非诺贝特治疗单侧输尿管结扎小鼠肾纤维化的机制研究

  目的  探讨非诺贝特在单侧输尿管结扎诱导的小鼠肾纤维化中的作用机制，为肾纤维化提供潜在的治疗靶点。  方法  成年雄性C57BL/6J小鼠31只，随机分为假手术组（Sham组，n=9）、单侧输尿管结扎组（UUO组，n=10）和单侧输尿管结扎+非诺贝特治疗组（UUO+Feno组，n=12）。UUO组及UUO+Feno组小鼠均结扎左侧输尿管，Sham组小鼠仅游离左侧输尿管，不做结扎处理。术后第二日起，UUO+Feno组每日给予10 mg/kg （终浓度为0.08 mg/mL）的非诺贝特溶液灌胃，持续14 d；其余两组每日灌胃等量生理盐水。术后第15 天灌胃结束后处死小鼠取材，检测血清肌酐和尿素氮，肾组织行HE染色、Masson染色和Sirius Red染色，检测肾组织羟脯氨酸含量，免疫组化染色检测肾组织α-平滑肌肌动蛋白（α-SMA）、Ⅰ型胶原（COLⅠ）蛋白表达，Western blot法检测肾组织α-SMA、COLⅠ蛋白表达变化，实时荧光定量（RT）- PCR法检测肾组织纤维化相关基因基质金属蛋白酶（MMP）2、MMP9、COLⅠA1、COLⅠA2、基质金属蛋白酶组织抑制因子-1（TIMP-1）、转化生长因子-β1（TGF-β1）、α-SMA mRNA的表达变化。  结果  与Sham组相比，UUO组小鼠血清肌酐及尿素氮水平升高（P<0.05）；与UUO组相比，UUO+Feno组小鼠血清肌酐及尿素氮水平降低（P<0.05）。HE染色、Masson染色、Sirius Red染色以及肾羟脯氨酸含量的结果均表明UUO组较Sham组胶原沉积明显增多，炎性细胞浸润明显，UUO+Feno组较UUO组胶原沉积程度显著减轻，炎性细胞浸润程度显著改善，对肾纤维化的启动阶段有抑制作用。免疫组化结果显示:与Sham组相比，UUO组 α -SMA、COLⅠ在肾组织中的表达水平升高（P<0.05）；与UUO相比，UUO+Feno组α-SMA、COLⅠ在肾组织中的表达水平降低（P<0.05）；RT-PCR和Western blot结果显示:与Sham组相比，UUO组纤维化相关因子的mRNA及α-SMA、COLⅠ蛋白表达水平均升高（P<0.05）；与UUO组相比，UUO+Feno组纤维化相关因子的mRNA及蛋白表达水平均降低（P< 0.05）。  结论  非诺贝特通过调控与肾脏纤维化相关因子的表达水平改善肾脏纤维化。

A Study on the Role and Mechanism of Fenofibrate in Mice Renal Fibrosis Induced by Unilateral Ureteral Obstruction

  Objective  To investigate the role and mechanism of fenofibrate in renal fibrosis induced by unilateral ureteral obstruction in mice, and to provide a potential therapeutic target for renal fibrosis.  Methods  31 adult male C57BL/6J mice were randomly divided into Sham operation group (Sham, n=9), unilateral ureteral obstruction group (UUO, n=10) and unilateral ureteral obstruction+ fenofibrate group (UUO+Feno, n=12). Mice in both the UUO group and UUO+Feno groups were ligated with left ureter, and the the mice in Sham group freed the left ureter without ligation. From the second day after the operation, the UUO+Feno group was daily intragastrically administrated with 10 mg/kg of fenofibrate normal saline solution (final concentration was 0.08 mg/mL) for 15 d, and the other two groups were intragastrically administrated with the same amount of normal saline solution. At 15th day postoperation after intragastric administration, mice were sacrificed, and the concentration of serum creatinine and blood urea nitrogen were detected, the kidney tissues were performed HE staining, Masson dyeing and Sirius Red staining, and the content of renal tissue hydroxyproline were determined. Besides, immunohistochemical staining was used to explore the expressions of α-smooth muscle actin (α-SMA), Collegan-Ⅰ (COL Ⅰ) protein in renal tissue, Western blot was carried out to observe the changes of the expression levels of kidney α-SMA and COL Ⅰ proteins, and real-time fluorescent quantitative (RT)-PCR method was performed to detect the changes of mRNA expression levels of renal tissue fibrosis related genes matrix metalloproteinase (MMP)2, MMP9, COLⅠA1, COLⅠA2, tissue inhibitors of metalloproteinases (TIMP)-1, transforming growth factor (TGF)-β1, α-SMA.  Results  Compared with the Sham group, the serum creatinine and blood urea nitrogen levels of UUO group increased (P<0.05); compared with UUO group, the serum creatinine and blood urea nitrogen levels of UUO+Feno group were significantly lower (P<0.05). The results of HE staining, Masson staining, Sirius Red staining and renal hydroxyproline content indicated that the collagen deposition in UUO+Feno group was significantly reduced compared with that in UUO group. Immunohistochemical staining results showed that, compared with UUO group, the expression levels of α-SMA, COL Ⅰin kidney tissues of UUO+Feno group were significantly reduced; Western blot and RT-PCR results showed that compared with the UUO group, the mRNA and protein expression levels of fibrotic factors were significantly reduced in the UUO+Feno group (P<0.05).  Conclusion  Fenofibrate reduced mice renal fibrosis caused by unilateral ureteral obstruction and its mechanism may be relate to its regulation effect on the expressions of renal tissue fibrosis related genes.