摘要
脑卒中是一类高发病率、高致死率和高致残率的中枢神经系统疾病。脑卒中包括缺血性脑卒中和出血性脑卒中。目前的研究认为,与脑卒中发生发展相关的病理机制包括：能量耗竭、兴奋性毒性、钙超载、氧化应激、炎症和凋亡等。虽然脑卒中的病理机制研究取得较大进展,但向临床应用的有效转化均失败。脑卒中尚缺乏理想有效的治疗药物,组织纤维蛋白酶原激活剂(tissue plasminogen activator, tPA)依然是当前治疗脑卒中的主要药物。但是,tPA严格的有效治疗时间窗及其不良反应等原因极大地限制了其在临床的应用。因此,研究、阐明脑卒中所致神经损伤的病理机制、寻找有效的生物靶点、研发理想的治疗药物显得尤为迫切。
ATP敏感性钾通道(ATP-sensitive potassium channel, K-ATP)是一种偶联细胞代谢与电活动、非电压依赖性的特殊钾离子通道,由内向整流钾通道(inwardly rectifying potassium channel, Kir)和磺酰脲受体(sulphonylurea receptor, SUR)亚单位以4：4的比例组合形成的异构八聚体,广泛分布于机体的多种组织与器官。长期以来,K-ATP通道与脑卒中相关性的研究大多关注于神经元Kir6.2/K-ATP通道,研究表明Kir6.2/K-ATP通道主要通过调节神经元膜电位的去极化而影响缺血性脑损伤。过去对脑卒中的研究主要集中于对神经元的保护。近年来,研究显示脑内的胶质细胞在脑缺血过程中扮演着重要的角色,其参与脑卒中的发生、发展以及后期的神经再生与修复。本实验室前期研究显示,Kir6.1/K-ATP通道高表达于脑内的星形胶质细胞、小胶质细胞和神经干细胞。但是,Kir6.1/K-ATP通道是否参与脑缺血损伤目前尚无研究报道。因此,本文工作主要研究Kir6.1/K-ATP通道在脑缺血再灌注损伤中的作用及机制,旨在为脑卒中的治疗提供新思路和新策略。
本文第一部分工作应用野生型(Wild-type, WT)小鼠与Kir6.1敲减(Kir6.1heterozygote knockout, Kir6.1+/-)小鼠制备大脑中动脉阻塞(middle cerebral artery occlusion, MCAO)导致的局灶性脑缺血模型,观察Kir6.1敲减对脑缺血再灌注急性期神经损伤的影响。研究结果显示,Kir6.1/K-ATP通道敲减加重小鼠局灶性脑缺血再灌注急性期的神经损伤,损伤机制与脑缺血后内质网应激与炎症反应的增强有关：基于第一部分研究发现Kir6.1/K-ATP通道在脑缺血炎症机制中的重要作用,第二部分工作研究调节BV-2细胞Kir6.1/K-ATP通道对氧糖剥夺／复糖复氧(oxygen-glucose deprivation/reperfusion, OGD/R)所致小胶质细胞炎症反应的影响。研究结果表明,K-ATP通道开放剂Cromakalim能够减轻OGD/R诱导的小胶质细胞的炎症反应,提示小胶质细胞Kir6.1/K-ATP通道是调节脑卒中炎症反应的重要靶点；第三部分基于星形胶质细胞在脑内的重要作用,研究星形胶质细胞Kir6.1/K-ATP通道对缺血性脑损伤过程的调节。研究结果显示,K-ATP通道开放剂Nicorandil能够减轻OGD所致星形胶质细胞的内质网应激与炎症反应。应用星形胶质细胞Kir6.1亚基特异性缺失的小鼠制备MCAO模型的研究发现,星形胶质细胞Kir6.1/K-ATP通道的特异性缺失加重脑缺血再灌注损伤。研究结果提示星形胶质细胞Kir6.1/K-ATP通道是缺血性脑损伤的重要靶标。
第一部分Kir6.1基因敲减对小鼠局灶性脑缺血再灌注急性期神经损伤的影响
目的：研究Kir6.1基因敲减对小鼠局灶性脑缺血再灌注急性期神经损伤的影响。
方法：应用2-3月龄WT及Kir6.1+/-雄性小鼠,采用线栓法建立MCAO模型,缺血1h分别再灌24h和72h。脑缺血急性期(再灌24h和72h),主要通过比较再灌不同时间小鼠的死亡率以及神经运动功能缺陷,应用2’3’5-氯化三苯基四氮唑(2'3'5-triphenyltetrazoliumchloride, TTC)染色法比较脑梗死体积的大小；伊文氏蓝(Evan's blue, EB)渗出法比较BBB通透性的改变；通过免疫组织化学染色法比较缺血脑组织内神经元、星形胶质细胞、小胶质细胞及血管基底膜的形态与数量的改变；运用蛋白质免疫印迹(Western blot)法检测基质金属蛋白酶9(matrix metalloproteinases9, MMP-9),内质网应激相关蛋白：糖调节蛋白78(glucose-regulated protein78, GRP78)、转录因子C/EBP同源蛋白(C/EBP-homologous protein, CHOP)和凋亡执行蛋白caspase-12以及炎症相关蛋白：核转录因子κB (nuclear factor-κB, NF-κB)、NOD样受体pyrin结合区域3(NOD-like receptor pyrin domain containing three, NLRP3)和caspase-1的表达变化；应用酶联免疫吸附法(enzyme-linked immunosorbent assay, ELISA)测定缺血脑组织中白介素-1beta (interleukin-1β, IL-1β)和肿瘤坏死因子-alpha (tumor necrosis factor-α、TNF-α)的含量。
结果：
1. Kir6.1基因敲减加重小鼠局灶性脑缺血再灌注所致的急性损伤
Kir6.1基因敲减加重小鼠脑缺血再灌注急性期的神经运动功能障碍,增加脑梗死体积；Kir6.1基因敲减加重小鼠脑缺血后BBB的破坏,增加缺血脑组织中激活型MMP-9的表达以及血管基底膜胶原蛋白Collagen-IV的降解；Kir6.1基因敲减增加小鼠脑缺血再灌注损伤后脑梗死区神经元的丢失,促进脑梗死周围区胶质细胞的增殖与活化。
2. Kir6.1基因敲减增强脑缺血再灌注损伤所致的内质网应激与炎症反应
Kir6.1基因敲减上调脑缺血再灌注损伤诱导的内质网应激相关蛋白,包括伴侣分子GRP78、转录因子CHOP和凋亡蛋白caspase-12;同时,Kir6.1基因敲减还增强脑缺血后的炎症反应,促进缺血脑组织内促炎因子TNF-a和IL-1β的释放。
结论：
1.Kir6.1/K-ATP通道敲减加重小鼠局灶性脑缺血再灌注所致的急性神经损伤。
2.Kir6.1/K-ATP通道敲减加重脑缺血再灌注损伤与增强脑缺血所致的内质网应激与炎症反应有关。
第二部分Kir6.1/K-ATP通道在氧糖剥夺/复糖复氧所致BV-2细胞炎症反应中的作用
目的：研究开放小胶质细胞K-ATP通道对氧糖剥夺/复糖复氧所致BV-2细胞炎症反应的影响。
方法：培养BV-2细胞,首先,通过细胞免疫荧光化学染色法验证BV-2细胞中K-ATP通道各亚基的表达；其次,通过Western blot检测氧糖剥夺模型下BV-2细胞K-ATP通道各亚基的表达变化,分别运用ELISA和Western blot方法比较氧糖剥夺模型下小胶质细胞炎症因子IL-1β的释放以及介导IL-1β产生的炎症小体的激活；最后,给予K-ATP通道开放剂Cromakalim预处理1h,运用ELISA和Western blot方法比较Cromakalim对OGD/R所致BV-2细胞炎症因子IL-1D的释放以及NLRP3和caspase-1表达的影响。
结果：
1.氧糖剥夺诱导BV-2细胞炎症小体的激活并促进IL-1p的产生
OGD/R下调BV-2细胞K-ATP通道的Kir6.1亚基；OGD/R上调BV-2细胞NLRP3和caspase-1的表达、促进炎症小体的激活,从而升高IL-1p的水平。
2.开放K-ATP通道减轻氧糖剥夺所致BV-2细胞的炎症反应
K-ATP通道开放剂Cromakalim预处理显著抑制OGD/R所致BV-2细胞IL-1p释放的增加；Cromakalim预处理抑制OGD/R诱导BV-2细胞NLRP3和caspase-1的表达上调、抑制炎症小体的激活,从而减少IL-1p的生成。
结论：
1.OGD/R导致BV-2细胞炎症小体的激活,促进IL-1p的产生与释放。
2.开放BV-2细胞Kir6.1/K-ATP通道抑制OGD/R所致炎症小体的激活,进而减少IL-1p的产生。
第三部分星形胶质细胞Kir6.1/K-ATP通道在缺血性脑损伤中的作用
目的：研究特异性表达于星形胶质细胞的Kir6.1/K-ATP通道在缺血性脑损伤中的作用。
方法：应用2-3月龄WT和Kir6.1敲除(Kir6.1homozygote knockout, Kir6.1-/-)的雄性小鼠,首先通过Real-time PCR筛选两基因型小鼠脑组织中表达差异的microRNAs;将WT小鼠应用线栓法制备MCAO模型,缺血1h再灌注24h后,观察脑缺血再灌注损伤对相关microRNAs表达的影响。其次,培养WT小鼠原代的星形胶质细胞,应用Real-time PCR方法比较OGD/R对与星形胶质细胞功能相关的miR-181a和miR-7表达的影响。同时,给予K-ATP通道开放剂Nicorandil预处理1h,分别运用ELISA和Western blot检测星形胶质细胞释放的炎症因子TNF-α、IL-1β和IL-10,内质网应激相关蛋白GRP78、CHOP和caspase-12以及与IL-1β生成相关的NLRP3和caspase-1的表达。最后,应用星形胶质细胞特异性敲除Kir6.1亚基的小鼠制备MCAO模型,通过神经运动功能评分的比较并结合TTC染色法比较脑梗死体积的大小,观察星形胶质细胞Kir6.1/K-ATP通道的特异性缺失对脑缺血再灌注损伤的影响,从在体水平验证星形胶质细胞Kir6.1/K-ATP通道在缺血性脑损伤中的作用。
结果：
1.Kir6.1基因缺失和缺血性脑损伤均下调miR-7
基础状态下,Kir6.1基因缺失下调脑内miR-7与miR-124水平；局灶性脑缺血再灌注损伤显著降低小鼠脑内miR-7.增加miR-181a与miR-290-5p的表达：OGD/R使星形胶质细胞内miR-7的表达减少、miR-181a的表达增加。
2.开放K-ATP通道减轻氧糖剥／复糖复氧夺所致星形胶质细胞的内质网应激与炎症反应
K-ATP通道开放剂Nicorandil预处理显著抑制OGD/R所致星形胶质细胞Kir6.1亚基和高迁移率组盒蛋白B1(high-mobility group box1,HMGB1)的表达下调；Nicorandil预处理减轻OGD/R所致内质网应激相关蛋白伴侣分子GRP78.转录因子CHOP和凋亡蛋白caspase-12的表达增加,因而减轻星形胶质细胞的内质网应激；通过降低Nicorandil预处理抑制OGD/R所致星形胶质细胞的活化,减少致炎因子TNF-a和IL-1p的释放；降低OGD/R所致星形胶质细胞NLRP3和caspase-1的表达增高,减少IL-1β的生成;Nicorandil抑制OGD/R所致星形胶质细胞miR-7的表达下调,miR-7靶向作用于NLRP3.负性调控NLRP3的表达。
3.星形胶质细胞Kir6.1/K-ATP通道特异性缺失加重缺血性脑损伤
特异性敲除星形胶质细胞Kir6.1/K-ATP通道,增加小鼠局灶性脑缺血再灌注损伤后的脑梗死体积,并加重脑缺血损伤所致的神经运动功能障碍。
结论：
1.开放Kir6.1/K-ATP通道抑制OGD/R所致星形胶质细胞的内质网应激与炎症反应。
