[논문]GPR88 효현제의 전처리에 의한 뇌졸중후 뇌손상 감소효과 연구

이서연; 박정화; 김민재; 최병태; 신화경

doi:10.5352/jls.2020.30.11.939

GPR88 효현제의 전처리에 의한 뇌졸중후 뇌손상 감소효과 연구
Pretreatment with GPR88 Agonist Attenuates Postischemic Brain Injury in a Stroke Mouse Model 원문보기

생명과학회지 = Journal of life science, v.30 no.11, 2020년, pp.939 - 946

이서연 (원광대학교 의과대학 약리학교실) , 박정화 (부산대학교 한의학전문대학원 한의과학과) , 김민재 (부산대학교 한의학전문대학원 한의과학과) , 최병태 (부산대학교 한의학전문대학원 한의과학과) , 신화경 (부산대학교 한의학전문대학원 한의과학과)

초록
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뇌졸중은 전 세계적으로 신경계 장애를 일으키는 주요 원인 중 하나이며, 뇌졸중 환자는 다양한 운동, 인지 및 정신 장애를 나타낸다. GPR88은 orphan G protein coupled receptor이며 striatal medium spiny neurons에서 높게 발현이 되며, GPR88이 결손이 된 경우 motor coordination과 motor learning에 문제가 발생하게 된다. 본 연구에서는 Western blot 및 real-time PCR을 사용하여 허혈성 마우스 모델에서 GPR88 발현이 감소함을 발견 하였다. 또한, 뇌에서 유래한 세 가지 유형의 세포들, 뇌혈관내피세포(brain microvascular endothelial cells), 미세 아교세포(microglial cells) 및 신경 세포들에서 GPR88의 발현정도를 확인한 결과, HT22 신경 세포에서 GPR88의 발현이 가장 높음을 관찰하였고, 뇌졸중과 유사한 실험조건인 oxygen glucose deprivation (OGD) 조건에 배양한 HT22 신경세포에서 GPR88의 발현이 감소하였다. 또한 GPR88 효현제인 RTI-13951-33 (10 mg/kg)을 전처리후에 뇌허혈을 유발하였을 때, infarct volume의 감소, vestibular-motor function 및 neurological score의 개선효과를 관찰할 수 있었다. 이러한 결과는 GPR88이 허혈성 뇌졸중을 포함한 CNS 질환의 치료를 위한 잠재적인 약물표적이 될 수 있음을 제시한다.

Abstract ▼ AI-Helper

Stroke is one of the leading causes of neurological disability worldwide and stroke patients exhibit a range of motor, cognitive, and psychiatric impairments. GPR88 is an orphan G protein-coupled receptor (GPCR) that is highly expressed in striatal medium spiny neurons; its deletion results in poor motor coordination and motor learning. There are currently no studies on the involvement of GPR88 in stroke or in post-stroke brain function recovery. In this study, we found a decrease in GPR88 protein and mRNA expression levels in an ischemic mouse model using Western blot and real-time PCR, respectively. In addition, we observed that, among the three types of cells derived from the brain (brain microvascular endothelial cells, BV2 microglial cells, and HT22 hippocampal neuronal cells), the expression of GPR88 was highest in HT22 neuronal cells, and that GPR88 expression was downregulated in HT22 cells under oxygen-glucose deprivation (OGD) conditions. Moreover, pretreatment with RTI- 13951-33 (10 mg/kg), a brain-penetrant GPR88 agonist, ameliorated brain injury following ischemia, as evidenced by improvements in infarct volume, vestibular-motor function, and neurological score. Collectively, our results suggest that GPR88 could be a potential drug target for the treatment of central nervous system (CNS) diseases, including ischemic stroke.

주제어

표/그림 (5)

