CAJ | 학술논문

目的观察体外抑制核因子-κB (NF-κB)对人眼角膜基质细胞分泌细胞因子的影响. 方法四甲基偶氮唑盐(MTT)法检测不同浓度的大黄素对人眼角膜基质细胞活性的影响.用脂多糖(LPS)刺激体外培养的基质细胞,将细胞分为正常对照组(n=6)、LPS组(n=24)和大黄素组(n=24).LPS组单纯给予LPS刺激,大黄素组于LPS刺激前先用大黄素预处理30min,其余处理同LPS组.分别用反转录聚合酶链反应(RT-PCR)和酶联免疫吸附实验(ELISA)检测LPS刺激对角膜基质细胞白细胞介素8(IL-8)基因表达和蛋白分泌的影响,Western blotting检测角膜基质细胞中κB抑制因子α(IκB-α)蛋白的表达. 结果所用浓度大黄素对体外培养的角膜基质细胞活性无影响.与刺激前相比,LPS刺激可引起人眼角膜基质细胞IL-8 mRNA表达增加,促进IL-8蛋白分泌、下调细胞内IκB-α蛋白水平,P＜0.01差异均有统计学意义.大黄素预处理可明显抑制LPS诱导的人眼角膜基质细胞表达IκB-α,下调细胞IL-8基因表达,减少IL-8蛋白分泌,明显低于同时间点LPS组的表达,P＜0.01差异具有统计学意义. 结论 LPS可引起人眼角膜基质细胞IκB-α的降解,促进NF-κB核转位,引起IL-8表达.体外抑制NF-κB,能减少人眼角膜基质细胞分泌IL-8.
목적 관찰체외억제핵인자-κB (NF-κB)대인안각막기질세포분비세포인자적영향. 방법 사갑기우담서염(MTT)법검측불동농도적대황소대인안각막기질세포활성적영향.용지다당(LPS)자격체외배양적기질세포,장세포분위정상대조조(n=6)、LPS조(n=24)화대황소조(n=24).LPS조단순급여LPS자격,대황소조우LPS자격전선용대황소예처리30min,기여처리동LPS조.분별용반전록취합매련반응(RT-PCR)화매련면역흡부실험(ELISA)검측LPS자격대각막기질세포백세포개소8(IL-8)기인표체화단백분비적영향,Western blotting검측각막기질세포중κB억제인자α(IκB-α)단백적표체. 결과 소용농도대황소대체외배양적각막기질세포활성무영향.여자격전상비,LPS자격가인기인안각막기질세포IL-8 mRNA표체증가,촉진IL-8단백분비、하조세포내IκB-α단백수평,P＜0.01차이균유통계학의의.대황소예처리가명현억제LPS유도적인안각막기질세포표체IκB-α,하조세포IL-8기인표체,감소IL-8단백분비,명현저우동시간점LPS조적표체,P＜0.01차이구유통계학의의. 결론 LPS가인기인안각막기질세포IκB-α적강해,촉진NF-κB핵전위,인기IL-8표체.체외억제NF-κB,능감소인안각막기질세포분비IL-8.
Objective To investigate the inhibition effects of nuclear factor-κB(NF-κB) on cytokine expression of cultured human corneal fibroblasts in vitro. Methods The cultured human corneal fibroblasts were used in the present experiment. Cell viability insensitive to emodin on cultured human corneal fibroblasts in vitro was assessed using the MTT assay.Cells were randomly divided into three groups:control group(group 0 hour,n=6),lipopolysaccharidel (LPS)group (n=24)and emodin pretreatment group(n=24). Cells of the emodin pretreatment group were incubated with emodin for 30 minutes before LPS challenged. The cultured human corneal fibroblasts were then challenged with LPS, the expression of interleukin-8(IL-8) mRNAs was detected by reverse transcription polymerase chain reaction analysis (RT-PCR) and the proteins of IL-8 secreted from these cells were assessed by enzyme-linked immunosorbent assays (ELISA). Western blotting analysis was used for detecting the protein expression of inhibitor of nuclear factor-κB-α(IκB-α)induced by LPS.At the same time,the effects of emodin on these expressions were also assessed in fibroblasts. Results The concentration of emodin used in this study was safe to these cultured human corneal fibroblasts in vitro. Both the protein release and mRNA expression of IL-8 in corneal fibroblasts increased significantly after challenged with LPS, however, the protein level of IκB-α significantly decreased after treated with LPS. These results indicated that LPS could mediate the activation of NF-κB and upregulate the expression of cytokines in corneal fibroblasts. Pretreated with emodin 30 min before challenged with LPS, the activation of NF-κB induced by LPS was markedly inhibited, and the mRNA expression as well as protein release of IL-8 were also inhibited(P<0.01).Conclusion These results suggest that LPS takes part in the degeneration of IκB-α and the expression of IL-8 in cultured human corneal fibroblasts in vitro. Emodin, an active component from the rhizome of Rheum palmatum, can inhibit the activation of NF-κB via decreasing this degeneration, which results in suppressing LPS-induced cytokine release from cultured human corneal fibroblasts.