CAJ | 학술논문

目的探讨马兜铃酸(AA)致肾小管上皮细胞损伤机制及sonic hedgehog(Shh)信号在损伤过程中发挥的作用.方法以溶剂作为对照组,AA分别以浓度1、10、100 mg/L作用于大鼠肾小管上皮细胞NRK-52E,培养24、48、72 h后,水溶性四氮唑(WST-1)法检测细胞增殖抑制率；Hoechst 33258染色观察胞核形态变化；实时定量PCR检测膜受体patched 1(Ptch1)、信号开关蛋白smoothened (Smo)、α平滑肌肌动蛋白(α-SMA)、E钙黏蛋白(E-cadherin)、转化生长因子(TGF)β1和Ⅲ型胶原(ColⅢ)mRNA表达；ELISA法检测Shh和TGF-β1含量；Western印迹法检测bcl-2、bax、α-SMA和E-cadherin蛋白表达.结果 WST-1结果显示,AA作用24 h可明显抑制NRK-52E细胞增殖,并呈浓度依赖性(r=0.817,P=0.047).Hoechst 33258染色发现,1、10 mg/L AA可诱导细胞凋亡,但浓度为100 mg/L时则诱导细胞坏死.10mg/L AA作用细胞24h后,bcl-2蛋白表达下调而bax蛋白表达上调,提示启动细胞凋亡机制可能是通过线粒体途径.Shh蛋白和Smo mRNA表达升高,而Ptch1 mRNA表达下降(P＜0.05),提示Shh信号活化.此外,TGF-β1、α-SMA和ColⅢ表达增加,而E-cadherin表达下降(P＜0.05),提示出现细胞转分化和胶原累积.结论 AA可明显抑制肾小管上皮细胞增殖,诱导其凋亡和坏死.在此过程中,Shh信号被活化,并诱导细胞表型转化进而出现纤维化.
목적 탐토마두령산(AA)치신소관상피세포손상궤제급sonic hedgehog(Shh)신호재손상과정중발휘적작용.방법 이용제작위대조조,AA분별이농도1、10、100 mg/L작용우대서신소관상피세포NRK-52E,배양24、48、72 h후,수용성사담서(WST-1)법검측세포증식억제솔；Hoechst 33258염색관찰포핵형태변화；실시정량PCR검측막수체patched 1(Ptch1)、신호개관단백smoothened (Smo)、α평활기기동단백(α-SMA)、E개점단백(E-cadherin)、전화생장인자(TGF)β1화Ⅲ형효원(ColⅢ)mRNA표체；ELISA법검측Shh화TGF-β1함량；Western인적법검측bcl-2、bax、α-SMA화E-cadherin단백표체.결과 WST-1결과현시,AA작용24 h가명현억제NRK-52E세포증식,병정농도의뢰성(r=0.817,P=0.047).Hoechst 33258염색발현,1、10 mg/L AA가유도세포조망,단농도위100 mg/L시칙유도세포배사.10mg/L AA작용세포24h후,bcl-2단백표체하조이bax단백표체상조,제시계동세포조망궤제가능시통과선립체도경.Shh단백화Smo mRNA표체승고,이Ptch1 mRNA표체하강(P＜0.05),제시Shh신호활화.차외,TGF-β1、α-SMA화ColⅢ표체증가,이E-cadherin표체하강(P＜0.05),제시출현세포전분화화효원루적.결론 AA가명현억제신소관상피세포증식,유도기조망화배사.재차과정중,Shh신호피활화,병유도세포표형전화진이출현섬유화.
Objective To investigate the molecular mechanism of aristolochic acid (AA)-induced renal tubular epithelial cells (NRK-52E) injury,and to elucidate possible role of sonic hedgehog (Shh) signaling in this process.Methods The proliferation inhibition rate was measured by WST-1 assay in vitro after NRK-52E cells were treated with AA (1,10,100 mg/L) for 12,24,and 48 h,respectively.Cell apoptosis was determined by Hoechst 33258 staining.mRNA expressions of Smo,Ptch1,α-SMA,E-cadherin,TGF-β1,and type Ⅲ collagen were detected by real-time PCR.The levels of Shh and TGF-β1 were detected by ELISA assay.The expressions of bcl-2,bax,α-SMA,and E-cadherin were examined by Western blotting.Results WST-1 assay showed that AA significantly inhibited the proliferation of NRK-52E cells after 24 h,which was in a dose-dependent pattern (r =0.817,P =0.047).Evidence from Hoechst 33258 staining revealed that lower concentration (1,10 mg/L)AA induced cell apoptosis,but higher concentration (100 mg/L) AA induced cell necrosis.In AA-treated cells (10 mg/L),apoptosis was induced,which was partly mediated by the mitochondrial pathway with decreased bcl-2 and enhanced bax expression.The over-expression of Shh protein and Smo mRNA,and down-regulation of Ptch1 mRNA expression were found,indicating that Shh signaling was activated.In addition,the expressions of α-SMA,TGF-β1,and type Ⅲ collagen were significantly increased,but E-cadherin expression was decreased,suggesting that epithelial-to-mesenchymal transition and fibrosis occurred.Conclusions AA can significantly inhibit proliferation,and induce apoptosis and necrosis in NRK-52E cells.In this process,Shh signaling is activated,which promotes epithelial-to-mesenchymal transition and fibrosis.