AMPK通过抑制炎性反应减轻小鼠肾脏缺血再灌注后纤维化  

AMPK attenuates inflammation to reduce fibrosis induced by acute ischemia reperfusion injury in mice

作　　者：周俊[1] 林文静[1] 林森[1] 黄振兴[1] 黄腾[1] 仲吉英[1] 杨承祥[1] 

机构地区：[1]佛山市第一人民医院麻醉科,528000

出　　处：《中华肾脏病杂志》2016年第6期450-456,共7页Chinese Journal of Nephrology

基　　金：广东省科技计划项目（2014A020212612）

摘　　要：目的探讨腺苷酸活化蛋白激酶（AMPK）抑制炎性反应在小鼠肾脏缺血再灌注损伤（IRI）后期纤维化中的作用。方法48只雄性C57BL／6小鼠，随机分为假手术组（Sham组）、IRI组、AMPK抑制剂＋IRI组（AMPK／IRI组）和生理盐水＋IRI组（NS／IRI组），每组各12只。肾脏缺血再灌注行双侧夹闭肾蒂30min后开放。肾灌注，Sham组分离双侧肾蒂但不夹闭。AMPK／IRI组和NS／IRI组小鼠IRI术前分别腹腔注射20mg／kgAMPK特异抑制剂CompoundC和等量生理盐水。术后2d每组随机各取6只小鼠，留取血液及肾组织标本，苏木精-伊红（HE）染色观察肾组织病理变化，全自动生化分析仪检测血尿素氮（BUN）及血肌酐（Scr）含量，Western印迹检测AMPK磷酸化水平，实时定量PCR法检测IL-1β、IL-6和TNF-α的mRNA表达。术后14d取每组剩余6只小鼠的肾组织标本，天狼星红苦味酸（Simsred）染色观察肾组织病理变化，免疫荧光、Western印迹检测肾组织胶原蛋白1（COL1）、d平滑肌肌动蛋白（α-SMA）和纤连蛋白（FN）的表达。结果与Sham组比较，IRI组、NS／IRI组小鼠术后2d的肾组织小管间质损害加重（P〈0．05），血BUN、Ser均升高（均P〈0．05），IL-1β、IL-6和TNF—α的mRNA表达均明显增多（均P〈0．05），肾组织AMPK磷酸化水平均增加（均P〈0．05）；术后14d的肾组织纤维化程度明显，COL1、α—SMA、FN表达均增加（均P〈0．05）。与IRI组比较，AMPK／IRI组术后2d肾小管损害明显加重（P〈0．05），血BUN、Scr均明显升高（均P〈0．05），IL-1β、IL-6和TNF-α的mRNA表达明显增多（均P〈0．05），AMPK磷酸化水平明显降低（P〈0．05）；术后14d的肾组织纤维化程度更明显，COLI、α—SMA、FN表达量增多（均P〈0．05）。结论在小鼠IRI引起的肾脏纤维化进程中AMPK可减轻纤维化，可能与减轻炎性反应有关。Objective To observe the effect of adenosine monophosphate activated protein kinase （AMPK） on attenuating inflammation in fibrosis induced by acute ischemia reperfusion injury （IRI） in mice. Methods Forty eight male C57BL/6 mice were randomly divided into four groups： sham operation group （sham group）, IRI group, AMPK inhibitor ＋ IRI group （AMPK/IRI group） and normal saline＋IRI group （NS/IRI group）, 12 mice each group. The mice with renal IRI were occluded for 30 rain through clipping bilateral renal pedicle, then released renal perfusion. Mice in sham group were performed the separation of renal pedicle without clipping. Mice in AMPK/IRI group and NS/IRI group were respectively intraperitoneal injected AMPK inhibitor and normal saline before IRI. At the 2 d after operation, 6 randomly-selected mice from each group were blooded by extraction eyeball to detect BUN and Scr. The renal histopathological changes were observed through HE staining. The mRNA expression of IL-1β, IL-6 and TNF-α was detected by real time PCR, and the level of AMPK phosphorylation was detected by Western blotting. At the 14 d after operation, Collagen 1 （COL1）, α- SMA and fibronectin （FN） were detected by immunofluorescence and Western blotting in 6 remained mice from each group. The degree of kidney fibrosis was observed through sirus red staining. Results Compared with those in sham group, tubular interstitial damage was aggravated （P 〈 0.05）, BUN and Scr were increased （P 〈 0.05）, the mRNA expression of IL-1α, IL-6 and TNF-α was increased at the 2 d after operation （all P 〈 0.05）, and the level of AMPK phosphorylation was activated in IRI group and NS/IRI group （all P 〈 0.05）; the degree of kidney fibrosis and the expression of COL1, α-SMA and FN were increased obviously at the 14 d （all P 〈 0.05）. Compared with those in IRI group, in AMPK/IRI group tubular interstitial damage was aggravated （P 〈 0.05）, BUN and Scr were increased （all P 〈 0.05）, the mRNA e

关 键 词：纤维化 肾疾病 再灌注损伤 细胞内信号肽和蛋白质类 炎症 

分 类 号：R692[医药卫生—泌尿科学]