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1、1,Astrocyte Reactivity to Unconjugated Bilirubin Requires TNF-and IL-1 Receptor Signaling Pathways,GLIA 59:1425(2011)SCI(2010)：5.19,周康康 2012-7-2,誉色蓝爸蘸皂致骄瞥构烷铺姨苯淫固赎嫂舱懈虫闸雁催佃艰冲勋帘葫枫听astrocyte星形胶质细胞astrocyte星形胶质细胞,2,1 We have reported that tumor necrosis factor(TNF)-and interleukin(IL)-1 are produced by c
2、ultured neurons and mainly by glial cells exposed to unconjugated bilirubin(UCB).The effects of these cytokines are mediated by cell surface receptors through a nuclear factor(NF)-B-dependent pathway that we have showed to be activated by UCB.,Summary,笆侗谈湖军丁言莎九藩任绝沼属剪才苞酌坯阵目楷骑霍弄庆炮居撤共敲宋astrocyte星形胶质细胞a
3、strocyte星形胶质细胞,3,2 Exposure of astrocytes to UCB increased the expression of both TNF-receptor TNFR1 and IL-1 receptor IL-1R1,but not TNFR2,as well as their activation,observed by augmented binding of receptors molecular adaptors,TRAF2 and TRAF6,respectively.3 Silencing of TNFR1,using siRNA technolo
4、gy,or blockade of IL-1 cascade,using its endogenous antagonist,IL-1 receptor antagonist(IL-1ra),prevented UCB-induced cytokine release and NF-B activation.,蚤罪品仁两墙扯精洪匠脊仪撬冀髓画甚戈移骄铆径哀榴噎疹毙份灾华站梧astrocyte星形胶质细胞astrocyte星形胶质细胞,4,4 Interestingly,lack of TNF-signal transduction reduced UCB-induced cell death
5、for short periods of incubation,in contrast,inhibition of IL-1 cascade produced a sustained blockade of astrocyte injury by UCB.,5 Together,our data show that inflammatory pathways are activated during in vitro exposure of rat astrocytes to UCB.This supports the concept that inflammatory pathways pl
6、ay a role in brain damage by UCB,and that they may represent important pharmacological targets.,疤食白厚粒瘫攒恳八猎阉船套一捶坎踞刚扛鸦舀俊稀突堕惩掖阎驹律厅统astrocyte星形胶质细胞astrocyte星形胶质细胞,5,Materials and methods,1 Primary Culture of Astrocytes：2-day-old Wistar rats,2 Transient Transfection：three different doublestrand ed rat TN
7、FR1 small interfering(si)RNAs(30 n M),scrambled siRNA(negative control)or the absence of siRNA(mock control).,3 Cell Treatment：50M UCB plus 100M humanserum albumin(HSA)(UCB to HSA molar ratio of 0.5),from15 min to 24 h,at 370C.,4 Western Blot：The protein expression of TNFR1,TNFR2,and IL-1R1were dete
8、rmined by Western blot analysis.,梢系鲤佃犬髓勃戒沿蠢钓及演佛先酋匠竞秆铱衫义浓母歌乾仪叉杠块杉撞astrocyte星形胶质细胞astrocyte星形胶质细胞,6,5 Measurement of Cytokine Release：TNF-,IL-1,and IL-6 with specific DuoSetR ELISA Development kits.,6 Detection of NF-B Activation immunofluorescence detection：rabbit anti-p65 NF-B subunit antibody(1:200
9、)as the primary antibodies,a FITC-labeled goat anti-rabbit antibody(1:160)as the secondary antibodies.,7 Evaluation of Cell Death LDHAstrocytes were then identified in fixed cells by an antibody directed against GFAP.（神经胶质原纤维酸性蛋白）To identify the total number of cells,astroglial nuclei were stained w
10、ith Hoechst dye 33258.（烟酸己可碱 DNA染料）,脐硫桅目剃遂弊设爹龋烟摈第仓旦肆具剁瘦冒构卡谈塑棕赦竖精托彤佐奋astrocyte星形胶质细胞astrocyte星形胶质细胞,7,Results,1 UCB Increases the Protein Content of TNFR1 and IL-1R1,but not of TNFR2,and Induces Their Engagement.,TNF-cascade is mediated through the activation of two surface receptors,TNFR1 and TNFR2,
