川陈皮素对脂多糖诱导的牙周膜细胞损伤和NOD样受体蛋白3炎性通路的影响

Effects of nobiletin on lipopolysaccharide induced periodontal ligament cells injury and NOD-like receptor protein-3 inflammatory pathway

目的 探讨川陈皮素(NOB)对脂多糖(LPS)诱导的人牙周膜细胞(hPDLCs)损伤的影响及其机制。方法 将体外培养的hPDLCs分为对照组(不做处理)、模型组(10μg·mL^(-1) LPS处理24 h)和低、中、高剂量实验组(模型组的基础上分别使用5,10和20μmol·L^(-1) NOB处理24 h),采用流式细胞术和酶联免疫吸附测定法(ELISA)分别检测各组细胞凋亡率和细胞上清液中肿瘤坏死因子-α(TNF-α)和白细胞介素(IL)-6含量,以蛋白质印迹法检测细胞中微管相关蛋白1(Beclin-1)、微管相关蛋白1轻链3(LC3)、NOD样受体蛋白3(NLRP3)、凋亡相关斑点样蛋白(ASC)、胱天蛋白酶(caspase)-1和IL-1β蛋白表达水平。结果 对照组、模型组和低、中、高剂量实验组细胞存活率分别为(96.13±5.33)%,(62.47±4.19)%,(68.25±4.83)%,(75.77±5.07)%和(83.60±5.88)%,凋亡率分别为(5.05±1.31)%,(33.86±2.27)%,(28.63±2.22)%,(23.35±2.04)%和(17.51±1.91)%,Beclin-1蛋白表达水平分别为0.18±0.02,0.26±0.03,0.36±0.03,0.47±0.04和0.65±0.04,LC3Ⅱ/Ⅰ比值分别为0.16±0.02,0.23±0.03,0.56±0.03,1.08±0.06和1.65±0.13,NLRP3蛋白表达水平分别为0.18±0.02,1.15±0.10,0.80±0.05,0.52±0.03和0.19±0.02,ASC蛋白表达水平分别为0.12±0.02,0.55±0.03,0.47±0.03,0.38±0.04和0.25±0.03,caspase-1蛋白表达水平分别为0.09±0.02,0.63±0.04,0.50±0.03,0.35±0.02和0.23±0.02,IL-1β蛋白表达水平分别为0.15±0.02,0.57±0.04,0.48±0.03,0.36±0.03和0.20±0.03;模型组与对照组比较,低、中、高剂量实验组与模型组比较,上述指标差异均有统计学意义(均P<0.05)。结论 NOB可减轻LPS诱导的hPDLCs损伤,其作用机制可能与增强细胞自噬和NLRP3炎性通路活化有关。

Objective To investigate the effects of nobiletin(NOB) on lipopolysaccharide(LPS)-induced injury of human periodontal ligament cells(hPDLCs) and its mechanism.Methods hPDLCs cultured and identified in vitro were divided into control group(no treatment) and model group(10 μg·mL^(-1) LPS treatment for 24 h) and L,M,H-experimental groups(treatment with 5,10 and 20 μmol·L^(-1) NOB for 24 h on the basis of model group).The apoptosis rate and the contents of tumor necrosis factor-α(TNF-α),interleukin(IL)-6 in cell supernatant were detected by flow cytometry and enzyme linked immunosorbent assay(ELISA).The expression level of microtubule-associated protein 1( Beclin-1),microtubule-associated protein 1 light chain 3( LC3),NOD-like receptor protein3( NLRP3),apoptosis related spotted protein( ASC),cysteine-containing aspartate protein hydrolase( Caspase-1)and IL-1β proteins were detected by Western blot.Results The cell survival rates of control group,model group and L,M,H-experimental groups were( 96.13 ± 5.33) %,( 62.47 ± 4.19) %,( 68.25 ± 4.83) %,( 75.77 ± 5.07) %,( 83.60 ± 5.88) %;apoptosis rates were( 5.05 ± 1.31) %,( 33.86 ± 2.27) %,( 28.63 ± 2.22) %,( 23.35 ± 2.04) %,( 17.51 ± 1.91) %;Beclin-1 protein expression levels were 0.18 ± 0.02,0.26 ± 0.03,0.36 ± 0.03,0.47 ± 0.04 and 0.65 ± 0.04,respectively;the ratio of LC3 Ⅱ/Ⅰ were 0.16 ± 0.02,0.23 ± 0.03,0.56 ± 0.03,1.08 ± 0.06 and 1.65 ± 0.13,respectively;the expression levels of NLRP3 protein were 0.18 ± 0.02,1.15 ± 0.10,0.80 ± 0.05,0.52 ± 0.03 and 0.19 ± 0.02 respectively;the expression levels of ASC protein were0.12 ± 0.02,0.55 ± 0.03,0.47 ± 0.03,0.38 ± 0.04 and 0.25 ± 0.03,respectively;the expression levels of Caspase-1 protein were 0.09 ± 0.02,0.63 ± 0.04,0.50 ± 0.03,0.35 ± 0.02 and 0.23 ± 0.02,respectively;the expression levels of IL-1β protein were 0.15 ± 0.02,0.57 ± 0.04,0.48 ± 0.03,0.36 ± 0.03 and 0.20 ± 0.03,respectively;there were significant differences in the above indexes between model group and control group,and between L,M,H-experimental group and model group( all P < 0.05).Conclusions NOB can reduce the injury of h PDLCs induced by LPS,and its mechanism may be related to enhancing autophagy and inhibiting the activation of NLRP3 inflammatory pathway.