2.星形胶质细胞Kir6.1/K-ATP通道的特异性缺失能够加重小鼠局灶性脑缺血再灌注损伤。
综上所述,本文工作的主要创新之处在于：
1、Kir6.1/K-ATP通道缺失加重小鼠局灶性脑缺血再灌注所致的急性神经损伤Kir6.1基因敲减加重脑缺血再灌注损伤,增强脑缺血所致的内质网应激与炎症反应。研究结果表明：Kir6.1/K-ATP通道是参与脑缺血急性期神经损伤的重要靶点,拓展了Kir6.1/K-ATP通道在脑缺血再灌注损伤中的研究。
2、Kir6.1/K-ATP通道抑制OGD/R所致小胶质细胞的炎症反应K-ATP通道开放剂Cromakalim预处理显著改善OGD/R所致小胶质细胞的炎症反应,抑制炎症因子IL-1β的释放以及IL-1p生成相关的炎症小体的激活。研究结果提示：Kir6.1/K-ATP通道可调节OGD/R所致小胶质细胞的炎症反应。
3、星形胶质细胞Kir6.1/K-ATP通道是调节脑缺血炎症反应的重要靶标开放Kir6.1/K-ATP通道抑制OGD/R所致星形胶质细胞的内质网应激与炎症反应：特异性敲除星形胶质细胞Kir6.1/K-ATP通道加重小鼠局灶性脑缺血再灌注损伤；Kir6.1/K-ATP通道缺失和脑缺血均可降低脑内miR-7的表达,靶向作用于NLRP3炎症小体、调节星形胶质细胞的炎症反应。研究结果表明,星形胶质细胞Kir6.1/K-ATP通道是参与脑卒中炎症反应调节的重要靶标,提示miR-7可能是Kir6.1/K-ATP通(?)的调节靶点。
Stroke is a central nervous system disease with highest morbidity, highest mortality and disability rate. Stroke is mainly classified into ischemic stroke and hemorrhagic stroke. Nowadays, the progressive injury following stroke originates from the complex pathologic mechamisms, including energy exhaustion, excitotoxicity, calcium overloading, oxidative stress, inflammation and apoptosis. Though there is some progress in animal experiments, it's not successful in the stroke translational researches. At present, there is no ideal drug for stroke. Currently, tissue plasminogen activator (tPA) is the main treatment drugs for stroke. However, this treatment is only appropriate for a very small number of patients, due to a narrow time window and adverse reactions. So, it is necessary to clarify the pathophysiological mechanisms of stroke, which is essential for drug development, all these are urgent for stroke treatment.
ATP-sensitive potassium (K-ATP) channels are an unsual and non-voltage dependent potassium channel, which provide a unique link between cellular energetics and electric excitability. They are eight heteromultimers composed of four inwardly rectifying potassium channel (Kir6.x) and four sulfonylurea receptors (SURs). These channels widely distribute all round the organism. For a long time, as to the study of K-ATP channels in stroke, there are only some researches about the roles of Kir6.2/K-ATP channels, which mainly express in neurons. These results showed that Kir6.2/K-ATP channels, which affected brain ischemic injury via potentiating the depolarization of cellular membrane potential. In the past few years, scientists focused on the neuroprotection of neurons against stroke. Recently, more and more studies clarified the importance of glia in stroke. In particular, astrocytes, which are abundant in the brain, appear to be important regulators of post-ischemic inflammatory responses and glial scar formation, all of them are significant in the neurogenesis and repair after stroke. In our previous studies, Kir6.1/K-ATP channels widely distribute in the brain, among astrocytes, microglia and neural stem cells, all these play essential roles in the development of stroke. However, whether Kir6.1/K-ATP associates with stroke or not? Up to now, there is no research about them in stroke. Therefore, this topic is to reveal the roles of Kir6.1/K-ATP channels in brain ischemic injury, and discovery new targets for stroke treatment.
So, in part I, we subjected both wild-type (WT) mice and Kir6.1heterozygote knockout (Kir6.1+/-) mice to cerebral ischemia/reperfusion injury with middle cerebral artery occlusion (MCAO) model, and to investigate the impacts of Kir6.1subunit knockdown on ischemic stroke. Our data reveals that the downregulation of Kir6.1/K-ATP channels is more susceptible to brain ischemia, which is related to enhancing ER stress and inflammatory responses during the brain ischemic injury. According to part Ⅰ, we found that Kir6.1/K-ATP channels played an essential part in post-ischemic inflammation. Then, in part Ⅱ, we focused on the roles of Kir6.1/K-ATP channels expressed in BV-2cell lines in OGD/R-induced inflammation. These results showed that opening Kir6.1/K-ATP channels could attenuate OGD/R-induced inflammation in BV-2cell lines, which implied that Kir6.1/K-ATP channels expressed in microglia were an important target for regulating post-ischemic inflammation. As to astrocytes play an important role in the brain. Accordingly, in part III, we concentrated on the roles of Kir6.1/K-ATP channels expressed in astrocytes in the brain ischemic injury. We found that opening Kir6.1/K-ATP channels expressed in astrocytes could inhibit OGD/R-induced ER stress and inflammation. Then, we subjected Kir6.1conditional knockout mice to MCAO model, and found astrocytes null for Kir6.1aggravated brain ischemic injury. These results suggested that Kir6.1/K-ATP channels expressed in astrocytes might be an essential target for stroke treatment.
Part I Effects of Kir6.1gene heterozygote knockout on the focal cerebral ischemia/reperfusion injury in mice
AIM:To investigate the effects of Kir6.1gene heterozygote knockout on focal cerebral ischemia/reperfusion injury in mice.
