Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
권호 표지
DOI QR코드

DOI QR Code

Yangkyuksanhwa-Tang Attenuates Ischemic Brain Injury in a Focal Photothrombosis Stroke Model

뇌허혈 마우스모델에서 양격산화탕이 뇌 손상 완화에 미치는 효과

  • Han, Do-Kyung (Department of Korean Medicine, School of Korean Medicine, Pusan National University) ;
  • Pak, Malk-Eun (Department of Korean Medical Science, School of Korean Medicine, Pusan National University) ;
  • Kwon, Ok-Sun (Department of Korean Medicine, School of Korean Medicine, Pusan National University) ;
  • Choi, Byung-Tae (Department of Korean Medicine, School of Korean Medicine, Pusan National University)
  • 한도경 (부산대학교 한의학전문대학원 한의학과) ;
  • 박맑은 (부산대학교 한의학전문대학원 한의과학과) ;
  • 권옥선 (부산대학교 한의학전문대학원 한의학과) ;
  • 최병태 (부산대학교 한의학전문대학원 한의학과)
  • Received : 2019.09.06
  • Accepted : 2019.10.10
  • Published : 2019.11.30
Download PDF
⟨ Previous Next ⟩

Abstract

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.

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.

Keywords

Introduction

Stroke is a serious global health problem as it is the second leading cause of death, and rehabilitation of permanent disability induced by the disease is important for patient management [15]. Recombinant tissue plasminogen activator is the only FDA-approved medication for patients with stroke, but it is effective only in a few patients because of its limited therapeutic time window [19, 21]. In addition, neuroprotective agents, radical scavengers, and calcium ion antagonists have been used for the treatment of ischemic stroke, but it is generally difficult to achieve favorable outcomes with these agents because of serious adverse effects and the single therapeutic target [16, 23].

Inflammatory response contributes significantly to the pathology of cerebral ischemia, and therefore, anti-inflammation is a preferred therapeutic strategy for stroke [8, 25]. Traditional herbal medicines have been widely used for patients with stroke in East Asia. The neuroprotective effect of these herbal medicines is mainly attributed to the inhibition of inflammatory cytokine production and the attenuation of the brain neuroinflammatory cascade [25, 28]. Restorative cell-based therapy has been reported to improve functional outcome in an experimental stroke model, and this therapy leads to the enhancement of endogenous neuronal progenitor cells [30].

Yangkyuksanhwa-Tang (YKSH) is a traditional Korean medicine based on Dongeuisoosebowon, a traditional Korean medicine literature; it is the most commonly used medicine for ischemic stroke in Korea [27]. YKSH is usually applied for Soyangin-type individuals, one of four types of individuals with a large spleen and small kidneys based on the Sasang Constitution theory [12]. Therefore, previous studies on this medicine have mainly investigated changes in the cytokine levels in the acute disease stage or peripheral blood mononuclear cells in Soyangin-type individuals with cerebral infarction [13, 14].

Animal models of stroke provide an essential tool for assessing novel therapeutic strategies for stroke, such as anti-inflammatory or neuroprotective prescription. 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.

Materials and Methods

Animals

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. All experimental protocols were approved by the Pusan National University Animal Care and Use Committee in accordance with the National Institutes of Health Guidelines (PNU-2017-1771). The mice were randomly divided into the following four groups (n=7): (1) a control group (Control), (2) a vehicle-treated group for focal cerebral ischemia (Vehicle) (3) a low-dose YKSH-treated group for focal cerebral ischemia (YKSH 25 mg), and (4) a high-dose YKSHtreated group for focal cerebral ischemia (YKSH 50 mg).

