Apelin对单侧输尿管结扎小鼠肾脏纤维化的影响研究

发布时间：2018-08-27 11:07

【摘要】：目的探讨生物活性肽Apelin对单侧输尿管结扎(UUO)小鼠肾脏纤维化的影响及相关机制,分析在肾脏纤维化时血清和肾脏局部Apelin/APJ系统的代偿性变化。方法 2014年1月选取7周龄雄性C57Bl/6j小鼠共60只,构建UUO的小鼠模型,并设立假手术组(Sham)作为对照。术后每天给小鼠腹腔注射Apelin-13(0.1μmol/kg)或等量0.9%氯化钠溶液(vehicle)。根据小鼠手术方式和术后用药情况分为4组(15只/组):Sham+vehicle组、Sham+Apelin组、UUO+vehicle组、UUO+Apelin组。术后2周将小鼠处死,取手术侧肾脏进行检测。采用Masson染色观察肾脏纤维化程度;采用实时荧光定量PCR(RT-PCR)法检测肾脏细胞外基质胶原〔纤维连接蛋白(Fibronectin)、Ⅰ型胶原(Collagen I)、Ⅲ型胶原(CollagenⅢ)〕表达水平以及肾脏Apelin、APJ表达水平;采用Western blotting法检测肾小管上皮细胞标志物上皮细胞钙黏蛋白(E-cadherin)、间充质细胞标志物α平滑肌肌动蛋白(α-SMA)表达水平以及转化生长因子β(TGF-β)/Smads信号通路的主要信号分子(TGF-β1、TGF-βR1、p-Smad2)表达水平;采用ELISA法检测小鼠血清Apelin表达水平。结果 4组小鼠肾脏纤维化程度比较,差异有统计学意义(P0.05)。UUO+vehicle组和UUO+Apelin组小鼠肾脏纤维化程度均高于Sham+vehicle组和Sham+Apelin组(P0.05);UUO+Apelin组小鼠肾脏纤维化程度低于UUO+vehicle组(P0.05)。4组小鼠肾脏Fibronectin、CollagenⅠ、CollagenⅢ表达水平比较,差异均有统计学意义(P0.05)。UUO+vehicle组和UUO+Apelin组小鼠肾脏Fibronectin、CollagenⅠ、CollagenⅢ表达水平高于Sham+vehicle组和Sham+Apelin组(P0.05);UUO+Apelin组小鼠肾脏Fibronectin、CollagenⅠ、CollagenⅢ表达水平低于UUO+vehicle组(P0.05)。4组小鼠肾脏E-cadherin、α-SMA表达水平比较,差异均有统计学意义(P0.05)。UUO+vehicle组和UUO+Apelin组小鼠肾脏E-cadherin表达水平低于Sham+vehicle组和Sham+Apelin组,α-SMA表达水平高于Sham+vehicle组和Sham+Apelin组(P0.05);UUO+Apelin组小鼠肾脏E-cadherin表达水平高于UUO+vehicle组,α-SMA表达水平低于UUO+vehicle组(P0.05)。4组小鼠肾脏TGF-β1、TGF-βR1、p-Smad2表达水平比较,差异均有统计学意义(P0.05)。UUO+vehicle组和UUO+Apelin组小鼠肾脏TGF-β1、TGF-βR1、p-Smad2表达水平均高于Sham+vehicle组和Sham+Apelin组(P0.05);UUO+Apelin组小鼠肾脏TGF-β1、TGF-βR1、p-Smad2表达水平均低于UUO+vehicle组(P0.05)。4组小鼠肾脏Apelin表达水平比较,差异无统计学意义(P0.05);4组小鼠肾脏APJ、血清Apelin表达水平比较,差异有统计学意义(P0.05)。UUO+vehicle组和UUO+Apelin组小鼠肾脏APJ表达水平高于Sham+vehicle组和Sham+Apelin组,血清Apelin表达水平低于Sham+vehicle组和Sham+Apelin组(P0.05);UUO+Apelin组小鼠肾脏APJ表达水平低于UUO+vehicle组,血清Apelin表达水平高于UUO+vehicle组(P0.05)。结论 Apelin通过干扰经典的TGF-β/Smads信号通路抑制UUO小鼠肾脏的肾小管上皮-间充质转分化,进而减轻肾脏纤维化。在肾脏纤维化时,小鼠血清Apelin表达水平降低,肾脏APJ表达水平升高,可能对慢性肾脏病的进展起到一定的代偿作用。
[Abstract]:Objective To investigate the effect of bioactive peptide Apelin on renal fibrosis in unilateral ureteral ligation (UUO) mice and its related mechanism, and to analyze the compensatory changes of Apelin/APJ system in serum and kidney during renal fibrosis. The mice were injected intraperitoneally with Apelin-13 (0.1 micromol/kg) or 0.9% sodium chloride solution (vehicle) every day after operation. The mice were divided into four groups (15 mice/group) according to the operation mode and the medication after operation: Sham+vehicle group, Sham+Apelin group, UUUO+vehicle group, UUUUO+Apelin group. The mice were killed 2 weeks after operation and the kidneys on the operation side were taken for detection. Masson staining was used to observe the degree of renal fibrosis; real-time fluorescence quantitative PCR (RT-PCR) was used to detect the expression of extracellular matrix collagen (fibroronectin), collagen type I (Collagen I), collagen type I I I (Collagen I I I)) and Apelin and