苯并(a)芘通过降低突触融合蛋白17和溶酶体关联膜蛋白2表达阻抑人脐静脉内皮细胞自噬流

Benzo(a)pyrene blocking up the autophagic flux in human umbilical vein endothelial cell by down-regulating the expressions of syntaxin 17 and lysosomal associated mem-brane protein 2

目的研究苯并(a)芘(BaP)影响人脐静脉内皮细胞(HUVEC)自噬的机制。方法HUVEC经BaP(2.5、5、10μmol/L)处理24 h后,分别采用间接免疫荧光法、Western blot、吖啶橙染色和单丹磺酰尸胺染色等技术方法,检测自噬体及其内容物降解、目标蛋白微管相关蛋白1轻链3(LC3)、选择性自噬接头蛋白p62、Beclin-1、自噬相关蛋白5(Atg5)、Atg7、Atg12、组织蛋白酶B(CTSB)、组织蛋白酶D(CTSD)、突触融合蛋白17(STX17)、溶酶体关联膜蛋白2(LAMP2)表达、溶酶体数量与功能,及相关上游关键调控蛋白丝氨酸-苏氨酸蛋白激酶(Akt)、细胞外调节蛋白激酶(ERK)、转录因子EB(TFEB)磷酸化水平。结果BaP暴露组HUVEC内,检测结果显示:(1)LC3 puncta水平、LC3Ⅱ/LC3Ⅰ比率增加,自噬起始关键蛋白(Beclin-1、Atg5、Atg7、Atg12)表达升高,Akt蛋白磷酸化则明显降低;(2)p62 puncta和p62蛋白水平明显升高;(3)溶酶体数量增加,并伴随着溶酶体特征性水解酶(CTSB、CTSD)表达升高,相应地ERK、TFEB磷酸化水平增加;(4)调控自噬体与溶酶体融合的关键蛋白STX17与LAMP2降低。结论BaP通过降低STX17与LAMP2表达水平,阻抑了HUVEC正常的自噬流。

Aim To study the mechanism of benzo(a)pyrene(BaP)affecting autophagy in human umbilical vein endothelial cell(HUVEC).Methods HUVECs were treated with BaP(2.5,5,10μmol/L)for 24 h.The degradation of autophagosome and its contents,the expressions of target proteins microtubule-associated protein 1 light chain 3(LC3),sequestosome 1(p62),Beclin-1,autophagy-related protein 5(Atg5),Atg7,Atg12,cathepsin B(CTSB),cathepsin D(CTSD),syntaxin 17(STX17),lysosomal associated membrane protein 2(LAMP2),the number and function of lysosomes,and the phosphorylation levels of related upstream key regulatory proteins serine-threonine protein kinase(Akt),extracellular regulated protein kinase(ERK)and transcription factor EB(TFEB)were detected re-spectively by indirect immunofluorescence,Western blot,acridine orange staining and monodansyl cadaverine staining.Results In HUVEC of BaP exposure group,the test results showed that:(1)The level of LC3 puncta and the ratio of LC3Ⅱ/LC3Ⅰincreased,the expressions of key autophagy initiation proteins(Beclin-1,Atg5,Atg7 and Atg12)in-creased,and the phosphorylation of Akt protein decreased significantly;(2)The levels of p62 puncta and p62 protein in-creased significantly;(3)The number of lysosomes increased,accompanied by the increased expressions of lysosomal char-acteristic hydrolases(CTSB,CTSD),and the phosphorylation levels of ERK and TFEB increased accordingly;(4)The key proteins STX17 and LAMP2 regulating the fusion of autophagy and lysosome decreased.Conclusion BaP inhibits the normal autophagic flux of HUVEC by reducing the expression levels of STX17 and LAMP2.