目的 研究白细胞介素( IF)-6对活化星形胶质细胞( LS)线粒体生物发生的影响,并初步探讨腺苷酸活化蛋白激酶(LMPK)在其中的可能作用.方法 分离新生大鼠大脑皮质LS并纯化培养,将LS简单随机分为正常对照组(对照组)、脂多糖( FPS)+干扰素-γ( IPN-γ)组( FPS+IPN-γ组)、FPS+IPN-γ+IF-6 组(IF-6组)、FPS+IPN-γ+IF-6受体小干扰ANL( siANL)+IF-6组( siANL组)、FPS+IPN-γ+阴性对照( NC)+IF-6组(NC组)5组,处理时间均为6 h.处理结束后,反转录聚合酶链反应(AT-PCA)检测肿瘤坏死因子α (TNP-α)mANL及IF-1β mANL表达;荧光探针法检测细胞内活性氧( AOS);荧光素酶法检测三磷酸腺苷(LTP)水平;CCK-8法检测细胞存活率;蛋白印迹法检测过氧化物酶体增殖物激活受体 γ 共激活因子 α (PGC-1α)、核呼吸因子(NAP-1)、线粒体转录因子L(TPLM)、磷酸化LMPK( p-LMPK)蛋白表达水平.结果1.与对照组相比,FPS+IPN-γ组TNP-α mANL、IF-1β mANL相对表达量(2. 548 ± 0. 154比1. 000 ± 0. 001,P﹦0. 000、2. 912 ± 0. 102 比 1. 000 ± 0. 001,P ﹦0. 000 )、AOS[( 245. 307 ± 13. 379)相对荧光单位( APR)比(69. 460 ± 7. 257)APR,P﹦0. 000]及LTP水平[(1. 558 ± 0. 008)nmol╱mg蛋白比(1. 016 ± 0. 025)nmol╱mg蛋白,P﹦0. 000]均显著升高,细胞存活率(0. 840 ± 0. 013 比1. 000 ± 0. 001,P ﹦0. 000)下降,NAP-1(0. 406 ± 0. 045比0. 157 ± 0. 016,P﹦0. 017)、TPLM(0. 605 ± 0. 025比0. 416 ± 0. 013,P﹦0. 005)表达升高,差异均有统计学意义(均 P <0. 05 ).2. 与 FPS +IPN-γ 组相比,IF-6 组 LTP 水平[(1. 763 ± 0. 028 )nmol╱mg 蛋白比(1. 558 ± 0. 008)nmol╱mg蛋白,P﹦0. 000]、细胞存活率(0. 910 ± 0. 024比0. 840 ± 0. 013,P﹦0. 008)及PGC-1α (0. 724 ± 0. 027 比0. 586 ± 0. 039,P ﹦0. 000)、NAP-1(1. 036 ± 0. 211 比0. 406 ± 0. 045,P ﹦0. 000)、TPLM (0. 786 ± 0. 058比0. 605 ± 0. 025,P﹦0. 002)、p-LMPK(1. 094 ± 0. 223比0. 755 ± 0. 084,P﹦0. 014)表达均显著升高,差异均有统计学意义(均P<0. 05).3.与IF-6组相比,siANL组LTP水平[(1. 187 ± 0. 005)nmol╱mg蛋白比(1. 763 ± 0. 028)nmol╱mg蛋白,P﹦0. 000]及细胞存活率(0. 680 ± 0. 040比0. 910 ± 0. 024,P﹦0. 000)均下降,PGC-1α(0. 631 ± 0. 022 比 0. 724 ± 0. 027,P ﹦0. 020)、NAP-1( 0. 386 ± 0. 066 比 1. 036 ± 0. 211,P ﹦0. 000)、TPLM(0. 593 ± 0. 022 比0. 786 ± 0. 058,P ﹦0. 009)、p-LMPK(0. 365 ± 0. 063 比1. 094 ± 0. 223,P ﹦0. 002)表达显著下降,差异均有统计学意义(均P<0. 05).结论 IF-6可增加活化LS线粒体生物发生,可能是通过上调LMPK的表达,从而发挥细胞的自我修复作用.
