【作者】 田静；

【导师】 彭佑铭；

【作者基本信息】 中南大学 ， 内科学， 2010， 博士

【摘要】 目的：观察白藜芦醇(Res)对TNF-α诱导类风湿关节炎成纤维样滑膜细胞(RA FLS)增殖及凋亡的影响,以及其对促凋亡因子Bcl-x1／Bcl-2相关死亡启动子(BAD)的作用,探讨Res对TNF-α诱导的RAFLS作用机制。方法：滑膜组织取自行膝关节置换术的活动性类风湿关节炎患者6例,分离出成纤维样滑膜细胞进行原代培养及鉴定。不同浓度的Res干预RA FLS后再用TNF-α(10ng.ml-1)刺激24h。MTT比色法测定Res对TNF-α诱导RA FLS生长的抑制作用。流式细胞仪检测法Res对RA FLS细胞周期及凋亡率的影响。Western-blot方法分析Res对TNF-α诱导RA FLS中磷酸化BAD蛋白表达的影响。结果：低浓度(12.5μM、25μM) Res对TNF-α诱导RA FLS增殖无明显的抑制作用,当Res浓度为50-200μM时,对TNF-α诱导RA FLS增殖具有明显抑制作用(P＜0.05),并呈时间和剂量依赖性。低浓度(12.5μM、25μM)Res对TNF-α诱导RA FLS细胞周期无影响,50-200μMRes对RA FLS细胞周期的影响表现为DNA合成前期的细胞(G1期)比例增加,DNA合成期(S期)细胞比例减少,DNA合成后期和分裂期(G2／M期)细胞比例亦减少。随着Res浓度增大,RA FLS增殖指数逐渐降低,并呈剂量依赖性。低浓度Res (12.5、25μM)组凋亡细胞百分比分别为(1.95±0.62)%、(2.37±0.76)%,与TNF-α组((1.52±0.73)%)比较,差异均没有统计学意义(P>0.05)；较高浓度Res(50、100、200μM)组凋亡细胞百分比分别为(5.92±1.23)%、(9.06±1.78)%、(12.26±3.01)%,随着Res浓度增高,凋亡细胞数增加,与TNF-a组比较,有显著性差异(P＜0.05)。TNF-α刺激RA FLS后,磷酸化BAD表达明显增加,当用不同浓度的Res干预后,磷酸化BAD表达减少,呈剂量依赖关系。结论：50-200μM Res通过阻滞RA FLS细胞周期,降低BAD磷酸化水平发挥抑制TNF-α诱导RA FLS增殖,促凋亡作用,Res具有治疗RA的潜在价值。目的：观察Res对TNF-α诱导的RAFLS主要炎性细胞因子IL-1β、MMP3产生的影响,从炎性细胞因子的角度来探讨Res的抗炎作用。方法：ELISA法检测各组细胞上清液中IL-1β、MMP-3的含量,逆转录-聚合酶链式反应(RT-PCR)检测TNF-α诱导的FLS中IL-1β、MMP-3的mRNA表达,Western-blot方法分析Res对TNF-α诱导RA FLS中IL-1β、MMP-3蛋白表达的影响。结果：在TNF-α刺激24h后,RA FLS上清液IL-1β、MMP-3的含量急剧升高,分别为(436.235±79.721)pg.ml-1, (89.950±14.458)ng.ml-1,与空白组比较,有显著性差异(P<0.01)。Res抑制TNF-α诱导RA FLS分泌IL-1β、MMP-3,呈剂量依赖性。Res在浓度为12.5μM时即可抑制TNF-α诱导RA FLS的IL-1β、MMP-3mRNA的表达水平,至100、200μM时抑制作用达到最高峰。Western-blot方法分析显示Res呈剂量依赖关系降低TNF-α诱导RA FLS中IL-1β、MMP-3的蛋白表达。结论：Res在mRNA和蛋白质水平抑制TNF-α诱导的RA FLS的IL-1β、MMP-3分泌,Res通过抑制细胞因子的产生发挥抗炎作用。目的：观察PI3K/Akt信号通路在TNF-α诱导RA FLS增殖及炎性因子产生中的作用,以及Res对TNF-α诱导RA FLS的PI3K/Akt信号通路影响,探讨Res对TNF-α诱导RA FLS发挥抗增殖及抗炎作用的分子机理。方法：间接免疫荧光细胞化学染色法检测RA FLS各组磷酸化Akt蛋白表达。Western-blot方法检测PI3K抑制剂LY294002 (20μM)对TNF-α诱导RA FLS中磷酸化BAD、IL-1β、MMP-3蛋白表达的影响以及Res对TNF-α诱导RA FLS中Akt、磷酸化Akt蛋白表达的作用。结果：荧光显微镜下观察RA FLS细胞,磷酸化Akt蛋白在TNF-Q组表达增强,而在空白组、LY294002组,磷酸化Akt蛋白基本没有表达,50μMRes组较TNF-α组,磷酸化Akt蛋白表达明显降低。Western-blot方法分析显示LY294002下调TNF-α诱导磷酸化BAD及炎性因子IL-1β、MMP3蛋白表达水平。50-200μM Res可显著降低TNF-Q诱导RA FLS磷酸化Akt蛋白水平,并呈剂量依赖关系。结论：TNF-α诱导的RA FLS增殖及IL-1β、MMP-3产生中存在PI3K/Akt信号通路的激活,LY294002可以阻断这一效应。50-200μMRes可降低Akt磷酸化水平,下调磷酸化BAD及IL-1β、MMP-3蛋白表达,抑制RA FLS增殖,发挥促凋亡及抗炎作用；Res可能在类风湿关节炎的治疗方面具有良好的临床应用前景。更多还原

