致密斑细胞COX-2的表达及AP-1、NFκB信号通路

Expression of Cyclooxygenase-2 in a Mouse Macula Densa CellLines and Signal Transduction of NF-κB and AP-1

目的评估低盐(LS)培养对小鼠致密斑(MMDD1)细胞环氧化酶-2(COX-2)表达及核因子-κB(NF-κB)和活化蛋白-1(AP-1)活性的影响。方法经脂质体转染含NF-κB或AP-1的报告质粒,采用瞬时表达方法检测正常盐(NS)与LS培养对NF-κB和AP-1转录活性的影响。采用RT-PCR检测MMDD1细胞COX-2表达的变化,Westernblot方法检测细胞内p-p38MAPK、p-p44/42、c-Jun、c-Fos和COX-2蛋白的表达。结果LS培养促进了MMDD1细胞COX-2mRNA和蛋白表达(P<0.01)。LS培养后,p38和p44/42的磷酸化程度显著上调(P<0.01),180min后达到高峰。p38抑制剂SB-203580、p44/42抑制剂PD-98059可降低LS诱导的COX-2表达(P<0.01)。LS培养促进了c-Jun、c-Fos蛋白表达(P<0.01),激活了AP-1和NF-κB的转录活性(P<0.01)。25μmol/LNF-κB抑制剂PDTC和20μmol/LAP-1抑制剂curcumin下调了LS诱导的NF-κB、AP-1活性(P<0.01)。25μmol/LPDTC、20μmol/Lcurcumin降低了LS诱导的COX-2mRNA和蛋白表达(P<0.01)。结论LS培养可促进MMDD1细胞COX-2的表达,其作用可能与促进p38MAPK、p44/42激酶的磷酸化,增加NF-κB和AP-1的活性有关。

Objective To elvaluate the effect of low salt （LS） on the expression of cyclooxygenase-2 （COX-2） and the activity of nuclear factor kappa B （NF-kB） and activator protein-1 （ AP-1 ） in the mouse macula densa derived （ MMDD1 ） cell line. Methods MMDD1 cells were transfected with luciferase reporter plasmid containing AP-1 or NF-kB. Luciferase reporter assay was used to evaluate the effect of normal salt （NS） and low salt （LS） on the activities of NF-kB and AP-1. The changes of COX-2 expression were examined by RT-PCR. The expression of p-p38 MAPK, p-p44/42, c-Jun, c-Fos, and COX-2 in MMDD1 cells were analyzed by Western blot. Results The expressions of COX-2 mRNA and protein in MMDD1 cells were significantly increased by LS （P 〈 0.01 ）. Phosphorylated p38 and p44/42 MAPkinase were significantly increased by treatment at 180 min （P 〈 0.01 ）. The up-regulated COX-2 protein expression with LS were significantly reduced with SB 203580 （p38 inhibitiors） and PD-98059 （ p44/42 inhibitiors） （P 〈0.01 ）. The expressions of c-Jun and c-Fos were increased by LS. The luciferase activities of AP-1 and NF-KB were stimulatedin LS （ P 〈 0. 01 ）, the up-regulated luciferase activities were attenuated by PDTC at 25 umol/L （ NF-kB inhibitor） and curcumin at 20 umol/L （ AP-1 inhibitor） （ P 〈 0. 01 ）. LS altered COX-2 mRNA abundance and protein expression were decreased in treatment with PDTC at 25 umol/L, curcumin at 20 umol/L （P 〈 0. 01 ）. Conclusion LS can induce the expression of COX-2 in MMDD1 cells, which may be involved in the activation of p38 MAPkinase, p44/42 kinase, AP-1, and NF-kB pathways.