CAJ | 학술논문

目的证实去甲斑蝥素(NCTD)具抗肾间质纤维化的作用,其抗肾间质纤维化的机制与靶向抑制蛋白磷酸酶2Ac(PP2Ac)对Smad3中间连接区(Smad3-L)的去磷酸化修饰有关.方法常规培养人肾小管上皮细胞(HK-2),转染PP2Ac shRNA质粒后,细胞分为5组:(1)空白组;(2)TGF-β1(5μg/L)组;(3) TGF-β1 +NCTD (2.5 mg/L)组;(4)TGF-β1+PP2Ac shRNA组;(5)TGF-β1+PP2Ac shRNA+NCTD组.采用实时荧光定量PCR和Western印迹检测PP2Ac、纤维连接蛋白(FN)、胶原蛋白Ⅰ (Col-Ⅰ)、α-平滑肌肌动蛋白(α-SMA)和E-钙黏蛋白(Ecadherin) mRNA及蛋白的表达.然后将HK-2细胞随机分为3组:(1)空白组;(2)TGF-β1组;(3)TGF-β1+NCTD组,采用免疫荧光检测pSmad3-L(Ser204)和pSmad3-L(Ser208)在HK-2细胞的分布,Western印迹检测HK-2细胞核蛋白pSmad3-L(Se204)和pSmad3-L(Ser208)的表达.结果 (1)TGF-β1促进HK-2细胞PP2Ac表达,同时上调FN、Col-Ⅰ和α-SMA的表达,下调E-cadherin 的表达.NCTD和PP2Ac shRNA均抑制PP2Ac的表达,下调FN、Col-Ⅰ和α-SMA表达,上调E-cadherin的表达.但NCTD与小干扰RNA共同作用对PP2Ac表达的抑制,以及对TGF-β1诱导的上述指标的改变程度同单纯PP2Ac小干扰RNA干预组无明显差异.(2)TGF-β1刺激HK-2细胞核内pSmad3-L(Ser204)、pSmad3-L(Ser208)表达明显增多,NCTD干预使pSmad3-L(Ser204)、pSmad3-L (Ser208)在细胞核内表达进一步增多.结论 NCTD通过抑制PP2Ac介导的Smad3-L区去磷酸化,发挥抗肾间质纤维化作用.
목적 증실거갑반모소(NCTD)구항신간질섬유화적작용,기항신간질섬유화적궤제여파향억제단백린산매2Ac(PP2Ac)대Smad3중간련접구(Smad3-L)적거린산화수식유관.방법 상규배양인신소관상피세포(HK-2),전염PP2Ac shRNA질립후,세포분위5조:(1)공백조;(2)TGF-β1(5μg/L)조;(3) TGF-β1 +NCTD (2.5 mg/L)조;(4)TGF-β1+PP2Ac shRNA조;(5)TGF-β1+PP2Ac shRNA+NCTD조.채용실시형광정량PCR화Western인적검측PP2Ac、섬유련접단백(FN)、효원단백Ⅰ (Col-Ⅰ)、α-평활기기동단백(α-SMA)화E-개점단백(Ecadherin) mRNA급단백적표체.연후장HK-2세포수궤분위3조:(1)공백조;(2)TGF-β1조;(3)TGF-β1+NCTD조,채용면역형광검측pSmad3-L(Ser204)화pSmad3-L(Ser208)재HK-2세포적분포,Western인적검측HK-2세포핵단백pSmad3-L(Se204)화pSmad3-L(Ser208)적표체.결과 (1)TGF-β1촉진HK-2세포PP2Ac표체,동시상조FN、Col-Ⅰ화α-SMA적표체,하조E-cadherin 적표체.NCTD화PP2Ac shRNA균억제PP2Ac적표체,하조FN、Col-Ⅰ화α-SMA표체,상조E-cadherin적표체.단NCTD여소간우RNA공동작용대PP2Ac표체적억제,이급대TGF-β1유도적상술지표적개변정도동단순PP2Ac소간우RNA간예조무명현차이.(2)TGF-β1자격HK-2세포핵내pSmad3-L(Ser204)、pSmad3-L(Ser208)표체명현증다,NCTD간예사pSmad3-L(Ser204)、pSmad3-L (Ser208)재세포핵내표체진일보증다.결론 NCTD통과억제PP2Ac개도적Smad3-L구거린산화,발휘항신간질섬유화작용.
Objective To investigate the inhibition of interstitial fibrosis by NCTD is related to the dephosphorylation of Smad3 linker region mediated by the inhibition of PP2Ac.Methods HK -2 cells were cultured and devided into 5 groups:(1) normal control group;(2) TGF-β1 group (5 μg/L);(3) TGF-β1 +NCTD group (2.5 mg/L);(4) TGF-β1 +PP2Ac shRNA group;(5)TGF-β1 +PP2Ac shRNA+ NCTD group.Real-time PCR and Western blot were used to detect the expression of PP2Ac,FN,Col-Ⅰ,α-SMA and E-cadherin.Additionally,the HK-2 cells were assigned to three groups:(1) normal control group;(2) TGF-β1 group;(3) TGF-β1 +NCTD group.Immunofluorescence were used to analysis the distribution of pSmad3-L(Ser204) and pSmad3-L(Ser208).Western blot analysis were used to detect the protein expression of pSmad3-L(Ser204) and pSmad3-L(Ser208).Results (1) TGF-β1 stimulated the expression of PP2Ac in HK-2 cells,increased the expression of FN,Col-Ⅰ and α-SMA,and decreased the expression of E-cadherin.Both NCTD and PP2Ac shRNA could inhibit PP2Ac expression accompanied with the downregulation of FN,Col-Ⅰ and α-SMA,and upregulation of E-cadherin.However,compared with PP2Ac shRNA transfected group,cells transfecting with PP2Ac shRNA and incubated with NCTD showed no obvious differences on the relief of the above indicators induced by TGF-β1 in HK2 cells.(2)The expression of pSmad3-L(Ser204) and pSmad3-L(Ser208) in the nucleus of HK2 cells stimulated by TGF-β1 was significantly elevated.The expression of pSmad3-L(Ser204) and pSmad3-L(Ser208) in the nucleus was further upregulated when treated with NCTD.Conclusions NCTD has anti-fibrosis effect and it may be due to the inhibition the dephosphorylation of Smad3 linker region mediated by the inhibition of PP2Ac.