【Abstract】 Objective To observe the effect of resveratrol (Res) on in vitro TNF-a-induced proliferation and apoptosis of human rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS), and further to investigate the effect of Res on the Bcl-2-associated death promoter (BAD) for the mechanism.Methods Synovial tisssues were obtained from patients with active RA according to the revised criteria of the American College of Rheumatology,who were undergoing knee joint replacement. RA FLS were stimulated with TNF-α(10ng.ml-1) for 24h in the presence or absence of resveratrol.The inhibition rate of RA FLS was examined by MTT assay. Cell cycle and the amount of apoptotic cells was measured by flow cytometry. Phosphorylated BAD protein expression was measured by western blot.Results In the lower concentration 12.5μM、25μM) of Res group, TNF-α-induced proliferation,cell phase and the apoptosis ratio of RA FLS have no obvious effections compared to TNF-a group (p>0.05), However in higher concentration(50μM、100μM、200μM), the living cells measured by MTT dose and time-dependently reduced, and the fraction of living cells in the S-phase and G2/M-phase decreased respectively, while that in G1-phase increased, the difference was statistically significant compared with the TNF-a group(P< 0.05).Flow cytometry demonstrated that the apoptosis rate increased in a dose-dependent manner among the higher concentration of Res groups. Higher concentration Res inhibited dose-dependently TNF-α-induced phosphorylation of BAD in RA FLS.Conclusions Higher concentration (50-200MM) Res can inhibit TNF-a-induced proliferation and induce apoptosis of RA FLS through blocking cell cycle and inhibition of BAD phosphorylation. Res may provide a new therapeutic approach in the treatment of RA.Objective To Observe the inhibition of Res on TNF-a-induced production of mainly inflammatory cytokines such as IL-1β、MMP3 in RA FLS, and further to explore the anti-inflammatory effects of Res from the perspective of inflammatory cytokines.Methods The levels of IL-1β、MMP-3 in cultural supernatants among groups were measured by enzyme-linked immunosorbent assay (ELISA). Messenger RNA expression of IL-1βand MMP-3 in RA FLS induced by TNF-a were analyzed using a reverse transcription-polymerase chain reaction (RT-PCR). Western-blot analysis was used to detect the expression of IL-1βand MMP-3 in RA FLS intervened by Res.Results After stimulating with TNF-a for 24h, the levels of IL-1βMMP-3 sharply increased in cultural supernatants of RA FLS, respectively(436.235±79.721)pg.ml-1, (89.950±14.458)ng.ml-1, contrast to blank group(P<0.05). Res inhibited TNF-a-induced production of IL-1βand MMP-3 on RA FLS in a dose-dependent manner. Res at concentration of 12.5μM can immediately inhibited the mRNAs expression of IL-1βand MMP-3, while going to the concentration of 100,200μM, inhibitions of Res reached a peak. The Western-blot analysis showed that Res inhibited TNF-α-induced proteins expression of IL-1βand MMP-3 on RA FLS in a dose-dependent manner.Conclusions Res inhibited TNF-α-induced secretion of IL-1βand MMP-3 in RA FLS on both the mRNA and the protein level. Res plays an anti-inflammatory role via preventing cytokines production.Objective To evaluate the role of PI3K/Akt signaling pathway in TNF-a-induced proliferation and production of proinflammatory cytokines on RA FLS, and the modulation of Res on PI3K/Akt signaling pathway, further to exploring the anti-inflammatory and anti-proliferative effects of Res for its molecular mechanism.Methods Indirect immunofluorescence staining was used to detect the expression of phosphorylated Akt among RA FLS groups. The proteins expression of phosphorylated BAD、IL-1β、MMP-3、phosphorylated Akt and Akt were examined by Western-blot method.Results Immunofluorescence staining showed that the expression of phosphorylated Akt increased significantly in TNF-αgroup, while there were no obvious expression of phosphorylated Akt both in blank group and PI3K inhibitor LY294002 group. However lower expression of phosphorylated Akt was seen in 50μMRes group compared with TNF-αgroup. The Western-blot method showed the PI3K inhibitor LY294002 decreased the proteins expression of TNF-α-induced phosphorylated BAD and IL-1βMMP-3. Higher concentration Res (50-200μM) also decreased significantly the expression of phosphorylated Akt dose-dependently.Conclusions Activation of PI3K/Akt signaling pathway exists in TNF-α-induced proliferation and production of IL-1β、MMP3 on RA FLS, which is hampered by LY294002.Res can reduce the expression of phosphorylated BAD and IL-1β、MMP-3 through inhibition of Akt phosphorylation. Res exerts an anti-proliferative and anti-inflammatory effects in TNF-α-induced RA FLS, indicating Res may have good clinical application prospect in the treatment of rheumatoid arthritis.更多还原