CAJ | 학술논문

目的探讨小檗碱对转化生长因子β1诱导的人胚肺成纤维细胞MRC-5转分化过程中细胞外羟脯氨酸和细胞内平滑肌肌动蛋白、纤连蛋白及丝裂原活化蛋白激酶信号通路相关蛋白磷酸化水平的影响.方法体外培养MRC-5细胞,采用 MTT法检测不同浓度小檗碱对细胞增殖的抑制作用,确定适合的作用范围;Western blot法检测不同浓度转化生长因子β1刺激MRC-5细胞转分化过程中平滑肌肌动蛋白、纤连蛋白表达水平,确定适宜的转化生长因子β1诱导浓度.不同浓度小檗碱预处理的MRC-5细胞经转化生长因子β1刺激后,采用比色法测定细胞外羟脯氨酸水平,对于细胞内平滑肌肌动蛋白、纤连蛋白表达水平以及丝裂原活化蛋白激酶信号通路相关蛋白p38 MAPK、SAPK/JNK、ERK1/2的磷酸化水平采用Western blot法进行检测.结果参考 MTT法检测结果,选定小檗碱适宜浓度(20、40、80μmol/L)进行研究.Western blot结果显示2 ng/ml浓度转化生长因子β1可明显诱导 MRC-5细胞转分化.小檗碱预处理的 MRC-5细胞,较单纯转化生长因子β1诱导细胞,培养液中羟脯氨酸水平明显降低.Western blot法检测结果显示,小檗碱预处理细胞较单纯转化生长因子β1诱导细胞,平滑肌肌动蛋白、纤连蛋白表达水平明显受到抑制,丝裂原活化蛋白激酶信号通路相关蛋白 p38 MAPK、SAPK/JNK、ERK1/2 磷酸化水平亦受到不同程度抑制.结论小檗碱预处理对转化生长因子β1诱导的MRC-5细胞转分化过程有抑制作用,其机制可能与小檗碱下调丝裂原活化蛋白激酶信号通路相关蛋白的磷酸化水平有关.
목적 탐토소벽감대전화생장인자β1유도적인배폐성섬유세포MRC-5전분화과정중세포외간포안산화세포내평활기기동단백、섬련단백급사렬원활화단백격매신호통로상관단백린산화수평적영향.방법 체외배양MRC-5세포,채용 MTT법검측불동농도소벽감대세포증식적억제작용,학정괄합적작용범위;Western blot법검측불동농도전화생장인자β1자격MRC-5세포전분화과정중평활기기동단백、섬련단백표체수평,학정괄의적전화생장인자β1유도농도.불동농도소벽감예처리적MRC-5세포경전화생장인자β1자격후,채용비색법측정세포외간포안산수평,대우세포내평활기기동단백、섬련단백표체수평이급사렬원활화단백격매신호통로상관단백p38 MAPK、SAPK/JNK、ERK1/2적린산화수평채용Western blot법진행검측.결과 삼고 MTT법검측결과,선정소벽감괄의농도(20、40、80μmol/L)진행연구.Western blot결과현시2 ng/ml농도전화생장인자β1가명현유도 MRC-5세포전분화.소벽감예처리적 MRC-5세포,교단순전화생장인자β1유도세포,배양액중간포안산수평명현강저.Western blot법검측결과현시,소벽감예처리세포교단순전화생장인자β1유도세포,평활기기동단백、섬련단백표체수평명현수도억제,사렬원활화단백격매신호통로상관단백 p38 MAPK、SAPK/JNK、ERK1/2 린산화수평역수도불동정도억제.결론 소벽감예처리대전화생장인자β1유도적MRC-5세포전분화과정유억제작용,기궤제가능여소벽감하조사렬원활화단백격매신호통로상관단백적린산화수평유관.
Objective The aim of this study is to investigate the effects of berberine (BBR) on transdifferentiation and phosphorylation level of MAPK signaling-related proteins induced by transforming growth factor-β1 (TGF-β1) in human fetal lung fibroblast cells MRC-5. Methods Suitable concentrations of BBR forin vitro treatment in this study were chosen according to the inhibition profile of cell proliferation measured by MTT assay. The expression levels of cell-transdifferentiation-related biomarkers such asα-smooth muscle-actin (α-SMA) and fibronectin (FN) were detected by Western blot and an effective concentration of TGF-β1 for cell transdifferentiation induction was selected. The expression of an extracelluar matrix protein, hydroxyproline, was analysed by colorimetric method and the expression ofα-SMA, FN, and the phosphorylation level of MAPK signaling-related molecules such as p38 MAPK, SAPK/JNK and ERK1/2 were determined by Western blot to evaluate the inhibitory effects of BBR on transdifferentiation induced by TGF-β1 in MRC-5 cells. Results According to the results of MTT assay, three concentraions of BBR (20, 40, 80μmol/L) were chosen for cell treatments in this study. Western blot analysis indicated that 2 ng/ml of TGF-β1 effectively induced transdifferentiation in MRC-5 cells and the concentration was used for the following treatments in this study. As compared to the cells treated with the single TGF-β1, the cells pretreated with BBR showed lower level of hydroxyproline in the supernatant. Also, Western blot analysis indicated that the expression ofα-SMA and FN as well as the phosphorylation level of p38 MAPK, SAPK/JNK and ERK1/2 were significantly suppressed by pretreatment of BBR in MRC-5 cells induced by TGF-β1. Conclusion BBR by pretreatment suppresses the transdifferentiation induced by TGF-β1 and down-regulates the phosphorylation level of MAPK signaling related proteins such as p38 MAPK, SAPK/JNK and ERK1/2 in MRC-5 cells.