姜黄素干预类风湿关节炎患者破骨细胞生成中NF-κB信号通路的机制研究

发布时间：2018-06-01 22:57

本文选题：姜黄素 + 类风湿关节炎　；参考：《南京中医药大学》2017年硕士论文

【摘要】：目的:观察姜黄素(Curcumin,Cur)对类风湿关节炎(rheumatoid arthritis,RA)患者破骨细胞生成的影响,探索核因子-κB(nuclear factor kappa B,NF-κB)信号通路在此过程中的调节机制,为姜黄素应用于RA骨破坏的防治提供理论依据。方法:(1)分离RA 患者外周血单个核细胞(peripheral blood mononuclear cells,PBMCs)诱导培养为破骨细胞,在培养过程中给予不同浓度姜黄素进行干预;(2)分组:Control组、Cur 2.5 μM 组、Cur 5μ 组、Cur 10 μM 组,姜黄素的浓度分别为 0、2.5 μmol/L、5 μmol/L、10 μmol/L;(3)诱导培养14天后进行抗酒石酸酸性磷酸酶(tartrate-resistant acid phosphatase,TRAP)染色标记成熟破骨细胞,并行细胞计数;(4)Western Blot检测各组细胞中IκBα、p-IκBα蛋白的表达;(5)Western Blot检测细胞浆和细胞核中NF-κB p65的表达;(6)细胞免疫荧光染色法观察NF-κB p65核易位。结果:(1)培养14天后,各组细胞均符合破骨细胞的形态学特征;(2)TRAP染色结果显示,与Control组相比,Cur 2.5 μM组、Cur 5 μM组和Cur 10 μM组TRAP阳性细胞数均明显减少,差异具有统计学意义(P0.05);(3)Western Blot结果显示,与Control组相比,Cur 2.5 μM组、Cur 5 μM组和Cur 10 μ组IκBα的蛋白表达水平明显升高,p-IκBα的表达水平明显下降,差异具有统计学意义(P0.05);(4)与Control组相比,Cur 2.5 μM组、Cur 5μ 组和Cur 10 μ组细胞浆中NF-κB p65的表达水平明显升高,而细胞核中NF-κB p65的表达水平明显下降,差异具有统计学意义(P0.05);(5)细胞免疫荧光染色结果显示,在未刺激状态下NF-κB p65主要分布于细胞浆中;经RANKL刺激后,NF-κB p65在细胞浆的表达减少,在细胞核的表达增加;经5 μM姜黄素干预后,与RANKL刺激后相比,NF-κB p65在细胞浆的表达增加,在细胞核的表达减少。结论:姜黄素通过抑制IκBα的磷酸化和IκBα的降解,减少了 NF-κB p65的核易位,从而抑制NF-κB活化,进而达到抑制RA患者破骨细胞生成的作用。
[Abstract]:Aim: to investigate the effect of curcuminia curcuminus on osteoclast formation in rheumatoid arthritis (RA) patients, and to explore the regulatory mechanism of NF- 魏 B(nuclear factor kappa BnNF- 魏 B signaling pathway in the process, and to provide theoretical basis for the application of curcumin in the prevention and treatment of RA bone failure. Methods the peripheral blood mononuclear cells (PBMCs) from patients with RA were isolated and induced to be osteoclasts. Different concentrations of curcumin were given in the process of culture. The concentration of curcumin was 2.5 渭 mol / L ~ 5 渭 mol / L ~ (5 渭 mol / L ~ (10) 渭 mol / L ~ (3). After 14 days of induction and culture, mature osteoclasts were stained with tartrate-resistant acid phosphatase TRAPs. The expression of I 魏 B 伪 -p-I 魏 B 伪 protein was detected by Western Blot. The expression of NF- 魏 B p65 in cytoplasm and nucleus was detected by Western Blot. The nuclear translocation of NF- 魏 B p65 was observed by cell immunofluorescence staining. Results after 14 days of culture, the cells in each group were in accordance with the morphological characteristics of osteoclasts. The results showed that compared with Control group, the number of TRAP positive cells in cur5 渭 M group and Cur 10 渭 M group decreased significantly compared with Control group. The difference was statistically significant (P 0.05). The results of Western Blot showed that compared with Control group, the protein expression level of cur5 渭 M group and Cur 10 渭 group significantly increased the expression level of p-I 魏 B 伪 in Cur 2.5 渭 M group and Cur 10 渭 group, and the expression level of p-I 魏 B 伪 decreased significantly. Compared with the Control group, the expression of NF- 魏 B p65 in the cytoplasm of Cur 2.5 渭 M group and Cur 10 渭 group was significantly increased, while the expression level of NF- 魏 B p65 in the nucleus decreased significantly. The results of immunofluorescence staining showed that NF- 魏 B p65 was mainly distributed in the cytoplasm under the condition of no stimulation, and the expression of NF- 魏 B p65 in the cytoplasm decreased and increased in the nucleus after RANKL stimulation. After treated with 5 渭 M curcumin, the expression of NF- 魏 B p65 in cytoplasm increased and decreased in nucleus after RANKL stimulation. Conclusion: curcumin can reduce the nuclear translocation of NF- 魏 B p65 by inhibiting the phosphorylation of I 魏 B 伪 and the degradation of I 魏 B 伪, thereby inhibiting the activation of NF- 魏 B and thus inhibiting osteoclast formation in RA patients.
【学位授予单位】：南京中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R593.22

【参考文献】