目的：探讨γ-分泌酶抑制剂DAPT对马兜铃酸（AA）诱导引起肾小管上皮细胞表型转化与胶原累积的作用及分子机制。方法将体外培养的大鼠肾小管上皮细胞NRK-52E分为正常细胞对照组、AA 10 mg·L-1组、AA 10 mg·L-1+DAPT1和10μmol·L-1组。24 h后，实时荧光定量PCR检测Notch信号关键分子Notch1、Jagged1和Numb、表型转化相关分子转化生长因子β1（TGF-β1）、E-钙黏着蛋白、α-平滑肌肌动蛋白（α-SMA）、骨形态发生蛋白7（Bmp7）和基质成分Ⅰ型胶原a1（Col1a1）和Ⅲ型胶原a1（Col3a1） mRNA的表达；细胞免疫荧光染色法检测Notch1、Jagged1、α-SMA和Col3a1蛋白的表达。结果与正常细胞对照相比，AA处理后，肾小管上皮细胞基质相关因子TGF-β1，α-SMA和Col3a1 mRNA表达上调，上皮标志物E-钙黏着蛋白mRNA的表达受到抑制，而且导致了Notch1、Jagged1 mRNA表达的上调和Numb mRNA表达的下调（P<0.05），提示AA促进肾小管上皮细胞表型转化与基质累积，同时激活了Notch信号通路。DAPT干预AA作用后，Notch1（P<0.01）和Jagged1（P<0.05）的mRNA表达下调，Numb mRNA表达上调（P<0.05），说明DAPT抑制了AA诱导的Notch信号通路活化。此外，与AA损伤组相比，DAPT也降低了TGF-β1，α-SMA，Col1a1和Col3a1 mRNA表达（P<0.05），提高BMP-7和E-钙黏着蛋白mRNA表达（P<0.05），提示DAPT抑制了AA诱导的上皮细胞的表型转化与基质累积。结论 DAPT抑制AA诱导的肾小管上皮细胞的表型转化与基质累积，其可能机制是DAPT靶向干预Notch信号的活化。%OBJECTIVE To investigate the effect ofγ-secretase inhibitor N-[N-(3,5-difluorophen⁃acetyl)-L-alanyl]-S-phenylglycine t-butyl ester(DAPT)on phenotypic transformation and matrix accu⁃mulation induced by aristolochic acid(AA) in renal tubular epithelial cells(NRK-52E)and explore the mechanism. METHODS NRK-52E cells were divided stochastically into normal cell control group,AA 10 mg·L-1 group and AA 10 mg·L-1+DAPT 1 and 10μmol·L-1 group. After 24 h,the mRNA expressions of Notch1,Jagged1,Numb,E-cadherin,transforming growth factor-β1(TGF-β1),α-smooth muscle actin(α-SMA),bone morphogenic protein 7 (Bmp7),typeⅠ a1 (Col1a1) and Ⅲ collagens a1 (Col3a1)were quantified by quantitative real-time RT-PCR. The protein expressions of Notch1,Jagged1,α-SMA,and Col3a1 in NRK-52E cel s were detected by immunofluorescence staining. RESULTS In NRK-52E cells,AA enhanced the expression of TGF-β1,α-SMA and Col3a1 mRNA(P<0.05),reduced the expression of E-cadherin mRNA(P<0.05),up-regulated the mRNA expression of Notch1 mRNA(P<0.01)and Jagged1(P<0.05),and down-regulated the mRNA expression of Numb mRNA(P<0.05) compared with normal cell control group,indicating that phenotypic transformation and matrix accumu⁃lation occurred in AA-treated NRK-52E cells,accompanied by activated Notch signaling. Treatment with DAPT inhibited Notch signaling by decreasing the expression of Notch1 and Jagged1 (P<0.05),and increasing the expression of Numb mRNA(P<0.05). Furthermore, DAPT also down-regulated the expression levels of TGF-β1,α-SMA,Col1a1 and Col3a1 mRNA(P<0.05), and up-regulated the expression level of Bmp7 and E-cadherin mRNA(P<0.05) compared with AA group,suggesting that DAPT inhibited phenotypic transformation and matrix accumulation in AA-treated NRK-52E cells. CONCLUSION AA induces phenotypic transformation and matrix accumulation in renal tubular epithelial cells,which is inhibited by DAPT treatment. The possible mechanism is that DAPT suppresses the activation of Notch signaling,resulting in the reduction of epithelial-to-mesenchymal transition and matrix deposition.
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