CAJ | 학술논문

目的探讨早期自噬激活对醛固酮诱导足细胞损伤的保护作用.方法体外培养永生性小鼠足细胞系,分别在醛固酮刺激6、12、24、48 h后应用Annexin V结合流式细胞术检测足细胞凋亡；透射电子显微镜检测足细胞凋亡小体和自噬小体；Western印迹检测自噬标志蛋白微管相关蛋白1轻链3(LC3)、凋亡相关蛋白caspase-3及足细胞分子nephrin的表达；实时定量PCR检测自噬相关基因Beclin-1的表达.结果醛固酮(10-7mol/L)呈时间依赖性诱导足细胞凋亡,下调nephrin表达.与对照组比较,醛固酮刺激24 h足细胞凋亡增加26.5％(P＜0.05),nephrin表达降低28.0％(P＜0.05).醛固酮(10-7mol/L)呈时间依赖性诱导足细胞发生自噬.与对照组比较,醛固酮刺激6h自噬相关基因Beclin-1表达显著上调(P＜0.05)；醛固酮刺激12h自噬标志蛋白LC3-Ⅱ/LC3-Ⅰ比值增加(P＜0.05).应用3-甲基腺嘌呤(3-MA)抑制自噬后,醛固酮诱导足细凋亡的时间提前至刺激后12h.3-MA处理组与醛固酮刺激24 h比较,足细胞凋亡增加39.0％ (P＜ 0.05),nephrin表达进一步下调19.5％(P＜ 0.05),并且caspase-3明显活化(P＜0.05).结论醛固酮可诱导足细胞发生自噬和凋亡；醛固酮刺激12h时自噬明显活化；刺激24 h时凋亡显著增加.早期自噬激活可抑制醛固酮诱导的足细胞凋亡,并减轻足细胞损伤.以足细胞自噬为靶点可能成为治疗肾小球疾病的新研究方向.
목적 탐토조기자서격활대철고동유도족세포손상적보호작용.방법 체외배양영생성소서족세포계,분별재철고동자격6、12、24、48 h후응용Annexin V결합류식세포술검측족세포조망；투사전자현미경검측족세포조망소체화자서소체；Western인적검측자서표지단백미관상관단백1경련3(LC3)、조망상관단백caspase-3급족세포분자nephrin적표체；실시정량PCR검측자서상관기인Beclin-1적표체.결과 철고동(10-7mol/L)정시간의뢰성유도족세포조망,하조nephrin표체.여대조조비교,철고동자격24 h족세포조망증가26.5％(P＜0.05),nephrin표체강저28.0％(P＜0.05).철고동(10-7mol/L)정시간의뢰성유도족세포발생자서.여대조조비교,철고동자격6h자서상관기인Beclin-1표체현저상조(P＜0.05)；철고동자격12h자서표지단백LC3-Ⅱ/LC3-Ⅰ비치증가(P＜0.05).응용3-갑기선표령(3-MA)억제자서후,철고동유도족세조망적시간제전지자격후12h.3-MA처리조여철고동자격24 h비교,족세포조망증가39.0％ (P＜ 0.05),nephrin표체진일보하조19.5％(P＜ 0.05),병차caspase-3명현활화(P＜0.05).결론 철고동가유도족세포발생자서화조망；철고동자격12h시자서명현활화；자격24 h시조망현저증가.조기자서격활가억제철고동유도적족세포조망,병감경족세포손상.이족세포자서위파점가능성위치료신소구질병적신연구방향.
Objective To explore the protection of early autophagy activation on podocyte injury induced by aldosterone.Methods In vitro cultured mouse podocyte clones (MPC5) were treated with aldosterone for 6,12,24,48 h respectively.Apoptosis of podocytes was detected by Annexin V combined with flow cytometry.After 24 h treatment with aldosterone,the existence of apoptotic body and autophagosome was observed by electron microscopy.The protein expressions of LC3,caspase-3 and nephrin were examined by Western blotting.The mRNA expression of Beclin-l was detected by real-time PCR.Results The induction of apoptosis and autophagy by aldosterone in podocytes was in timedependent mannner.After 24 h treatment with aldosterone,the apoptosis was increased by 26.5％ (P ＜ 0.05)and the expression of nephrin was decreased by 28.0％ (P ＜ 0.05) compared to control group.Aldosterone remarkably induced the expression of Beclin-1 at 6 h and promoted the transformation of LC3-Ⅰ to LC3-Ⅱat 12 h (P ＜ 0.05).Compared to simple aldosterone treatment,the apoptosis rate of podocyte was increased by 39.0％ (P ＜ 0.05) and the expression of nephrin was declined by 19.5％ (P ＜ 0.05) after 3-methyladenine (3-MA) pre-treatment.Conclusions Aldosterone can induce autophagy and apoptosis in podocytes.Autophagy occurs earlier (12 h) than apoptosis (24 h).The occurrence of autophagy can inhibit the apoptosis,so the autophagy pathway may be a new research topic of glomerular disease treatment.