中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
CHINESE JOURNAL OF EXPERIMENTAL SURGERY
2014年
9期
1913-1915
,共3页
徐希德%龚佩佩%施炜%陈建%倪兰春%樊健
徐希德%龔珮珮%施煒%陳建%倪蘭春%樊健
서희덕%공패패%시위%진건%예란춘%번건
有丝分裂原和应激活化蛋白激酶1%脑损伤%星形胶质细胞%炎症
有絲分裂原和應激活化蛋白激酶1%腦損傷%星形膠質細胞%炎癥
유사분렬원화응격활화단백격매1%뇌손상%성형효질세포%염증
Mitogen-and stress-activated protein kinase-1%Phosphorylation%Astrocytes%Inflammation
目的 探讨有丝分裂原和应激活化蛋白激酶1(MSK1)在中枢神经系统炎性反应过程中的表达及其意义.方法 将90只SD大鼠分实验组(35只)、假手术组(35只)及对照组(20只).实验组以脂多糖(LPS)侧脑室注射建立中枢神经系统炎症模型,假手术组以生理盐水注入侧脑室建立模型,模型建立后观察时间点设置为0、3、6、12h、1、3、5d,采集标本.分别采用Western blot、免疫荧光双染等方法观察脑损伤及炎性反应过程中,MSK1及磷酸化MSK1(p-MSK1)蛋白表达的时空变化和细胞定位及其与炎症相关凶子诱导性一氧化氮合酶(iNOS)、肿瘤坏死因子-α(TNF-α)及星形胶质细胞标志物胶质纤维酸性蛋白(GFAP)的相关性.结果 在炎性受损的大脑皮层中,p-MSK1主要表达在星形胶质细胞及神经元中;0、3、6、12h、1、3、5d时段采集标本进行Western blot检测,p-MSK1(Set360位点)相对表达量分别为0.746 ±0.075、0.988±0.101、1.126±0.097、1.096±-0.103、1.168±0.095、0.757±0.082、0.726±0.090,p-MSK1(Thr581位点)相对表达量分别值为0.298±0.034、0.441 ±0.052、0.679±0.075、0.831±0.107、0.888±0.103、0.426±0.051、0.419±0.057;6～ 24 h与对照组比较差异有统计学意义(P＜0.05),MSK1的磷酸化主要位于苏氨酸581号位的磷酸化位点.1d时行免疫荧光染色量化分析,MSK1及p-MSK1在大脑皮层中表达与正常组比较差异有统计学意义(P＜0.05).此外,在LPS刺激1d后的大脑皮层中,iNOS、TNF-α及GFAP等表达均显著增加,1、3、6、9、12h、1、3d时段采集标本进行Western blot检测,iNOS相对表达量分别为0.463±0.056、0.925±0.095、0.889±0.101、1.224±0.121、1.375 ±0.119、1.095 ±0.106、0.862±0.105;TNF-α相对表达量分别为0.501±0.053、0.425±0.041、0.670±0.082、0.995±0.110、1.007±0.108、0.845±0.093、0.817 ±0.095;GFAP相对表达量分别为0.385±0.037、0.403 ±0.061、0.695±0.084、0.791±0.075、1.092±0.107、1.195±0.108、1.092±0.107;与p-MSK1表达有相关.免疫荧光双标法结果表明,LPS侧脑室注射1d后,皮层中p-MSK1表达阳性的星形胶质细胞与TNF-α有共定位.结论 MSK1磷酸化与中枢神经系统炎性反应密切相关,可通过调控其表达,抑制星形胶质细胞炎性因子的产生,发挥中枢神经系统炎性反应的调节作用,减轻中枢神经系统进一步损伤.
