Effect of Shikonin on proliferation and osteogenic differentiation of human periodontal ligament stem cells(hPDLSCs)under simulated inflammatory microenvironment
Objective To investigate the effects of Shikonin on the proliferation and osteogenic differentia-tion of human periodontal ligament stem cells(hPDLSCs)in a lipopolysaccharide-induced inflammatory microenvironment.Methods hPDLSCs were isolated and cultured from healthy human periodontal tis-sues,andthe cell surface markers CD29,CD105,CD34and CD45 were identified by flow cytometry.CCK-8 method was used to detect the effects of different concentrations of Shikonin on the proliferative activity of hPDLSCs in normal and simulated inflammatory microenvironments.Firstly,the suitable concentration range of Shikonin to promote the proliferation of human periodontal stem cells were screened out.And then periodontal stem cells were taken for subsequent experiments:blank group(simple medium),Shikonin group(experimental drug concentration Shikonin),LPS group(Porphyromonas gingivalis lipopolysaccha-ride,P.g-LPS,10 μg/mL)and Shikonin+LPS group(experimental drug concentration Shikonin+P.g-LPS).The levels of IL-6 and IL-18 in the cell supernatant were detected by enzyme-linked immunosorbent assay at 3 d;alkaline phosphatase staining and alkaline phosphatase activity test was performed at 7 d;and alizarin red staining was performed to detect the differentiation ability of osteogenic bone at 21 d;and RT-PCR was performed to detect the expression of osteogenic genes,such as osteocalcin,RUNT-associated transcription factor 2,and alkaline phosphatase,at 168 h.Results Isolated cultured hPDLSCs were mainly bundled long shuttle-shaped.Flow cytometry identified the results of surface markers CD29(99.91％),CD105(92.34％),CD34(0.76％)and CD45(0.32％).The results confirmed that the isolated cells conformed to stem cell characteristics.The results of CCK-8 assay showed that the relative cell viability of hPDLSCs was elevated in the 0.062 5,0.125,0.25 and 0.5 μmol/L groups of Shikonin compared with blank group under normal state(P＜0.05);and that the activity of hPDLSCs in the 0.5 μmol/L group of Shikonin was the best under the simulated inflammatory environment(P＜0.05).Meanwhile,0.5 μmol/L concen-tration of Shikonin promoted cell migration and decreased the levels of IL-6 and IL-18 in the inflammatory state(P＜0.05).And 0.5 μmol/L concentration of Shikonin had a significant effect on the osteogenic dif-ferentiation of hPDLSCs in the inflammatory state,which could enhance the osteocalcin,RUNT-related transcription factor 2 and alkaline phosphatase and other osteogenic genes in the inflammatory state(P＜0.05).Conclusion In the simulated inflammatory microenvironment,proper concentrations of Shikonin can reduce the levels of IL-6 and IL-18 in theinflammatory environment,and can promotethe proliferation,migration and osteogenic differentiation of humanperiodontal stem cells,which can be used as a potential candidate drug for the prevention and treatment of chronic periodontitis.
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