茯苓酸对梗阻性肾积水间质纤维化的作用

Study of protective effect of pachymic acid on obstructive hydronephrosis interstitial fibrosis

目的 探讨茯苓酸（PA）改善梗阻性肾积水后导致的肾间质纤维化的作用及其机制.方法 将28只C57雄性小鼠按照随机列表法分为4组：A组：假手术组;B组：模型组;C组：茯苓酸小剂量组;D组：茯苓酸大剂量组.建模及给药7d后,处死小鼠并取材,苏木素-伊红（HE）染色观察肾脏病理损伤变化,天狼猩红染色检测间质胶原纤维的表达,免疫组织化学染色及实时定量聚合酶链反应（Real-time PCR）检测间质中胶原纤维蛋白-1（COI-1）、纤维粘连蛋白（FN）和平滑肌肌动蛋白（α-SMA）的表达,Western blot检测转化生长因子-β1（TGF-β1）,磷酸化Smad2/3（pSmad2/3）,基质金属蛋白酶-9（MMP-9）的表达.结果 输尿管梗阻（UUO）小鼠给予茯苓酸治疗7d后,HE染色显示肾脏病理改变较对照组减轻.天狼猩红染色可见肾间质纤维化程度较对照组明显减轻,且大剂量组减轻更明显.免疫组织化学结果显示COI-1、FN、α-SMA在梗阻后表达明显升高,给予茯苓酸治疗可降低基质中COI-1、FN、α-SMA的表达.Real-time PCR结果显示,在模型组、小剂量茯苓酸组、大剂量茯苓酸组COI-1 mRNA水平相对表达量分别为4.20±1.42、2.30±0.76、1.70 ±0.70;FN在mRNA水平相对表达量分别为5.20 ±2.22、3.00±1.70、1.70±1.06;α-SMA在mRNA水平相对表达量分别4.60±1.23、2.30 ±0.98、1.40 ±0.70.给药组与模型组比较,差异有统计学意义（P＜0.05）.Western blot结果显示茯苓酸可下调TGF-β1、pSmad2/3、MMP-9的表达.结论茯苓酸可改善单侧肾积水导致的间质纤维化,可能是通过下调TGF-β1、pSmad2/3、MMP-9的表达来实现的.

Objective To investigate protective effects of pachymic acid （PA） on renal interstitial fibrosis induced by hydronephrosis of obstructive models and its potential mechanism.Methods Twentyeight male C57 mice were divided into four groups according to random list method,each containing seven mice.A：sham operation group; B：model group; C：a small dose pachymic acid group; D：a high dose pachymic acid group.All mice were sacrificed on 7 days after operation and tubular injury was observed using hematoxylin and eosin （HE） staining.Sirius red staining was performed to detect the expression of collagen in interstitial,and immunohistochemistry and real-time polymerase chain reaction （Real-time PCR） were used to assay mRNA expressions of collagen-1 （COI-1）,fibronectin （FN） and α-smooth muscle actin （α-SMA）.Transforming growth factor-β1 （TGF-β1）,pSmad2/3,matrix metalloproteinase-9 （MMP-9） at protein levels were measured using Western blotting.Results Seven days after the treatment of pachymic acid in unilateral ureteral obstruction （UUO） model,HE staining indicated that renal tubular injury was ameliorated in the PA treated group,and the results of Sirius red staining showed that renal interstitial fibrosis were significantly reduced compared to model group,and it is more obvious in high dose group.The results of immunohistochemistry indicated that COI-1,FN,α-SMA expressions were significantly increased in model group,and the treatment of pachymic acid significantly inhibited the expressions of COI-1,FN and α-SMA in matrix.Meanwhile,mRNA levels of COI-1 detected by Real-time PCR in model group,the small dose of pachymic acid group and the large dose of pachymic acid group were 4.20 ± 1.42,2.30 ± 0.76,1.70 ± 0.70.Expressions of FN in three groups were 5.20 ± 2.22,3.00 ± 1.70,1.70 ± 1.06 and α-SMA expressions were 4.60 ± 1.23,2.30 ± 0.98,1.40 ± 0.70.There was statistical difference compared with the model group.The results of Western blotting showed pachymic acid could down-regulate expressions of TGF-β1,pSmad2/3 and MMP-9.Conclusion Pachymic acid could improve interstitial fibrosis induced by unilateral hydronephrosis,which might be achieved through the approach of suppressing the expressions of TGF-β1,pSmad2/3 and MMP-9.