摘要
目的:探讨葛根素(PR)对炎症条件下人牙髓干细胞(hDPSCs)成骨分化的影响及机制。方法:分离培养hDPSCs,CCK-8法检测PR对hDPSCs增殖活力的影响。将hDPSCs分为对照组、脂多糖(LPS)组、p38 MAPK抑制剂SB203580组(20μmol/L)、20μmol/L PR组、40μmol/L PR组、40μmol/L PR+20μmol/L SB203580组,采用LPS诱导建立体外hDPSCs炎症模型。通过乳酸脱氢酶(LDH)测定法、茜素红染色和碱性磷酸酶(ALP)活性测定评估PR对LPS诱导的hDPSCs细胞毒性和成骨分化能力的影响,实时荧光定量PCR(RT-qPCR)检测细胞中炎症(IL-6、IL-1β、TNF-α)和成骨分化相关基因(RUNX2、OCN、BSP)表达,Western blot检测细胞中丝裂原活化蛋白激酶(MAPK)/核因子-κB(NF-κB)通路相关蛋白表达。结果:20和40μmol/L的PR可显著增加hDPSCs的增殖活力,增强LPS刺激的hDPSCs中ALP活性、矿化结节和成骨分化相关基因的表达,降低LDH活性和炎症相关基因表达,抑制IκBα的磷酸化降解和NF-κB p65、p38 MAPK的磷酸化(均P<0.05)。40μmol/L PR与SB203580对MAPK信号通路的抑制作用接近,两者联用对MAPK/NF-κB信号通路的抑制作用和炎症状态下hDPSCs成骨分化的促进作用明显优于两者单独应用。结论:PR可减轻LPS诱导的hDPSCs炎症损伤,促进炎症状态下hDPSCs成骨分化;其作用机制可能与抑制MAPK/NF-κB信号通路有关。
Objective:To investigate the effects and mechanism of puerarin(PR)on the osteogenic differentiation of human dental pulp stem cells(hDPSCs)under inflammatory condition.Methods:hDPSCs were in vitro cultured,and divided into control group,lipopolysaccharide(LPS,inflammatory model of hDPSCs in vitro)group,p38 MAPK inhibitor SB203580 group(20μmol/L),20μmol/L PR group,40μmol/L PR group,and 40μmol/L PR+20μmol/L SB203580 group.The cell proliferation was detected by CCK-8 assay.PR on the LPS-induced cytotoxicity and osteogenic differentiation capacity of hDPSCs were assessed by Lactate dehydrogenase(LDH)assay,alizarin red staining and alkaline phosphatase(ALP)activity assays respectively.RT-qPCR was performed to detect the expression of IL-6,IL-1β,TNF-α,RUNX2,OCN and BSP of the cells.Western blot was performed to detect the expression of mitogen-activated protein kinase(MAPK)/nuclear factor-κB(NF-κB)signaling pathway-related proteins in the cells.Results:20 and 40μmol/L PR increased the proliferation activity,ALP activity,mineralized nodule and osteogenic differentiation-related gene expression,decreased LDH activity and inflammation-related gene expression,inhibited phosphorylation and degradation of IκBαand phosphorylation of NF-κB p65 and p38 MAPK(all P<0.05).The inhibitory effects of 40μmol/L PR and SB203580 on the MAPK signaling pathway were similar,and the combination of the two showed stronger inhibitory effects on the MAPK/NF-κB signaling pathway and the promotion of osteogenic differentiation of hDPSCs under inflammatory condition than either alone.Conclusion:PR can alleviate LPS-induced inflammatory injury of hDPSCs and promote the osteogenic differentiation of hDPSCs under inflammatory state;its mechanism may be related to the inhibition of MAPK/NF-κB signaling pathway.
作者
支旺
温惠慧
崔志强
ZHI Wang;WEN Huihui;CUI Zhiqiang(Department of Stomatology,Peking University Medical Lu'an Hospital,China Changzhi,046204;Department of Stomatology,Heji Hospital,Changzhi Medical College)
出处
《实用口腔医学杂志》
CAS
CSCD
北大核心
2023年第5期648-654,共7页
Journal of Practical Stomatology
关键词
牙髓炎
葛根素
人牙髓干细胞
脂多糖
成骨分化
丝裂原活化蛋白激酶
核因子-κB
Pulpitis
Puerarin
Human dental pulp stem cells
Lipopolysaccharide
Osteogenic differentiation
Mitogen-activated protein kinase
Nuclear factor-κB
