红景天苷对LPS刺激人牙周膜细胞增殖和分泌表达的影响

Effect of salidroside on the proliferation and secretion of human periodontal ligament cells stimulated by lipopolysaccharide

目的研究红景天苷(salidroside,SAL)对大肠杆菌脂多糖(lipopolysaccharide,LPS)刺激后体外培养人牙周膜细胞(human periodontal ligament cells,h PDLCs)的增殖及对分泌表达Toll样受体(Toll-like receptor 4,TLR 4)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白介素-1β(interleukin-1β,IL-1β)、白细胞介素-6(interleukin-6,IL-6)、骨保护素(osteoprotegerin,OPG)及核因子-κB受体活化因子配体(receptor activator of NF-κB ligand,RANKL)的影响,为红景天苷在牙周炎治疗方面提供初步的实验依据。方法组织块法原代培养正常h PDLCs;以2 mg/L LPS刺激第4代细胞,加入不同浓度的红景天苷作用12、24、48 h;采用MTT方法检测细胞的增殖;采用Western blot、ELISA、qRT-PCR等方法检侧TLR-4、TNF-α、IL-1β、IL-6、OPG及RANKL的表达水平,并进行统计学分析。结果 MTT结果显示低浓度的红景天苷(≤10μmol/L)均能促进细胞增殖,并存在浓度依赖性,其中0.5μmol/L对h PDLCs增殖作用最明显(P<0.05),高浓度的红景天苷(>10μmol/L)不能促进牙周膜细胞增殖;Western blot、ELISA及qRT-PCR结果显示:0.5μmol/L红景天苷可使LPS刺激的h PDLCs的TLR4、TNF-α、IL-1β、IL-6及RANK表达水平随时间延长而逐渐降低,其中48 h降低的最为明显,存在显著性差异(P<0.05);另外,LPS刺激对OPG的表达几乎没有影响,而0.5μmol/L红景天苷作用可以显著上调OPG的表达水平。结论红景天苷可减轻LPS刺激后h PDLCs的细胞损伤,其机制可能通过调节与TLR4相关信号通路,降低炎症因子TNF-α、IL-1β、IL-6及与骨吸收相关因子RANKL、OPG的表达,从而抑制炎症反应和骨吸收。

Objective To investigate the effect of salidroside（ SAL） on the proliferation and secretion of Toll-like receptor 4（ TRL4）,tumor necrosis factor-α（ TNF-α）,interleukin-1β（ IL-1β）,interleukin-6（ IL-6）,osteoprotegerin（ OPG） and receptor activator of NF-κB ligand（ RANKL） in human periodontal ligament cells（ h PDLCs） treated by lipopolysaccharide（ LPS） and to provide preliminary experimental basis for treatment of periodontitis by salidroside. Methods h PDLCs were cultured by tissue cultivation in vitro and treated with 2 mg / L LPS and various concentrations of salidroside for 12,24 and 48 h. Cell proliferation of h PDLCs was detected by MTT assay. The expression levels of TRL4 TNF-α,IL-1β,IL-6,RANKL and OPG in h PDLCs were examined by Western blot,ELISA or qRT-PCR analysis. The data were processed by statistical analysis. Results Salidroside at low concentrations（ ≤10μmol /L） significantly promoted h PDLCs proliferation in dose-dependent manner; 0.5 μmol /L of salidroside had the greatest effect whereas high concentrations of salidroside（ 〉10 μmol / L） had no obvious effect on cell proliferation of h PDLCs; The expression of TLR4,TNF-α,IL-1β,IL-6 and RANKL was significantly increased by LPS stimulation. After treatment with 0.5 μmol / L of salidroside,the expression levels of TLR4,TNF-α,IL-1β,IL-6 and RANKL were significantly inhibited and the obvious effect occurred at48 h（ P〈0.05）; In addition,the expression of OPG was nearly unchanged by LPS,but significantly upregulated by 0.5 μmol / L of salidroside. Conclusion Salidroside could attenuate LPS-induced cell injury in h PDLCs through inhibiting TLR4 signaling pathways and the production of inflammation factors including TNF-α,IL-1β,IL-6 and RANKL and promoting OPG expression which thereby inhibited inflammatory reaction and bone resorption.