Preparation of YKSH

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., Rehmannia glutinosa (Gaertner) Libosch. (8 g), Forsythia koreana Nakai (8 g), Lonicera japonica Thunb. (8 g), Mentha arvensis var. piperascens Makinv. (4 g), Saposhnikovia divaricata Schiskin (4 g), Gardenia jasminoides J. Ellis (4 g), Gypsum (4 g), Anemarrhena asphodeloides Bunge (4 g), and Schizonepeta tenuifolia var. japonica (Maxim.) Kitag. (4 g); total 48 g] were immersed in 650 ml of water and extracted for 2.5 hr by heating. The extract was subsequently filtered twice through Whatman No. 2 paper (Advantech, Milpitas, CA, USA) and evaporated under reduced pressure using freeze dry systems (7960042; Labconco Corp., Kansas City, MO, USA). The final quantity of extracted YKSH was approximately 6.35 g. The yield of YKSH was 13.2%. YKSH (25 or 50 mg/mouse) was orally administered once per day for 5 days before focal cerebral ischemia induction. The low dose of YKSH 25 mg was calculated based on the patient’s daily dose and body weight and converted to the weight of the mouse. Double dose of YKSH 50 mg was treated as the high dose.

Induction of focal cerebral ischemia

Focal cerebral ischemia was induced by photochemically induced thrombosis. The mice were anesthetized with 2% isoflurane in O2 (20%) and N2O (80%). They received an intraperitoneal injection of rose bengal (0.1 ml of 10 mg/ml rose bengal in 0.9% saline; Sigma-Aldrich, St. Louis, MO, USA), 5 min before illumination. The mice were then fixed on a stereotaxic frame (David Kopf Instruments, Tujunga, CA, USA), and then the skull was exposed. A fiber optic bundle containing a KL 1500 LED cold light source (Carl Zeiss, Jena, Germany) was positioned onto the right sensorimotor cortex of the exposed skull (2.4 mm lateral to the bregma) and illuminated for 15 min. The incision was sutured after illumination, and the mice were restored under a heating lamp and returned to their cage.

Behavioral tests

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 fore paws 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. 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. Motor coordination and balance ability were verified using a rotarod apparatus (Panlab S.L.U., Barcelona, Spain). After the adaptation trials, each mouse was made to 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.

Measurement of infarct volume

At 8 days after focal cerebral ischemia induction, mice anesthetized with 8% chloral hydrate solution (400 mg/kg; Sigma-Aldrich) received intracardiac perfusion with phosphate-buffered saline solution (PBS) and then with 4% paraformaldehyde. The brain was removed and post-fixed in the same fixative for 4 hr at 4℃, and then immersed in 30% sucrose for 48 hr at 4℃ for cryoprotection. Frozen 30-μm thick brain sections were obtained to determine infarct volume Nissl staining (cresyl violet acetate, c5042-10G; SigmaAldrich). Images were captured using a stereo microscope (Stemi 305, Carl Zeiss). Infarct volume was quantified using i-Solution software (IMT i-solution, Inc., Vancouver, BC, Canada) with brain sections of bregma 2.65 mm. The value was determined using the area (µm2 ) of the injured region.

Immunohistochemistry

Frozen 30-μm thick coronal sections were incubated in a blocking buffer (containing 0.3% Triton X-100, 5% normal serum, and 1× PBS) for 1 hr at room temperature. The sections were then incubated with the primary antibody overnight at 4℃. The antibody against Ki67 (ab15580) was supplied by Abcam (Cambridge, UK). The antibodies against doublecortin (DCX, sc-8066) and tumor necrosis factor-α (TNF-α, sc-8301) were supplied by Santa Cruz Biotechnology (Dallas, TX, USA), and those against glial fibrillary acidic protein (GFAP, z0334) was supplied by Dako Inc. (Glostrup, Denmark). The antibody against ionized calcium-binding adapter molecule 1 (Iba-1, 019-19741) was procured from Fujifilm Wako Pure Chemical Cop. (Osaka, Japan) and that against myeloperoxidase (MPO, AF 3667) from R&D systems (Minneapolis, MN, USA). After washing with Tween 20 in PBS (PBST), the sections were incubated with the secondary fluorescent antibodies, Alexa Fluor 594 (A11037, rabbit) and 488 (A11001, mouse) purchased from Invitrogen Thermo Fisher Scientific (Carlsbad, CA, USA) for 2 hr at room temperature in dark. The sections were then washed three times with PBST. Hoechst 33342 (H3570, Invitrogen Thermo Fisher Scientific) staining was performed for 30 min. After washing in PBST, slides were mounted with a mounting medium (S3023; Dako North America Inc., Carpinteria, CA, USA) and images were captured using a fluorescence microscope (Carl Zeiss Imager M1, Carl Zeiss). The cell number were measured at 200x magnification using i-solution (IMT i-solution Inc.) after acquiring images using fluorescence.