METHODS:We subjected two to three-month-old WT and Kir6.1+/-male mice to cerebral ischemia by MCAO model with a modified intralumenal filament technique as described previously. They were all peformed by ischemia1h and reperfusion24h or72h. The mortality rate and neurological deficits were measured in each group after cerebral ischemia. The infarct size was determined by staining coronal brain slices with235-triphenyltetrazoliumchloride (TTC), the extravasation of Evan's blue (EB) made known the BBB disruption. Moreover, we observed the changes of neurons, astrocytes, microglia and neurovascular substrates by immunohistochemistry staining, and applied Western blot assay to analyse expression of matrix metalloproteinases9(MMP-9), ER stress related proteins, including glucose-regulated protein78(GRP78), C/EBP-homologous protein (CHOP), caspase-12, and inflammatory related proteins, such as nuclear factor κB (NF-κB), NOD-like receptor pyrin domain containing three (NLRP3) and caspase-1. Furthermore, we detected the contents of interleukin-lbeta (IL-1β) and tumor necrosis factor-alpha (TNF-a) in the ischemic brain tissues by enzyme-linked immunoabsorbent assay (ELISA).
RESULTS:
1. Kir6.1gene knockdown exacerbated acute neural injury induced by cerebral ischemia/reperfusion injury in mice
Kir6.1gene knockdown aggravated the neurological disorder and infarct size after brain ischemic injury in mice. Kir6.1gene knockdown exacerbated BBB extravasation, increased expression of activated MMP-9and advanced Collagen-IV degradation. Kir6.1gene knockdown exacerbated neuronal death in the core infarct area, and promoted glial responses in the peri-infarct area after cerebral ischemia/reperfusion injury.
2. Kir6.1gene knockdown exacerbated brain ischemic injury via enhancing ER stress and post-ischemic inflammation
Kir6.1gene knockdown enhanced ER stress induced by ischemic injury, by upregulating ER stress-related proteins, including molecular chaperone GRP78, transcription factor CHOP and apoptosis executor caspase-12. Kir6.1gene knockdown intensified post-ischemic inflammation, and promoted production of inflammatory factors TNF-a and IL-1β.
CONCLUSIONS:
1. Kir6.1/K-ATP channels knockdown aggravates the brain ischemic injury induced by focal cerebral ischemia/reperfusion in mice.
2. Kir6.1/K-ATP channels knockdown exacerbates cerebral ischemia and reperfusion injury via advancing ER stress and post-ischemic inflammation.
Part II Roles of Kir6.1/K-ATP channels in OGD/R-induced inflammation in BV-2cell lines
AIM:To investigate the roles of K-ATP channels expressed in microglia in the OGD/R-induced inflammation.
METHODS:BV-2cell lines were used in the following experiments. Firstly, we used immunofluorescence images to validate K-ATP channels subunits expressed in BV-2cell lines. Secondly, we analysed the changes of K-ATP channels subunits after OGD/R by Western blot, and then observed inflammatory cytokine IL-1β release and inflammasome activation, which cleaved proIL-1β into mature IL-1β, by ELISA and Western blot assays respectively. Finally, we pretreated BV-2cell lines with K-ATP channels opener (Cromakalim)1h before OGD, compared the production of IL-1β and the expression of NLRP3and caspase-1by ELISA and Western blot assays respectively.
RESULTS:
1. Oxygen-glucose deprivation induced inflammasome activation and promoted IL-1β production in BV-2cell lines
OGD/R induced K-ATP channels Kir6.1subunit downregulation. OGD/R upregulated NLRP3and caspase-1, and induced inflammasome activation, which lead to an increase of IL-ip release from BV-2cell lines.
2. Opening K-ATP channels inhibited OGD/R-induced inflammation in BV-2cell lines
K-ATP channels opener (Cromakalim) pretreatment could ameliorate OGD/R-induced IL-1β release from BV-2cell lines. Pretreatment with Cromakalim could inhibit OGD/R-induced upregulation of NLRP3and caspase-1, and impeded activation of inflammasome, all of these resulted in the decrease of IL-1β.
CONCLUSIONS:
1. OGD/R results in activation of inflammasome and increases IL-1β production in BV-2cell lines.
2. Opening Kir6.1/K-ATP channels inhibites inflammasome activation, which decreases production of IL-1β in BV-2cell lines.
Part III Roles of Kir6.1/K-ATP channels expressed in astrocytes in the brain ischemic injury
AIM:To investigate the effects of Kir6.1/K-ATP channels specificly expressed in astrocytes on brain ischemic injury.
METHODS:We selected two to three-month-old WT and Kir6.1homozygote knockout (Kir6.1-/-) male mice, and screened the expression of microRNAs between both genetype mice by Real-time PCR analyses. Next, we subjected WT mice to brain ischemia by MCAO model as described previously and analysed changes of related microRNAs. Thirdly, we isolated primary astrocytes from the cortex of WT mice, and observed the expression of miR-181a and miR-7after OGD by Real-time PCR assays. Moreover, we pretreated primary astrocyte with Nicorandil1h before OGD, and detected release of pro-inflammatory factors TNF-α, IL-1β and IL-10by ELISA. Expression of GRP78, CHOP, caspase-12, NLRP3and caspase-1, which were relevant to ER stress and inflammation, were determined by Western blot. Finally, in order to verify the influence of Kir6.1/K-ATP channels expressed in astrocytes deficiency on brain ischemic injury in vivo, we introduced Kir6.1conditional knockout mice and subjected them to MCAO model. We utilized the neurological score and TTC staining to assess the neurological disorder and infarct size.
RESULTS:
1. Either Kir6.1gene deficiency or brain ischemic injury caused downregulation of miR-7
At the baseline, Kir6.1knockout caused downregulation of miR-7and miR-124. Brain ischemia induced downregulation of miR-7, upregulation of miR-181a and miR-290-5p. Moreover, OGD/R caused downregulation of miR-7and upregulation of miR-181a in primary astrocytes.
2. Opening K-ATP channels decreased ER stress and inflammation induced by OGD/R in primary astrocytes
Pretreatment with K-ATP channels opener (Nicorandil) could inhibited OGD/R-induced downregulation of Kir6.1subunit and high-mobility group boxl (HMGB1). Nicorandil decreased expression of ER stress-related proteins, including molecular chaperone GRP78, transcription factor CHOP and apoptosis executor caspase-12. So it could attenuate ER stress induced by OGD/R model. Nicorandil decreased OGD/R-induced TNF-α and IL-1β release from astrocytes, it could also inhibit OGD/R-induced upregulation of NLRP3and caspase-1, impeded inflammasome activation and decreased production of IL-1β.Nicorandil downregulated OGD/R-induced the increase of miR-7, and targeted the NLRP3inflammasome, which inversely correlated to the expression of NLRP3.
3. Kir6.1/K-ATP channels deficiency in astrocytes aggravated cerebral ischemia/reperfusion injury
Compared to contol group mice, there was larger infarct size in the mice, which were deficient by Kir6.1/K-ATP channels expressed in astrocytes. Moreover, it's also accompanied by severer neurological disorder.
CONCLUSIONS:
1. Opening Kir6.1/K-ATP channels attenuates OGD/R-induced ER stress and inflammatory responses in primary astrocytes.
2. Kir6.1/K-ATP channels deficiency in astrocytes aggravates focal cerebral ischemia/reperfusion injury in mice.
SUMMARY:
1. Kir6.1/K-ATP channels deficiency exacerbates acute neural injury induced by focal cerebral ischemia/reperfusion
Kir6.1gene knockdown aggravated cerebral ischemia/reperfusion injury, and enhanced ER stress and post-ischemic inflammation. These results indicate that Kir6.1/K-ATP channels are important targets for the acute brain ischemic injury, which expands the study of Kir6.1/K-ATP channels to stroke.
2. Kir6.1/K-ATP channels inhibites OGD/R-induced inflammation in BV-2cell lines
Cromakalim opening Kir6.1/K-ATP channels decreased OGD/R-induced inflammatory responses in BV-2cell lines, including inhibiting IL-1β release and inflammasome activation. These results suggest Kir6.1/K-ATP channels expressed in microglia might be a new target for OGD/R-induced inflammation.
3. Kir6.1/K-ATP channels expressed in astrocytes are an important target for post-ischemic inflammation in brain ischemia
Opening Kir6.1/K-ATP channels could inhibite inflammation induced by OGD/R in astrocytes. Kir6.1conditional knockout in astrocytes could exacerbate cerebral ischemia/reperfusion injury. Both Kir6.1/K-ATP channels deficiency and cerebral ischemia could induce downregulation of miR-7. While miR-7targeted NLRP3inflammasome and regulated astrocytic inflammation. These results indicate that Kir6.1/K-ATP channels expressed in astrocytes are an essential target for the post-ischemic inflammation of stroke, and it also implies that miR-7might be a regulator of Kir6.1/K-ATP channels.
引文
1. Donnan GA, Fisher M, Macleod M, and Davis SM. Stroke. Lancet.2008; 371:1612-23.
2. Wang Y, Liu PP, Li LY, Zhang HM, and Li T. Hypothermia reduces brain edema, spontaneous recurrent seizure attack, and learning memory deficits in the kainic acid treated rats. CNS Neurosci Ther.2011; 17:271-80.
3. Green AR. Pharmacological approaches to acute ischaemic stroke: reperfusion certainly, neuroprotection possibly. Br J Pharmacol.2008; 153 Suppl 1:S325-38.
4. Broussalis E, Killer M, McCoy M, Harrer A, Trinka E, and Kraus J. Current therapies in ischemic stroke. Part A. Recent developments in acute stroke treatment and in stroke prevention. Drug Discov Today.2012; 17:296-309.
