[논문]뇌허혈 마우스모델에서 양격산화탕이 뇌 손상 완화에 미치는 효과

한도경; 박맑은; 권옥선; 최병태

doi:10.5352/jls.2019.29.11.1258

뇌허혈 마우스모델에서 양격산화탕이 뇌 손상 완화에 미치는 효과
Yangkyuksanhwa-Tang Attenuates Ischemic Brain Injury in a Focal Photothrombosis Stroke Model 원문보기

생명과학회지 = Journal of life science, v.29 no.11, 2019년, pp.1258 - 1266

한도경 (부산대학교 한의학전문대학원 한의학과) , 박맑은 (부산대학교 한의학전문대학원 한의과학과) , 권옥선 (부산대학교 한의학전문대학원 한의학과) , 최병태 (부산대학교 한의학전문대학원 한의학과)

초록
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양격산화탕은 9가지의 약재로 구성된 처방으로 한의학적 뇌졸중 치료에 가장 널리 사용되는 처방 중 하나이며, 주로 사상체질이론의 소양인 뇌졸중 치료에 적용된다. 본 연구는 실험동물을 이용한 뇌졸중에 대한 양격산화탕의 효과에 대한 연구가 전무하여, photothrombosis로 유발된 허혈성 마우스모델을 이용하여 양격산화탕의 효과를 살펴 보았다. 동물행동학적 변화와 더불어 뇌손상에 미치는 영향을 뇌경색 용적에 대한 조직학적 검색 및 신경염증과 신생세포에 대한 면역조직화학적 검색으로 살펴 보았다. 동물행동학적 결과로 보아, 양격산화탕은 뇌허혈에 의해 손상된 운동기능, 즉 wire grip과 rotarod test에 의한 운동조정과 균형 능력 등에 대한 기능적 회복을 보였으며, 이는 조직학적 검색으로 관찰된 뇌경색 용적의 축소를 동반하였다. 면역조직화학적 결과를 보면, 양격산화탕은 tumor necrosis factor-${\alpha}$와 myeloperoxidase 면역반응세포의 수를 현저히 감소시켰다. 이와 반대로 양격산화탕은 glial fibrillary acidic protein와 ionized calcium-binding adapter molecule 1 면역반응세포의 수를 현저히 증가시켰다. 또한 양격산화탕은 Ki67/doublecortin 면역반응세포의 수를 현저히 증가시켰다. 이상의 결과로 보아, 양격산화탕은 항염증, astrocyte와 microglia의 활성화 및 신경세포의 증식을 통해 뇌경색 용적을 감소시키며, 이는 뇌허혈성 운동장애에 대한 완화 효과로 이어 지는 것을 알 수 있다. 따라서 양격산화탕은 뇌손상에 대한 신경기능적 완화효과를 보여 줌으로서 뇌졸중 환자에 대한 유효한 치료제로 사료된다.

Abstract ▼ AI-Helper

Yangkyuksanhwa-Tang (YKSH), consisting of nine different herbs, is commonly used in Soyangin-type individuals with stroke, based on the Sasang Constitution Theory in Korea. However, no evidence has yet confirmed a beneficial effect of YKSH in ischemic stroke treatment. In this study, we investigated the effects of YKSH on ischemic brain injury in a mouse model of cerebral ischemia. Focal cerebral ischemia in mice was induced by photothrombosis, and behavioral recovery was evaluated. Infarct volume, inflammation, and newly generated cells were evaluated by histology and immunochemistry. YKSH treatment resulted in a significant recovery from the motor impairments induced by focal cerebral ischemia, as determined with wire grip and rotarod tests. YKSH treatment also decreased the infarct volume and the number of cells positive for tumor necrosis factor-${\alpha}$ and myeloperoxidase when compared with a vehicle-treated control group. By contrast, YKSH treatment considerably increased the number of cells positive for glial fibrillary acidic protein and ionized calcium-binding adapter molecule 1, as well as the number of cells doubly positive for Ki67/doublecortin when compared with the vehicle-treated group. These results suggest that YKSH treatment attenuated the infarct size by anti-inflammatory action, astrocyte and microglia activation, and neuronal proliferation, thereby facilitating neurofunctional recovery from a cerebral ischemic assault. YKSH could therefore be a potential treatment for neurofunctional restoration of the injured brains of patients with stroke.