Statistical analyses

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. The results with a P-value < 0.05 were interpreted as statistically significant.

Results

Effects of YKSH on post-stroke behavior

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. 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). In the cylinder test, percentage of right paw touch was significantly high in all the groups of focal cerebral ischemia compared with that in the control (control vs. vehicle or YSKH 25 mg or YKSK 50 mg at 3 days; 50.4±3.2% vs. 68.6±3.1%, p<0.01 or 71.4±3.4%, p<0.01 or 76.4±5.0%, p<0.001, control vs. vehicle or YSKH 25 mg or YKSK 50 mg at 7 days; 48.2±2.5% vs. 64.6±2.8%, p<0.01 or 65.7±5.8%, p<0.05 or 66.1±4.3%, p<0.05), but there were no significant changes among the groups of focal cerebral ischemia (Fig. 1B). In the rotarod test, focal cerebral ischemia resulted in a significantly short latency time compared with that in the control (control vs. vehicle; 80.0±2.1 sec vs. 53.6±3.8 sec, p<0.001), but the YKSH 50 mg group required a considerably longer time than the vehicle group required at 7 days after cerebral ischemic induction (vehicle vs. YKSH 50 mg; 53.6±3.8 sec vs. 69.2±5.2 scores, p<0.05) (Fig. 1C). These results suggest that YKSH treatment can improve neuromotor function in a focal cere- bral ischemia model.

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.

Effects of YKSH on infarct volume

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. vehicle; 0.0±0.0 μm2 vs. 2.2±0.3 μm2, p<0.05), the YKSH 50 mg group showed lower infarct volume than that of the vehicle and YKSH 25 mg groups (vehicle vs. YKSH 50 mg; 2.2±0.3 μm2 vs. 1.8±0.2 μm2, p< 0.05) (Fig. 2). These results demonstrated that YKSH treatment, especially at a high dose, decreased the infarct volume and this might lead to behavioral change.

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, YangkyuksanhwaTang.

Effects of YKSH on neuroinflammation

To verify whether YKSH reduces the level of inflammation induced by focal cerebral ischemia, TNF-α and MPO staining was performed. The number of TNF-α positive cells was significantly increased in the focal cerebral ischemia model compared with that in the control (control vs. vehicle; 27.1±2.3 cells vs. 107.0±5.0 cells, p<0.001), but the YSH 25 mg group showed a significant change compared with that in the vehicle group (control vs. YKSH 25 mg; 107.0±5.0 cells vs. 94.2±2.1 cells, p<0.05) (Fig. 3B). The number of MPO positive cells showed a significant increase in the vehicle group compared with that in the control group (control vs. vehicle; 34.9±2.9 cells vs. 88.2±5.1 cells, p<0.001). However, these cells showed a considerable decrease in both YKSH-treated groups compared with that in the vehicle group (vehicle vs. YKSH 25 mg or YKSH 50 mg; 88.2±5.1 cells vs. 50.7±3.4 cells, p<0.001 or 52.6±1.5 cells, p<0.001) (Fig. 3C). These results demonstrated that YKSH treatment decreased neuroinflammation induced by focal cerebral ischemia.

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.

