摘要
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、AA10 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)m RNA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 m RNA表达上调,上皮标志物E-钙黏着蛋白m RNA的表达受到抑制,而且导致了Notch1、Jagged1 m RNA表达的上调和Numb m RNA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m RNA表达下调,Numb m RNA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 m RNA表达(P<0.05),提高BMP-7和E-钙黏着蛋白m RNA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 of γ-secretase inhibitor N-[N-（3,5-difluorophenacetyl）-L-alanyl]-S-phenylglycine t-butyl ester（DAPT）on phenotypic transformation and matrix accumulation induced by aristolochic acid（AA） in renal tubular epithelial cells（NRK-52E）and explore the mechanism. METHODS NRK-52 E cells were divided stochastically into normal cell control group,AA10 mg·L^-1group and AA 10 mg·L-1＋DAPT 1 and 10 μmol·L^-1group. 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）,type Ⅰ 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-52 E cels were detected by immunofluorescence staining. RESULTS In NRK-52 E 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 accumulation occurred in AA-treated NRK-52 E 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- 52 E 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 s
出处
《中国药理学与毒理学杂志》
CAS
CSCD
北大核心
2016年第3期209-214,共6页
Chinese Journal of Pharmacology and Toxicology
基金
浙江省自然科学基金(LQ16H310005)
浙江省自然科学基金(LY16H050007)
温州市科技计划项目(Y20130149)~~
关键词
DAPT
马兜铃酸
表型转化
基质累积
NOTCH信号
DAPT
aristolochic acid
phenotype transformation
matrix accumulation
Notch signaling
