去甲斑蝥素通过抑制蛋白磷酸酶2Ac抗肾间质纤维化

Norcantharidin inhibits renal interstitial fibrosis through down-regulating protein phosphatase-2Ac

目的研究蛋白磷酸酶2Ac（PP2Ac）和肾间质纤维化的关系，探讨去甲斑蝥素（NCTD）对单侧输尿管梗阻（UUO）大鼠模型肾间质纤维化的作用及其机制。方法16只雄性SD大鼠随机分成4组：假手术组（Sham组）、UUO3d组、UUO7d组及UUO14d组，每组各4只，分别于术后1d、3d、7d、14d处死。免疫组化法检测肾组织PP2Ac、纤连蛋白（FN）、I型胶原（C01．I）的表达。20只雄性SD大鼠随机分成4组，每组5只：假手术组（Sham组）、UUO模型组、NCTD低剂量治疗组（0．05mg·kg-1·d。）及NCTD高剂量治疗组（0．1mg·kg-1·d-1）。从建立UUO模型前1d始，治疗组分别予以NCTD0．05mg·kg·d。和0．1mg·kg-1·d。腹腔注射，Sham组和UUO模型组仅予以等量生理盐水腹腔注射，术后第14天处死大鼠。免疫组化、Western印迹和实时荧光定量PCR法检测肾组织FN、Col—I、d—SMA、E．cadherin和PP2Ac蛋白及mRNA的表达。结果（1）与Sham组相比，随着梗阻时间的延长，PP2Ac的表达逐渐增多，UUO14d表达最高，同时伴随FN、C01．I的表达升高（均P〈0．05）。相关分析结果显示，PP2Ac与FN、Col—I表达呈正相关（r=0．894和r=0．887，均P〈0．05）。（2）与Sham组相比，UUO模型组FN、C01．I、a-SMA表达明显上调，而E．cadherin表达明显下降，同时伴PP2Ac表达上调（均P〈0．05）；与UUO模型组相比，NCTD组剂量依赖性抑制FN、C01-I的表达，下调a．SMA的表达，上调E—eadherin的表达，同时抑制PP2Ae的表达（均P〈0．05）。结论PP2Ae与肾间质纤维化程度呈正相关；NCTD可抑制肾问质纤维化，其机制可能与抑制PP2Ae的表达有关。

Objective To observe the relationship between protein phosphatase-2Ac （PP2Ac） and renal interstitial fibrosis, and to investigate the effects and the underlying mechanism of norcantharidin （NCTD） on renal fibrosis in unilateral ureteral obstruction （UUO） rats. Methods Sixteen male Sprague-Dawley rats were randomly divided into four groups： Sham operation group, UUO day 3 group, UUO day 7 group and UUO day 14 group. The rats were sacrificed at day 1, 3, 7, 14 respectively and kidneys were harvested. Immunohistochemical staining were used to detect the expression of fibroneetin （FN）, type I collagen （Col-I） and PP2Ac. Another twenty male Sprague-Dawley rats were also randomly divided into four groups： Sham operation group, UUO group, low dose NCTDtreatment group （0.05 mg. kg-l. d-l） and high dose NCTD treatment group （0.1 mg. kg-l. d-l）. The rats in NCTD treatment groups were injected with norcantharidin into abdominal cavity 1 day before operation, while the Sham and UUO group were injected with equal normal saline. Rats were sacrificed at day 14 after surgery and the kidneys were harvested. Immunohistochemical staining, Western blotting and real- time PCR were used to detect the expression of FN, Col-I, a-SMA, E-cadherin and PP2Ac. Results （1）Compared with sham group, the expression of PP2Ac, FN and Col-I increased with the development of ureteral obstruction （all P 〈 0.05）. The expression of PP2Ac was positively correlated with the expression of FN and Col- I （r = 0.894 and 0.887, all P 〈 0.05）. （2）Compared with sham group, the expression of FN, Col-I, a-SMA increased and the E-cadherin decreased in UUO group, the expression of PP2Ac also increased （all P 〈 0.05）. After the treatment of NCTD, the above changes were all alleviated in a dose dependent manner, and the expression of PP2Ac was down-regulated （all P〈 0.05）. Conclusion NCTD can ameliorate tubulointerstitial fibrosis and its anti- fibrosis effect may be related to its inhibition to PP2Ac.