CAJ | 학술논문

目的研究N-乙酰基-丝氨酰-天门冬氨酰-赖氨酰-脯氨酸(Ac-SDKP)通过对乙酰化微管蛋白α(Ac-Tub α)的调控,抑制矽肺肌成纤维细胞转化的作用及机制.方法构建矽肺大鼠模型,分为对照组(4、8周)、矽肺模型组(4、8周)、Ac-SDKP抗纤维化治疗组和预防治疗组;原代肺成纤维细胞分为对照组、血管紧张素(Ang)Ⅱ诱导组、Ac-SDKP预处理组、血管紧张素Ⅱ受体1(AT1)阻滞剂(缬沙坦)预处理组、组蛋白去乙酰化酶(HDAC)6选择性抑制剂(TCS HDAC6 20b)预处理组.免疫组化及免疫荧光染色法观察Ac-Tub α、α-平滑肌肌动蛋白(α-SMA)在肺组织或细胞中的表达与定位.免疫印迹法检测肺组织或细胞中Ⅰ型胶原、α-SMA、Ac-Tubα、HDAC6蛋白的表达.结果免疫组化结果显示,矽肺模型组矽结节和间质纤维化区域Ac-Tubα表达缺如,α-SMA阳性表达.Ac-SDKP能够抑制Ⅰ型胶原、α-SMA、HDAC6蛋白的表达,其中抗纤维化治疗组与矽肺模型8周组比较下降至48.39％、52.63％和70.18％,预防治疗组与矽肺模型8周组比较下降至32.26％、64.91％和54.39％;在Ac-SDKP抗纤维化治疗组和预防治疗组Ac-Tub α蛋白表达为矽肺模型8周组的3.00和2.90倍,以上结果经方差分析差异均有统计学意义(P＜0.05).Ang Ⅱ诱导组Ac-Tubα表达减弱为对照组的44.44％.而AngⅡ诱导组的α-SMA、HDAC6和Ⅰ型胶原表达增多,分别是对照组的1.66、3.56和4.00倍(P＜0.05).予以Ac-SDKP、缬沙坦或TCSHDAC6 20b预处理,能明显抑制该变化.结论 Ac-SDKP抑制矽肺大鼠肌成纤维细胞转化和胶原沉积,发挥抗矽肺纤维化的作用,可能与下调HDAC6和促进Ac-Tub α的表达有关.
목적 연구N-을선기-사안선-천문동안선-뢰안선-포안산(Ac-SDKP)통과대을선화미관단백α(Ac-Tub α)적조공,억제석폐기성섬유세포전화적작용급궤제.방법 구건석폐대서모형,분위대조조(4、8주)、석폐모형조(4、8주)、Ac-SDKP항섬유화치료조화예방치료조;원대폐성섬유세포분위대조조、혈관긴장소(Ang)Ⅱ유도조、Ac-SDKP예처리조、혈관긴장소Ⅱ수체1(AT1)조체제(힐사탄)예처리조、조단백거을선화매(HDAC)6선택성억제제(TCS HDAC6 20b)예처리조.면역조화급면역형광염색법관찰Ac-Tub α、α-평활기기동단백(α-SMA)재폐조직혹세포중적표체여정위.면역인적법검측폐조직혹세포중Ⅰ형효원、α-SMA、Ac-Tubα、HDAC6단백적표체.결과 면역조화결과현시,석폐모형조석결절화간질섬유화구역Ac-Tubα표체결여,α-SMA양성표체.Ac-SDKP능구억제Ⅰ형효원、α-SMA、HDAC6단백적표체,기중항섬유화치료조여석폐모형8주조비교하강지48.39％、52.63％화70.18％,예방치료조여석폐모형8주조비교하강지32.26％、64.91％화54.39％;재Ac-SDKP항섬유화치료조화예방치료조Ac-Tub α단백표체위석폐모형8주조적3.00화2.90배,이상결과경방차분석차이균유통계학의의(P＜0.05).Ang Ⅱ유도조Ac-Tubα표체감약위대조조적44.44％.이AngⅡ유도조적α-SMA、HDAC6화Ⅰ형효원표체증다,분별시대조조적1.66、3.56화4.00배(P＜0.05).여이Ac-SDKP、힐사탄혹TCSHDAC6 20b예처리,능명현억제해변화.결론 Ac-SDKP억제석폐대서기성섬유세포전화화효원침적,발휘항석폐섬유화적작용,가능여하조HDAC6화촉진Ac-Tub α적표체유관.
Objective To explore the inhibition effect and mechanism of N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) on myofibroblast differentiation via regulating acetylated tubulin α (Ac-Tub α) in vivo and in vitro.Methods Silicotic model were made by SiO2 douched and divided into 6 groups as follows:control (4w,8w) group,silicotic model (4w,8w) group and post-or pre-treatment by Ac-SDKP group.Pulmonary fibroblasts were divided into 5 groups:(1) control;(2) Ang Ⅱ;(3) Ang Ⅱ+Ac-SDKP;(4) Ang Ⅱ+Valsartan;(5)Ang Ⅱ+ TCS histone deacetylase (HDAC) 6 20b.The localization of Ac-Tub α and α-smooth muscle actin(SMA) were observed by immunohistochemical (IHC) and immunofluorescence staining.The protein levels of Ac-Tub α,α-SMA,collagen type Ⅰ (col Ⅰ) and HDAC6 were measured by western blot.Results In silicotic nodules and interstitial fibrosis area,positive expression of α-SMA,a classical marker of myofibroblast,was observed by IHC,accompanied with absence expression of Ac-Tub α.Furthermore,Ac-SDKP post-treatment could attenuate the levels of col Ⅰ,α-SMA and HDAC6 to 48.39％,52.63％ and 70.18％ compared with the silicotic 8w group respectively.And in Ac-SDKP pre-treatment group,compared with the silicotic 8w group,these protein levels were decreased to 32.26％,64.91％ and 54.39％ respectively (P＜0.05).The up-regulation of Ac-Tub α was found in Ac-SDKP post-and pre-treatment and increased to 3.00 and 2.90 folds compared with the silicotic 8w group.Compared with control group,the levels of α-SMA,HDAC6 and col Ⅰ in Ang Ⅱ group were up-regulated to 1.66,3.56 and 4.00 folds accompanied with down-regulation of Ac-Tub by 44.44％ (P＜0.05).Pre-treatment with Valsartan,TCS HDAC6 20b or Ac-SDKP could inhibited all this changes induced by Ang Ⅱ in vitro.Conclusion Ac-SDKP can inhibit the myofibroblast differentiation and collagen deposition via suppress HDAC6 and up-regulate the expression of Ac-Tub α in vivo and in vitro.