Effects of YKSH on the activation of astrocytes and microglia

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. vehicle; 25.3±3.5 cells vs. 81.2±1.8 cells, p<0.001). These cells showed a considerable increase with YKSH treatment compared with that in the vehicle group (vehicle vs. YKSH 25 mg or YKSH 50 mg; 81.2±1.8 cells vs. 113.4±4.3 cells, p<0.001 or 188.1±7.4 cells, p<0.001) (Fig. 4B). The number of Iba-1 positive cells was considerably increased in the vehicle group compared with that in the control group (control vs. vehicle; 25.3±3.5 cells vs. 81.2±1.8 cells, p<0.001), and these cells showed a considerable increase in the YKSH 50 mg group compared with that in the vehicle group (vehicle vs. YKSH 50 mg; 81.2±1.8 cells vs. 190.46±11.1 cells, p<0.01) (Fig. 4C). Both GFAP and Iba-1 positive cells were significantly increased in the YKSH 50 mg group compared with those in the YKSH 25 mg group (GFAP, YKSH 25 mg vs. YKSH 50 mg; 113.4±4.3 cells vs. 188.1±7.4 cells, p<0.05, Iba-1, YKSH 25 mg vs. YKSH 50 mg; 118.9±4.3 cells vs. 190.5±11.1 cells, p<0.05). These results demonstrated that YKSH treatment induced the activation of astrocytes and microglia.

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, YangkyuksanhwaTang.

Effects of YKSH on proliferation of new neuron

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. vehicle vs. YKSH 25 mg; 31.4±3.3 cells vs. 29.8±2.8 cells vs. 29.8±2.1 cells), but a significant increase in Ki67/DCX double-positive cells was observed in the YKSH 50 mg group compared with that in the vehicle group (vehicle vs. YKSH 50 mg; 29.8±3.3 cells vs. 43.0±1.7 cells, p<0.05) (Fig. 5). This result suggests that high dose YKSH treatment is effective for the proliferation and differentiation of new neurons.

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-positive cells was considerably higher in the high-dose YKSH-treated group than in the vehicle-treated group. Data are expressed as mean ± SEM. # < 0.05 vs. vehicle group; $ < 0.05 vs. YKSH 25 mg group. Scale bar = 100 μm. DCX, doublecortin; YKSH, Yangkyuksanhwa-Tang.

Discussion

Several traditional medicines, combining various medicinal plants, have been widely used, and the beneficial effects of these traditional medicines differ from the overall effects achieved with individual plants [3]. Prescription drugs prepared using a variety of herbal medicines can have additive effects because two or more compounds target different pharmacological sites for the same disease, resulting in a synergistic effect [3, 31]. In the present study, we investigated the potential neuroprotective effects of YKSH in a focal cerebral ischemia model. Our findings indicated that treatment with YKSH alleviates infarct volume and induces anti-inflammation, astrocyte and microglia activation, and neuronal cell differentiation, and thereby improves recovery of behavioral impairments.

Motor function impairments after stroke are easily recognized; they impose significant challenges for treatment and patient care [17]. Neurological deficits and motor impairments are closely related to cerebral infarction volume [11]. Therefore, in the present study, we performed behavioral tests for motor functions and infarct volume. Pretreatment with YKSH, especially at a high dose, significantly restored vestibular motor function, motor coordination, and balance ability. Moreover, high-dose YKSH treatment significantly reduced the volume of cerebral infarction.

In the process of ischemic stroke, hypoxia and inflammation disrupt blood–brain barrier and increase its permeability via loss of tight junction proteins in the microvasculature [9, 22]. TNF-α is one of the key initiators or mediators of inflammatory reactions in the progression of stroke, and the loss of tight junction proteins in the microvasculature are mediated by this cytokine [9, 18, 22]. MPO is secreted by activated neutrophils and macrophages/microglia, and the activity of this enzyme in the cerebral tissue is considered an indicator of neutrophil infiltration after stroke injury [2].

