低强度牵张应力对异丙肾上腺素诱导下牙周膜成纤维细胞炎症反应的影响

Effect of low magnitude tension on the inflammatory response of periodontal ligament cells induced by isoproterenol

目的 :探讨低强度牵张力对异丙肾上腺素(isoproterenol,ISO)诱导下人牙周膜成纤维细胞炎症反应的影响及其分子机制。方法:体外培养人牙周膜成纤维细胞(periodontal ligaments cells,PDLCs),给予一定浓度(0.01、0.1、1μmol/L)ISO刺激24 h,设置空白对照组,同时施加不同强度(5%、10%、15%形变量)的牵张力,利用实时荧光定量PCR(RTqPCR)检测细胞内IL-1β、IL-6 mRNA的表达。设置空白対照组、ISO刺激组(0.1μmol/L)、低强度牵张力组(5%形变量)和ISO+低强度牵张力组,利用蛋白免疫印迹法(Western blot)检测内质网应激相关p-PERK、PERK、p-eIF2α、eIF2α、ATF4蛋白表达量的变化。应用细胞转染技术敲低PDLCs中PERK基因的表达,RT-qPCR检测低表达PERK的牙周膜成纤维细胞在ISO及5%形变量牵张力作用下IL-1β及IL-6 mRNA的表达。采用SPSS 22.0软件包对数据进行统计学分析。结果:ISO诱导可显著上调牙周膜成纤维细胞中IL-1β及IL-6 mRNA的表达(P<0.05);与对照组相比,5%形变量牵张力抑制ISO诱导下PDLCs中IL-1β和IL-6 mRNA的表达,差异有统计学意义(P<0.05);Western印迹结果显示,5%形变量牵张力可抑制ISO诱导引起的PERK、eIF2α磷酸化及ATF4的表达(P<0.05)。与阴性对照组相比,ISO诱导下PERK沉默的PDLCs中IL-1β及IL-6 mRNA表达显著降低(P<0.05)。结论:5%形变量牵张力可能通过内质网应激PERK通路抑制ISO诱导下牙周膜成纤维细胞的炎症反应。

PURPOSE: To investigate the effect of tension on the inflammatory response of human periodontal ligament cells(PDLCs) induced by isoproterenol(ISO) and its molecular mechanism. METHODS: Human PDLCs were cultured in vitro and stimulated with a certain concentration of ISO(0.01, 0.1, 1 μmol/L) for 24 h. Cyclic tensile strain with different degrees of elongation(5%, 10% and 15%) were applied. The expression of IL-1β and IL-6 mRNA in PDLCs was detected by real-time quantitative PCR(RT-qPCR). The protein expression of p-PERK, PERK, p-eIF2α, eIF2α and ATF4 related to ER stress was detected by Western blot. The expression of PERK gene in PDLCs was knocked down by cell transfection technique, and the expression of IL-1β and IL-6 mRNA in PDLCs with low expression of PERK was detected by RTqPCR under the stimulation of ISO and low magnitude tension. Statistical analysis was conducted with SPSS 22.0 software package. RESULTS: ISO induction could significantly up-regulate the IL-1β and IL-6 mRNA expression in PDLCs(P<0.05). Compared with the control group, the expression of IL-1β and IL-6 mRNA in PDLCs induced by ISO was inhibited by low magnitude tension, and the difference was statistically significant(P<0.05). Western blot results showed that low magnitude tension could inhibit the ISO-stimulated phosphorylation of PERK and eIF2α and the expression of ATF4(P<0.05). Compared with the negative control group, IL-1β and IL-6 mRNA expression was decreased in the ISO-stimulated PDLCs silenced by PERK gene. CONCLUSIONS: Tension with 5% degrees of elongation may inhibit ISO-stimulated periodontal inflammatory response through endoplasmic reticulum stress pathway.