探討Chemokines RANTES與MIF在中風的病理角色及治療策略-從臨床到基礎之研究

Abstract

腦中風在十大死因中排行第三位，在惡性腫瘤和心臟疾病之後，根據WHO的調查顯示，每年約有1500萬人發生腦中風，而腦中風後造成的死亡及長久的行動不便，無法工作又或生活無法自理，這些問題不僅對個人甚至家庭及社會造成沈重的經濟負擔，在逐漸高齡化的社會中，腦中風是不容忽視的疾病。 然而，造成腦中風的致病原因及腦部損傷的機轉十分的複雜，雖然投入大量的研究，但仍舊無法了解透徹。在中風患者中，缺血性中風佔多數，約80%。血栓溶解劑（t-PA）被用在缺血性中風後的治療，但是使用血栓溶解劑會受到黃金三小時的限制，當中風發生後經過的時間愈長，使用血栓溶解劑的效果就會隨之減小，甚至會導致出血的副作用增加，不利於神經功能的恢復。因此，研究新的治療方法用於缺血性中風，以改善其預後是十分重要且急迫的。 在臨床方面，我們以人類細胞激素檢測盤（human cytokine array）來測定使用血栓溶解劑前後中風病患血漿中細胞激素的改變。由此，我們發現MIF與RANTES在缺血性中風發生時，濃度明顯的上升，而在使用血栓溶解劑後24小時也顯著地降低其濃度，而其他的細胞激素則沒有如此的表現。因此，我們認為這兩個細胞激素應該與缺血性中風有緊密的關聯，之後，又以ELISA針對MIF和RANTES進行更多的檢體檢測，再次確認這兩個細胞激素在中風時的濃度變化。 此外我們也以大鼠的腦中風模式（tMCAO），在24小時後觀察兩種細胞激素對於中風後腦部受損體積的體積的影響，發現MIF會增加神經損傷，而RANTES則是有神經保護的作用，但這兩種激素在缺氧缺糖（oxygen glucose deprivation）的離體神經細胞實驗中卻沒有促使神經死亡之作用，因此推斷MIF和RANTES在中風病理並非是直接的對腦神經細胞產生毒性或是保護因子，而是有其他細胞參與其中。 接著，為了研究MIF和RANTES的機轉，我們使用大鼠的血腦障壁實驗（Evans blue assay）及離體的大鼠腦內皮細胞實驗中發現MIF會顯著的造成中風後血腦障壁的破壞，使得內皮細胞間的緊密連接（tight junctions）減少，因而導致中風後的損傷加重。反之，RANTES在中風後會降低血腦障壁的通透性，推測是RANTES產生保護效果的原因之一。 在現代醫療的進步下，中風後的死亡率在過去的十年中不斷降低，但社會朝向高齡化的方向發展，造成中風的人口不斷攀升，中風伴隨而來的神經功能受損變成了另一個嚴重且迫切需要解決的問題。目前，在中風後的治療上，雖然各大藥廠皆投入大量心血研發，但市場上仍然需要更有效之藥物。我們運用各種實驗釐清MIF和RANTES兩種細胞激素在腦中風時所扮演的角色，期盼我們的研究能提供一個未來藥物研發的方向。

Stroke is the third leading cause of death, after cancer and heart disease. WHO reported that about 15 million people have a stroke each year. The post-stroke disability leads to serious financial issues for patients, their families and the whole society. About 80% of stroke belongs to ischemic stroke, which occurs by the obstruction in cerebral blood vessel and causes neuronal damage. The best and common treatment for ischemic stroke is intravenous injection of t-PA within 3 hours of stroke onset; however, the benefit of t-PA is time-dependent, there is no significant benefit after 4.5 hours of stroke onset. Therefore, developing the new strategy to treat stroke is important and urgent. Macrophage migration inhibitory factor (MIF) and Regulated on Activation, Normal T Cell Expressed and Secreted (RANTES) are well-known pro-inflammatory cytokines, but the roles in ischemic stroke remain controversial. Here we found that, the human plasma levels of MIF and RANTES are elevated after ischemia. In addition, the rodent model of ischemic stroke also showed the significant elevation of MIF and RANTES in serum. We thus examined the roles of these two cytokines in ischemic stroke. Male Wistar rats were used in this study. Rats were subjected to two common carotid arteries and right middle cerebral artery occlusion (tMCAO) for 50 min, and then treated with MIF, RANTES or ISO-1 (MIF antagonist) following occlusion/reperfusion. Twenty- hours later, infarct sizes were quantified with TTC stain and Modified Neurological Severity Score was used to investigate the motor function of rats. The experimental results show that exogenous administration of MIF promotes neuronal death and aggravates neurological deficit in experimental stroke. In contrast, treatment of RANTES can significantly improve the neurological score and reduce infarct volume. Blood-brain barrier (BBB), a highly selective permeability barrier, prevents harmful substances such as inflammatory molecules from entering the brain. However, stroke can disrupt the BBB and lead to the brain damage. To investigate the details in ischemic stroke, Evans blue assay was used to examine the BBB function after administration of MIF and RANTES in tMCAO models. It was found that, MIF aggravated BBB leakage in ischemic stroke models by decreasing the integrity of tight junctions. However, ISO-1 (MIF antagonist) could reduce the BBB leakage after stroke. On the other hand, RANTES may protect neurons in cerebral ischemia by reducing the BBB permeability. Our studies indicate that administration of MIF antagonist and RANTES during ischemic stroke exerts significant neuroprotection in rodent models by reducing the BBB leakage following ischemia/reperfusion. MIF and RANTES may be a good drug target for developing drugs for the treatment of ischemic stroke.