脂多糖和转化生长因子-β1对牙髓细胞Toll样受体4的表达及信号通路的影响

摘要

Objective To explore the expression of Toll like receptor 4(TLR4) on the pulp cells and the change of related signaling molecules under the condition of concomitant lipopolysaccharide (LPS) and transforming growth factor-pi (TGF-pi) during the course of pulpitis. Methods After treated by LPS and TGF-pl, the expression of TLR4 on pulp cells was detected by flow cytometry (FCM). The expressions of signaling molecules evolutionarily conserved signaling intermediate in Toll pathways (ECSIT) and nuclear factor-KB (NF-kB) were detected by real-time polymerase chain reaction (real-time PCR) and Western blot. The secretion of interleukin-6 (IL-6) was detected by enzymelinked immunosorbent assay (ELISA). Furthermore, the change of corresponding targets in inflamed pulp cells from clinical samples were detected by real-time PCR. Results After treated by LPS and TGF-pi in vitro, there was no change in the expression of TLR4 on pulp cells, but the secretion of proinflammatory cytokines IL-6 increased. LPS and TGF-pi could also increase the expression of signal downstream ECSIT and actived NF-kB. Furthermore, the expression of TLR4 mRNA had no increase in inflamed pulp cells from clinical samples, while the expression of TGF-β1, ECSIT and IL-6 mRNA increased through real-time PCR. Conclusion During the course of pulpitis, although the expression of TLR4 on pulp cells was inhibited by increased expression of TGF-pi, the TLR4 pathway was still activated. This effect could be caused through activation of ECSIT mediated by LPS, which might inhibit the TGF-β1 pathway.%目的 研究牙髓炎症过程中,在促炎因子脂多糖(LPS)和抑炎因子转化生长因子-β1 (TGF-β1)同时存在的情况下,牙髓细胞表面Toll样受体4(TLR4)的表达水平及相关信号分子的变化情况.方法 LPS、TGF-β1作用于体外培养的牙髓细胞,用流式细胞术检测牙髓细胞表面TLR4的表达；实时荧光定量聚合酶链反应(real-time PCR)、Western blot方法检测相关信号分子的表达水平,包括进化保守的Toll信号中介分子(ECSIT)和核转录因子-κB (NF-κB)；酶联免疫吸附试验(ELISA)检测前炎症因子白细胞介素-6 (IL-6)的分泌水平；然后进一步通过real-time PCR法检测临床炎症牙髓组织中相应指标的变化.结果 体外培养的牙髓细胞在LPS、TGF-β1共同作用下,细胞表面TLR4的表达水平没有明显变化,但是IL-6分泌增加,ECSIT表达增加,NF-κB入核增加.临床标本的real-time PCR结果表明:炎症状态下的牙髓组织中TGF-β1 mRNA表达增加,TLR4 mRNA表达没有明显变化,ECSIT及IL-6 mRNA表达增加.结论 牙髓炎症发展过程中,虽然牙髓组织中TGF-β1表达增加,抑制细胞表面TLR4的表达,但TLR4的信号通路仍然被活化,主要机制可能是LPS引起信号分子ECSIT的活化,从而抑制TGF-β1信号通路的活化.