三氧化二砷对高迁移率族蛋白诱导的大鼠滑膜细胞增殖的抑制作用

Inhibitory effect of arsenic trioxide on proliferation of rat synoviocyte stimulated by HMGB1

目的:探讨三氧化二砷(AS2O3)对高迁移率族蛋白1(HMGB1)诱导的大鼠滑膜细胞(RSC-364细胞)增殖的抑制作用及其机制。方法:将常规培养的RSC-364细胞分为正常对照组、HMGB1组及HMGB1加0、5、10、20、40和80μmol·L-1AS2O3实验组;MTT法检测不同浓度AS2O3对RSC-364细胞生长的影响,RT-PCR检测STAT1mRNA的表达变化,免疫细胞化学和流式细胞术检测STAT1、cyclinD1和增殖细胞核抗原(PCNA)蛋白表达的变化。结果:①MTT结果显示,AS2O3可抑制HMGB1诱导的RSC-364细胞的增殖,80μmol·L-1AS2O3作用24h抑制率可达88.18%,其抑制作用呈时间和浓度依赖性(P<0.05或P<0.01);②与对照组比较,HMGB1组STAT1mRNA及STAT1、PCNA、cyclinD1蛋白表达量增强,AS2O3组STAT1mRNA及STAT1、PCNA、cyclinD1蛋白降低,并呈剂量依赖性(P<0.01);③STAT1与cyclinD1蛋白表达呈正相关关系(r=0.842,P=0.001)。结论:AS2O3能够抑制HMGB1诱导的滑膜细胞的增殖,其机制可能与抑制STAT1的活性从而下调细胞周期调节蛋白cyclinD1的表达有关。

Objective To investigate the inhibitory effect of arsenic trioxid （AS2O3） on the proliferation of RSC- 364 synoviocyte lines stimulated by HMGB1 and its possible mechanism. Methods RSC-364 synoviocytes were cultivated with standard medium as control group or with medium supplemented with HMGB1 （HMGB1 group） or HMGB1 and 0, 5, 10, 20, 40, 80μmol· L^-1 AS2O3 （experimental groups）, respectively. MTT assay was carried out to study the inhibitory effects of AS203 with different concentrations on the growth of RSC-364. RT- PCR was used to detect the mRNA expression of STAT1. Proteins of phospho-STAT1, cyclin D1 and proliferation cell nuclear antigen （PCNA） were detected by immunoeytoehemistry and FCM. Results ① MTT data indicated AS2O3 could inhibit the growth of RSC-364 stimulated by HMGB1 in a time-dependent and dose-dependent manner. ②Compared with control group, HMGB1 up-regulated the expression of phospho-STAT1 protein and mRNA as well as PCNA protein. Both mRNA and protein levels of phospho-STAT1 were decreased in the RSC-364 cells exposed to AS2O3 in a dose-dependent manner; AS2O3 also down-regulated the expression of PCNA protein. ③HMGB1 up-regulated the expression of cyclin D1, while AS2O3 decreased the expression of cyclin D1 . ④There was positive correlation between phospho-STAT1 and cyclin D1 （r= 0. 842, P= 0. 001）;. Conclusion Arsenic trioxide could inhibit the proliferation of RSC-364 synoviocytes, partly by down-regulating the expression of phospho-STAT1/cyclin D1 .