摘要
目的:探讨糖皮质激素对关节软骨细胞衰老和自噬影响,并分析自噬在其中的作用及机制。方法:培养SD大鼠软骨细胞,阿利辛染色和collagen II免疫组化进行鉴定。使用不同浓度的地塞米松作用于软骨细胞不同的时间后,Lyso Tracker Red和MDC染色观察自噬囊泡的生成,Western blot检测LC3、beclin-1、P62、p70S6K和4EBP1蛋白表达的变化。β-半乳糖苷酶染色分析糖皮质激素对软骨细胞衰老的影响,并使用自噬抑制剂来探讨自噬对地塞米松作用下软骨细胞衰老的作用。结果:地塞米松可以显著提高软骨细胞的自噬水平,尤其是在作用4 d后,LC3-II表达随着地塞米松浓度的增加而升高,而P62和beclin-1的表达也验证自噬水平的升高。Lyso Tracker Red和MDC染色发现软骨细胞中自噬泡的生成随着地塞米松浓度的升高而增加。m TOR信号通路上的p70S6K和4EBP1的磷酸化均被地塞米松抑制。更重要的是,地塞米松可以引起软骨细胞的衰老,而使用3-MA抑制自噬后,细胞进一步衰老。结论:糖皮质激素激活软骨细胞中的自噬,并同时诱导软骨细胞的衰老,而自噬在这过程中对细胞的衰老可能具有代偿性的保护作用。同时糖皮质激素抑制m TOR信号通路,可能与自噬的激活相关。
AIM： To explore the effect of glucocorticoid on autophagy and senescence in the chondroeytes. METHODS ： The collagen II in the normal chondrocytes isolated from the SD rats was checked. After stimulation with glu- cocorticoid, LysoTracker Red staining, MDC staining and Western blot were used to detect the level of autophagy in the chondrocytes. The mTOR pathway related molecules were investigated by Western blot. Cell senescence was analyzed by SA-β-gal staining. RESULTS ： A dose-dependent increase in the number of autophagic vacuoles was observed in the dexa- methasone-treated chondrocytes, which was demonstrated by the LysoTracker Red and MDC staining. The expression of LC3-Ⅱ and beclin-1 was increased by dexamethasone, especially in the cells treated with dexamethasone for 4 d. However, P62 expression was decreased. SA-β-gal staining showed that the percentage of cell senescence was increased by dexam- ethasone. Surprisingly, the cell senescence induced by dexamethasone was exacerbated by the autophagic inhibitor 3-MA. CONCLUSION： Autophagy induced by dexamethasone protects chondrocyte from senescence. The mTOR pathway may be involved in the autophagy activation.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2015年第10期1737-1743,共7页
Chinese Journal of Pathophysiology
基金
温州市科技局项目(No.Y20140582)
关键词
糖皮质激素
软骨细胞
自噬
衰老
Glucocorticoids
Chondrocyte
Autophagy
Senescence
