Objectives: Human periodontal ligament cells (hPDLCs) are important source of periodontal tissue reconstruction. Under chronic inflammation, the multi-directional differentiation potential and chemotaxis in hPDLCs are decreased. Therefore, inhibiting inflammatory microenvironment and improving the functional characteristics of stem cells can better promote periodontal tissue reconstruction. This study was to investigate the effect of astaxanthin (AST) on lipopolysaccharide (LPS)-induced inflammation in hPDLCs and the underlying mechanisms.
Methods: hPDLCs were isolated and cultured in vitro, and vimentin and keratin immunocytochemical staining were used to identify hPDLCs. CCK-8 assay was used to measure the effects of AST (1, 5, 10, 20, 50, 100, and 200 μmol/L) on proliferation of hPDLCs. Quantitative RT-PCR (RT-qPCR) and ELISA were used to measure the mRNA and protein expression of inflammatory factors (IL-6, IL-1β, and TNF-α) in the control (Con) group, the LPS group, and the LPS+AST (5, 10, 20, and 50 μmol/L) group. Western blotting was used to detect the protein expression of IKBα, phosphorylated IKBα (p-IKBα), and p65 in the Con group, the LPS group, the AST (20 μmol/L) group, and the LPS+AST (20 μmol/L) group. After 10 μmol/L PDTC treatment, the mRNA and protein expressions of IL-6, IL-1β, and TNF-α were detected by RT-qPCR and ELISA.
Results: Cell morphology and immunocytochemical staining showed that the cells were in line with the characteristics of hPDLCs. Treatment with AST could promote the proliferation of hPDLCs, which reached the peak at 20 μmol/L. The mRNA and protein expressions of IL-6, IL-1β, and TNF-α in the LPS group were higher than those in the Con group (all P<0.05). Compared with the LPS group, the mRNA and protein expressions of IL-6, IL-1β, and TNF-α in the LPS+AST (5, 10, 20, and 50 μmol/L) group were down-regulated (all P<0.05). Compared with the Con group, the levels of IKBα and p65 in cytoplasm of the LPS group were significantly downregulated (both P<0.05), and the levels of p-IKBα in cytoplasm and p65 in nucleus of the LPS group were significantly up-regulated (both P<0.05). Compared with the LPS group, the levels of IKBα and p65 in cytoplasm of the LPS+AST (20 μmol/L) group were significantly upregulated (both P<0.05), and the levels of p-IKBα in cytoplasm and p65 in nucleus of the LPS+AST (20 μmol/L) group were significantly downregulated (both P<0.05). The mRNA and protein expressions of IL-6, IL-1β, and TNF-α in the LPS+PDTC (10 μmol/L) group were lower than those in the LPS group (all P<0.05).
Conclusions: AST promotes the proliferation of hPDLCs, which is related to suppression of LPS-induced the secretion of inflammatory factors via inhibiting the activation of NF-κB signaling pathway.
目的: 人牙周膜细胞(human periodontal ligament cells，hPDLCs)是牙周组织重建的重要来源。在慢性炎症时，hPDLCs的多向分化潜能及趋化能力降低。因此，抑制炎症微环境，提高干细胞的功能特性，能更好地促进牙周组织重建。本研究探讨虾青素(astaxanthin，AST)对脂多糖(lipopolysaccharide，LPS)诱导的hPDLCs炎症因子分泌的作用及其机制。方法: 在体外分离、培养hPDLCs，采用免疫化学细胞染色法行波形蛋白和角蛋白染色以鉴定hPDLCs。采用CCK-8法检测1、5、10、20、50、100、200 μmol/L AST对hPDLCs增殖的影响，定量RT-PCR(RT-qPCR)和ELISA法检测对照(Con)组、LPS组、LPS+AST(5、10、20、50 μmol/L)组炎症因子IL-6、IL-1β及TNF-α mRNA和蛋白质的表达，蛋白质印迹法检测Con组、LPS组、AST(20 μmol/L)组、LPS+AST(20 μmol/L)组IKBα、磷酸化的IKBα(p-IKBα)和p65蛋白质的表达。经NF-κB抑制剂PDTC(10 μmol/L)处理后，采用RT-qPCR和ELISA法检测IL-6、IL-1β及TNF-α mRNA和蛋白质的表达。结果: 细胞形态及免疫化学细胞染色结果表明培养的细胞符合hPDLCs特性。AST能够促进hPDLCs的增殖，且在20 μmol/L时达到峰值。LPS组细胞IL-6、IL-1β及TNF-α的mRNA和蛋白质表达水平均高于Con组(均P<0.05)；LPS+AST(5、10、20、50 μmol/L)组细胞IL-6、IL-1β及TNF-α的mRNA和蛋白质的表达水平均低于LPS组(均P<0.05)。与Con组相比，LPS组细胞质中IKBα和p65水平显著下调(均P<0.05)，细胞质中p-IKBα和细胞核中p65水平显著上调(均P<0.05)；与LPS组相比，LPS+AST(20 μmol/L)组细胞质中IKBα和p65水平显著上调(均P<0.05)，细胞质中p-IKBα和细胞核中p65水平显著下调(均P<0.05)。LPS+PDTC (10 μmol/L)组IL-6、IL-1β及TNF-α的mRNA和蛋白质的表达水平均低于LPS组(均P<0.05)。结论: AST能促进hPDLCs增殖，可能与通过NF-κB信号通路抑制LPS介导的炎症因子分泌有关。.
Keywords: astaxanthin; human periodontal ligament cells; inflammatory factors; lipopolysaccharide.