주제어

표/그림 (5)

그림 Fig. 1. Effects of YKSH on motor function impairment in a focal photothrombosis stroke model. (A) Wire grip test, (B) cylinder test, and (C) rotarod test (n=7). Treatment with YKSH exerted beneficial effects on the motor function as indicated by changes in the wire grip score and rotarod latency time compared with those of the vehicle group. Data are expressed as mean ± SEM. * < 0.05, ** < 0.01, *** < 0.001 vs. control group; ^# < 0.05 vs. vehicle group; ^$ < 0.05 vs. YKSH 25 mg group. YKSH, Yangkyuksanhwa-Tang.
그림 Fig. 2. Effects of YKSH on infarct volume in a focal photothrombosis stroke model. (A) Photomicrograph and (B) histogram depicting infarct volume at bregma 2.65 mm (n=7). Treatment with high-dose YKSH showed lower infarct volume than that of the vehicle group. Data are expressed as mean ± SEM. * < 0.05 vs. control group. Scale bar = 100 μm. YKSH, Yangkyuksanhwa-Tang.
그림 Fig. 3. Effects of YKSH on TNF-α and MPO positive cells in a focal photothrombosis stroke model. (A) Photomicrograph and histogram depicting (B) TNF-α and (C) MPO positive cells in the infarct region (n=7). The number of TNF-α and MPO positive cells was considerably lower in the YKSH-treated group than in the vehicle-treated group. Data are expressed as mean ± SEM. *** < 0.001 vs. control group; ^# < 0.05, ^### < 0.001 vs. vehicle group. Scale bar = 100 μm. TNF-α, tumor necrosis factor-α; MPO, myeloperoxidase; YKSK, Yangkyuksanhwa-Tang.
그림 Fig. 4. Effects of YKSH on GFAP and Iba-1 positive cells in a focal photothrombosis stroke model. (A) Photomicrograph and histogram depicting (B) GFAP and (C) Iba-1 positive cells in the infarct region (n=7). The number of GFAP and IBa-1 positive cells was considerably higher in the high-dose YKSH-treated group than in the vehicle-treated group. Data are expressed as mean ± SEM. *** < 0.001 vs. control group; ^## < 0.01, ^### < 0.001 vs. vehicle group; ^$ < 0.05 vs. YKSH 25 mg group. Scale bar = 100 μm. GFAP, glial fibrillary acidic protein; Iba-1, ionized calcium-binding adapter molecule 1; YKSH, Yangkyuksanhwa-Tang.
그림 Fig. 5. Effects of YKSH on Ki67/Dcx double-positive cells in a focal photothrombosis stroke model. (A) Photomicrograph and histogram depicting (B) Ki67/Dcx double-positive cells in the subventricular zone (n=7). The number of Ki67/Dcx double-pos-itive cells was considerably higher in the high-dose YKSH-treated group than in the vehicle-treated group. Data are expressed as mean ± SEM. ^# < 0.05 vs. vehicle group; ^$ < 0.05 vs. YKSH 25 mg group. Scale bar = 100 μm. DCX, doublecortin; YKSH, Yangkyuksanhwa-Tang.

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가설 설정

To our knowledge, there is no pharmacological evidence, in an animal model of stroke, that YKSH is effective for ischemic stroke to alleviate cerebral infarction, inflammatory response, and neuronal progenitor cells. Therefore, in this study, we investigated whether YKSH shows neuroprotective effect in a mouse stroke model by ameliorating neuroinflammation and activating neural progenitor cells, and subsequently helps recover behavioral impairments.

