서 증가되는 양상을 확인하였다 (P <0.05).
결 론 : MPTP에 의한 도파민성 신경세포 손상에 대한 양릉천 침처치의 신경보호 효과는 세포외 기질중의 하 나인 laminin의 발현 정도를 조절하여 비롯되는 것으로 사료된다.
Key word : 침, 양릉천(GB34), Laminin, 파킨슨병, MPTP, 신경보호
Ⅰ. Introduction
Parkinson’s disease (PD) is a neurological disorder characterized by chronic and
⋅교신저자 : Dr. Hi-Joon Park, Department of Meridian &
Acupoint, College of Korean Medicine, Kyung Hee University, 1 Hoegi-dong, Dongdaemun-gu, Seoul 130-701, Republic of Korea, Tel. +82-2-961-9435, E-mail: [email protected]
⋅Acknowledgement: This research was supported by the Kyung Hee University Research Fund in 2005. (KHU 20051044)
⋅투고 : 2008/03/17 심사 : 2008/03/19 채택 : 2008/03/24
progressive degeneration of the dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc). The pathogenesis of PD remains obscure, but there are increasing evidences that impairment of mitochondrial function, oxidative damage, inflammation, excitotoxicity and protein misfolding are contributing factors, all of which are linked
1,2). A number of therapeutic targets for inflammation and oxidative stress are efficacious in the PD animal model.
However, despite overwhelming experimental evidence, selective targets derived from each of these major cell death pathways have not directly led to a ‘cure’ in clinical trials.
Acupuncture has been widely utilized as a clinical treatment for various neurological disorders in East Asian medicine. Recently, acupuncture has been reported to have the neuroprotective effects and alleviate behavioral deficits in animal models with PD
3-6).
Laminins are the major non-collagenous component of the basal lamina, such as those on which cells of an epithelium sit
7). They are a family of glycoprotein that is an integral part of the structural scaffolding of basement membranes. Each laminin molecule is a heterotrimer assembled form alph-, beta-, and gamma-chain.
Laminins form independent networks and are associated with type IV collagen networks via entactin, and perlecan. They
also bind to cell membranes through integrin receptors and other plasma membrane molecules, such as the dystroglycan glycoprotein complex
7). Through these interactions, laminin critically contribute to cell attachment and differentiation, cell shape and movement, maintenance of tissue phenotype, and promotion of tissue survival
7-8).
Matrix metalloproteinases (MMPs) comprise a family of zinc-dependent endopeptidases that includes over 25 distinct members. The primary role of MMPs is to dynamically degrade and altered extracellular matrix (ECM) structure and function
9). The aberrant, excessive activity of MMPs contributes directly to neuron apoptosis and brain damage
10-13). Gu’s colleague demonstrated that MMP-9 degrades the extracellular matrix protein, laminin and this degradation induces neuronal apoptosis in a transient focal cerebral ischemia model in mice
14). MMP-3, another member of matrix metalloproteinases family, is released from stressed DA neuronal cells and activates microglia
15). These results demonstrated that MMPs plays an important role in DA neuronal degeneration through the degradation of laminin, and the microglial activation.
In this study, we investigated the effects of acupuncture on dopaminergic neuronal degeneration induced by MPTP treatment.
In order to confirm the protective effect of
acupuncture, behavioral test of mice and laminin and tyrosine hydroxylase (TH) expression were examined.
Ⅱ. Materials and Methods
1. Animals and MPTP administration
A eight-week-old male C57BL/6 mice (Orient, Korea), weighing 20–25 g, were used in all the experiments and were maintained on a 12-h light–dark schedule with free access to food and water according to the guidelines of NIH and Korean Academy of Medical Sciences. At the beginning of the experiment, the mice received an intraperitoneal (i.p.) injection of MPTP-HCl (30 mg/kg of free base;
Sigma, St. Louis, MO, USA) in saline at 24 h intervals for 5 days
16). The normal group was injected with saline only on the same day. The animals were killed at 7 days after the last MPTP or saline injection.
2. Experimental groups and acupuncture treatment
As to acupuncture group (n=5-7), stainless needles (0.2 mm in diameter) were inserted about 1-2 mm in depth into the GB34 acupuncture points. The needles were twisted twice a second for 60 seconds and then removed. The stimulated areas
correspond to acupuncture points in humans. The normal group and MPTP treated group (n=5-7) was also immobilized for 60 seconds.
3. Immunohistochemistry
The animals were killed by anesthesia with chloral hydrate (300 mg/kg) on the day 7 after the last MPTP or saline injection and then perfused transcardially with 4% paraformaldehyde in 0.05 M phosphate buffer (PB). The brains were removed, post-fixed, and cryoprotected.
Immunohistochemistry was performed as described previously
17-18)on free-floating cryomicrotome-cut sections (40 μm thick) that encompassed entire ST and SN. After incubation with 3% H
2O
2in 0.05 M phosphate-buffered saline (PBS), followed by 0.3% Triton X-100, and 3% bovine serum albumin (BSA) in 0.1M PBS, the sections were stained overnight at a room temperature using the primary antibodies:
laminin (1:1000, Sigma, St. Louis, MO, USA), TH (1:1000; Biotechnologies, Santa Cruz CA, USA) for dopaminergic neurons;
The Vectastain elite ABC kit (Vector Laboratories, Burlingame, CA, USA) was used for amplification.
4. Statistical analysis
The data were expressed as the mean ±
SEM. A statistical analysis was performed with an analysis of variance (ANOVA).
The significance was determined through the Newman–Keuls post hoc test. In all analyses, differences were considered significant at P <0.05.