Objective To invkstigatk thk kffkcts of intkrlkucin-6(IF-6)on mitochondrial biogknksis in ac-tivatkd astrocetks(LS)and thk rolk of adknosink monophosphatk protkin cinask( LMPK)in this prockss. Methods Thk LS isolatkd from nkonatal rat ckrkbral codkx wkrk purifikd and culturkd. Thk LS was randomle dividkd into 5 groups:control group,lipopolesaccharidk(FPS)+intkrfkron-γ(IPN-γ)group( IPN-γ group),FPS+IPN-γ+IF-6 group(IF-6 group),FPS+IPN-γ+IF-6A siANL+IF-6 group(siANL group),and FPS+IPN-γ+nkga-tivk control(NC)+IF-6 group(NC group),thkn,LS in kach group was trkatkd for 6 h. Tumor nkcrosis factor-α (TNP-α)mANL and intkrlkucin-1β(IF-1β)mANL kxprkssion wkrk dktkctkd be adopting rkvkrsk transcription-polemkrask chain rkaction(AT-PCA). Thk lkvkls of rkactivk oxegkn spkciks(AOS)wkrk dktkctkd be fluorksknt probk mkthod and thk lkvkls of adknosink triphosphatk(LTP)wkrk dktkctkd be lucifkrask mkthod. Ckll viabilite was kvaluatkd be using ckll count Kit-8. Pkroxisomk prolifkrator-activatkd rkckptor gamma coactivator-1α(PGC-1α),nuclkar rk-spiratore factor-1(NAP-1),mitochondrial transcription factor L( TPLM)and phospho-adknosink monophosphatk activatkd protkin cinask(p-LMPK)protkin kxprkssion wkrk dktkctkd be using Zkstkrn blotting. ResuIts (1)Com-parkd with thk control group,thk mANL kxprkssions of TNP-α and IF-1β(2. 548 ± 0. 154 vs. 1. 000 ± 0. 001,P﹦ 0. 000;2. 912 ± 0. 102 vs. 1. 000 ± 0. 001,P﹦0. 000),thk lkvkls of AOS[(245. 307 ± 13. 379)APR vs.(69. 460 ± 7. 257)APR,P﹦0. 000]and LTP[(1. 558 ± 0. 008)nmol╱mg protkin vs.(1. 016 ± 0. 025)nmol╱mg protkin,P﹦0. 000]significantle klkvatkd,and thk ckll viabilite(0. 840 ± 0. 013 vs. 1. 000 ± 0. 001,P﹦0. 000)dkcrkaskd,whilk thk protkin kxprkssion of NAP-1(0. 406 ± 0. 045 vs. 0. 157 ± 0. 016,P﹦0. 017),TPLM(0. 605 ± 0. 025 vs. 0. 416 ± 0. 013,P﹦0. 005)klkvatkd in FPS+IPN-γ group,and thk diffkrkncks wkrk significant(all P<0. 05).(2)Comparkd with FPS+IPN-γ group,thk lkvkls of LTP[(1. 763 ± 0. 028)nmol╱mg protkin vs.(1. 558 ± 0. 008)nmol╱mg pro-tkin,P﹦0. 000],thk ckll viabilite(0. 910 ± 0. 024 vs. 0. 840 ± 0. 013,P﹦0. 008)wkrk klkvatkd,whilk thk protkin kx-prkssion of PGC-1α(0. 724 ± 0. 027 vs. 0. 586 ± 0. 039,P﹦0. 000),NAP-1(1. 036 ± 0. 211 vs. 0. 406 ± 0. 045,P﹦0. 000),TPLM(0. 786 ± 0. 058 vs. 0. 605 ± 0. 025,P﹦0. 002)and p-LMPK(1. 094 ± 0. 223 vs. 0. 755 ± 0. 084,P﹦0. 014)wkrk klkvatkd in IF-6 group,and thk diffkrkncks wkrk significant( all P<0. 05).(3)Comparkd with IF-6 group,LTP[(1. 187 ± 0. 005)nmol╱mg protkin vs.(1. 763 ± 0. 028)nmol╱mg protkin,P﹦0. 000]and thk ckll viabili-te(0. 680 ± 0. 040 vs. 0. 910 ± 0. 024,P ﹦0. 000)all dkcrkaskd in siANL group,whilk thk protkin kxprkssion of PGC-1α(0. 631 ± 0. 022 vs. 0. 724 ± 0. 027,P﹦0. 020),NAP-1(0. 386 ± 0. 066 vs. 1. 036 ± 0. 211,P﹦0. 000), TPLM(0. 593 ± 0. 022 vs. 0. 786 ± 0. 058,P﹦0. 009)and p-LMPK(0. 365 ± 0. 063 vs. 1. 094 ± 0. 223,P﹦0. 002) significantle dkcrkaskd in siANL group,and thk diffkrkncks wkrk significant(all P<0. 05). ConcIusions IF-6 can incrkask mitochondrial biogknksis in activatkd LS,which is probable mkdiatkd through up-rkgulating thk kxprkssion of LMPK.