A possible role of microglia-derived nitric oxide by lipopolysaccharide in activation of astroglial pentose-phosphate pathway via the Keap1/Nrf2 system.
A possible role of microglia-derived nitric oxide by lipopolysaccharide in activation of astroglial pentose-phosphate pathway via the Keap1/Nrf2 system.
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DOI：
10.1186/s12974-016-0564-0
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发表时间：
2016-05-04
影响因子：
9.3
通讯作者：
Suzuki N
中科院分区：
文献类型：
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作者：
Iizumi T;Takahashi S;Mashima K;Minami K;Izawa Y;Abe T;Hishiki T;Suematsu M;Kajimura M;Suzuki N
toll样受体4 （TLR4）在卒中诱导炎症的病理生理中起关键作用。星形胶质细胞和小胶质细胞均表达TLR4，缺血脑内产生的内源性配体可诱导炎症反应。TLR4激活产生的活性氧（ROS）、一氧化氮（NO）和炎症细胞因子在脑卒中后神经元损伤中起有害作用。虽然星形胶质细胞在脂多糖（LPS）刺激TLR4时表现出促炎反应，但它们也可能通过激活戊糖磷酸途径（PPP）来发挥细胞保护作用，通过谷胱甘肽过氧化物酶减少氧化应激。我们研究了星形胶质细胞通过激活PPP减少氧化应激的机制，使用TLR4刺激和缺氧与小胶质细胞协同作用。体外实验使用Sprague-Dawley大鼠制备的细胞进行。星形胶质细胞培养中共存的小胶质细胞用l-亮氨酸甲酯（LME）化学消除。将细胞暴露于LPS （0.01 μg/mL）或缺氧（1% O2）中12-15小时。用[1- 14c]葡萄糖和[6-14C]葡萄糖测定PPP活性。分别用2′，7′-二氯双氢荧光素和二氨基荧光素- fm双醋酸酯测定ROS和NO的生成。核因子-红细胞2相关因子2 （Nrf2）是应激条件下调节葡萄糖6-磷酸脱氢酶（PPP的限速酶）的主要转录因子，使用免疫组织化学评估其参与情况。暴露于LPS的培养星形胶质细胞诱导PPP通量增加20%，这些作用似乎与Nrf2有关。然而，共存的小胶质细胞的化学耗竭消除了PPP和Nrf2星形胶质核易位的增加。LPS诱导含有小胶质细胞的星形胶质细胞产生ROS和NO，而不诱导缺乏小胶质细胞的星形胶质细胞产生ROS和NO。脂多糖增加了谷胱甘肽耗竭后星形胶质细胞ROS的产生。U0126是一种丝裂原活化蛋白激酶的上游抑制剂，可以消除lps诱导的NO产生，而ROS的产生则不受影响。U0126还消除了星形胶质细胞-小胶质细胞培养中lps诱导的PPP激活，表明小胶质细胞来源的NO介导星形胶质细胞PPP激活。缺氧诱导星形胶质细胞PPP激活，不依赖于小胶质细胞- no通路。天然Nrf2激活剂萝卜硫素能消除ROS和NO的产生，证实了星形胶质细胞的保护机制。星形胶质细胞与小胶质细胞可能在脑卒中中发挥抗氧化应激的细胞保护作用。本文的在线版本（doi:10.1186/s12974-016-0564-0）包含补充材料，可供授权用户使用。
Toll-like receptor 4 (TLR4) plays a pivotal role in the pathophysiology of stroke-induced inflammation. Both astroglia and microglia express TLR4, and endogenous ligands produced in the ischemic brain induce inflammatory responses. Reactive oxygen species (ROS), nitric oxide (NO), and inflammatory cytokines produced by TLR4 activation play harmful roles in neuronal damage after stroke. Although astroglia exhibit pro-inflammatory responses upon TLR4 stimulation by lipopolysaccharide (LPS), they may also play cytoprotective roles via the activation of the pentose phosphate pathway (PPP), reducing oxidative stress by glutathione peroxidase. We investigated the mechanisms by which astroglia reduce oxidative stress via the activation of PPP, using TLR4 stimulation and hypoxia in concert with microglia. In vitro experiments were performed using cells prepared from Sprague–Dawley rats. Coexisting microglia in the astroglial culture were chemically eliminated using l-leucine methyl ester (LME). Cells were exposed to LPS (0.01 μg/mL) or hypoxia (1 % O2) for 12–15 h. PPP activity was measured using [1-14C]glucose and [6-14C]glucose. ROS and NO production were measured using 2′,7′-dichlorodihydrofluorescein diacetate and diaminofluorescein-FM diacetate, respectively. The involvement of nuclear factor-erythroid-2-related factor 2 (Nrf2), a cardinal transcriptional factor under stress conditions that regulates glucose 6-phosphate dehydrogenase, the rate-limiting enzyme of PPP, was evaluated using immunohistochemistry. Cultured astroglia exposed to LPS elicited 20 % increases in PPP flux, and these actions of astroglia appeared to involve Nrf2. However, the chemical depletion of coexisting microglia eliminated both increases in PPP and astroglial nuclear translocation of Nrf2. LPS induced ROS and NO production in the astroglial culture containing microglia but not in the microglia-depleted astroglial culture. LPS enhanced astroglial ROS production after glutathione depletion. U0126, an upstream inhibitor of mitogen-activated protein kinase, eliminated LPS-induced NO production, whereas ROS production was unaffected. U0126 also eliminated LPS-induced PPP activation in astroglial–microglial culture, indicating that microglia-derived NO mediated astroglial PPP activation. Hypoxia induced astroglial PPP activation independent of the microglia–NO pathway. Elimination of ROS and NO production by sulforaphane, a natural Nrf2 activator, confirmed the astroglial protective mechanism. Astroglia in concert with microglia may play a cytoprotective role for countering oxidative stress in stroke. The online version of this article (doi:10.1186/s12974-016-0564-0) contains supplementary material, which is available to authorized users.