芍药苷调节LKB1-AMPK信号通路对LPS诱导的牙周膜干细胞凋亡和成骨分化的影响

Influences of Paeoniflorin on LPS-Induced Apoptosis and Osteogenic Differentiation of Periodontal Ligament Stem Cells by Regulating LKB1-AMPK Signaling Pathway

目的:探讨芍药苷(PF)调节肝激酶B1(LKB1)-腺苷酸活化蛋白激酶(AMPK)信号通路对脂多糖(LPS)诱导的牙周膜干细胞(PDLSCs)凋亡和成骨分化的影响。方法:收集对数生长期PDLSCs,分为对照组、LPS组(10μg/mL),LPS+PF低浓度(PF-L,10μg/mL LPS+5μmoL/L PF)组、LPS+PF中浓度(PF-M,10μg/mL LPS+10μmoL/L PF)组、LPS+PF高浓度(PF-H,10μg/mL LPS+20μmoL/L PF)组、LPS+PF-H+LKB1抑制剂(radicicol,10μg/mL LPS+20μmoL/L PF+10μmoL/L radicicol)组。ELISA检验细胞中炎症因子[白细胞介素(IL)-1β、肿瘤坏死因子-α(TNF-α)、IL-6]水平;茜素红染色观察矿化结节变化;碱性磷酸酶(ALP)检测细胞成骨分化;流式细胞仪检测细胞凋亡;qRT-PCR检测成骨基因(OPN、RUNX2及OCN)表达水平;Western blot检测LKB1-AMPK通路蛋白及凋亡蛋白(caspase-1)表达水平。结果:与对照组相比,LPS组矿化结节减少,细胞凋亡率及caspase-1表达、TNF-α、IL-1β、IL-6含量显著增加,ALP水平及OPN、RUNX2、OCN表达、LKB1与AMPK磷酸化水平显著降低(P<0.05);与LPS组相比,LPS+PF-L组、LPS+PF-M组、LPS+PF-H组矿化结节明显增多,细胞凋亡率及caspase-1表达、TNF-α、IL-1β、IL-6含量显著降低,ALP水平及OPN、RUNX2、OCN表达、LKB1与AMPK磷酸化水平显著增加,均呈现剂量依赖性(P<0.05);但radicicol逆转了PF-H对LPS诱导的PDLSCs凋亡和成骨分化的作用(P<0.05)。结论:PF通过上调LKB1-AMPK信号通路,抑制LPS诱导的PDLSCs凋亡、促进成骨分化。

Objective:To investigate the influences of paeoniflorin(PF)on lipopolysaccharide(LPS)-induced apoptosis and osteogenic differentiation of periodontal ligament stem cells(PDLSCs)by regulating liver kinase B1(LKB1)-adenylate-activated protein kinase(AMPK)signaling pathway.Methods:PDLSCs in logarithmic growth phase were collected and separated into control group,LPS group(10μg/mL),LPS+PF low concentration(PF-L,10μg/mL LPS+5μmol/L PF)group,LPS+PF medium concentration(PF-M,10μg/mL LPS+10μmol/L PF)group,LPS+PF high concentration(PF-H,10μg/mL LPS+20μmol/L PF)group,and LPS+PF-H+LKB1 inhibitor(radicicol,10μg/mL LPS+20μmol/L PF+10μmol/L radicicol)group.ELISA was used to detect the levels of inflammatory factors[interleukin(IL)-1β,tumor necrosis factor-α(TNF-α),IL-6]in cells;alizarin red staining was used to observe the changes of mineralized nodules;alkaline phosphatase(ALP)was used to detect the cell osteogenic differentiation;cell apoptosis was detected by flow cytometry;the expression levels of osteogenic genes(OPN,RUNX2 and OCN)were detected by qRT-PCR;Western blot was used to detect the expression levels of LKB1-AMPK pathway protein and apoptosis protein(caspase-1).Results:Compared with the control group,the mineralized nodules were reduced in the LPS group,the apoptosis rate,the expression of caspase-1,the contents of TNF-α,IL-1βand IL-6 increased obviously,the level of ALP,the expression of OPN,RUNX2 and OCN,and the phosphorylation levels of LKB1 and AMPK decreased obviously(P<0.05);compared with the LPS group,the mineralized nodules were reduced in the LPS+PF-L group,the LPS+PF-M group,and the LPS+PF-H group,the apoptosis rate,the expression of caspase-1,the contents of TNF-α,IL-1βand IL-6 decreased obviously,the level of ALP,the expression of OPN,RUNX2 and OCN,and the phosphorylation levels of LKB1 and AMPK increased obviously,all were dose-dependent(P<0.05);however,radicicol reversed the effects of PF-H on LPS-induced apoptosis and osteogenic differentiation of PDLSCs(P<0.05).Conclusion:PF inhibits LPS-induced apoptosis of PDLSCs and promotes osteogenic differentiation by up-regulating LKB1-AMPK signaling pathway.