神經膠細胞在神經性發炎角色之探討

Role of Glial cells in Neuroinflammation

盧大宇(Dah-Yuu Lu)

指導教授 ： 符文美

國立臺灣大學/醫學院/藥理學研究所/博士(2007年)

https://doi.org/10.6342/NTU.2007.01656

摘要

Microglia在中樞神經系統缺氧狀態下會形成活化態；之前的研究也已證實缺血性中風與神經細胞的死亡和缺氧與microglia的活化有關。這可能是因為在缺氧狀態下活化的microglia會釋放具神經毒性的發炎物質，而加速在缺氧狀態下的神經細胞死亡。第一型缺氧誘導因子（hypoxia-inducible factor-1， HIF-1)，是感測細胞氧的含量，而做出適當的反應，調節生理功能使組織適應低氧狀態。然而microglia在缺氧狀態時HIF-1的表現及作用，目前仍然不清楚。在本論文我們研究缺氧對mciroglia HIF-1alpha表現的影響及HIF-1alpha對於發炎因子iNOS的調控機轉。不論是初級microglia或是microglia細胞株BV-2，在缺氧狀態下都會引發iNOS及NO表現增加。此外，microglia在缺氧狀態下HIF-1α的穩定性會增加，而增加HIF-1alpha的蛋白質表現，促進HIF-1alpha進到細胞核內進行轉譯而增加iNOS的表現。在缺氧狀態下會促進microglia的Akt及mammalian target of rapamycin (mTOR)的磷酸化，使用LY294002、wortmannin及rapamycin會抑制缺氧造成iNOS的表現。phosphatidylinositol 3-kinase (PI3-kinase)/Akt/mTOR的訊息傳遞路徑，會調節iNOS的表現；因此在腦部缺氧狀態下，不只會直接造成神經細胞的傷害，可能也會經由活化microglia，而加速神經細胞的受損。本論文發現microglia在缺氧狀態下會增加HIF-1α的穩定性，且透過PI3-kinase/Akt/mTOR的訊息傳遞路徑調節iNOS的表現。
Stromal cell-derived factor-1alpha (SDF-1alpha)及其專一性的受體CXCR4表現在中樞神經系統的各種細胞，並調節各種不同的生理功能。例如：SDF-1alpha在小腦granule cells會促進其移行和增生，SDF-1alpha也是microglia的趨化劑 (chemoattractant)，會因濃度差而影響microglia的migration。另外SDF-1alpha也會刺激astrocytes的細胞激素(cytokines)和glutamate的釋放。當中樞神經系統缺血性中風時會增加SDF-1alpha及CXCR4的表現，且在HIV感染中樞神經系統時microglia細胞膜上的CXCR4表現是一個關鍵因素。但目前對於microglia的CXCR4下游的訊息傳遞路徑仍不清楚。本論文因此探討在SDF-1alpha刺激microglia產生IL-6的訊息傳遞路徑。在之前報告中IL-6是一個會影響神經細胞存活和死亡及調節BBB通透性的proinflammatory cytokines。將microglia 處理SDF-1alpha時會隨著時間和濃度而增加IL-6的表現，前處理CXCR4的抑制劑AMD3100會拮抗SDF-1alpha促進IL-6的表現。此外，SDF-1alpha會增加microglia的ERK和Akt之活化，前處理LY294002和PD98059可以拮抗SDF-1alpha對IL-6的表現。SDF-1alpha也會促進microglia IKKalpha/beta(Ser 180/181)及IkappaBalpha的磷酸化，前處理PDTC、TPCK及Bay 11-7082可以拮抗SDF-1alpha促進IL-6的表現。因此，SDF-1alpha是經由CXCR4 receptor，活化pERK和pAkt，IKKalph/beta、IkappaBalpa的磷酸化，造成NF-kappaB進到細胞核內調控IL-6的表現。