The therapeutic success of YKSH can be partially attributed to its inhibitory effects on inflammatory cytokines or enhancing effect on anti-inflammatory cytokines [3, 13]. YKSH treatment of Soyangin-type patients with cerebral infarction has been reported to significantly reduce the plasma levels of TNF-α, IL-4, and IL-6 [13]. It also inhibited IL-1α, IL-1β, and IL-8 production in peripheral blood mononuclear cells in these patients [14]. In the present study, TNF-α and MPO were highly expressed in the infarct regions of mice with focal cerebral ischemia compared with those in the controls, but treatment with YKSH prevented the increase in TNF-α and MPO positive cells.

Activation of glial cells, including astrocytes and microglia, mediates the production of several inflammatory cascades in the development of cerebral ischemia [6, 7, 10]. Reactive astrocytes are the main components of prominent pathological features of ischemic stroke, such as astrogliosis and glial scar formation, acting as a barrier for axonal regeneration [5]. 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.

Astrocyte scars have been regarded as critical inhibitors of the regrowth of central nervous system (CNS) axon, but recent studies have shown that glial scar formation aids central nervous system axon regeneration, rather than prevent it [1]. Glial scars permit and support robust levels of appropriately stimulated CNS axon regeneration during the acute phase after injury by limiting damage [1, 26]. Contrary to the present concept as being destructive in neurological diseases, microglia also maintain CNS homeostasis and protect the CNS under pathological conditions of stroke by promoting neurogenesis and suppressing inflammation [4, 29]. Thus, the activation of astrocytes and microglia observed in the early phase of ischemic stroke can be considered a mechanism to limit brain damage.

Remodeling of the injured brain after stroke can potentially induce functional recovery via stimulation of endogenous neurogenesis from the SVZ and the subgranular zone [24, 30]. Appropriate therapeutic drugs can be developed for ischemic stroke if the proliferation of endogenous neural progenitor cells and their maturation can be stimulated. In the present study, treatment with YKSH induced the proliferation of neural progenitor cells in the SVZ and promoted the differentiation of newly generated cells into DCX positive neurons, elucidating a beneficial mechanism of YKSH treatment for ischemic stroke. In summary, treatment with YKSH attenuated focal photothrombosis-induced motor function impairments along with the attenuation of infarct volume in an ischemic stroke model. YKSH also suppressed photothrombosis-induced TNF-α and MPO expression and glial activation, and increased the differentiation of newly generated cells in the SVZ. Therefore, our results suggest that YKSH might be a therapeutic agent for neurological disorders associated with cerebral ischemic impairment.

Acknowledgement

This work was supported by a 2-Year Research Grant of Pusan National University.