目的 探討有絲分裂原和應激活化蛋白激酶1(MSK1)在中樞神經繫統炎性反應過程中的錶達及其意義.方法 將90隻SD大鼠分實驗組(35隻)、假手術組(35隻)及對照組(20隻).實驗組以脂多糖(LPS)側腦室註射建立中樞神經繫統炎癥模型,假手術組以生理鹽水註入側腦室建立模型,模型建立後觀察時間點設置為0、3、6、12h、1、3、5d,採集標本.分彆採用Western blot、免疫熒光雙染等方法觀察腦損傷及炎性反應過程中,MSK1及燐痠化MSK1(p-MSK1)蛋白錶達的時空變化和細胞定位及其與炎癥相關兇子誘導性一氧化氮閤酶(iNOS)、腫瘤壞死因子-α(TNF-α)及星形膠質細胞標誌物膠質纖維痠性蛋白(GFAP)的相關性.結果 在炎性受損的大腦皮層中,p-MSK1主要錶達在星形膠質細胞及神經元中;0、3、6、12h、1、3、5d時段採集標本進行Western blot檢測,p-MSK1(Set360位點)相對錶達量分彆為0.746 ±0.075、0.988±0.101、1.126±0.097、1.096±-0.103、1.168±0.095、0.757±0.082、0.726±0.090,p-MSK1(Thr581位點)相對錶達量分彆值為0.298±0.034、0.441 ±0.052、0.679±0.075、0.831±0.107、0.888±0.103、0.426±0.051、0.419±0.057;6～ 24 h與對照組比較差異有統計學意義(P＜0.05),MSK1的燐痠化主要位于囌氨痠581號位的燐痠化位點.1d時行免疫熒光染色量化分析,MSK1及p-MSK1在大腦皮層中錶達與正常組比較差異有統計學意義(P＜0.05).此外,在LPS刺激1d後的大腦皮層中,iNOS、TNF-α及GFAP等錶達均顯著增加,1、3、6、9、12h、1、3d時段採集標本進行Western blot檢測,iNOS相對錶達量分彆為0.463±0.056、0.925±0.095、0.889±0.101、1.224±0.121、1.375 ±0.119、1.095 ±0.106、0.862±0.105;TNF-α相對錶達量分彆為0.501±0.053、0.425±0.041、0.670±0.082、0.995±0.110、1.007±0.108、0.845±0.093、0.817 ±0.095;GFAP相對錶達量分彆為0.385±0.037、0.403 ±0.061、0.695±0.084、0.791±0.075、1.092±0.107、1.195±0.108、1.092±0.107;與p-MSK1錶達有相關.免疫熒光雙標法結果錶明,LPS側腦室註射1d後,皮層中p-MSK1錶達暘性的星形膠質細胞與TNF-α有共定位.結論 MSK1燐痠化與中樞神經繫統炎性反應密切相關,可通過調控其錶達,抑製星形膠質細胞炎性因子的產生,髮揮中樞神經繫統炎性反應的調節作用,減輕中樞神經繫統進一步損傷.
목적 탐토유사분렬원화응격활화단백격매1(MSK1)재중추신경계통염성반응과정중적표체급기의의.방법 장90지SD대서분실험조(35지)、가수술조(35지)급대조조(20지).실험조이지다당(LPS)측뇌실주사건립중추신경계통염증모형,가수술조이생리염수주입측뇌실건립모형,모형건립후관찰시간점설치위0、3、6、12h、1、3、5d,채집표본.분별채용Western blot、면역형광쌍염등방법관찰뇌손상급염성반응과정중,MSK1급린산화MSK1(p-MSK1)단백표체적시공변화화세포정위급기여염증상관흉자유도성일양화담합매(iNOS)、종류배사인자-α(TNF-α)급성형효질세포표지물효질섬유산성단백(GFAP)적상관성.결과 재염성수손적대뇌피층중,p-MSK1주요표체재성형효질세포급신경원중;0、3、6、12h、1、3、5d시단채집표본진행Western blot검측,p-MSK1(Set360위점)상대표체량분별위0.746 ±0.075、0.988±0.101、1.126±0.097、1.096±-0.103、1.168±0.095、0.757±0.082、0.726±0.090,p-MSK1(Thr581위점)상대표체량분별치위0.298±0.034、0.441 ±0.052、0.679±0.075、0.831±0.107、0.888±0.103、0.426±0.051、0.419±0.057;6～ 24 h여대조조비교차이유통계학의의(P＜0.05),MSK1적린산화주요위우소안산581호위적린산화위점.1d시행면역형광염색양화분석,MSK1급p-MSK1재대뇌피층중표체여정상조비교차이유통계학의의(P＜0.05).차외,재LPS자격1d후적대뇌피층중,iNOS、TNF-α급GFAP등표체균현저증가,1、3、6、9、12h、1、3d시단채집표본진행Western blot검측,iNOS상대표체량분별위0.463±0.056、0.925±0.095、0.889±0.101、1.224±0.121、1.375 ±0.119、1.095 ±0.106、0.862±0.105;TNF-α상대표체량분별위0.501±0.053、0.425±0.041、0.670±0.082、0.995±0.110、1.007±0.108、0.845±0.093、0.817 ±0.095;GFAP상대표체량분별위0.385±0.037、0.403 ±0.061、0.695±0.084、0.791±0.075、1.092±0.107、1.195±0.108、1.092±0.107;여p-MSK1표체유상관.면역형광쌍표법결과표명,LPS측뇌실주사1d후,피층중p-MSK1표체양성적성형효질세포여TNF-α유공정위.결론 MSK1린산화여중추신경계통염성반응밀절상관,가통과조공기표체,억제성형효질세포염성인자적산생,발휘중추신경계통염성반응적조절작용,감경중추신경계통진일보손상.
