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

doi:10.3969/j.issn.1006-6233.2023.01.001

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摘要 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。

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关键词 ：芍药苷, LKB1-AMPK信号通路, 脂多糖, 牙周膜干细胞, 凋亡, 成骨分化

Abstract：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-1β and 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-1β and 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.

Key words： Paeoniflorin LKB1-AMPK signaling pathway Lipopolysaccharide Periodontal ligament stem cells Apoptosis Osteogenic differentiation

基金资助:甘肃省科技计划项目,(编号:21JR11RA006)

通讯作者: 李健学

引用本文:

梅妹, 苟雅萍, 郭艳, 杨彦伟, 郭娅, 李健学. 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[J]. 河北医学, 2023, 29(1): 1-6.
MEI Mei, et al. Influences of Paeoniflorin on LPS-Induced Apoptosis and Osteogenic Differentiation of Periodontal Ligament Stem Cells by Regulating LKB1-AMPK Signaling Pathway. HeBei Med, 2023, 29(1): 1-6.

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http://www.hbyxzzs.cn/CN/10.3969/j.issn.1006-6233.2023.01.001 或 http://www.hbyxzzs.cn/CN/Y2023/V29/I1/1

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