그림 Fig. 1. Downregulation of GPR88 after transient MCAO in C57 BL/6 mice. Six-week-old C57BL/6 mice underwent transient focal cerebral ischemia by middle cerebral artery occlusion (MCAO) for 60 min, followed by reperfusion for 4 hr, 24 hr, and 48 hr (MCAO/R). (A) Ipsilateral brain tissues were lysed and analyzed for GPR88 expression by Western blotting (n=5 each). Quantification graph was presented as mean ± SEM. *p<0.05 and ***p<0.001 versus sham group. (B) GPR88 mRNA expression at 60 min MCAO/24 hr reperfusion by real-time PCR. Data was presented as mean ± SEM (n=4). ***p<0.001 versus sham group.
그림 Fig. 2. GPR88 expression in three types of brain cells and under OGD conditions. (A) Western blots showing GPR88 expression in BV2 microglial cells, HT22 neuronal cells, and brain microvascular endothelial cells (mEC). Data was presented as mean ± SEM (n=4). **p<0.01 versus BV2 cells. (B, C) HT22 cells under OGD conditions for 24 hr were analyzed for GPR88 mRNA expression (B) by RT-PCR (n=3) and protein expression (C) by Western blot (n=3). Data was presented as mean ± SEM. *p<0.05 and ***p<0.001 versus control group.
그림 Fig. 3. Cell viability reduction in HT22 under OGD conditions. (A) HT22 cells were cultured under OGD conditions for 24 hr. Magnification: ×200; scale bar=200 μm. (B) Cell viability was determined via MTT assay. Three independent experiments with triplicates were performed. Data are presented as mean ± SEM. ***p<0.001 versus the control group.
그림 Fig. 4. Effect of GPR88 agonist on brain injury. (A) Mice were intraperitoneally injected with the GPR88 agonist RTI- 13951-33 hydrochloride (5 or 10 mg/kg, n=5 each) or PBS (vehicle group, n=5) 30 min prior to ischemic insult. Representative photographs of brain sections stained with TTC. (B) Quantification of direct infarct volume 24 hr postischemia. *p<0.05 versus vehicle group.
그림 Fig. 5. Effect of GPR88 agonist on brain function and behavior following ischemic brain injury. Neurological score (A) and wire-grip tests (B) were performed to evaluate the recovery of neurological deficits and vestibular motor functions after ischemic brain injury. Data was presented as mean ± SEM (n=5~7 each). *p<0.05, ***p< 0.001 versus vehicle group.

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가설 설정

In this study, we investigated the possible role of GPR88 in ischemic stroke by determining the expression levels of GPR88 in ischemic mice and neural cells. We then used a GPR88 agonist, RTI-13951-33 [8, 9], and examined its effects on ischemic brain damage, particularly in terms of infarct volume, neurological deficits, and motor function, using a focal cerebral ischemia mouse model.

제안 방법

Louis, MO, USA) was used as an internal control. Chemiluminescence was quantified using an ImageQuant LAS 4000 apparatus (GE Healthcare Life Sciences, Uppsala, Sweden), and band intensity was analyzed using Image J software (NIH, Bethesda, MD. USA).
For the cerebral ischemia and reperfusion model, middle cerebral artery occlusion (MCAO) using the intraluminal filament technique was performed. The creation of the MCAO model was performed by inserting a 7-0 monofilament (Doccol Corporation, Redlands, CA) coated with silicone into the internal carotid artery to occlude the middle cerebral artery. The monofilament was withdrawn 60 min after occlusion to achieve reperfusion.
The monofilament was withdrawn 60 min after occlusion to achieve reperfusion. The mice were sacrificed, and their brains were removed 4 hr, 24 hr, and 48 hr after occlusion. For the permanent ischemic occlusion model, focal cerebral ischemia was induced by photothrombosis of the cortical microvessels, as previously described [12].

대상 데이터

C57BL/6 mice (six-week-old, male) were purchased from Nara Biotechnology (Seoul, Korea). Mice were housed under a 12-hr light/dark cycle and allowed ad libitum access to food and water.
The primer sequences used were as follows: mouse GPR88 (Forward 5'–GCCCCA AATCAAGCAGGCAA–3', Reverse 5'–CCACGATTCTTC TTCCTCGCA–3') and m18S (Forward 5'–GTAACCCGTT GAACCCCATT–3, Reverse 5–CCATCCAATCGGTAGTA GCG–3').

데이터처리

The statistical differences between two groups were compared using a Student's t-test.

이론/모형

Wire-Grip Test. Vestibular-motor function was evaluated 24 hr after the ischemic injury using the wire-grip test. Each mouse was suspended on a metal wire and forced to hang with both forepaws.

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용어 설명: PCR은 세균 특이성이 있는 primer를 이용하여 적은 수의 세균이 있을지라도 쉽게 검출할 수 있는 유용한 방법이며, 이를 이용하여 구강 내 치면세균막이나 타액에서 직접 세균을 검출할 수 있게 되었다[8].

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