5. Oliver KR, Wainwright A, Edvinsson L, Pickard JD, and Hill RG. Immunohistochemical localization of calcitonin receptor-like receptor and receptor activity-modifying proteins in the human cerebral vasculature. J Cereb Blood Flow Metab.2002; 22:620-9.
6. Astrup J, Siesjo BK, and Symon L. Thresholds in cerebral ischemia-the ischemic penumbra. Stroke.1981; 12:723-5.
7. Baron JC. Mapping the ischaemic penumbra with PET:implications for acute stroke treatment. Cerebrovasc Dis.1999; 9:193-201.
8. Heiss WD, Kracht LW, Thiel A, Grond M, and Pawlik G. Penumbral probability thresholds of cortical flumazenil binding and blood flow predicting tissue outcome in patients with cerebral ischaernia. Brain.2001; 124:20-9.
9. Warach S. Measurement of the ischemic penumbra with MRI:it's about time. Stroke.2003; 34:2533-4.
10. Hossmann KA. Cerebral ischemia:models, methods and outcomes. Newopharmacology.2008; 55:257-70.
11. Kinouchi H, Sharp FR, Hill MP, Koistinaho J, Sagar SM, and Chan PH. Induction of 70-kDa heat shock protein and hsp70 mRNA following transient focal cerebral ischemia in the rat. J Cereb Blood Flow Metab.1993; 13: 105-15.
12. Block F, Peters M, and Nolden-Koch M. Expression of IL-6 in the ischemic penumbra. Neuroreport.2000; 11:963-7.
13. Matsushita K, Matsuyama T, Kitagawa K, Matsumoto M, Yanagihara T, and Sugita M. Alterations of Bcl-2 family proteins precede cytoskeletal proteolysis in the penumbra, but not in infarct centres following focal cerebral ischemia in mice. Neuroscience.1998; 83:439-48.
14. Lo EH. A new penumbra:transitioning from injury into repair after stroke. Nat Med.2008; 14:497-500.
15. Robertson CA, McCabe C, Gallagher L, Lopez-Gonzalez Mdel R, Holmes WM, Condon B, et al. Stroke penumbra defined by an MRI-based oxygen challenge technique:2. Validation based on the consequences of reperfusion. J Cereb Blood Flow Metab.2011; 31:1788-98.
16. Lo EH. T time in the brain. Nat Med.2009; 15:844-6.
17. Iadecola C and Anrather J. The immunology of stroke:from mechanisms to translation. Nat Med.2011; 17:796-808.
18. Ekdahl CT, Kokaia Z, and Lindvall O. Brain inflammation and adult neurogenesis:the dual role of microglia. Neuroscience.2009; 158:1021-9.
19. Jin R, Yang G, and Li G. Inflammatory mechanisms in ischemic stroke:role of inflammatory cells. J Leukoc Biol.2010; 87:779-89.
20. Tower DB and Young OM. The activities of butyrylcholinesterase and carbonic anhydrase, the rate of anaerobic glycolysis, and the question of a constant density of glial cells in cerebral cortices of various mammalian species from mouse to whale. JNeurochem.1973; 20:269-78.
21. Sofroniew MV and Vinters HV. Astrocytes:biology and pathology. Acta Neuropathol.2010; 119:7-35.
22. Sofroniew MV. Molecular dissection of reactive astrogliosis and glial scar formation. Trends Neurosci.2009; 32:638-47.
23. Shichita T, Ago T, Kamouchi M, Kitazono T, Yoshimura A, and Ooboshi H. Novel therapeutic strategies targeting innate immune responses and early inflammation after stroke. J Neurochem.2012; 123 Suppl 2:29-38.
24. Noma A. ATP-regulated K+ channels in cardiac muscle. Nature.1983; 305: 147-8.
25. Inagaki N, Gonoi T, Clement JPt, Namba N, Inazawa J, Gonzalez G, et al. Reconstitution of IKATP:an inward rectifier subunit plus the sulfonylurea receptor. Science.1995; 270:1166-70.
26. Ng KE, Schwarzer S, Duchen MR, and Tinker A. The intracellular localization and function of the ATP-sensitive K+channel subunit Kir6.1. J Membr Biol 2010; 234:137-47.
27. Bajgar R, Seetharaman S, Kowaltowski AJ, Garlid KD, and Paucek P. Identification and properties of a novel intracellular (mitochondrial) ATP-sensitive potassium channel in brain. J Biol Chem.2001; 276: 33369-74.
28. Babenko AP and Bryan J. Sur domains that associate with and gate KATP pores define a novel gatekeeper. JBiol Chem.2003; 278:41577-80.
29. Inagaki N, Gonoi T, Clement JP, Wang CZ, Aguilar-Bryan L, Bryan J, et al. A family of sulfonylurea receptors determines the pharmacological properties of ATP-sensitive K+ channels. Neuron.1996; 16:1011-7.
30. Nichols CG. KATP channels as molecular sensors of cellular metabolism. Nature.2006; 440:470-6.
31. Isomoto S, Kondo C, Yamada M, Matsumoto S, Higashiguchi O, Horio Y, et al. A novel sulfonylurea receptor forms with BIR (Kir6.2) a smooth muscle type ATP-sensitive K+ channel. J Biol Chem.1996; 271:24321-4.
32. Seino S and Miki T. Physiological and pathophysiological roles of ATP-sensitive K+ channels. Prog Biophys Mol Biol.2003; 81:133-76.
33. Sun HS and Feng ZP. Neuroprotective role of ATP-sensitive potassium channels in cerebral ischemia. Acta Pharmacol Sin.2013; 34:24-32.
34. Quinn KV, Giblin JP, and Tinker A. Multisite phosphorylation mechanism for protein kinase A activation of the smooth muscle ATP-sensitive K+channel. Oirc Res.2004; 94:1359-66.
35. Pasyk EA, Kang Y, Huang X, Cui N, Sheu L, and Gaisano HY. Syntaxin-1A binds the nucleotide-binding folds of sulphonylurea receptor 1 to regulate the KATP channel. J Biol Chem.2004; 279:4234-40.
36. Chen PC, Bruederle CE, Gaisano HY, and Shyng SL. Syntaxin 1A regulates surface expression of beta-cell ATP-sensitive potassium channels. Am J Physiol Cell Physiol 2011; 300:C506-16.
37. Cui N, Kang Y, He Y, Leung YM, Xie H, Pasyk EA, et al. H3 domain of syntaxin 1A inhibits KATP channels by its actions on the sulfonylurea receptor 1 nucleotide-binding folds-1 and -2. J Biol Chem.2004; 279: 53259-65.
38. Roper J and Ashcroft FM. Metabolic inhibition and low internal ATP activate K-ATP channels in rat dopaminergic substantia nigra neurones. Pflugers Arch. 1995; 430:44-54.
39. Ohno-Shosaku T and Yamamoto C. Identification of an ATP-sensitive K+ channel in rat cultured cortical neurons. Pflugers Arch.1992; 422:260-6.
40. Zawar C, Plant TD, Schirra C, Konnerth A, and Neumcke B. Cell-type specific expression of ATP-sensitive potassium channels in the rat hippocampus. JPhysiol 1999; 514 (Pt 2):327-41.
41. Ashford ML, Boden PR, and Treherne JM. Tolbutamide excites rat glucoreceptive ventromedial hypothalamic neurones by indirect inhibition of ATP-K+ channels. Br J Pharmacol. 1990; 101:531-40.
42. Trapp S and Ballanyi K. KATP channel mediation of anoxia-induced outward current in rat dorsal vagal neurons in vitro. J Physiol.1995; 487 (Pt 1): 37-50.
43. Thomzig A, Laube G, Pruss H, and Veh RW. Pore-forming subunits of K-ATP channels, Kir6.1 and Kir6.2, display prominent differences in regional and cellular distribution in the rat brain. J Comp Neurol.2005; 484:313-30.
44. Thomzig A, Wenzel M, Karschin C, Eaton MJ, Skatchkov SN, Karschin A, et al. Kir6.1 is the principal pore-forming subunit of astrocyte but not neuronal plasma membrane K-ATP channels. Mol Cell Neurosci.2001; 18:671-90.
45. Zhou F, Yao HH, Wu JY, Ding JH, Sun T, and Hu G. Opening of microglial K(ATP) channels inhibits rotenone-induced neuroinflammation. J Cell Mol Med.2008; 12:1559-70.
46. Blanco-Rivero J, Gamallo C, Aras-Lopez R, Cobeno L, Cogolludo A, Perez-Vizcaino F, et al. Decreased expression of aortic KIR6.1 and SUR2B in hypertension does not correlate with changes in the functional role of K(ATP) channels. Eur J Pharmacol.2008; 587:204-8.
47. Asemu G, Papousek F, Ostadal B, and Kolar F. Adaptation to high altitude hypoxia protects the rat heart against ischemia-induced arrhythmias. Involvement of mitochondrial K(ATP) channel. J Mol Cell Cardiol. 1999; 31: 1821-31.