LPS常被用來活化microglia及研究神經性發炎的藥物。在本論文中我們探討YC-1對於LPS誘發microglia活化，造成神經性發炎的作用及YC-1之作用機轉。LPS會刺激初級microglia及microglia細胞株BV-2大量的釋放發炎物質iNOS和COX-2。YC-1可以有效的抑制LPS造成microglia 活化，抑制iNOS、COX-2及proinflammatory cytokines TNF-alpha和IL-1beta的表現；在之前的報告中NF-kappaB是會調節大部分的發炎物質的表現；LPS會刺激NF-kappaB活化，進到細胞核內進行轉譯作用。Microglia前處理YC-1也會抑制LPS刺激 NF-kappaB的活化。
在大白鼠的substantia nigra注射LPS會刺激microglia活化，引發dopaminergic neurons 的神經死亡。在本論文中我們發現LPS造成dopaminergic neurons 的神經死亡，可以被YC-1所拮抗。在substantia nigra注射LPS 24 小時會促進microglia活化，iNOS、COX-2的表現，且在給予LPS 7天後會減少striatum的dopamine含量及促進substantia nigra dopaminergic neurons 的神經死亡，而增加amphetamine誘導的運動失調作用。同時注射YC-1及LPS可以有效的降低LPS造成的microglia活化，iNOS、COX-2及proinflammatory cytokines IL-1、TNF-alpha的表現，另外每天腹腔注射(IP)給予YC-1 7天也可以減少LPS造成striatum dopamine含量的減少及substantia nigra dopaminergic neurons 的神經死亡，並改善amphetamine誘導的運動失調作用。因此我們發現YC-1能有效的對抗LPS造成的神經性發炎，保護神經細胞減少發炎物質的傷害。這些結果可以提供新藥開發的方向，尋找治療神經性發炎造成的神經損傷之新藥物。
在腦部缺血性中風時，MMP的增加和腦部組織的傷害有極大的相關性。在本論文我們發現astrocytes在缺氧狀態下會活化，造成MMP-13的表現增加，MMP-13會促進BBB endothelial permeability。在缺氧狀態下，astrocytes會增加MMP-13在細胞內表現並促進MMP-13分泌增加；astrocytes在缺氧狀態處理8小時並收集conditioned medium (Hx-CM)，將之加到adult rat brain endothelial cells (ARBECs)中24小時，endothelial permeability會明顯增加，且會被前處理anti-MMP-13的抗體所拮抗。在astrocytes細胞轉殖c-Fos、c-Jun的AS-ODN會抑制缺氧處理astrocytes的MMP-13的表現且也會抑制astrocytes的 Hx-CM造成的endothelial permeability增加。同樣地，在endothelial cells外給合成的MMP-13 也會造成endothelial permeability增加、並且會有tight junction protein被分解的現象。因此我們的結果發現，在缺氧狀態下astrocytes會表現增加MMP-13，且會被c-Fos、c-Jun所調控，並且MMP-13會造成endothelial permeability增加。此外，astrocytes產生的MMP-13造成endothelial permeability增加，可能是經由破壞endothelial cells的tight junction而來的。
microglia和astrocytes 在中樞神經系統是生理功能之調節非常的重要；會影響神經細胞的分化、生長，並且在病理狀態下過度活化也會影響神經細胞的存活。本論文探討刺激microglia和astrocytes活化（包括缺氧和LPS），會促進microglia iNOS、COX-2、IL-1beta、TNF-alpha 及IL-6的表現 及astrocytes MMP-13的表現。因此glial cells可能是一個研究神經退化性疾病的另一個標的，了解glial cells的活化及其造成的神經性發炎，可以提供開發藥物治療退化性神經疾病的方針。

