Congman Xie1,2, Min Lin3,4, Haonan Tian3,4, Lin Zhang3,5, Aishu Ren6,7. 1. Department of Orthodontics, Stomatological Hospital of Chongqing Medical University, Chongqing 401147. hyacinth_xie@stu.cqmu.edu.cn. 2. Chongqing Key Laboratory of Oral Diseases and Biomedical Sciences, Chongqing 401147. hyacinth_xie@stu.cqmu.edu.cn. 3. Department of Orthodontics, Stomatological Hospital of Chongqing Medical University, Chongqing 401147. 4. Chongqing Key Laboratory of Oral Diseases and Biomedical Sciences, Chongqing 401147. 5. Chongqing Municipal Key Laboratory of Oral Biomedical Engineering of Higher Education, Chongqing 401147, China. 6. Department of Orthodontics, Stomatological Hospital of Chongqing Medical University, Chongqing 401147. 500220@hospital.cqmu.edu.cn. 7. Chongqing Municipal Key Laboratory of Oral Biomedical Engineering of Higher Education, Chongqing 401147, China. 500220@hospital.cqmu.edu.cn.
Abstract
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.
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.
Entities:
Keywords:
astaxanthin; human periodontal ligament cells; inflammatory factors; lipopolysaccharide