48. Mayanagi K, Gaspar T, Katakam PV, and Busija DW. Systemic administration of diazoxide induces delayed preconditioning against transient focal cerebral ischemia in rats. Brain Res.2007; 1168:106-11.
49. Shimizu K, Lacza Z, Rajapakse N, Horiguchi T, Snipes J, and Busija DW. MitoK(ATP) opener, diazoxide, reduces neuronal damage after middle cerebral artery occlusion in the rat. Am J Physiol Heart Circ Physiol.2002; 283:H1005-11.
50. Sun HS, Feng ZP, Miki T, Seino S, and French RJ. Enhanced neuronal damage after ischemic insults in mice lacking Kir6.2-containing ATP-sensitive K+ channels. JNeurophysiol.2006; 95:2590-601.
51. Sun HS, Feng ZP, Barber PA, Buchan AM, and French RJ. Kir6.2-containing ATP-sensitive potassium channels protect cortical neurons from ischemic/anoxic injury in vitro and in vivo. Neuroscience.2007; 144: 1509-15.
52. Liu X, Wu JY, Zhou F, Sun XL, Yao HH, Yang Y, et al. The regulation of rotenone-induced inflammatory factor production by ATP-sensitive potassium channel expressed in BV-2 cells. Neurosci Lett.2006; 394:131-5.
53. Uluc K, Miranpuri A, Kujoth GC, Akture E, and Baskaya MK. Focal cerebral ischemia model by endovascular suture occlusion of the middle cerebral artery in the rat. J Vis Exp.2011;
54. Guo M, Lin V, Davis W, Huang T, Carranza A, Sprague S, et al. Preischemic induction of TNF-alpha by physical exercise reduces blood-brain barrier dysfunction in stroke. J Cereb Blood Flow Metab.2008; 28:1422-30.
55. Yang JZ, Huang X, Zhao FF, Xu Q, and Hu G. Iptakalim enhances adult mouse hippocampal neurogenesis via opening Kir6.1-composed K-ATP channels expressed in neural stem cells. CNS Neurosci Ther.2012; 18: 737-44.
56. Lim A, Park SH, Sohn JW, Jeon JH, Park JH, Song DK, et al. Glucose deprivation regulates KATP channel trafficking via AMP-activated protein kinase in pancreatic beta-cells. Diabetes.2009; 58:2813-9.
57. Ogawa S, Kitao Y, and Hori O. Ischemia-induced neuronal cell death and stress response. Antioxid Redox Signal.2007; 9:573-87.
58. Shibata M, Hattori H, Sasaki T, Gotoh J, Hamada J, and Fukuuchi Y. Activation of caspase-12 by endoplasmic reticulum stress induced by transient middle cerebral artery occlusion in mice. Neuroscience.2003; 118: 491-9.
59. Tajiri S, Oyadomari S, Yano S, Morioka M, Gotoh T, Hamada JI, et al. Ischemia-induced neuronal cell death is mediated by the endoplasmic reticulum stress pathway involving CHOP. Cell Death Differ.2004; 11: 403-15.
60. Zhang L, Zhang ZG, and Chopp M. The neurovascular unit and combination treatment strategies for stroke. Trends Pharmacol Sci.2012; 33:415-22.
61. Kim HJ, Jeong JS, Kim SR, Park SY, Chae HJ, and Lee YC. Inhibition of endoplasmic reticulum stress alleviates lipopolysaccharide-induced lung inflammation through modulation of NF-kappaB/HIF-1 alpha signaling pathway. Sci Rep.2013; 3:1142.
62. Macrez R, Ali C, Toutirais O, Le Mauff B, Defer G, Dirnagl U, et al. Stroke and the immune system:from pathophysiology to new therapeutic strategies. Lancet Neurol.2011; 10:471-80.
63. Denes A, Thornton P, Rothwell NJ, and Allan SM. Inflammation and brain injury:acute cerebral ischaemia, peripheral and central inflammation. Brain Behav Immun.2010; 24:708-23.
64. Kane GC, Lam CF, O'Cochlain F, Hodgson DM, Reyes S, Liu XK, et al. Gene knockout of the KCNJ8-encoded Kir6.1 K(ATP) channel imparts fatal susceptibility to endotoxemia. Faseb J.2006; 20:2271-80.
65. Boutin H, LeFeuvre RA, Horai R, Asano M, Iwakura Y, and Rothwell NJ. Role of IL-1 alpha and IL-lbeta in ischemic brain damage. JNeurosci.2001; 21:5528-34.
66. Denes A, Ferenczi S, Halasz J, Kornyei Z, and Kovacs KJ. Role of CX3CR1 (fractalkine receptor) in brain damage and inflammation induced by focal cerebral ischemia in mouse. J Cereb Blood Flow Metab.2008; 28:1707-21.
67. Schroder K and Tschopp J. The inflammasomes. Cell.2010; 140:821-32.
68. Abulafia DP, de Rivero Vaccari JP, Lozano JD, Lotocki G, Keane RW, and Dietrich WD. Inhibition of the inflammasome complex reduces the inflammatory response after thromboembolic stroke in mice. J Cereb Blood Flow Metab.2009; 29:534-44.
69. Cassel SL and Sutterwala FS. Sterile inflammatory responses mediated by the NLRP3 inflammasome. Eur J Immunol.2010; 40:607-11.
70. Savage CD, Lopez-Castejon G, Denes A, and Brough D. NLRP3-Inflammasome Activating DAMPs Stimulate an Inflammatory Response in Glia in the Absence of Priming Which Contributes to Brain Inflammation after Injury. Front Immunol.2012; 3:288.
71. Rothwell N, Allan S, and Toulmond S. The role of interleukin 1 in acute neurodegeneration and stroke:pathophysiological and therapeutic implications. JClin Invest.1997; 100:2648-52.
72. Schilling M, Besselmann M, Muller M, Strecker JK, Ringelstein EB, and Kiefer R. Predominant phagocytic activity of resident microglia over hematogenous macrophages following transient focal cerebral ischemia:an investigation using green fluorescent protein transgenic bone marrow chimeric mice. Exp Neurol.2005; 196:290-7.
73. Eleftherianos I, Won S, Chtarbanova S, Squiban B, Ocorr K, Bodmer R, et al. ATP-sensitive potassium channel (K(ATP))-dependent regulation of cardiotropic viral infections. Proc Natl Acad Sci USA.2011; 108:12024-9.
74. Gade AR, Kang M, and Akbarali HI. Hydrogen sulfide as an allpsteric modulator of ATP-sensitive potassium channels in colonic inflammation. Mol Pharmacol.2013; 83:294-306.
75. Simard JM, Chen M, Tarasov KV, Bhatta S, Ivanova S, Melnitchenko L, et al. Newly expressed SUR1-regulated NC(Ca-ATP) channel mediates cerebral edema after ischemic stroke. Nat Med.2006; 12:433-40.
76. Tricarico D, Mele A, Liss B, Ashcroft FM, Lundquist AL, Desai RR, et al. Reduced expression of Kir6.2/SUR2A subunits explains KATP deficiency in K+-depleted rats. Neuromuscul Disord.2008; 18:74-80.
77. Mariathasan S, Weiss DS, Newton K, McBride J, O'Rourke K, Roose-Girma M, et al. Cryopyrin activates the inflammasome in response to toxins and ATP. Nature.2006; 440:228-32.
78. Yamasaki K, Muto J, Taylor KR, Cogen AL, Audish D, Bertin J, et al. NLRP3/cryopyrin is necessary for interleukin-lbeta (IL-lbeta) release in response to hyaluronan, an endogenous trigger of inflammation in response to injury. JBiol Chem.2009; 284:12762-71.
79. Halle A, Hornung V, Petzold GC, Stewart CR, Monks BG, Reinheckel T, et al. The NALP3 inflammasome is involved in the innate immune response to amyloid-beta. Nat Immunol.2008; 9:857-65.
80. Zhou R, Tardivel A, Thorens B, Choi I, and Tschopp J. Thioredoxin-interacting protein links oxidative stress to inflammasome activation. Nat Immunol.2010; 11:136-40.
81. Martinon F, Petrilli V, Mayor A, Tardivel A, and Tschopp J. Gout-associated uric acid crystals activate the NALP3 inflammasome. Nature.2006; 440: 237-41.
82. Dostert C, Petrilli V, Van Bruggen R, Steele C, Mossman BT, and Tschopp J. Innate immune activation through Nalp3 inflammasome sensing of asbestos and silica. Science.2008; 320:674-7.
83. Feldmeyer L, Keller M, Niklaus G, Hohl D, Werner S, and Beer HD. The inflammasome mediates UVB-induced activation and secretion of interleukin-1 beta by keratinocytes. Curr Biol.2007; 17:1140-5.
84. Rajamaki K, Nordstrom T, Nurmi K, Akerman KE, Kovanen PT, Oorni K, et al. Extracellular acidosis is a novel danger signal alerting innate immunity via the NLRP3 inflammasome. J Biol Chem.2013;
85. Rubartelli A. Redox control of NLRP3 inflammasome activation in health and disease. J Leukoc Biol.2012; 92:951-8.
86. Eng LF, Ghirnikar RS, and Lee YL. Glial fibrillary acidic protein: GFAP-thirty-one years (1969-2000). Neurochem Res.2000; 25:1439-51.