Ⅲ. Results
1. The effect of acupuncture on tyrosine hydroxylase (TH) expression in the striatum and substantia nigra (SN) after treatment of MPTP
TH expression in the striatum and SN
were determined by immunohistochemistry.
The striatal density of TH was decreased in MPTP group compare to normal group, and these patterns of TH density was recovered in the acupuncture with MPTP group, but there is no significance(Fig. 1).
TH positive neuron in the SN was markedly decreased by MPTP injection compared to the normalgroup (P <0.05).
However acupuncture with MPTP group had significantly more TH-positive neurons in the SN compared to the MPTP group (P <0.05, Fig. 2).
A B C
0 3 0 6 0 9 0 1 2 0 1 5 0
No r ma l MP T P MP T P + A c u
No. of TH positive neuron
*
***
Fig. 1. Prevention of MPTP-elicited striatal DA neuronal degeneration in acupuncture treated mice.
Representative photomicrograph of TH-positive DA neuronal fibers in striatum 7-day post MPTP treatment of both MPTP and MPTP + Acupuncture treated mice (each n=5, P<0.05). A: Saline treated group (Normal), B: MPTP injected group (MPTP), C: Acupuncture treated group with MPTP injection (MPTP+Acu). All data are means ± SEM. **P < 0.005: Normal condition vs MPTP treatment for 7 days.
A B C
MPTP MPTP+Acu
Fig. 2. Prevention of MPTP-elicited SN DA neuronal degeneration in acupuncture treated mice.
Representative photomicrograph of TH-positive DA neuron in SNpc 7-day post MPTP treatment of both MPTP and MPTP + Acupunture treated mice (each n=5, P<0.05). A: Saline treated group (Normal), B: MPTP injected group (MPTP), C: Acupuncture treated group with MPTP injection (MPTP+Acu). All data are means ± SEM. ##P <
0.005: Normal condition vs MPTP treatment for 7 days, *P < 0.05: MPTP treatment for 7 days vs MPTP treatment with acupuncture.
2. The effect of acupuncture on laminin expression in the substantia nigra (SN) after treatment of MPTP
The expression of laminin in the SN was determined by immunohistochemistry. The expression of laminin in the SN was dramatically decreased in 3 days of MPTP treatment and then recovered slightly in 7 days and 14 days compare to the 3 days
of MPTP treatment. But there is no difference in each MPTP groups (Fig. 3).
laminin positive neuron in the SN was markedly decreased by MPTP injection compared to the normal group (P <0.05).
However, acupuncture with MPTP group
had significantly more laminin-positive
neurons in the SN compared to the MPTP
group(P <0.05, Fig. 4).
M3d M7d M14d
Fig. 3. Immunostaining of laminin in SNpc.
After 5days of consecutive treatment of MPTP, the number of laminin-reactive neuron was dramatically decreased, even though TH-positive neurons are still maintained (each n=5, P<0.05). M3d, M7d and M14d represent the time point at 3, 7 and 14 days after first MPTP injection. A: Saline treated group (Normal), B: MPTP injected group (MPTP), C: Acupuncture treated group with MPTP injection (MPTP+Acu). All data are means ± SEM. *P < 0.05: Normal condition vs MPTP treatment for 3 days.MPTP MPTP+Acu
Fig. 4. Representative photomicrograph of laminin-reactive DA neuron in SNpc 7-day post MPTP
treatment of both MPTP and MPTP + Acupunture treated mice.
Acupuncture group showed restoration of laminin-reactive neurons after MPTP treated mice (n=5, P <0.05). A: Saline treated group (Normal), B: MPTP injected group (MPTP), C: Acupuncture treated group with MPTP injection (MPTP+Acu). All data are means ± SEM. ## P < 0.005: Normal condition vs MPTP treatment for 7 days, *P < 0.05: MPTP treatment for 7 days vs MPTP treatment with acupuncture.Ⅳ. Discussion
Recent studies have indicated that inflammation plays important
role in neurodegenerative diseases such as PD and Alzheimer’s disease
19,20). After the initial injury, factors released from damaged neurons induce microgliosis, which is the hallmark of inflammation in the brain
21). Reactive microgliosis may enhance the neurotoxicity by producing excessive superoxide, NO, and proinflammatory cytokines
22-24). This microglia-dependent inflammatory process may underlie the progressive neurodegenerative diseases such as PD. Recently, it is suggested that the ECM plays a role in neurodegenerative condition.
Expression of matrix metalloproteinases (MMPs) was reported in PD and other inflammation-related neurodegenerative diseases
25-26). ECM molecules, such as laminin and fibronectin, are substrates of MMPs and can be modified or cleaved by MMPs to release soluble forms. Kim et al.
suggested MMP-3 evoked microglial activation that accelerates a dopaminergic neuron in SNpc
27).
Laminin is an extracellular matrix protein that involved in epithelial morphogenesis, preneuronal cell migration and neurite outgrowth
28,29). Moreover intraneuronal laminin promotes neurite outgrowth and axonal specification
30-33)and has been used in
vascular remodeling to identify the formation of new blood vessels
34-35).
Recently, acupuncture has been demonstrated to possess the neuroprotective effects and alleviate behavioral deficits in animal models with PD
3-5). It has been also reported that acupuncture promotes the expressions of brain-derived neurotrophic factor (BDNF) and its receptor trkB in the substantia nigra (SN)
4,5)and inhibits microglial activation and inflammatory events in the PD animal models
6). In this study, we demonstrated that acupuncture at GB34 protects dopaminergic neuronal damage in the MPTP-induced mice PD model via the restoration of intra-neuronal laminin. This result supports the clinical value of acupoint, GB34 which has been used to modulate movement related symptoms in Traditional Korean Medicine
36)
