CAJ | 학술논문

目的：探讨环杷明干预Hedgehog（HH）信号对马兜铃酸（AA）致肾小管上皮细胞表型转化和基质累积的影响。方法：根据干预措施将体外培养的大鼠肾小管上皮细胞NRK-52E分为溶剂对照组、AA损伤组（分别用终浓度为1、5和10 mg／L的AA处理细胞）和环杷明干预组（10 mg／L AA基础上加入1、5和10μmol／L环杷明）。细胞培养24 h后，用real-time PCR检测HH信号关键分子Ptch1和Smo、表型转化相关分子α-SMA和E-cad-herin、ZO-1、BMP-7和基质成分I型和III型胶原mRNA的表达；ELISA法检测Shh和TGF-β1的含量；细胞免疫荧光染色检测Ptch1、Smo、E-cadherin、α-SMA和III型胶原蛋白表达。结果：AA不仅增加了TGF-β1、α-SMA和III型胶原的表达，降低了E-cadherin和ZO-1的表达，而且诱导了Shh和Smo mRNA表达的升高和Ptch1 mRNA表达的下降，提示AA促进小管上皮细胞表型转化和胶原累积，同时也激活了HH信号通路。环杷明干预AA作用后，Smo mRNA或蛋白表达下调，Ptch1 mRNA表达升高，这说明环杷明抑制了AA诱导的HH信号通路的活化。此外，环杷明也降低TGF-β1、α-SMA、I型和III型胶原的表达，提高BMP-7、ZO-1和E-cadherin的表达，这提示环杷明抑制了AA所致的上皮细胞的表型转化和基质累积。结论：环杷明可抑制AA所致的肾小管上皮细胞表型转化和基质累积，可能是通过靶向抑制HH信号的活化来实现的。
목적：탐토배파명간예Hedgehog（HH）신호대마두령산（AA）치신소관상피세포표형전화화기질루적적영향。방법：근거간예조시장체외배양적대서신소관상피세포NRK-52E분위용제대조조、AA손상조（분별용종농도위1、5화10 mg／L적AA처리세포）화배파명간예조（10 mg／L AA기출상가입1、5화10μmol／L배파명）。세포배양24 h후，용real-time PCR검측HH신호관건분자Ptch1화Smo、표형전화상관분자α-SMA화E-cad-herin、ZO-1、BMP-7화기질성분I형화III형효원mRNA적표체；ELISA법검측Shh화TGF-β1적함량；세포면역형광염색검측Ptch1、Smo、E-cadherin、α-SMA화III형효원단백표체。결과：AA불부증가료TGF-β1、α-SMA화III형효원적표체，강저료E-cadherin화ZO-1적표체，이차유도료Shh화Smo mRNA표체적승고화Ptch1 mRNA표체적하강，제시AA촉진소관상피세포표형전화화효원루적，동시야격활료HH신호통로。배파명간예AA작용후，Smo mRNA혹단백표체하조，Ptch1 mRNA표체승고，저설명배파명억제료AA유도적HH신호통로적활화。차외，배파명야강저TGF-β1、α-SMA、I형화III형효원적표체，제고BMP-7、ZO-1화E-cadherin적표체，저제시배파명억제료AA소치적상피세포적표형전화화기질루적。결론：배파명가억제AA소치적신소관상피세포표형전화화기질루적，가능시통과파향억제HH신호적활화래실현적。
AIM:To investigate the effect of cyclopamine on Hedgehog (HH) signaling, phenotypic transfor-mation and matrix accumulation induced by aristolochic acid (AA) in renal tubular epithelial cell NRK-52E.METHODS:NRK-52E cells were randomly divided into control group (treated with solvent only), AA group (treated with AA at con-centrations of 1, 5, 10 mg/L) and cyclopamine group (treated with AA at concentration of 10 mg/L plus cyclopamine at concentrations of 1, 5, 10μmol/L).After cultured for 24 h, the mRNA expression of Ptch1, Smo,α-SMA, E-cadherin, ZO-1, BMP-7, type I collagen and type III collagen was quantified by real-time PCR.The protein levels of Shh and TGF-β1 were detected by ELISA .Immunofluorescence staining was used to evaluate the expression of Ptch 1, Smo,α-SMA, E-cadherin and type III collagen in the NRK-52E cells.RESULTS: AA increased the expression of TGF-β1, α-SMA and type III collagen, decreased the expression of E-cadherin and ZO-1 protein, and down-regulated the expression of Ptch1, Shh and Smo mRNA in the NRK-52E cells, indicating that AA activated HH signaling , and phenotypic transformation and matrix accumulation occurred in AA-treated NRK-52E cells.Treatment with cyclopamine inhibited HH signaling by decrea-sing Smo expression and increasing Ptch 1 expression.Moreover, cyclopamine also down-regulated the expression of TGF-β1,α-SMA, type I collagen and III collagen , and up-regulated the expression of BMP-7, ZO-1 and E-cadherin.CON-CLUSION:AA induces phenotypic transformation and matrix accumulation in renal tubular epithelial cells , which can be inhibited by cyclopamine treatment .The possible mechanism is that cyclopamine suppresses the activation of HH signaling , resulting in the reduction of epithelial-to-mesenchymal transition and matrix deposition .