垂盆草提取物对马兜铃酸致大鼠肾小管上皮细胞损伤的改善作用  

Protective effect of Sedum sarmentosum Bunge extract against aristolochic acid-induced rat renal tubular epithelial cell injury

作　　者：陆红[1] 陈必成[2] 胡丽萍[1] 洪炜龙[2] 林成成[2] 白永恒[2] 

机构地区：[1]温州医科大学附属第一医院医学检验中心,浙江温州325000 [2]温州医科大学附属第一医院外科实验室,浙江温州325000

出　　处：《中国药理学与毒理学杂志》2013年第6期988-994,共7页Chinese Journal of Pharmacology and Toxicology

基　　金：浙江省自然科学基金(LQ12H050001);浙江省自然科学基金(LY12H050004);温州市科技计划项目(Y20110028);温州市科技计划项目(Y20110072)~~

摘　　要：目的研究马兜铃酸(AA)致肾小管上皮细胞损伤的分子机制及垂盆草提取物(SSBE)对AA损伤的干预机制。方法将体外培养的大鼠肾小管上皮细胞NRK-52E加入AA 1,5,10,50和100 mg·L-1;或AA 10 mg·L-1+SSBE 10,50,100,500和1000 mg·L-1,培养24 h后,采用倒置相差显微镜观察细胞形态变化;Hoechst 33258染色和流式细胞仪检测NRK-52E细胞凋亡;ELISA和免疫细胞荧光染色检测巨噬细胞移动抑制因子(MIF)的表达;实时荧光定量RT-PCR和Western蛋白质印迹法检测凋亡相关基因c-Myc和p53 mRNA和蛋白的表达。结果随着AA浓度的增加,NRK-52E细胞损伤愈加明显。与正常对照组相比,AA 10和50 mg·kg-1诱导细胞凋亡,并促进了c-Myc和p53明显表达(P<0.05);AA 10和50 mg·kg-1损伤肾小管上皮细胞后,MIF的分泌和表达量明显增加(P<0.05)。与AA 10 mg·L-1组相比,SSBE 1000 mg·L-1不仅减轻了AA所致的损伤,而且明显下调了MIF的表达(P<0.05)。同时,SSBE1000 mg·L-1也明显抑制了c-Myc和p53的表达(P<0.05)。结论 AA损伤肾小管上皮细胞,诱导细胞凋亡,并促进MIF的表达,进而影响炎症应激反应过程中巨噬细胞的浸润和活化。SSBE的干预可明显降低AA所致的损伤,这可能与MIF的表达下调有关。OBJECTIVE To investigate the protective effect of Sedum sarmentosum Bunge extract （SSBE） on aristolochic acid （AA）-induced renal tubular epithelial cell injury, and to explore its possible molecular mechanisms. METHODS In vitro, NRK-52E cells were divided randomly into 3 groups, solvent control group, AA 1, 5, 10, 50, 100 mg·L-1 group and AA 10 mg·L-1＋SSBE 10, 50, 100, 500 and 1000 mg·L-1. After cells were cultured for 24 h, the morphology of NRK-52E cells was observed with inverted/phase contrast microscopy. Hoechst 33258 staining and flow cytometry analysis were used to determine cell apoptosis and necrosis. The expression of macrophage migration inhibitory factor （MIF） was measured by ELISA and immunofluorescent analysis. Using real-time RT-PCR and western blot, mRNA and protein expressions of p53 and c-Myc were quantified. RESULT Injury obviously increased in AA-treated cells by microscopy and Hoechst 33258 staining, and it aggravated with the passage of time and increase of concentrations. Compared with normal control group, AA 10 and 50 mg·L-1 induced apoptosis of NRK-52E cells, and promoted the expression of p53 and c-Myc （P〈0.05）, The secretion and expression of MIF protein significantly increased in NRK-52E cells in AA 10 and 50 mg·L-1 groups（P〈0.05）. Compared with AA 10 mg·L-1 group, with SSBE 1000 mg·L-1 treatment, injury in NRK-52E cells was alleviated, and expression of MIF protein also decreased （P〈0.05）. In addition, SSBE 1000 mg·L-1 decreased expression of c-Myc and p53 in concentration dependent manner （P〈0.05）. CONCLUSION AA-induced injury in NRK-52E cells involves in cellular apoptosis and proliferation. AA promotes the expression of MIF, which regulates the gathering and activation of macrophages in inflammatory response. AA-induced injury can be reduced with the treatment of SSBE by down-regulating expression of MIF.

关 键 词：垂盆草 植物提取物 马兜铃酸 巨噬细胞 巨噬细胞游走抑制因子 细胞凋亡 

分 类 号：R285.5[医药卫生—中药学]