References

  1. Anderson, M. A., Burda, J. E., Ren, Y., Ao, Y., O'Shea, T. M., Kawaguchi, R., Coppola, G., Khakh, B. S., Deming, T. J. and Sofroniew, M. V. 2016. Astrocyte scar formation aids central nervous system axon regeneration. Nature 532, 195-200. https://doi.org/10.1038/nature17623
  2. Breckwoldt, M. O., Chen, J. W., Stangenberg, L., Aikawa, E., Rodriguez, E., Qiu, S., Moskowitz, M. A. and Weissleder, R. 2008. Tracking the inflammatory response in stroke in vivo by sensing the enzyme myeloperoxidase. Proc. Natl. Acad. Sci. USA. 105, 18584-18589. https://doi.org/10.1073/pnas.0803945105
  3. Burns, J. J., Zhao, L., Taylor, E. W. and Spelman, K. 2010. The influence of traditional herbal formulas on cytokine activity. Toxicology 278, 140-159. https://doi.org/10.1016/j.tox.2009.09.020
  4. Chen, Z. and Trapp, B. D. 2016. Microglia and neuroprotection. J. Neurochem. 136 Suppl 1, 10-17. https://doi.org/10.1111/jnc.13062
  5. Choudhury, G. R. and Ding, S. 2016. Reactive astrocytes and therapeutic potential in focal ischemic stroke. Neurobiol. Dis. 85, 234-244. https://doi.org/10.1016/j.nbd.2015.05.003
  6. de Lima, N. M., Ferreira, E. O., Fernandes, M. Y., Lima, F. A., Neves, K. R., do Carmo, M. R. and de Andrade, G. M. 2017. Neuroinflammatory response to experimental stroke is inhibited by boldine. Behav. Pharmacol. 28, 223-237. https://doi.org/10.1097/FBP.0000000000000265
  7. Fernandes, M. Y. D., Carmo, M. R. S. D., Fonteles, A. A., Neves, J. C. S., Silva, A. T. A. D., Pereira, J. F., Ferreira, E. O., Lima, N. M. R., Neves, K. R. T. and Andrade, G. M. 2019. (-)-alpha-bisabolol prevents neuronal damage and memory deficits through reduction of proinflammatory markers induced by permanent focal cerebral ischemia in mice. Eur. J. Pharmacol. 842, 270-280. https://doi.org/10.1016/j.ejphar.2018.09.036
  8. Gu, Y., Chen, J. and Shen, J. 2014. Herbal medicines for ischemic stroke: combating inflammation as therapeutic targets. J. Neuroimmune Pharmacol. 9, 313-339. https://doi.org/10.1007/s11481-014-9525-5
  9. Huang, J., Li, Y., Tang, Y., Tang, G., Yang, G. Y. and Wang, Y. 2013. CXCR4 antagonist AMD3100 protects blood-brain barrier integrity and reduces inflammatory response after focal ischemia in mice. Stroke 44, 190-197. https://doi.org/10.1161/STROKEAHA.112.670299
  10. Iadecola, C. and Anrather, J. 2011. The immunology of stroke: from mechanisms to translation. Nat. Med. 17, 796-808. https://doi.org/10.1038/nm.2399
  11. Jeffers, M. S. and Corbett, D. 2018. Synergistic effects of enriched environment and task-specific reach training on poststroke recovery of motor function. Stroke 49, 1496-1503. https://doi.org/10.1161/STROKEAHA.118.020814
  12. Lee, J. M. 2003. Dongeui Soosebowon. Yeogang, Seoul, Republic of Korea.
  13. Jeong, H. J., Hong, S. H., Park, H. J., Kweon, D. Y., Lee, S. W., Lee, J. D., Kim, K. S., Cho, K. H., Kim, H. S., Kim, K. Y. and Kim, H. M. 2002. Yangkyuk-Sanhwa-Tang induces changes in serum cytokines and improves outcome in focal stroke patients. Vascul. Pharmacol. 39, 63-68. https://doi.org/10.1016/S1537-1891(02)00217-3
  14. Jeong, H. J., Lee, H. J., Hong, S. H., Kim, H. M. and Um, J. Y. 2007. Inhibitory effect of Yangkyuk-Sanhwa-Tang on inflammatory cytokine production in peripheral blood mononuclear cells from the cerebral infarction patients. Int. J. Neurosci. 117, 525-537. https://doi.org/10.1080/00207450600773590
  15. Langhorne, P., Bernhardt, J. and Kwakkel, G. 2011. Stroke rehabilitation. Lancet 377, 1693-1702. https://doi.org/10.1016/S0140-6736(11)60325-5
  16. Lapchak, P. A., Chapman, D. F. and Zivin, J. A. 2001. Pharmacological effects of the spin trap agents N-t-butyl-phenylnitrone (PBN) and 2,2,6, 6-tetramethylpiperidine-N-oxyl (TEMPO) in a rabbit thromboembolic stroke model: combination studies with the thrombolytic tissue plasminogen activator. Stroke 32, 147-153. https://doi.org/10.1161/01.STR.32.1.147