87. Lewis DK, Thomas KT, Selvamani A, and Sohrabji F. Age-related severity of focal ischemia in female rats is associated with impaired astrocyte function. Neurobiol Aging.2012; 33:1123 e1-16.
88. Saugstad JA. MicroRNAs as effectors of brain function with roles in ischemia and injury, neuroprotection, and neurodegeneration. J Cereb Blood Flow Metab.2010; 30:1564-76.
89. Kloosterman WP and Plasterk RH. The diverse functions of microRNAs in animal development and disease. Dev Cell.2006; 11:441-50.
90. Vemuganti R. The MicroRNAs and Stroke:No Need to be Coded to be Counted. Transl Stroke Res.2010; 1:158-60.
91. Rink C and Khanna S. MicroRNA in ischemic stroke etiology and pathology. Physiol Genomics.2011; 43:521-8.
92. Weng H, Shen C, Hirokawa G, Ji X, Takahashi R, Shimada K, et al. Plasma miR-124 as a biomarker for cerebral infarction. Biomed Res.2011; 32: 135-41.
93. Ouyang YB, Lu Y, Yue S, Xu LJ, Xiong XX, White RE, et al. miR-181 regulates GRP78 and influences outcome from cerebral ischemia in vitro and in vivo. Neurobiol Dis.2012; 45:555-63.
94. Ouyang YB, Lu Y, Yue S, and Giffard RG. miR-181 targets multiple Bcl-2 family members and influences apoptosis and mitochondrial function in astrocytes. Mitochondrion.2012; 12:213-9.
95. Zhang S, Zhou F, Ding JH, Zhou XQ, Sun XL, and Hu G. ATP-sensitive potassium channel opener iptakalim protects against MPP-induced astrocytic apoptosis via mitochondria and mitogen-activated protein kinase signal pathways. JNeurochem.2007; 103:569-79.
96. Zhang S, Ding JH, Zhou F, Wang ZY, Zhou XQ, and Hu G Iptakalim ameliorates MPP+-induced astrocyte mitochondrial dysfunction by increasing mitochondrial complex activity besides opening mitoK(ATP) channels. JNeurosci Res.2009; 87:1230-9.
97. Grun D, Wang YL, Langenberger D, Gunsalus KC, and Rajewsky N. microRNA target predictions across seven Drosophila species and comparison to mammalian targets. PLoS Comput Biol.2005;1:e13.
98. Cheng LC, Pastrana E, Tavazoie M, and Doetsch F. miR-124 regulates adult neurogenesis in the subventricular zone stem cell niche. Nat Neurosci.2009; 12:399-408.
99. Makeyev EV, Zhang J, Carrasco MA, and Maniatis T. The MicroRNA miR-124 promotes neuronal differentiation by triggering brain-specific alternative pre-mRNA splicing. Mol Cell.2007; 27:435-48.
100. Ponomarev ED, Veremeyko T, Barteneva N, Krichevsky AM, and Weiner HL. MicroRNA-124 promotes microglia quiescence and suppresses EAE by deactivating macrophages via the C/EBP-alpha-PU.1 pathway. Nat Med. 2011; 17:64-70.
101. Jeyaseelan K, Lim KY, and Armugam A. MicroRNA expression in the blood and brain of rats subjected to transient focal ischemia by middle cerebral artery occlusion. Stroke.2008; 39:959-66.
102. Tan KS, Armugam A, Sepramaniam S, Lim KY, Setyowati KD, Wang CW, et al. Expression profile of MicroRNAs in young stroke patients. PLoS One. 2009; 4:e7689.
103. Laterza OF, Lim L, Garrett-Engele PW, Vlasakova K, Muniappa N, Tanaka WK, et al. Plasma MicroRNAs as sensitive and specific biomarkers of tissue injury. Clin Chem.2009; 55:1977-83.
104. Sepramaniam S, Armugam A, Lim KY, Karolina DS, Swaminathan P, Tan JR., et al. MicroRNA 320a functions as a novel endogenous modulator of aquaporins 1 and 4 as well as a potential therapeutic target in cerebral ischemia JBiol Chem.2010; 285:29223-30.
105. Yin KJ, Deng Z, Hamblin M, Xiang Y, Huang H, Zhang J, et al. Peroxisome proliferator-activated receptor delta regulation of miR-15a in ischemia-induced cerebral vascular endothelial injury. J Neurosci.2010; 30: 6398-408.
106. Selvamani A, Sathyan P, Miranda RC, and Sohrabji F. An antagomir to microRNA Let7f promotes neuroprotection in an ischemic stroke model. PLoS One.2012; 7:e32662.
107. Buller B, Liu X, Wang X, Zhang RL, Zhang L, Hozeska-Solgot A, et al. MicroRNA-21 protects neurons from ischemic death. FEBS J.2010; 277: 4299-307.
108. Junn E, Lee KW, Jeong BS, Chan TW, Im JY, and Mouradian MM. Repression of alpha-synuclein expression and toxicity by microRNA-7. Proc Natl Acad Sci USA.2009; 106:13052-7.
109. Doxakis E. Post-transcriptional regulation of alpha-synuclein expression by mir-7 and mir-153. JBiol Chem.2010; 285:12726-34.
110. Kefas B, Godlewski J, Comeau L, Li Y, Abounader R, Hawkinson M, et al. microRNA-7 inhibits the epidermal growth factor receptor and the Akt pathway and is down-regulated in glioblastoma. Cancer Res.2008; 68: 3566-72.
111. Lu ZJ, Liu S Y, Yao YQ, Zhou YJ, Zhang S, Dai L, et al. The effect of miR-7 on behavior and global protein expression in glioma cell lines. Electrophoresis.2011; 32:3612-20.
112. Kong D, Piao YS, Yamashita S, Oshima H, Oguma K, Fushida S, et al. Inflammation-induced repression of tumor suppressor miR-7 in gastric tumor cells. Oncogene.2012; 31:3949-60.
113. Liu Y, Kintner DB, Begum G, Algharabli J, Cengiz P, Shull GE, et al. Endoplasmic reticulum Ca2+ signaling and mitochondrial Cyt c release in astrocytes following oxygen and glucose deprivation. J Neurochem.2010; 114:1436-46.
114. Fradejas N, Pastor MD, Burgos M, Beyaert R, Tranque P, and Calvo S. Caspase-11 mediates ischemia-induced astrocyte death:involvement of endoplasmic reticulum stress and C/EBP homologous protein. JNeurosci Res. 2010; 88:1094-105.
115. Liu G, Zhao J, Chang Z, and Guo G. CaMKII Activates ASK1 to Induce Apoptosis of Spinal Astrocytes Under Oxygen-Glucose Deprivation. Cell Mol Neurobiol 2013; 33:543-9.
116. Alberdi E, Wyssenbach A, Alberdi M, Sanchez-Gomez MV, Cavaliere F, Rodriguez JJ, et al. Ca(2+)-dependent endoplasmic reticulum stress correlates with astrogliosis in oligomeric amyloid beta-treated astrocytes and in a model of Alzheimer's disease. Aging Cell.2013; 12:292-302.
117. Begum G, Kintner D, Liu Y, Cramer SW, and Sun D. DHA inhibits ER Ca2+ release and ER stress in astrocytes following in vitro ischemia. J Neurochem. 2012; 120:622-30.
118. Tamura Y, Tanabe K, Kitagawa W, Uchida S, Schreiner GF, Johnson RJ, et al. Nicorandil, a K(atp) channel opener, alleviates chronic renal injury by targeting podocytes and macrophages. Am J Physiol Renal Physiol.2012; 303:F339-49.
1. Lee RC, Feinbaum RL, and Ambros V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell. 1993; 75:843-54.
2. Wightman B, Ha I, and Ruvkun G. Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans. Cell.1993; 75:855-62.
3. Reinhart BJ, Slack FJ, Basson M, Pasquinelli AE, Bettinger JC, Rougvie AE, et al. The 21-nucleotide let-7 RNA regulates developmental timing in Caenorhabditis elegans. Nature.2000; 403:901-6.
4. Grun D, Wang YL, Langenberger D, Gunsalus KC, and Rajewsky N. microRNA target predictions across seven Drosophila species and comparison to mammalian targets. PLoS Comput Biol.2005; 1:e13.
5. Kloosterman WP and Plasterk RH. The diverse functions of microRNAs in animal development and disease. Dev Cell.2006; 11:441-50.
6. Saugstad JA. MicroRNAs as effectors of brain function with roles in ischemja and injury, neuroprotection, and neurodegeneration. J Cereb Blood Flow Metab.2010; 30:1564-76.
7. Vemuganti R. The MicroRNAs and Stroke:No Need to be Coded to be Counted. Transl Stroke Res.2010; 1:158-60.
8. Broussalis E, Killer M, McCoy M, Harrer A, Trinka E, and Kraus J. Current therapies in ischemic stroke. Part B. Future candidates in stroke therapy and experimental studies. Drug Discov Today.2012; 17:296-309.
