CAJ | 학술논문

目的：观察大鼠肾脏缺血再灌注( ischemia reperfusion, I/R)损伤中肾小管上皮细胞的自噬激活情况,探讨其对肾脏I/R损伤发挥保护性作用的分子机制。方法将40只雄性Wistar大鼠随机分为4组：假手术组( Sham)、I/R组、氯喹干预组( I/R+CQ)和雷帕霉素干预组( I/R+Rap)。常规方法建立大鼠肾脏I/R损伤模型,再灌注24 h后留取各组大鼠血和肾脏标本。血标本用于尿素氮( BUN)和血肌酐( Scre)含量检测；肾组织标本行HE染色,观察病理学损伤情况；TUNEL法检测肾小管上皮细胞凋亡的改变；采用免疫组化法检测Beclin-1和Caspase-3蛋白表达,透射电镜观察大鼠肾脏自噬泡的形成情况。结果与I/R组相比,I/R+CQ组BUN和Scre明显升高(P<0．05,P<0．01),肾组织损伤积分增加(P<0．01),TUNEL阳性细胞数增多(P<0．05),Beclin-1蛋白表达减弱(P<0．01),Caspase-3蛋白表达增强(P<0．01),电镜下自噬泡数量较少(P<0．05)；I/R+Rap组BUN和Scre显著降低(P<0．01),肾组织损伤积分减低(P<0．05),TUNEL阳性细胞数减少(P<0．05),Beclin-1蛋白表达增强(P<0．01),Caspase-3蛋白表达减弱(P<0．01),自噬泡数量明显增多(P<0．01)。结论自噬激活在肾脏I/R损伤过程中可通过抑制凋亡而发挥的保护作用,其机制可能涉及Beclin-1及Caspase-3等蛋白的表达调控。
목적：관찰대서신장결혈재관주( ischemia reperfusion, I/R)손상중신소관상피세포적자서격활정황,탐토기대신장I/R손상발휘보호성작용적분자궤제。방법장40지웅성Wistar대서수궤분위4조：가수술조( Sham)、I/R조、록규간예조( I/R+CQ)화뢰파매소간예조( I/R+Rap)。상규방법건립대서신장I/R손상모형,재관주24 h후류취각조대서혈화신장표본。혈표본용우뇨소담( BUN)화혈기항( Scre)함량검측；신조직표본행HE염색,관찰병이학손상정황；TUNEL법검측신소관상피세포조망적개변；채용면역조화법검측Beclin-1화Caspase-3단백표체,투사전경관찰대서신장자서포적형성정황。결과여I/R조상비,I/R+CQ조BUN화Scre명현승고(P<0．05,P<0．01),신조직손상적분증가(P<0．01),TUNEL양성세포수증다(P<0．05),Beclin-1단백표체감약(P<0．01),Caspase-3단백표체증강(P<0．01),전경하자서포수량교소(P<0．05)；I/R+Rap조BUN화Scre현저강저(P<0．01),신조직손상적분감저(P<0．05),TUNEL양성세포수감소(P<0．05),Beclin-1단백표체증강(P<0．01),Caspase-3단백표체감약(P<0．01),자서포수량명현증다(P<0．01)。결론자서격활재신장I/R손상과정중가통과억제조망이발휘적보호작용,기궤제가능섭급Beclin-1급Caspase-3등단백적표체조공。
Purpose To observe the autophagy activation of renal tubular epithelial cells during rat renal ischemia-reperfusion ( I/R) injury, and then explore the possible mechanisms of the protective role of autophagy. Methods Forty male Wistar rats were randomly divided into the following four groups: (1) sham operation group (Sham), (2) I/R, (3) chloroquine intervention group (I/R+CQ), (4) rapamycin intervention group (I/R +Rap). The specimens of blood and kidney were collected after reperfusion of 24 hours. The serum creatinine ( Scre) and blood urea nitrogen ( BUN) levels were measured from blood samples. Tissue samples of the kidney were stained with HE to observe the pathological changes. Apoptotic cells in the kidney sections were detected using the termi-nal deoxynucleotidyl transferase dUTP nick end labeling ( TUNEL) assay. Immunohistochemistry was used to detect the expression of Beclin-1 and Caspase-3. The structures of autophagic vacuoles were revealed by transmission electron microscopy. Results Compared to I/R group, the I/R+CQ group presented higher levels of BUN and Scre (P<0. 05, P<0. 01), higher renal tissue injury scores (P<0. 01), increased TUNEL-positive cells (P<0. 05), down-regulation of Beclin-1 (P<0. 01) and up-regulation of Caspase-3 (P<0. 01), as well as reduced number of autophagic vacuoles (P<0. 05). On the contrary, I/R+Rap group exhibited lower levels of BUN and Scre (P<0. 01), decreased renal tissue injury scores (P<0. 05), increased TUNEL-positive cells (P<0. 05), up-regula-tion of Beclin-1 (P<0. 01) and down-regulation of Caspase-3 (P<0. 01), as well as increased autophagic vacuoles (P<0. 05). Conclusion Activated autophagy provided a protective role in the kidney I/R injury through inhibiting apoptosis, and the mechanisms may be involved in the regulation of Beclin-1 and Caspase-3.