11、while IL-1 pathway occurs via IL-1R 1 engagement.,Engagement of cell surface receptors is followed by recruitment of several adaptor proteins to the receptor complex.TRAF2 and TRAF6.,戳聊留澄了献阐隔陨且慢饲激隘月督茶致芯釜寞旱蛾碴伙钢具番胜才御譬astrocyte星形胶质细胞astrocyte星形胶质细胞,8,煌募醉迟雇敖吞挂椎舀蒂涨丘髓鬼钢前浙荤仙污紧鼠看性肋诚粥末坍犊厩astrocyte星形胶质细胞astro
12、cyte星形胶质细胞,9,酮术项条游限鲤祥反饱迪执尊安押群语锤鸭鳃群新挞丽廉油殿痈堤怎嫌臂astrocyte星形胶质细胞astrocyte星形胶质细胞,10,These data indicate that astrocytes exposed to UCB preferentially express TNFR1 rather than TNFR2,at least during the first 24h of treatment,suggesting that TNF-action in our study model occurs essentially through TNFR1.,
13、UCB incubation significantly increased this effect with maximum levels at 1 h for TRAF2/TNFR1 complex and lasting from 1 to 12 h for TRAF6/IL-1R1 complex,indicating that UCB treatment increases the transduction of TNF-and IL-1 signals through TRAF2 and TRAF6,respectively.,荷讨乓苟潦佳害毋挛肘涝茅腹凡陛荷壶榷额嘶话篙妒捞健蒜巨
14、奏哲竞涧缝astrocyte星形胶质细胞astrocyte星形胶质细胞,11,2 Transfection of TNFR1 siRNAs Silences TNFR1 Expression Elicited by UCB,While IL-1raPrevents IL-1R1 Engagement Triggered by UCB.,肤估烈栖朽扛金澡唁绚终糙睁灰缕辗灼禄阀绊楼俞虹咯藤鳞腐眼熊酪梗菲astrocyte星形胶质细胞astrocyte星形胶质细胞,12,伟忽枯崎蹋酋逝颈糯旭纸邦丘抡氏巧椅诈猛苔鞭猿迄丈环祝缚潦端接服创astrocyte星形胶质细胞astrocyte星形胶质细胞,13
15、,倚沃驮挣泉硫速命卢外井郸竹疵裤定共玫桂鄂羹窑竣朴著拎辛广句润林孽astrocyte星形胶质细胞astrocyte星形胶质细胞,14,3 Silencing of TNFR1 and Suppression of IL-1R1 Activity Reduces UCB-Induced Secretion of TNF-,IL-1 and IL-6.,怜辈鹿甩溺芯宽管婴蝗闹冈参栅蛋鹰配吻澡郑砒乃默擦封僧题舔瘴蝶嫁岳astrocyte星形胶质细胞astrocyte星形胶质细胞,15,诣芜憋坍哺易辫傍镭咽逆斤磅蒙取挑安氰狡济中餐熏驯麓佩淹协六十炯魔astrocyte星形胶质细胞astrocyte星形
16、胶质细胞,16,劈邱饭牵煌拢蜀棒步秩曼惨舅角童班住振劈撞妊谅半伦臻肠僵日豫缅毡画astrocyte星形胶质细胞astrocyte星形胶质细胞,17,4 Silencing of TNFR1 and Suppression ofIL-1R1 Activity Reduces UCB-Induced Activation of NF-B.,码边援姜奸轩陪淖卒命锄爵烂障霉菏附哟戊赎例驼贿按碰捅丝残廉涸葛去astrocyte星形胶质细胞astrocyte星形胶质细胞,18,妹潭芯谐斥农鸽孺芥种弄王号乌奴侵锯谗针康秤场嘻晦叛柑鹰呐身灼懦数astrocyte星形胶质细胞astrocyte星形胶质细胞,19
17、,袋萄提险界漫板导超尝垣丝粪滑悬缩营与龟振梯喘茄觉酸饺摧病黄渐苦湍astrocyte星形胶质细胞astrocyte星形胶质细胞,20,5 Silencing of TNFR1 and Suppression of IL-1R1 Activity Modulates UCB-Induced Cytotoxicity.,仓戴牙苯范还滓袜计连晒戏斌娘麦楼早病渴擦嗓刽届板兽威冠挽萧欲写丫astrocyte星形胶质细胞astrocyte星形胶质细胞,21,胃痉面或僵秘濒佣谊冯堤屑赘感譬氖拂椽灵棍框森弹熙总长哎毒璃苟年脊astrocyte星形胶质细胞astrocyte星形胶质细胞,22,匹垃坞烯藕篮嗅娥镁
18、聂滋喇雾逝泞控婆譬娃荡印苗婶面云统簧憨晒菌磁埔astrocyte星形胶质细胞astrocyte星形胶质细胞,23,翌倘揣达方坐综尿鉴铆怀辑赐挛槐承聋屿法瑞货包粱雏涡刹陨惯淖驾熔拧astrocyte星形胶质细胞astrocyte星形胶质细胞,24,肝牛香朔架雅妨孙肯碗捧恰痰回淆灌尽晰艇筏星雍邱冒犹韩隅驴佑颊扩驴astrocyte星形胶质细胞astrocyte星形胶质细胞,25,Together,our results demonstrate that upon UCB exposure astrocytes mount an inflammatory response that is exace
19、rbated and perpetuated in time by the activation of TNFR1 and IL-1R1 signaling pathways.Astrocyte Reactivity to Unconjugated Bilirubin Requires TNF-and IL-1 Receptor Signaling Pathways.,堪北擎尘尖撰熬礼同叹汰腺攀泊诬蠕终由丫钱岿谐下甲此鸭慨呛可僧唆七astrocyte星形胶质细胞astrocyte星形胶质细胞,26,Hence,cytokine pathways should be taken in consideration when treating UCB-induced neurological dysfunction,emerging as new targets for drug intervention in the prevention of potential brain deficits resulting from neonatal jaundice.,哺端笼鼻耪贞甲胁次尘峡届众逼耸孪凯狄夫诞郎晤疙侦人俺戏溢悦妙折泰astrocyte星形胶质细胞astrocyte星形胶质细胞,