  17. Li, S. 2017. Spasticity, Motor Recovery, and Neural Plasticity after Stroke. Front. Neurol. 8, 120.
  18. Liguz-Lecznar, M., Zakrzewska, R. and Kossut, M. 2015. Inhibition of Tnf-alpha R1 signaling can rescue functional cortical plasticity impaired in early post-stroke period. Neurobiol. Aging 36, 2877-2884. https://doi.org/10.1016/j.neurobiolaging.2015.06.015
  19. Lindekleiv, H., Berge, E., Bruins Slot, K. M. and Wardlaw, J. M. 2018. Percutaneous vascular interventions versus intravenous thrombolytic treatment for acute ischaemic stroke. Cochrane Database Syst. Rev. 10, CD009292.
  20. Liu, L., Vollmer, M. K., Fernandez, V. M., Dweik, Y., Kim, H. and Dore, S. 2018a. Korean Red Ginseng pretreatment protects against long-term sensorimotor deficits after ischemic stroke likely through Nrf2. Front. Cell. Neurosci. 12, 74.
  21. Liu, S., Feng, X., Jin, R. and Li, G. 2018b. Tissue plasminogen activator-based nanothrombolysis for ischemic stroke. Expert Opin. Drug Deliv. 15, 173-184. https://doi.org/10.1080/17425247.2018.1384464
  22. Minagar, A., Long, A., Ma, T., Jackson, T. H., Kelley, R. E., Ostanin, D. V., Sasaki, M., Warren, A. C., Jawahar, A., Cappell, B. and Alexander, J. S. 2003. Interferon (IFN)-beta 1a and IFN-beta 1b block IFN-gamma-induced disintegration of endothelial junction integrity and barrier. Endothelium 10, 299-307. https://doi.org/10.1080/10623320390272299
  23. Minnerup, J., Sutherland, B. A., Buchan, A. M. and Kleinschnitz, C. 2012. Neuroprotection for stroke: current status and future perspectives. Int. J. Mol. Sci. 13, 11753-11772. https://doi.org/10.3390/ijms130911753
  24. Parent, J. M., Vexler, Z. S., Gong, C., Derugin, N. and Ferriero, D. M. 2002. Rat forebrain neurogenesis and striatal neuron replacement after focal stroke. Ann. Neurol. 52, 802-813. https://doi.org/10.1002/ana.10393
  25. Peng, T., Jiang, Y., Farhan, M., Lazarovici, P., Chen, L. and Zheng, W. 2019. Anti-inflammatory effects of traditional Chinese medicines on preclinical in vivo models of brain ischemia-reperfusion-injury: prospects for neuroprotective drug discovery and therapy. Front. Pharmacol. 10, 204. https://doi.org/10.3389/fphar.2019.00204
  26. Sandvig, I., Augestad, I. L., Haberg, A. K. and Sandvig, A. 2018. Neuroplasticity in stroke recovery. The role of microglia in engaging and modifying synapses and networks. Eur. J. Neurosci. 47, 1414-1428. https://doi.org/10.1111/ejn.13959
  27. Shin, M. G., Song, I. B and Son, S. K. 2001. Effects of Yanggyuksanhwa-Tang on cerebral blood flow and ischemic brain damage in rats. Journal of Sasang Constitutional Medicine 13, 165-176.
  28. Widmann, C., Gandin, C., Petit-Paitel, A., Lazdunski, M. and Heurteaux, C. 2018. The traditional Chinese medicine MLC 901 inhibits inflammation processes after focal cerebral ischemia. Sci. Rep. 8, 18062. https://doi.org/10.1038/s41598-018-36138-0
  29. Zhang, X., Zhao, H. H., Li, D. and Li, H. P. 2019. Neuroprotective effects of matrix metalloproteinases in cerebral ischemic rats by promoting activation and migration of astrocytes and microglia. Brain Res. Bull. 146, 136-142. https://doi.org/10.1016/j.brainresbull.2018.11.003
  30. Zhang, Z. G. and Chopp, M. 2009. Neurorestorative therapies for stroke: underlying mechanisms and translation to the clinic. Lancet Neurol. 8, 491-500. https://doi.org/10.1016/S1474-4422(09)70061-4
  31. Zimmermann, G. R., Lehar, J. and Keith, C. T. 2007. Multi-target therapeutics: when the whole is greater than the sum of the parts. Drug Discov. Today 12, 34-42. https://doi.org/10.1016/j.drudis.2006.11.008