芦丁减轻LPS诱导的牙周膜干细胞炎症、成骨分化和成脂作用

Rutin reduces the inflammation,osteogenic differentiation and adipogenesis of periodontal ligament stem cells induced by LPS

目的探究芦丁对脂多糖(LPS)诱导的牙周膜干细胞(PDLSCs)成骨分化、成脂作用和TLR4信号通路的影响。方法通过分离培养获取PDLSCs,将PDLSCs细胞分别用不同浓度LPS(0,0.1,1和5μg/ml)和不同浓度芦丁(0,0.01,0.1,1,5,10,50和100μmol/L)培养14 d,通过形态学观察、碱性磷酸酶(ALP)活性测定、ALP染色和qPCR测定LPS和芦丁对PDLSCs成骨分化的影响;茜素红S染色评估钙沉积物的形成。再将PDLSCs细胞分为对照组、芦丁组、LPS组和LPS+芦丁组。ELISA试剂盒测定LPS对PDLSCs中炎性细胞因子和氧化应激的影响;蛋白质印迹分析技术测定LPS对PDLSCs中TLR4及NF-κB通路蛋白表达水平的影响。结果ALP和茜素红S染色结果显示,与0μg/ml LPS相比,0.1μg/ml LPS组ALP的表达水平显著降低、钙沉积物的形成显著减少(P<0.01)。在芦丁浓度为10μmol/L时ALP的活性最高;与10μmol/L的芦丁相比,50μmol/L和100μmol/L芦丁处理下ALP活性均显著降低(P<0.05)。与对照组相比,LPS组TNF-α、IL-6、IL-1β、ROS、NO、iNOS、PPARγ、LPL、p-NF-κB p65、MyD88和TLR4的表达水平显著升高(P<0.01);与LPS组相比,LPS+芦丁组TNF-α、IL-6、IL-1β、ROS、NO、iNOS、PPARγ、LPL、p-NF-κB p65、MyD88和TLR4的表达水平显著降低(P<0.01)。结论芦丁通过抑制PDLSCs中TLR4/NF-κB途径中关键因子的表达水平,从而减轻LPS诱导的炎症、成骨分化和成脂作用。

Objective To explore the effects of rutin on LPS-induced osteogenic differentiation,adipogenesis of periodontal ligament stem cells(PDLSCs)and TLR4 signaling pathway.Methods PDLSCs were obtained through isolation and culture.PDLSCs cells were cultured with different concentrations of LPS(0,0.1,1 and 5μg/ml)or different concentrations of rutin(0,0.01,0.1,1,5,10,50 and 100μmol/L)for 14 d,respectively.The effects of LPS and rutin on the osteogenic differentiation of PDLSCs were determined by morphological observation,alkaline phosphatase(ALP)activity assay,ALP staining and qPCR.And the alizarin red S staining was used to evaluate the formation of calcium deposits.The PDLSCs were divided into control group,rutin group,LPS group and LPS+rutin group.ELISA kit was used to determine the effect of LPS on inflammatory cytokines and oxidative stress in PDLSCs.Western blotting was used to determine the influence of LPS on the expression protein levels of TLR4 and NF-κB pathways in PDLSCs.Results The results of ALP and Alizarin red S staining showed that compared with 0μg/ml LPS group,the expression level of ALP was significantly reduced in 0.1μg/ml LPS group,and the formation of calcium deposits was significantly reduced at 14 d(P<0.01).When the concentration of rutin was 10μmol/L,the activity of ALP was the highest(P<0.05).Compared with 10μmol/L rutin,the activity of ALP was significantly decreased after culture with 50μmol/L and 100μmol/L rutin(P<0.05).Compared with control group,the expression levels of TNF-α,IL-6,IL-1β,ROS,NO,iNOS,PPARγ,LPL,p-NF-κB p65,MyD88 and TLR4 were significantly increased in LPS group(P<0.01).Compared with LPS group,the expression levels of TNF-α,IL-6,IL-1β,ROS,NO,iNOS,PPARγ,LPL,p-NF-κB p65,MyD88 and TLR4 in LPS+rutin group were significantly reduced(P<0.01).Conclusion Rutin can reduce LPS-induced inflammation,osteogenic differentiation and adipogenesis of PDLSCs by inhibiting the overexpression of key factors in the TLR4/NF-κB pathway.