摘要
目的通过观察塞来昔布（CXB）对常染色体显性多囊肾病（ADPKD）动物模型Han：SPRD大鼠的作用，从而研究CXB延缓肾衰竭进展的机制。方法选取3周龄杂合（cy／＋）Han：SPRD大鼠共57只，随机分成3组（每组19只）进行实验。按照CXB在饲料中的含量分为对照组（0mg·kg^-1·d^-1）、小剂量组（3mg·kg^-1·d^-1）和大剂量组（10mg·kg^-1·d^-1）。在大鼠4、6、8、10、12和16周龄时测定血清BUN、Scr。ELISA法检测16周龄大鼠血浆6-酮前列腺素F1α（6-keto—PGF-1α）和血栓烷B2（TXB2）的含量。实时荧光定量PCR法检测大鼠肾组织肿瘤坏死因子α（TNF—α）的mRNA水平。免疫荧光共聚焦显微镜检测TNF—α和环氧化酶2（COX-2）的蛋白表达。Western印迹检测16周龄大鼠TNF—α的蛋白表达量。结果对照组BUN、Scr持续升高，分别于6、8周龄时即大于正常范围；至16周龄时，BUN为（26．56±9．19）mmol／L，Scr为（95．08±67．54）μmol／L；CXB小剂量组和大剂量组均能减缓BUN、Scr上升速度，减小其上升幅度。16周龄大鼠CXB小剂量组血浆中6-keto—PGF-α和TXB2[（1831．68±233．31）ng／L和（156．62±9．29）ng／L1和大剂量组两者的含量[（1148．57±105.80）ng／L和（157．87±10．16）ng／L]均显著低于对照组[（2792．26±830．48）ng／L和（248．88±93．72）ng／L]（均P〈0．05）。实时荧光定量PCR结果示，16周龄大鼠肾组织小剂量组和大剂量组TNF—α mRNA水平均显著低于对照组（均P〈0．05）。免疫荧光共聚焦显微镜显示，对照组TNF-α和COX-2在小管间质区高表达，小剂量组和大剂量组荧光强度显著减弱。与对照组比较，CXB小剂量组和大剂量组大鼠肾组织蛋白表达量显著减少，差异有统计学意义（均P〈0．05）。结论CXB可能通过抑制COX-2的活性，阻止膜磷脂释放花生四烯酸代谢产物6-keto—PGF-1α和TXB2，减少炎性因子TNF-α的含量，正反馈避免诱导COX-2的高表达，发挥抗炎作用来延缓肾衰竭的进展。
Objective To investigate the amelioration mechanism of renal dysfunction by Celeeoxib （CXB） through the observation of CXB on Han： SPRD rats with ADPKD. Methods Fifty-seven 3-week-old male Han：SPRD heterozygous （Cy/＋） rats were randomly divided into 3 groups （n=19）. One group was fed with normal forage （control group）, another two groups were fed with low dosage （3 mg·kg^-1·d^-1） and high dosage （10 mg·kg^-1·d^-1） administration of CXB respectively. The BUN and Scr were determined respectively at 3, 5, 7, 9, 12 and 16 weeks. The content of 6-keto-PGF-1α and TXB2 was measured by enzyme-linked immunosorbent assay （ELISA）. The expression of TNF-α mRNA was detected by real-time RT-PCR assay. The coexpression of TNF-α and COX-2 was examined by double immunofluorescence labeling technique and laser scanning confocal microscopy. The expression of TNF-α protein was detected by Western blotting. Results BUN and Scr increased continuously in control group and exceeded the normal at 6-week-old and at 8-week-old respectively. At 16-week-old, BUN and Scr were （26.56±9.19） mmol/L and （95.08±67.54）μmol/L. After being treated with CXB, the progression of BUN and Scr was reduced in both low and high dosage group. The content of 6-keto-PGF-1α and TXB2 in low [（1831.68±233.31） ng/L and （156.62±9,29） ng/L] and high dosage group [（1148.57±105.80） ng/L and （157.87±10.16）ng/L] was significantly lower than those in control group [（2792.26±830.48） ng/L and （248.88±93.72） ng/L]. TNF-α mRNA levels in low[（2.52±0.01）×10^3} and high dosage group[（2.48±0.02）×10^3] were both decreased as compared to control group[（6.17±0.19）×10^3]. The co-expression of TNF-α and COX-2 distributed widely in tubulointerstitial area in control group and only few in low dosage group. More TNF-α protein in CXB-treated group was detected than that in control group. Conclusion CXB can slow down disease progression in Han： SPRD rats with renal dysfunction through anti-inflammatory effect, including inhibition of COX-2 activity, blockage of 6-keto-PGF-1α and TXB 2 release, and down-regulation of COX-2 over expression by positive feedback.
出处
《中华肾脏病杂志》
CAS
CSCD
北大核心
2009年第10期765-770,共6页
Chinese Journal of Nephrology
基金
国家自然科学基金（30330640,30271523,30570867）志谢衷心感谢美国Mayo Clinic Department of Nephrology and Hypertention MD Qi Qian惠赠Han：SPRD大鼠
衷心感谢第二军医大学电镜室汤莹老师对免疫荧光共聚焦扫描所提供的帮助和指导
