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目的探讨炎性介质骨桥蛋白(OPN)的表达和核转录因子在慢性环孢素A(CsA)肾毒性中的作用。方法雄性Sprague-Dawley大鼠喂食低盐(0.05%钠盐)饲料下分为两组,正常对照组给予皮下注射橄榄油(1 mL·kg-1·d-1);毒性组给予皮下注射CsA(15 mg·kg-1·d-1)4周。通过观察炎性细胞浸润(ED-1)和肾小管间质纤维化,比较两组大鼠的肾小管间质损伤程度;采用RNA印迹、免疫组织化学染色方法检测OPN在mRNA和蛋白水平的表达;用凝胶电泳迁移率实验(EMSA)分析法和免疫印迹法测定核转录因子(NF-κB和AP-1)的结合活性和IκB蛋白的表达。结果毒性组大鼠表现为肾小管间质带状纤维化[(38.9±3.3)%/5 mm2vs(0±0)%/5 mm2,P<0.01]和大量ED-1阳性细胞浸润[(89±9)vs(7±2),P<0.01]。与对照组相比,毒性组大鼠OPN mRNA和蛋白的表达增加[(729±37)%vs(103±4)%,P<0.01],分布于肾小管上皮细胞,其主要位于肾小管间质损伤部位;毒性组核转录因子NF-κB[(218±19)%vs(116±15)%,P<0.01]和AP-1[(735±225)%vs(101±4)%,P<0.01]结合活性增加,而IκB蛋白的表达减少[(9±7)%vs(105±7)%,P<0.01]。直线相关分析示OPN mRNA的表达与肾小管间质纤维化程度(r=0.959,P<0.001)和核转录因子的结合活性呈正相相关(NF-κB:r=0.773,P<0.01;AP-1:r=0.619,P=0.01)。结论炎性介质OPN和核转录因子参与了慢性CsA肾毒性肾小管间质的损伤。
Objective To investigate the expression of osteopontin (OPN) and the role of nuclear transcription factor in chronic cyclosporine A (CsA) nephrotoxicity. Methods Male Sprague-Dawley rats were divided into two groups by low-salt (0.05% sodium salt) diet. The normal control group was subcutaneously injected with olive oil (1 mL · kg-1 · d-1) (15 mg · kg-1 · d-1) for 4 weeks. The level of tubulointerstitial injury was compared between the two groups by observing inflammatory cell infiltration (ED-1) and tubulointerstitial fibrosis. The expression of OPN mRNA and protein was detected by Northern blotting and immunohistochemical staining The binding activity of nuclear transcription factor (NF-κB and AP-1) and the expression of IκB protein were determined by EMSA and Western blot. Results Toxicity group showed tubulointerstitial fibrosis [(38.9 ± 3.3)% / 5 mm2 vs (0 ± 0)% / 5 mm2, P <0.01] and a significant amount of ED- ± 9) vs (7 ± 2), P <0.01]. Compared with the control group, the expression of OPN mRNA and protein in the toxic group increased [(729 ± 37)% vs (103 ± 4)%, P <0.01] and distributed in renal tubular epithelial cells, The levels of nuclear factor NF-κB [(218 ± 19)% vs (116 ± 15)%, P <0.01] and AP-1 [(735 ± 225)% vs (101 ± 4)%, P <0.01], but decreased the expression of IκB protein [(9 ± 7)% vs (105 ± 7)%, P <0.01]. Linear correlation analysis showed that the expression of OPN mRNA was positively correlated with the degree of tubulointerstitial fibrosis (r = 0.959, P <0.001) and nuclear transcription factor binding activity (NF-κB: r = 0.773, 1: r = 0.619, P = 0.01). Conclusion The inflammatory mediators OPN and nuclear transcription factor are involved in the renal tubulointerstitial damage of chronic CsA.