CAJ | 학술논문

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)、UUO 3 d组、UUO 7 d组及UUO 14 d组,每组各4只,分别于术后1d、3d、7d、14 d处死.免疫组化法检测肾组织PP2Ac、纤连蛋白(FN)、Ⅰ型胶原(Col-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(0.05 mg·kg-1·d-1)及NCTD高剂量治疗组(0.1 mg· kg-1·d-1).从建立UUO模型前1d始,治疗组分别予以NCTD 0.05 mg· kg-1·d-1和0.1 mg·kg-1·d-1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、α-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,UUO 14 d表达最高,同时伴随FN、Col-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、Col-Ⅰ、α-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、Col-Ⅰ的表达,下调α-SMA的表达,上调E-cadherin的表达,同时抑制PP2Ac的表达(均P＜0.05).结论 PP2Ac与肾间质纤维化程度呈正相关;NCTD可抑制肾间质纤维化,其机制可能与抑制PP2Ac的表达有关.
목적 연구단백린산매2Ac(PP2Ac)화신간질섬유화적관계,탐토거갑반모소(NCTD)대단측수뇨관경조(UUO)대서모형신간질섬유화적작용급기궤제.방법 16지웅성SD대서수궤분성4조:가수술조(Sham조)、UUO 3 d조、UUO 7 d조급UUO 14 d조,매조각4지,분별우술후1d、3d、7d、14 d처사.면역조화법검측신조직PP2Ac、섬련단백(FN)、Ⅰ형효원(Col-Ⅰ)적표체.20지웅성SD대서수궤분성4조,매조5지:가수술조(Sham조)、UUO모형조、NCTD저제량치료조(0.05 mg·kg-1·d-1)급NCTD고제량치료조(0.1 mg· kg-1·d-1).종건립UUO모형전1d시,치료조분별여이NCTD 0.05 mg· kg-1·d-1화0.1 mg·kg-1·d-1복강주사,Sham조화UUO모형조부여이등량생리염수복강주사,술후제14천처사대서.면역조화、Western인적화실시형광정량PCR법검측신조직FN、Col-Ⅰ、α-SMA、E-cadherin화PP2Ac단백급mRNA적표체.결과 (1)여Sham조상비,수착경조시간적연장,PP2Ac적표체축점증다,UUO 14 d표체최고,동시반수FN、Col-Ⅰ적표체승고(균P＜0.05).상관분석결과현시,PP2Ac여FN、Col-Ⅰ표체정정상관(r=0.894화r=0.887,균P＜0.05).(2)여Sham조상비,UUO모형조FN、Col-Ⅰ、α-SMA표체명현상조,이E-cadherin표체명현하강,동시반PP2Ac표체상조(균P＜0.05);여UUO모형조상비,NCTD조제량의뢰성억제FN、Col-Ⅰ적표체,하조α-SMA적표체,상조E-cadherin적표체,동시억제PP2Ac적표체(균P＜0.05).결론 PP2Ac여신간질섬유화정도정정상관;NCTD가억제신간질섬유화,기궤제가능여억제PP2Ac적표체유관.
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 1,3,7,14respectively and kidneys were harvested.Immunohistochemical staining were used to detect the expression of fibronectin (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 NCTD treatment group (0.05 mg· kg-1· d-1) and high dose NCTD treatment group (0.1 mg· kg-1· d-1).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 14after surgery and the kidneys were harvested.Immunohistochemical staining,Western blotting and realtime PCR were used to detect the expression of FN,Col-Ⅰ,α-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,α-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.