脂多糖通过促进透明质酸受体CD44向核转移介导牙周膜细胞白细胞介素6释放

Lipopolysaccharide mediates interleukin-6 release from periodontal ligament cells by promoting hyaluronan receptor CD44 transfer to nuclear

目的初步探讨炎症状态下人牙周膜细胞(hPDLC)炎症反应的分子作用机制。方法利用细胞免疫荧光法鉴定原代培养hPDLC波形丝蛋白、角蛋白,使用不同浓度(0、0.1、10和100μmol/L)的脂多糖(LPS)刺激hPDLC,流式细胞术检测细胞凋亡水平。选择不同的LPS刺激时间,酶联免疫检测方法(ELISA)检测细胞培养上清中透明质酸受体CD44的表达。使用ELISA、蛋白免疫印迹(Western blot)及细胞免疫荧光法等实验技术检测hPDLC在炎症环境下细胞质与细胞核中CD44、基质金属蛋白酶14(MMP-14)的表达变化,微量样本多指标流式蛋白定量技术(CBA)检测炎症因子的表达。进一步在炎症环境下加入MMP-14、Importinβ抑制剂,使用ELISA法检测细胞核内CD44的表达及定量反转录聚合酶链反应(RT-PCR)法检测细胞白细胞介素6(IL-6)mRNA表达水平。结果不同浓度(0.1、1、10、100μmol/L)LPS不会对hPDLC的凋亡产生影响。在1μmol/L LPS刺激hPDLC 4 h后,MMP-14荧光强度表达明显增强(233.75),对照组为107.53,差异有统计学意义(t=10.10,P<0.001)。CD44荧光强度为106.7,对照组为84.58,差异有统计学意义(t=3.13,P=0.02)。使用MMP-14抑制剂能够明显抑制细胞培养上清中CD44的表达,LPS+MMP-14抑制剂组较LPS组CD44表达明显下调,差异有统计学意义(t=5.03,P=0.001)。LPS刺激hPDLC会导致细胞核内CD44与Importinβ表达量增加,差异有统计学意义(P<0.001)。LPS刺激后细胞核内CD44与Importinβ也有上调表达的趋势,LPS会导致IL-6表达量增加,差异有统计学意义(P<0.001),并且LPS加入Importinβ抑制剂后细胞中IL-6 mRNA的表达量较LPS处理组明显减少,差异有统计学意义(t=16.79,P<0.001)。结论LPS能够导致hPDLC CD44、Importinβ与IL-6的上调表达,核内CD44上调表达显著,MMP-14、Importinβ抑制剂能够抑制炎症环境下CD44入核,抑制IL-6上调表达。提示在炎症状态下MMP-14与CD44参与hPDLC炎症反应,CD44能够向细胞核内转移介导IL-6的释放。

Objective To investigate the molecular mechanism of inflammatory response in human periodontal ligament cells(hPDLCs)under inflammatory conditions.Methods Vimentin and keratin were identified by immunofluorescence.hPDLCs were stimulated with different concentrations(0,0.1,10,100μmol/L)of lipopolysaccharide(LPS),and apoptosis was detected by flow cytometry.The expression of hyaluronan receptor CD44 in cell culture supernatant was detected by enzyme-linked immune sorbent assay(ELISA)after the LPS stimulation.The expression of CD44 and matrix metalloproteinase(MMP)-14 in the cytoplasm and nucleus of hPDLCs in inflammatory environment was detected by ELISA,Western blot and cell immunofluorescence.The expression of inflammatory factors was detected by Cytometric Bead Array(CBA).Furthermore,MMP-14 and Importinβinhibitor were added in the inflammatory environment,and the expression of CD44 and interleukin-6(IL-6)mRNA was detected by ELISA and RT-PCR,respectively.Results LPS did not affect the apoptosis of hPDLCs at different concentrations(0.1,1,10,100μmol/L).After 4 h stimulation with 1μmol/L LPS,the fluorescence intensity of MMP-14 in hPDLCs significantly increased(233.75),while that in control group was 107.53,and the difference was statistically significant(t=10.10,P<0.001).CD44 fluorescence intensity was 106.7 in the LPS group and 84.58 in the control group,and the difference was statistically significant(t=3.13,P=0.02).MMP-14 inhibitor could significantly inhibit the expression of CD44 in cell culture supernatant,and the expression of CD44 in the LPS+MMP-14 inhibitor group was significantly lower than that in the LPS group,and the difference was statistically significant(t=5.03,P=0.001).LPS stimulation of hPDLCs increased the expression of CD44 and Importin β in the nucleus,and the difference was statistically significant(P<0.001).After LPS stimulation,the expression of CD44 and Importin β in the nucleus was also up-regulated.LPS increased the expression of IL-6,and the difference was statistically significant(P<0.001),and the expression of IL-6 mRNA in the cells treated with LPS plus Importin β inhibitor was significantly lower than that in the LPS treatment group.The differences were statistically significant(t=16.79,P<0.001).Conclusions LPS can up-regulate the expression of CD44,Importin β and IL-6 in hPDLCs.MMP-14 and Importin β inhibitor can inhibit the nuclear translocation of CD44 and the up-regulated expression of IL-6 in inflammatory environment.These results suggested that MMP-14 and CD44 participated in the inflammatory response of hPDLCs under inflammatory conditions,and CD44 can transfer to the nucleus and mediate the release of IL-6.