9. Kikuchi K, Uchikado H, Morioka M, Murai Y and Tanaka E. Clinical neuroprotective drugs for treatment and prevention of stroke. Int J Mol Sci.2012; 13:7739-61.
10. O'Carroll D and Schaefer A. General principals of miRNA biogenesis and regulation in the brain. Neuropsychopharmacology.2013; 38:39-54.
11. Lee Y, Kim M, Han J, Yeom KH, Lee S, Baek SH, et al. MicroRNA genes are transcribed by RNA polymerase II. Embo J.2004; 23:4051-60.
12. Lee Y, Ahn C, Han J, Choi H, Kim J, Yim J, et al. The nuclear RNase III Drosha initiates microRNA processing. Nature.2003; 425:415-9.
13. Yi R, Qin Y, Macara IG, and Cullen BR. Exportin-5 mediates the nuclear export of pre-microRNAs and short hairpin RNAs. Genes Dev.2003; 17: 3011-6.
14. Hutvagner G, McLachlan J, Pasquinelli AE, Balint E, Tuschl T, and Zamore PD. A cellular function for the RNA-interference enzyme Dicer in the maturation of the let-7 small temporal RNA. Science.2001; 293:834-8.
15. Khvorova A, Reynolds A, and Jayasena SD. Functional siRNAs and miRNAs exhibit strand bias. Cell.2003; 115:209-16.
16. Schwarz DS, Hutvagner G, Du T, Xu Z, Aronin N, and Zamore PD. Asymmetry in the assembly of the RNAi enzyme complex. Cell.2003; 115: 199-208.
17. Krol J, Loedige I, and Filipowicz W. The widespread regulation of microRNA biogenesis, function and decay. Nat Rev Genet.2010; 11: 597-610.
18. Brodersen P and Voinnet O. Revisiting the principles of microRNA target recognition and mode of action. Nat Rev Mol Cell Biol.2009; 10:141-8.
19. Ruby JG, Jan CH, and Bartel DP. Intronic microRNA precursors that bypass Drosha processing. Nature.2007; 448:83-6.
20. Okamura K, Hagen JW, Duan H, Tyler DM, and Lai EC. The mirtron pathway generates microRNA-class regulatory RNAs in Drosophila. Cell. 2007; 130:89-100.
21. Babiarz JE, Ruby JG, Wang Y, Bartel DP, and Blelloch R. Mouse ES cells express endogenous shRNAs, siRNAs, and other Microprocessor-independent, Dicer-dependent small RNAs. Genes Dev.2008; 22:2773-85.
22. Yi R, Pasolli HA, Landthaler M, Hafner M, Ojo T, Sheridan R, et al. DGCR8-dependent microRNA biogenesis is essential for skin development Proc Natl Acad Sci U S A.2009; 106:498-502.
23. Babiarz JE, Hsu R, Melton C, Thomas M, Ullian EM, and Blelloch R. A role for noncanonical microRNAs in the mammalian brain revealed by phenotypic differences in Dgcr8 versus Dicerl knockouts and small RNA sequencing. Rna.2011; 17:1489-501.
24. Berezikov E, Chung WJ, Willis J, Cuppen E, and Lai EC. Mammalian mirtron genes. Mol Cell.2007; 28:328-36.
25. Cheloufi S, Dos Santos CO, Chong MM, and Hannon GJ. A dicer-independent miRNA biogenesis pathway that requires Ago catalysis. Nature.2010; 465:584-9.
26. Cifuentes D, Xue H, Taylor DW, Patnode H, Mishima Y, Cheloufi S, et al. A novel miRNA processing pathway independent of Dicer requires Argonaute2 catalytic activity. Science.2010; 328:1694-8.
27. O'Carroll D and Schaefer A. General Principals of miRNA Biogenesis and Regulation in the Brain. Neuropsychopharmacology.
28. Nudelman AS, DiRocco DP, Lambert TJ, Garelick MG, Le J, Nathanson NM, et al. Neuronal activity rapidly induces transcription of the CREB-regulated microRNA-132, in vivo. Hippocampus.2010; 20:492-8.
29. Impey S, Davare M, Lesiak A, Fortin D, Ando H, Varlamova O, et al. An activity-induced microRNA controls dendritic spine formation by regulating Racl-PAK signaling. Mol Cell Neurosci.2010; 43:146-56.
30. Yin KJ, Deng Z, Hamblin M, Xiang Y, Huang H, Zhang J, et al. Peroxisome proliferator-activated receptor delta regulation of miR-15a in ischemia-induced cerebral vascular endothelial injury. J Neurosci.2010; 30: 6398-408.
31. Suzuki HI, Arase M, Matsuyama H, Choi YL, Ueno T, Mano H, et al. MCPIP1 ribonuclease antagonizes dicer and terminates microRNA biogenesis through precursor microRNA degradation. Mol Cell.2011; 44: 424-36.
32. Suzuki HI, Arase M, Matsuyama H, Choi YL, Ueno T, Mano H, et al. MCPIP1 ribonuclease antagonizes dicer and terminates microRNA biogenesis through precursor microRNA degradation. Mol Cell.44:424-36.
33. Heo I, Joo C, Kim YK, Ha M, Yoon MJ, Cho J, et al. TUT4 in concert with Lin28 suppresses microRNA biogenesis through pre-microRNA uridylation. Cell.2009; 138:696-708.
34. Cazalla D, Yario T, and Steitz JA. Down-regulation of a host microRNA by a Herpesvirus saimiri noncoding RNA. Science.2010; 328:1563-6.
35. Plante I, Ple H, Landry P, Gunaratne PH, and Provost P. Modulation of microRNA Activity by Semi-microRNAs. Front Genet.2012; 3:99.
36. Poliseno L, Salmena L, Zhang J, Carver B, Haveman WJ, and Pandolfi PP. A coding-independent function of gene and pseudogene mRNAs regulates tumour biology. Nature.2010; 465:1033-8.
37. Plante I, Ple H, Landry P, Gunaratne PH, and Provost P. Modulation of microRNA Activity by Semi-microRNAs. Front Genet.3:99.
38. Sen CK, Gordillo GM, Khanna S, and Roy S. Micromanaging vascular biology:tiny microRNAs play big band. J Vasc Res.2009; 46:527-40.
39. Castoldi G, Di Gioia CR, Bombardi C, Catalucci D, Corradi B, Gualazzi MG, et al. MiR-133a regulates collagen 1A1:potential role of miR-133a in myocardial fibrosis in angiotensin II-dependent hypertension. J Cell Physiol. 2012; 227:850-6.
40. Guo L, Qiu Z, Wei L, Yu X, Gao X, Jiang S, et al. The microRNA-328 regulates hypoxic pulmonary hypertension by targeting at insulin growth factor 1 receptor and L-type calcium channel-alpha 1C. Hypertension.2012; 59:1006-13.
41. Guo L, Qiu Z, Wei L, Yu X, Gao X, Jiang S, et al. The microRNA-328 regulates hypoxic pulmonary hypertension by targeting at insulin growth factor 1 receptor and L-type calcium channel-alpha 1C. Hypertension.59: 1006-13.
42. Schaar DG, Medina DJ, Moore DF, Strair RK, and Ting Y. miR-320 targets transferrin receptor 1 (CD71) and inhibits cell proliferation. Exp Hematol. 2009; 37:245-55.
43. Jamieson WR, Koerfer R, Yankah CA, Zittermann A, Hayden RI, Ling H, et al. Mitroflow aortic pericardial bioprosthesis--clinical performance. Eur J Cardiothorac Surg.2009; 36:818-24.
44. Zhu H, Shyh-Chang N, Segre AV, Shinoda G, Shah SP, Einhorn WS, et al. The Lin28/let-7 axis regulates glucose metabolism. Cell.2011; 147:81-94.
45. Frost RJ and Olson EN. Control of glucose homeostasis and insulin sensitivity by the Let-7 family of microRNAs. Proc Natl Acad Sci U S A. 2011; 108:21075-80.
46. Feng B, Chen S, McArthur K, Wu Y, Sen S, Ding Q, et al. miR-146a-Mediated extracellular matrix protein production in chronic diabetes complications. Diabetes.60:2975-84.
47. Wijesekara N, Zhang LH, Kang MH, Abraham T, Bhattacharjee A, Warnock GL, et al. miR-33a modulates ABCA1 expression, cholesterol accumulation, and insulin secretion in pancreatic islets. Diabetes.2012; 61:653-8.
48. Grueter CE, van Rooij E, Johnson BA, DeLeon SM, Sutherland LB, Qi X, et al. A cardiac microRNA governs systemic energy homeostasis by regulation of MED13. Cell.2012; 149:671-83.
49. Patella F and Rainaldi G. MicroRNAs mediate metabolic stresses and angiogenesis. Cell Mol Life Sci.2012; 69:1049-65.
50. Goedeke L and Fernandez-Hernando C. Regulation of cholesterol homeostasis. Cell Mol Life Sci.2012; 69:915-30.
51. Gao W, He HW, Wang ZM, Zhao H, Lian XQ, Wang YS, et al. Plasma levels of lipometabolism-related miR-122 and miR-370 are increased in patients with hyperlipidemia and associated with coronary artery disease. Lipids Health Dis.2012; 11:55.