Objective It is generally accepted that inflammation has a role in the progression of many central nervous system (CNS) diseases,although the mechanisms remain unclear.Among mitogenactivated protein kinase (MAPK) targets,mitogen-and stress-activated protein kinase (MSK1) has been thought to be involved in the pathology of inflammatory gene expression.In this study,the roles of MSK1 activation in neuroinflammation were investigated.Methods Ninety SD rats were divided into three groups:brain inflammation group (n =35),control group (n =20) and treatment control group (n =35).The model of brain inflammation was prepared in rats by the intracerebroventricular injection of lipopolysaccharide (LPS).The expression and the cellular location of p-MSK1 protein were assayed by Western blotting and immunofluorescent staining.Results The model of brain inflammation was prepared in rats by the intracerebroventricular injection of Specific upregulation of p-MSK1 in astrocytes was observed and increased in inflamed cerebral cortex.By western blot detection,corresponding to time of 1,3,6,9,12 h,1,3 d,value of p-MSK1 (Ser360) is 0.746 ±0.075,0.988 ±0.101,1.126 ±0.097,1.096 ±0.103,1.168 ±0.095,0.757 ± 0.082,0.726 ± 0.090,and value of p-MSK1 (Thr581) is 0.298 ± 0.034,0.441 ±0.052,0.679 ±0.075,0.831 ±0.107,0.888 ±0.103,0.426 ±0.051,0.419 ±0.057; From 6 h to 24 h,expression was significant (P ＜ 0.05),then progressively reduced.Phosphorylated MSK1 (p-MSK1 Thr581) was induced significantly after intracerebral injection of LPS into the lateral ventricles of the rat brain.By western blot detection,corresponding to time of 1,3,6,9,12 h,1,3 d,value of inducible nitric oxide synthase (iNOS) is 0.463 ±0.056,0.925 ±0.095,0.889 ±0.101,1.224 ±0.121,1.375 ±0.119,1.095 ±0.106,0.862 ±0.105; value of tumor necrosis factor (TNF)-α is 0.501 ±0.053,0.425 ±0.041,0.670 ±0.082,0.995 ±0.110,1.007 ±0.108,0.845 ±0.093,0.817 ±0.095 ; value of GFAP is 0.385 ± 0.037,0.403 ± 0.061,0.695 ± 0.084,0.791 ± 0.075,1.092 ±0.107,1.195 ±0.108,1.092 ±0.107; At 1 day after LPS stimulation,iNOS,TNF-α expression,and the astrocyte marker glial fibrillary acidic protein (GFAP) were increased significantly.Conclusion Collectively,these results suggest that MSK1 phosphorylation is associated with the regulation of LPS-induced brain injury.