제안 방법

Mice that were unable to remain on the wire for less than 30 s were assigned a score of zero. The cylinder test was conducted to evaluate sensorimotor function. A mouse was made to enter a transparent acrylic cylinder (9 cm × 9 cm × 15 cm), and the frequency of use of its left, right, and both paws was expressed as percentage during 20 trials.
After the adaptation trials, each mouse was madeto stand on a rotarod wheel rotating at a speed of 20 rpm for 2 min, with three trials per day, and the duration that a mouse was able to hold itself on the rod was recorded. These behavioral tests were performed at 3 and 7 days after focal cerebral ischemia induction.
To investigate the effects of YKSH on infarct volume, infarct volume was determined by cresyl violet staining at 8 days after focal cerebral ischemia. Although a significantly higher infarct volume was observed in the focal cerebral ischemia model (control vs.
To investigate whether YKSH can recover damaged neural function induced by focal cerebral ischemia, the wire grip, cylinder, and rotarod tests were conducted. In the wire grip test, the scores showed a significantly less change in the vehicle group compared with that in the control (control vs.
To verify the proliferative effect of YKSH on neural progenitor cells, Ki67 and DCX staining was performed in the subventricular zone (SVZ). There were no significant changes among the control, vehicle, and YKSH 25 mg groups (control vs.
To verify whether YKSH induces the activation of astrocytes and microglia, GFAP and Iba-1 staining was performed. The number of GFAP positive cells was significantly increased in the vehicle group compared with that in the control (control vs.
To verify whether YKSH reduces the level of inflammation induced by focal cerebral ischemia, TNF-α and MPO staining was performed.

대상 데이터

The composition of YKSH was based on Dongeuisoosebowon (Jema 2003). Herbs were purchased from Hwalim Nature Drug (Pusan, Korea) and were authenticated by Prof. J. U. Baek, Department of Korean Medicine, School of Korean Medicine, Pusan National University. The herbs constituting YKSH [viz.
Male C57BL/6 mice (aged 5 weeks, weighing 15-20 g) were obtained from Dooyeol Biotech (Seoul, Korea). The mice were housed at 22℃ under alternating 12-hr light/dark cycle; they were allowed access to food and water ad libitum.

데이터처리

All data are expressed as mean ± standard error of the mean (SEM) and analyzed using the SigmaStat statistical program version 11.2 (Systat Software, San Jose, CA, USA).
Statistical comparisons were performed using a one-way analysis of variance (ANOVA) with repeated measures and Tukey’s post hoc test of least significant difference.

이론/모형

To measure vestibular motor function, the wire-grip test was conducted. A mouse was placed on a 45 cm long wire suspended across two upright poles (height: 45 cm), and then scored as follows: 1, mouse failed to hold on to the wire with both forepaws and hind paws together; 2, mouse held on to the wire with both forepaws and hind paws but not the tail; 3, mouse used its tail along with both forepaws and hind paws; 4, mouse moved along the wire on all four paws plus tail; and 5, mouse scored four points and also ambulated down one of the posts that were used to support the wire.

성능/효과

In the wire grip test, the scores showed a significantly less change in the vehicle group compared with that in the control (control vs. vehicle; 4.6±0.3 scores vs. 2.6±0.5 scores, p<0.05) and these scores significantly improved in the YKSH 50 mg group compared with those in the vehicle group at 3 days after cerebral ischemic induction (vehicle vs. YKSH 50 mg; 2.6±0.5 scores vs. 4.3±0.3 scores, p<0.05) (Fig. 1A).
Several beneficial effects of herbal medicines are accompanied by attenuation of astrocyte and microglia/ macrophage activation [20, 28]. Our results showed that the expression of astroglial and microglial marker GFAP and Iba-1 was increased in ischemic brain compared with that in the control, as reported previously. However, treatment with YKSH induced a significant increase in the expression of these markers.

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