地塞米松对人晶状体上皮细胞核转录因子κB及其抑制蛋白α的表达和细胞凋亡的影响

Effects of dexamethasone on expression of nuclear factor kappa B/inhibitor kappa B alpha and apoptosis of lens epithelial cells

背景长期全身或眼局部应用糖皮质激素可诱导皮质类固醇性白内障，但其作用机制尚不明确。目的研究地塞米松作用于人晶状体上皮细胞（LECs）后对LECs中核转录因子κB（NF—κB）／NF—κB抑制蛋白κ（IκBκ）表达的影响及LECs凋亡的发生情况，了解皮质类固醇性白内障的发病机制。方法取人LECs系（HLE283）在含质量分数20％胎牛血清的DMEM中进行培养和传代。将不同浓度的地塞米松（0．01、0．1、1、10、100μmol／L）分别加入DMSO无血清DMEM培养液中作为不同浓度地塞米松组，不含地塞米松的DMSO无血清DMEM培养液培养的LECs作为空白对照组。采用逆转录聚合酶链反应（RT—PCR）、Western blot、流式细胞术等方法，分别在基因水平、蛋白水平检测LECs中NF—κB／IκB α浓度的改变及LECs凋亡率的变化。结果扩增的基因片段与所设计片段大小一致。地塞米松作用后NF—α核蛋白在LECs中的表达量随着地塞米松浓度的增加而下降，差异有统计学意义（F=36．077，P=0．004）；IκBα蛋白的表达随着地塞米松浓度的增加而上升，差异有统计学意义（F=35．741，P=0．002）。在同浓度地塞米松组，NF—κB核蛋白在LECs中的表达量随作用时间的不同差异有统计学意义（F=16．606，P=0．01），与地塞米松作用24h时的表达量比较，36h和48h时的NF—κB核蛋白表达量明显下降，差异有统计学意义（P=0．002；P=0．01）。与地塞米松作用24h时的表达量比较，36h和48hIκBα蛋白在LECs中的表达量明显增加，差异均有统计学意义（P=0．014；P=0．002）。流式细胞仪检测显示LECs的凋亡率随着地塞米松浓度的增加明显上升，与空白对照组相比差异有统计学意义（P〈0．05）。结论地塞米松通过上调IκBα的表达而过度抑制了NF—κB的活性，并导致LECs凋亡，其作用呈浓度依赖性，这可能是皮质类固醇性白内障发生发展的细胞学和分子生物学机制之一。

Background Researches demonstrated that the long-term application of glucocorticoids can induce cataraα. However, its molecular mechanism is unclear. Objeαive Present study was to investigate the effeαs of dexamethasone on the regulation of nuclear faαor kappa B（NF-κB）/ inhibitor kappa B alpha（IκBα） line on human lens epithelial cells （LECs） and the LECs apoptosis. Methods Human LECs line （ HLE2B3 ） were cultured and passaged in DMEM containing 20% fetal bovine serum and treated by different concentrations of dexamethasone （0.01,0. 1,1,10,100 μmoL/L） for 24,36 and 48 hours respeαively. The LECs cultured in free-serum DMEM without dexamethasone were as blank control group. The expressions of IκBa in the LECs were examined by reverse transcription polymerase chain reaαion （RT-PCR） and Western blot, and the expressions of NF-κB neucleoprotein in LECs were deteαed by Western blot after exposure to dexamethasone. The apoptosis rate of LECs was determined by flow cytometer. Results Agarose gel eleαrophoresis showed that the amplified gene fragment was coincident to designed one. The expressing level of NF-κB neucleoprotein in LECs was significantly lowed with the increase of dexamethasone concentration （ F = 36. 077, P = 0. 004 ） , and that of IκBα was evidently ascended （ F = 35. 741 ,P=0. 002）. In the same concentration of dexamethasone group,the expression of NF-κB in LECs showed the considerable alteration in different duration after treated of dexamethasone with the lowest expressing level in 36 hours,and significant differences were found in the expressing level between 24 hours and 36 hours （P = 0. 002 ） and between 24 hours and 48 hours（ P=0. 01 ）. The differences of expression of IκBα in LECs appeared the same pattern to NF-κB neucleoprotein. Flow cytometry showed that the apoptosis rate of LECs was obviously enhanced after aαion of dexamethasone in a dose-dependent manner, showing a significant difference among different groups （F= 73. 261, P=0.001）. Conclusion It is implied that dexamethasone results in the pathogenesis and development of glucocorticoid cataraα by up-regulating the expression of IκBα in LECs and suppressing the aαivity of NF-κB and herein induce the apoptosis of LECs at concentration-and time-dependent manner. This might be one of cellular and biological mechanisms of glucocorticoid cataraα formation.