52. Chen T, Huang Z, Wang L, Wang Y, Wu F, Meng S, et al. MicroRNA-125a-5p partly regulates the inflammatory response, lipid uptake, and ORP9 expression in oxLDL-stimulated monocyte/macrophages. Cardiovasc Res.2009; 83:131-9.
53. Gerin I, Clerbaux LA, Haumont O, Lanthier N, Das AK, Burant CF, et al. Expression of miR-33 from an SREBP2 intron inhibits cholesterol export and fatty acid oxidation. J Biol Chem.2010; 285:33652-61.
54. Rayner KJ, Suarez Y, Davalos A, Parathath S, Fitzgerald ML, Tamehiro N, et al. MiR-33 contributes to the regulation of cholesterol homeostasis. Science. 2010; 328:1570-3.
55. Weber M, Baker MB, Moore JP, and Searles CD. MiR-21 is induced in endothelial cells by shear stress and modulates apoptosis and eNOS activity. Biochem Biophys Res Commun.2010; 393:643-8.
56. Weber M, Baker MB, Moore JP, and Searles CD. MiR-21 is induced in endothelial cells by shear stress and modulates apoptosis and eNOS activity. Biochem Biophys Res Commun.393:643-8.
57. Ji R, Cheng Y, Yue J, Yang J, Liu X, Chen H, et al. MicroRNA expression signature and antisense-mediated depletion reveal an essential role of MicroRNA in vascular neointimal lesion formation. Circ Res.2007; 100: 1579-88.
58. Harris TA, Yamakuchi M, Ferlito M, Mendell JT, and Lowenstein CJ. MicroRNA-126 regulates endothelial expression of vascular cell adhesion molecule 1. Proc Natl Acad Sci USA.2008; 105:1516-21.
59. Zampetaki A, Kiechl S, Drozdov I, Willeit P, Mayr U, Prokopi M, et al. Plasma microRNA profiling reveals loss of endothelial miR-126 and other microRNAs in type 2 diabetes. Circ Res.2010; 107:810-7.
60. Poliseno L, Tuccoli A, Mariani L, Evangelista M, Citti L, Woods K, et al. MicroRNAs modulate the angiogenic properties of HUVECs. Blood.2006; 108:3068-71.
61. Dentelli P, Rosso A, Orso F, Olgasi C, Taverna D, and Brizzi MR microRNA-222 controls neovascularization by regulating signal transducer and activator of transcription 5A expression. Arterioscler Thromb Vasc Biol. 2010; 30:1562-8.
62. Fasanaro P, Greco S, Lorenzi M, Pescatori M, Brioschi M, Kulshreshtha R, et al. An integrated approach for experimental target identification of hypoxia-induced miR-210. JBiol Chem.2009; 284:35134-43.
63. Schembri F, Sridhar S, Perdomo C, Gustafson AM, Zhang X, Ergun A, et al. MicroRNAs as modulators of smoking-induced gene expression changes in human airway epithelium. Proc Natl Acad Sci USA.2009; 106:2319-24.
64. Izzotti A, Calin GA, Arrigo P, Steele VE, Croce CM, and De Flora S. Downregulation of microRNA expression in the lungs of rats exposed to cigarette smoke. Faseb J.2009; 23:806-12.
65. Izzotti A, Larghero P, Longobardi M, Cartiglia C, Camoirano A, Steele VE, et al. Dose-responsiveness and persistence of microRNA expression alterations induced by cigarette smoke in mouse lung. Mutat Res.2011; 717:9-16.
66. Shan H, Zhang Y, Lu Y, Zhang Y, Pan Z, Cai B, et al. Downregulation of miR-133 and miR-590 contributes to nicotine-induced atrial remodelling in canines. Cardiovasc Res.2009; 83:465-72.
67. Chen J, Yang T, Yu H, Sun K, Shi Y, Song W, et al. A functional variant in the 3'-UTR of angiopoietin-1 might reduce stroke risk by interfering with the binding efficiency of microRNA 211. Hum Mol Genet.2010; 19:2524-33.
68. Siegel C, Li J, Liu F, Benashski SE, and McCullough LD. miR-23a regulation of X-linked inhibitor of apoptosis (XIAP) contributes to sex differences in the response to cerebral ischemia. Proc Natl Acad Sci U S A. 2011; 108:11662-7.
69. Jeyaseelan K, Lim KY, and Armugam A. MicroRNA expression in the blood and brain of rats subjected to transient focal ischemia by middle cerebral artery occlusion. Stroke.2008; 39:959-66.
70. Dharap A, Bowen K, Place R, Li LC, and Vemuganti R. Transient focal ischemia induces extensive temporal changes in rat cerebral microRNAome. J Cereb Blood Flow Metab.2009; 29:675-87.
71. Gan CS, Wang CW, and Tan KS. Circulatory microRNA-145 expression is increased in cerebral ischemia Genet Mol Res.2012; 11:147-52.
72. Lukiw WJ and Pogue AI. Induction of specific micro RNA (miRNA) species by ROS-generating metal sulfates in primary human brain cells. J Inorg Biochem.2007; 101:1265-9.
73. Selvamani A, Sathyan P, Miranda RC, and Sohrabji F. An antagomir to microRNA Let7f promotes neuroprotection in an ischemic stroke model. PLoS One.2012; 7:e32662.
74. Sepramaniam S, Armugam A, Lim KY, Karolina DS, Swaminathan P, Tan JR., et al. MicroRNA 320a functions as a novel endogenous modulator of aquaporins 1 and 4 as well as a potential therapeutic target in cerebral ischemia. JBiol Chem.2010; 285:29223-30.
75. Witwer KW, Sisk JM, Gama L, and Clements JE. MicroRNA regulation of IFN-beta protein expression:rapid and sensitive modulation of the innate immune response. J Immunol.2010; 184:2369-76.
76. Bonauer A, Carmona G, Iwasaki M, Mione M, Koyanagi M, Fischer A, et al. MicroRNA-92a controls angiogenesis and functional recovery of ischemic tissues in mice. Science.2009; 324:1710-3.
77. Buller B, Liu X, Wang X, Zhang RL, Zhang L, Hozeska-Solgot A, et al. MicroRNA-21 protects neurons from ischemic death. FEBS J.2010; 277: 4299-307.
78. Yin KJ, Deng Z, Huang H, Hamblin M, Xie C, Zhang J, et al. miR-497 regulates neuronal death in mouse brain after transient focal cerebral ischemia. Neurobiol Dis.2010; 38:17-26.
79. Ouyang YB, Lu Y, Yue S, Xu LJ, Xiong XX, White RE, et al. miR-181 regulates GRP78 and influences outcome from cerebral ischemia in vitro and in vivo. Neurobiol Dis.2012; 45:555-63.
80. Ouyang YB, Lu Y, Yue S, and Giffard RG miR-181 targets multiple Bcl-2 family members and influences apoptosis and mitochondrial function in astrocytes. Mitochondrion.2012; 12:213-9.
81. Shi G, Liu Y, Liu T, Yan W, Liu X, Wang Y, et al. Upregulated miR-29b promotes neuronal cell death by inhibiting Bcl2L2 after ischemic brain injury. Exp Brain Res.2012; 216:225-30.
82. Sepramaniam S, Armugam A, Lim KY, Karolina DS, Swaminathan P, Tan JR, et al. MicroRNA 320a functions as a novel endogenous modulator of aquaporins 1 and 4 as well as a potential therapeutic target in cerebral ischemia. JBiol Chem.285:29223-30.
83. Ouyang YB, Lu Y, Yue S, Xu LJ, Xiong XX, White RE, et al. miR-181 regulates GRP78 and influences outcome from cerebral ischemia in vitro and in vivo. Neurobiol Dis.45:555-63.
84. Baroukh N, Ravier MA, Loder MK, Hill EV, Bounacer A, Scharfmann R, et al. MicroRNA-124a regulates Foxa2 expression and intracellular signaling in pancreatic beta-cell lines. JBiol Chem.2007; 282:19575-88.
85. Langlois RA, Shapiro JS, Pham AM, and tenOever BR. In vivo delivery of cytoplasmic RNA virus-derived miRNAs. Mol Ther. 2012; 20:367-75.
86. Shahzad MM, Mangala LS, Han HD, Lu C, Bottsford-Miller J, Nishimura M, et al. Targeted delivery of small interfering RNA using reconstituted high-density lipoprotein nanoparticles. Neoplasia.2011; 13:309-19.
87. Pan X, Thompson R, Meng X, Wu D, and Xu L. Tumor-targeted RNA-interference:functional non-viral nanovectors. Am J Cancer Res.2011; 1:25-42.
88. Czech MP, Aouadi M, and Tesz GJ. RNAi-based therapeutic strategies for metabolic disease. Nat Rev Endocrinol.2011; 7:473-84.
89. Toloue MM and Ford LP. Antibody targeted siRNA delivery. Methods Mol Biol.2011; 764:123-39.
90. Tan A, De La Pena H, and Seifalian AM. The application of exosomes as a nanoscale cancer vaccine. Int J Nanomedicine.2010; 5:889-900.