塞来昔布对Han：SPRD大鼠肾细胞外基质重塑的影响

目的 研究塞来昔布（CXB）对Han：SPRD大鼠肾细胞外基质（ECM）重塑的影响，探讨其抑制多囊肾肾间质纤维化的作用机制。 方法 选取杂合（cy/+）交配后第4代雄性、3周龄、体质量（68.5±16.6） g的Han：SPRD大鼠共57只，随机分成对照组（CXB：0 mg&#8226;kg-1&#8226;d-1）、CXB小剂量组（CBX：3 mg&#8226;kg-1&#8226;d-1）、CXB大剂量组（CBX：10 mg&#8226;kg-1&#8226;d-1），每组19只。另选19只SD大鼠作为正常对照。饲料中加入CXB喂食13周后，处死动物。倒置显微镜下分析肾组织纤维化指数；实时荧光定量PCR检测肾组织胶原Ⅳ（COLⅣ）、基质金属蛋白酶（MMP）2、金属蛋白酶2组织抑制剂（TIMP）和转化生长因子（TGF）β1 mRNA的表达；免疫荧光共聚焦扫描法检测COLⅣ、MMP-2、TIMP-2、TGF-β1与PCNA共染的蛋白丰度；蛋白免疫印迹法检测TGF-β1的表达。 结果 与对照组相比，小剂量组和大剂量组均能显著减少肾囊肿指数（42.90±6.56和47.10±7.28比64.80±62.71，均P < 0.05）、纤维化指数（11.20±2.63和10.10±3.30比16.30±4.16，均P < 0.05）和间质炎性细胞的浸润（2.60±0.26和2.80±0.31比3.70±0.33，均P < 0.05）。小剂量组和大剂量组COLⅣ、TIMP-2和TGF-β1 mRNA的表达量显著低于对照组（均P < 0.05 ），而MMP-2 mRNA的表达量显著高于对照组（均P < 0.05）。小剂量组和大剂量组COLⅣ荧光强度较对照组显著减弱，差异有统计学意义（20.30±5.11比61.40±4.51，P < 0.01；27.50±6.73比61.40±4.51，P < 0.05）。小剂量组和大剂量组MMP-2/TIMP-2比值较对照组增高，差异有统计学意义（4.88±1.52 和3.63±1.67比0.35±0.13，均P < 0.05）。小剂量组和大剂量组TGF-β1蛋白表达比对照组均显著减少。 结论 塞来昔布可能通过下调TGF-β1、增加MMP-2/TIMP-2比值、促进胶原Ⅳ的降解来抑制肾小管间质的纤维化。

Effect of celecoxib on extracellular matrix remodeling in Han:SPRD rats

Objective To investigate the effect of celecoxib (CXB) on extracellular matrix (ECM) remodeling in a model of autosomal dominant polycystic kidney (ADPKD).Methods Fifty seven Han:SPRD heterozygous (Cy/+) rats were randomly divided into 3 groups:control group,small dosage CXB group (3 mg·kg~(-1)·d~(-1)) and large dosage CXB group (10 mg·kg-1· d-1).Renal cystic index (CT),fibrosis index and inflammatory cells infiltration in interstitium were analyzed.The mRNA expression of typeⅣ collagen (COLⅣ),matrix matalloproteinase-2 (MMP-2),metalloproteinase-2 tissue inhibitor (TIMP-2) and transforming growth factor β1 (TGF-β1) was detected by real-time PCR.The co-expression of COL Ⅳ,MMP-2,TIMP-2,TGF-β1 and proliferating cell nuclear antigen (PCNA) was analyzed by double immunofluorescence labeling technique and laser scanning confocal microscopy.The protein expression of TGF-β1 was detected by Western blotting.Results CT (42.90±6.56 & 47.10±7.28 vs 64.80±6.71,all P＜0.05),fibrosis index (11.20±2.63 & 10.10±3.30 vs 16.30±4.16,all P＜0.05) and inflammatory cells infiltration (2.60±0.26 & 2.80±0.31 vs 3.70±0.33,all P＜0.05) in interstitium all decreased in small and large dosage group compared to control group.COLⅣ,TIMP-2 and TGF-β1 mRNA level decreased in both small and large dosage groups as well.Contrarily MMP-2 mRNA level increased in both groups(all P＜0.05).The co-expression of COLⅣ distributed widely in tubulointerstitial area in control group but only few in small (20.30±5.11 vs 61.40±4.51,P＜0.01) and large dosage group (27.50±6.73 vs 61.40±4.51,P＜0.05).MMP-2/TIMP-2 ratio increased in small dosage group (4.88±1.52 vs 0.35±0.13,P＜0.05) and large dosage group (3.63±1.67 vs 0.354±0.13,P＜0.05) as compared to control group.Western blotting showed the expression of TGF-β1 decreased in both small and large dosage groups (all P ＜0.05).ConclusionCXB can inhibit tubulointerstitial fibrosis in Han:SPRD rats by inhibiting the expression of TGF-β1,promoting COLⅣ degradation and increasing MMP-2/TIMP-2 ratio.