脂联素介导 APPL1／AMPK 信号通路对小鼠肾脏缺血再灌注损伤后期纤维化的影响

目的：研究脂联素（APN）介导 APPL1／AMPK 信号通路对小鼠肾脏急性缺血再灌注损伤后期纤维化的影响。方法雄性 C57BL／6小鼠48只，随机分为假手术组（Sham 组）、肾脏急性缺血再灌注组（IRI 组）、肾脏急性缺血再灌注＋APN 组（APN／IRI 组）和肾脏急性缺血再灌注＋生理盐水组（NS／IRI 组），每组各12只。 APN／IRI 组在术前15 min、术后每24 h 分别经腹腔注射 APN 蛋白球状片段5μg／g，共3次；NS／IRI 组在相应时点经腹腔注射等量生理盐水。 IRI 组均行双侧夹闭肾蒂30 min 后开放肾灌注，Sham 组分离肾蒂但不夹闭。各组大鼠分别在术后2 d 随机各取6只小鼠，留取血液及肾组织标本。 PAS 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 blot 检测肾脏组织 APPL1蛋白表达和 AMPK 磷酸化水平。术后14 d 每组取剩余6只小鼠肾脏标本，天狼星红苦味酸（Sirus red）染色观察肾组织病理变化，real time PCR 检测α平滑肌肌动蛋白（α－SMA）和转化生长因子β1（TGF －β1）的 mRNA 表达。结果术后2 d，与 Sham 组相比，IRI组、APN／IRI 组和 NS／IRI 组小鼠肾组织小管间质损害加重，血 BUN、Scr 升高（P ＜0．05），APPL1蛋白表达和AMPK 磷酸化水平明显增高（P ＜0．05）；与 IRI 组相比，APN／IRI 组小鼠肾小管损害明显减轻，血 BUN、Scr 明显降低（P ＜0．05），APPL1蛋白表达和 AMPK 磷酸化水平明显增高（P ＜0．05）。术后14 d，与 Sham 组相比，IRI 组、APN／IRI 组和 NS／IRI 组小鼠肾组织纤维化程度明显增高，α－SMA 和 TGF －β1的 mRNA 的表达明显增多（P ＜0．05）；与 IRI 组相比，APN／IRI 组小鼠肾组织纤维化程度明显降低，α－SMA 和 TGF －β1的 mRNA 的表达量明显减少（P ＜0．05）。结论APN 介导 APPL1／AMPK 信号通路减轻小鼠肾脏急性缺血再灌注损伤后期纤维化。

APN attenuates late fibrosis induced by acute ischemia reperfusion injury in mice via APPL1/AMPK signaling pathway

Objective To investigate the attenuation by APN on late fibrosis induced by acute ischemia reperfu-sion injury in mice via APPL1 /AMPK signaling pathway.Methods Forty -eight male C57BL/6 mice were randomly di-vided into sham operation group (Group Sham), acute ischemia -reperfusion group (Group IRI), acute ischemia -reper-fusion plus APN group (Group APN/IRI) and acute ischemia -reperfusion plus NS group (Group NS/IRI).The gAPN (5 μg/g) and physiological saline were injected intraperitoneally 15 min before operation and every 24 h after operation in Group APN/IRI and Group NS/IRI, respectively.Renal acute ischemia reperfusion group were performed open renal per-fusion after bilateral renal pedicle clip for 30 min was performed in Group IRI, as separation of renal pedicle without clip was performed in Group Sham.On the 2nd day after the operation, 6 mice were randomly selected from each groups were sacrificed for blood and kidney samples.The renal histopathological changes were observed through Schiff periodic acid staining (PAS).The contents of BUN and Scr were determined.The APPL1 protein expression and the phosphorylate lev-el of AMPK were detected by Western blot.On the 14th day after the operation, 6 mice from each group were sacrificed for kidney samples.The αsmooth muscle actin (α-SMA) and transforming growth factor beta 1 (TGF -β1 ) mRNA were determined by real -time PCR.The degree of kidney fibrosis was observed through Sirus red staining.Results Com-pared with Group Sham, tubular interstitial damage were significantly aggravated and the levels of BUN and Scr were in-creased in Group IRI, APN/IRI group and NS/IRI group on the 2nd day after the operation (P <0.05), so were the in-creases APPL1 protein and the phosphorylate level of AMPK (P <0.05).Compared with Group IRI, tubular interstitial damage were significantly attenuated and the levels of BUN and Scr were significantly reduced in Group APN/IRI group on the 2nd day after the operation (P <0.05), so were the increases APPL1 protein and the phosphorylate level of AMPK (P <0.05).Compared with Group Sham, the degree of kidney fibrosis and the expression of α-SMA and TGF -β1 were significantly increased on the 14th day after the operation (P <0.05).Compared with Group IRI, the degree of kidney fi-brosis and the expression of α-SMA and TGF -β1 were significantly reduced on the 14th day after the operation (P <0.05).Conclusion APN attenuates late fibrosis induced by acute ischemia reperfusion injury in mice via APPL1/AMPK signaling pathway.