APJ; Western blotting was used to detect the epithelial fineness of renal tubules. The expression levels of E-cadherin, alpha-smooth muscle actin (alpha-SMA) and major signal molecules (TGF-beta, TGF-beta R1, p-Smad2) in the transforming growth factor beta (TGF-beta) / Smads signaling pathway were detected by ELISA. Results The expression levels of Apelin in serum of mice in four groups were detected by ELISA. The degree of renal fibrosis in UUO + vehicle group and UUO + Apelin group was higher than that in Sham + vehicle group and Sham + Apelin group (P 0.05); the degree of renal fibrosis in UUO + Apelin group was lower than that in UUO + vehicle group (P 0.05). The expression levels of Fibronectin, Collagen I and Collagen III in UUO+vehicle group and UUO+Apelin group were higher than those in Sham+vehicle group and Sham+Apelin group (P 0.05), while the expression levels of Fibronectin, Collagen I and Collagen III in UUO+Apelin group were lower than those in UUO+vehicle group (P 0.05). The expression level of E-cadherin in kidney of UUO+vehicle group and UUO+Apelin group was lower than that of Sham+vehicle group and Sham+Apelin group, and the expression level of alpha-SMA was higher than that of Sham+vehicle group and Sham+Apelin group (P 0.05). The expression levels of TGF-beta 1, TGF-beta 1 and p-Smad2 in the kidneys of UUO+vehicle group and UUO+Apelin group were lower than those of UUO+vehicle group (P 0.05). The expression levels of TGF-beta 1, TGF-beta 1 and p-Smad2 in the kidneys of UUO+vehicle group and UUUO+Apelin group were higher than those of Sham+vehicle group and Sham+Apelin group (P 0.05). The expression levels of GF-beta R1 and p-Smad2 were lower in UUO+vehicle group than in UUO+vehicle group (P 0.05). There was no significant difference in the expression levels of Apelin between the four groups (P 0.05). The expression levels of APJ in kidney and serum were significantly higher in UUO+vehicle group and UUO+vehicle group than in Sham+vehicle group and Sham+vehicle group (P 0.05). The expression of Apelin was lower in the + Apelin group than in the Sham + vehicle group and Sham + Apelin group (P 0.05). The expression of APJ in the kidney of UUO + Apelin group was lower than that of UUO + vehicle group, and the expression of Apelin in the serum was higher than that of UUO + vehicle group (P 0.05). Conclusion Apelin inhibited tubular epithelial-mesenchymal hypertrophy in the kidney of UUUO mice by interfering with the classical TGF-beta/Smads signaling pathway. In the course of renal fibrosis, the expression of Apelin in serum and APJ in kidney are decreased, which may play a compensatory role in the progression of chronic kidney disease.
【作者单位】：首都医科大学附属北京友谊医院肾内科;
【基金】：国家自然科学基金资助项目(81570660,81300607) 北京市自然科学基金课题(7132091)
【分类号】：R692

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