慢病毒介导shRNA靶向下调过氧化物酶体增殖物激活受体α对全氟十二烷酸致大鼠肝细胞BRL 3A氧化损伤的影响

Effect of lentivirus-mediated shRNA targeting down-regulation of PPARαon perfluorododecanoic acid induced oxidative damage in rat hepatocytes BRL 3A

目的通过观察慢病毒介导shRNA靶向下调大鼠肝细胞BRL 3A中过氧化物酶体增殖物激活受体(peroxisome proliferator-activated receptor,PPAR)α的表达,研究PPARα在全氟十二烷酸(perfluorododecanoic acid,PFDoA)致大鼠肝脏氧化损伤中的作用。方法构建PPARα慢病毒兼容shRNA干扰载体Lenti-iPα和阴性对照载体Lenti-NC,与慢病毒包装辅助质粒共转染293FT细胞进行慢病毒包装,收集慢病毒原液后浓缩并测定病毒滴度。分为NC-组(加阴性对照病毒,不加PFDoA)、NC+组(加阴性对照病毒,加75μmol/L PFDoA)、iPα-组(加干扰病毒,不加PFDoA)、iPα+组(加干扰病毒,加75μmol/L PFDoA)。使用慢病毒处理大鼠肝BRL 3A细胞96 h,最后24 h再对NC+组、iPα+进行75μmol/L PFDoA暴露。观察BRL 3A细胞中PPARα的干扰情况及PPARα的表达在PFDoA引起活性氧簇(reactive oxygen species,ROS)变化中所起的作用。结果慢病毒介导shRNA成功实现靶向下调PPARα的表达,与NC-组相比,iPα-组的大鼠肝细胞系BRL 3A中ROS平均荧光强度为12043.42±808.58,显著升高(P<0.05);PPARα下游靶蛋白酰基辅酶A硫酯酶(acyl-CoA thioesterases,Acot)1的基因和蛋白表达水平(分别为0.43±0.04和0.34±0.08)均显著下降(P<0.05)。PFDoA处理后,与NC+组相比,iPα+组大鼠肝细胞BRL 3A中ROS平均荧光强度为12386.25±356.36,显著上升(P<0.05);Acot1的基因和蛋白表达水平(分别为0.85±0.10和0.33±0.04)显著下降(P<0.05)。结论PPARα及其下游靶蛋白Acot1在大鼠肝细胞系BRL 3A中可能起到清除ROS的作用,避免外源物质对肝脏的氧化损伤。

OBJECTIVE To investigate the role of peroxisome proliferator-activated receptor alpha(PPARα)in perfluorododecanoic acid(PFDoA)-induced liver oxidative damage in rats by observing lentivirus-mediated shRNA targeting and down-regulating PPARαexpression in rat hepatocytes BRL 3 A.METHODS A PPARαlentivirus-compatible shRNA interference vector Lenti-iPαand a negative control vector Lenti-NC were constructed,and co-transfected with lentivirus packaging helper plasmids into 293 FT cells for lentivirus packaging.The lentivirus stock solution was collected,concentrated and the virus titer was determined.The experimental grouping was as follows,NC-group(infected with negative control lentivirus,without PFDoA exposure),NC+group(infected with negative control lentivirus,75μmol/L PFDoA exposure),iPα-group(infected with interference lentivirus,without PFDoA exposure),iPα+group(infected with interference lentivirus,75μmol/L PFDoA exposure).Rat hepatocytes BRL 3 A cells were treated with lentivirus for 96 h,and then exposed with 75μmol/L PFDoA in the NC+group and iPα+group in the last 24 h.The interference of PPARαin BRL 3 A cells and the role of PPARαin reactive oxygen species(ROS)changes caused by PFDoA were observed.RESULTS Lentivirus-mediated shRNA successfully achieved targeted downregulation of PPARαexpression in BRL 3 A cells.Compared with the NC-group,the mean fluorescence intensity of ROS in rat hepatocytes BRL 3 A in the iPα-group was 12043.42±808.58,significantly increased(P<0.05);The transcription levels of acyl-CoA thioesterases(Acot)1 gene and its protein expression levels were 0.43±0.04 and 0.34±0.08,respectively,both significantly decreased(P<0.05).After PFDoA treatment,compared with NC+group,the mean fluorescence intensity of ROS in iPα+group was 12386.25±356.36,which also increased significantly(P<0.05).The transcription levels of Acot1 gene and its protein expression levels were 0.85±0.10 and 0.33±0.04,respectively,which also decreased significantly(P<0.05).CONCLUSION PPARαand its downstream target protein Acot1 may play a role in scavenging ROS in rat hepatocytes BRL 3 A,keeping hepatocytes from oxidative damage caused by foreign substances to the liver.