關鍵字

微膠細胞 ； 星狀細胞 ； 缺氧 ； 細胞激素 ； 神經性發炎

並列摘要

Exposure to hypoxia caused microglia activation and animal studies have shown that neuronal cell death is related to microglia activation following cerebral ischemia. Thus, it is likely that toxic inflammatory mediators produced by activated microglia under hypoxic condition may exacerbate neuronal injury following cerebral ischemia. The hypoxia-inducible factor-1 (HIF-1) is primarily involved in the sense and adaption of cells to changes in the O2 level. However, the role of HIF-1 in microglia activation under hypoxia has not yet been defined. We investigated the signaling pathways of HIF-1α involved in the regulation of hypoxia-induced overexpression of inducible NO synthase (iNOS) in microglia. Exposure of primary rat microglia cultures as well as established microglia cell line BV-2 to hypoxia induced the expression of iNOS, indicating that hypoxia could lead to the inflammatory activation of microglia. iNOS induction was accompanied with NO production. Moreover, the molecular analysis of these events indicated that iNOS expression was regulated by the phosphatidylinositol 3-kinase (PI3-kinase)/AKT/mammalian target of rapamycin (mTOR) signaling pathway and activation of HIF-1α. Thus, during cerebral ischemia, hypoxia may not only directly damage neurons, but also promote neuronal injury indirectly via microglia activation. These results suggest that hypoxia induced iNOS expression via HIF-1α in microglia.
The chemokine stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4 receptor expressed in a variety of neural cells, and this signaling results in diverse biological effects. It enhances migration and proliferation of cerebellar granule cells, chemoattracts microglia, and stimulates cytokine production and glutamate release by astrocytes. However, the signaling pathways of CXCR4 in glial cells are not clear. We investigated the signaling pathways involved in IL-6 production caused by SDF-1α in microglia. SDF-1αcaused a concentration- and time-dependent increase in IL-6 production. SDF-1α-mediated IL-6 production was attenuated by CXCR4 antagonist (AMD3100), phosphatidylinositol 3-kinase inhibitor (LY294002 and wortmannin), NF-κB inhibitor (PDTC), IκB protease inhibitor (TPCK) or IκBα phosphorylation inhibitor (Bay 11-7082). Stimulation of microglia with SDF-1α increased IκB kinaseα/β (IKKα/β) phosphorylation, IκBα phosphorylation, IκBα degradation, p65 translocation from the cytosol to the nucleus, and κB-luciferase activity. SDF-1α-mediated increase of IKK α/β phosphorylation, κB-luciferase activity was inhibited by LY294002. These results suggest that SDF-1αincreased IL-6 production in microglia via the CXCR4 receptor/PI3K/Akt and NF-κB signaling pathway.
LPS has been reported to cause marked microglia activation. We here investigated the inhibitory effect of YC-1 against LPS-induced inflammatory responses in microglia. To understand the inhibitory effects of YC-1 on LPS-induced neuroinflammation, primary microglia culture and microglia cell line BV-2 were used. To examine the action mechanism of YC-1, LPS-induced nitric oxide (NO) and prostaglandin E2 (PGE2) production, iNOS, COX-2 and cytokine expression were analyzed by Griess reaction, ELISA, Western blotting and RT-PCR, respectively. The effect of YC-1 on LPS-induced nuclear factor kappa B (NF-κB) activation was studied by NF-κB reporter assay and immunocytochemistry. YC-1 inhibited LPS-induced production of NO and PGE2 in a concentration-dependent manner. The protein and mRNA expression of iNOS and COX-2 in response to LPS application were also decreased by YC-1. In addition, YC-1 effectively reduced LPS-induced mRNA expression of proinflammatory cytokines of TNF-αand IL-1β. Furthermore, YC-1 also inhibited LPS-induced NF-κB activation in microglia.
It has been reported that intranigral injection of LPS leads to a marked microglia activation and neuronal death of dopaminergic neurons in substantia nigra (SN). As mentioned above YC-1 effectively attenuates LPS-induced proinflammatory responses in microglia. We further investigated whether YC-1 exerts neuroprotective effect in LPS-induced neurotoxicity in a rat experimental model. It was found that intranigral injection with LPS concomitant with YC-1 inhibited the microglia activation and the lose of tyrosine hydroxylase (TH)-positive dopaminergic neurons. In addition, the induction of iNOS and COX-2 was examined following LPS injection. YC-1 treatment decreased the expression of iNOS, COX-2 and pro-inflammatory cytokines in SN. Taken together, our results provide evidence that YC-1 is able to inhibit LPS-induced iNOS and COX-2 expression and NF-κB activation in microglia. In addition, YC-1 protected dopaminergic neurons against LPS-induced neurotoxicity by a decrease in the number of activated microglia, suggesting that the reduction in microglia-mediated release of inflammatory mediators may contribute to the anti-inflammatory effect.
Matrix metalloproteinases (MMPs) are involved in tissue destruction produced by the neuroinflammatory response that follows ischemic stroke. We here found that ischemia-reperfusion increased MMP-13 expression, which co-localized with astrocytes as shown by immunohistochemistry. To further assess the mechanisms involved in the induction of MMP-13 in astrocytes, we used primary culture of rat astrocyte. Exposure to hypoxia rapidly upregulated the mRNA level of MMP-13, c-Fos and c-Jun within 0.5 to 8 h. Hypoxia-induced MMP-13 overexpression was reduced by transfection of AS-ODN (antisense oligonucleotide) of c-Fos and c-Jun. Conditioned medium (CM) collected from astrocytes exposed to hypoxia increased blood-brain barrier (BBB) permeability using adult rat brain endothelial cell (ARBEC) culture. Anti-MMP-13 antibody antagonized CM-induced hyperpermeability in ARBEC culture insert systems. Transfection of AS-ODN of c-Fos, c-Jun or MMP-13 significantly decreased the hyperpermeability effect of CM. Treatment of recombinant MMP-13 protein in ARBEC culture also increased BBB permeability. In addition, recombinant MMP-13 protein caused a destruction of the tight junction protein of ARBECs. Taken together, these data suggest that hypoxia induced MMP-13 expression in astrocytes, which may be an important factor in the pathogenesis of blood-brain barrier following ischemic insult.
Microglia and astrocytes play a key role in the regulation of neuroal development, growth as well as degeneration. Our results provide evidence that environment stress (hypoxia or LPS) increased iNOS、COX-2、IL-1β、TNF-α、IL-6 and MMP expression. Therefore, glial cells may be a good target to develop new drugs for the treatment of neurodegeneration. In addition, YC-1 inhibits microglia activation and cytokines expression and may be a good candidate to treat neuroinflammation.

並列關鍵字

microglia ； astrocyte ； hypoxia ； cytokine ； neuroinflammation

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