Effects Of Melatonin On Osteogenic Differentiation And Oxidative Stress Of Human Periodontal Ligament Stem Cells In Inflammatory Microenvironment

Objective:To explore whether melatonin(Mela)can protect human periodontal ligament stem cells(h PDLSCs)from Osteogenic potential in the presence of the inflammatory cytokine tumor necrosis factor-alpha(TNF-α)and reduce the production of reactive oxygen species(ROS),and its potential mechanism was initially explored,providing theoretical basis for melatonin as a candidate drug for the treatment of periodontitis.Methods:(1)1/3 of the periodontal ligament tissue in the root of the tooth was scraped,the primary human periodontal ligament stem cells were isolated and cultured by the tissue block enzyme digestion method,and the expression of cell surface antigens CD146,CD90 and CD45 was detected by flow cytometry,and the multidirectional differentiation ability of osteogenic and adipogenic cells were detected;(2)Alkaline phosphatase activity assay was used to detect the effect of different concentrations of melatonin on the osteogenic differentiation ability of h PDLSCs under inflammatory microenvironment,and the optimal concentration was determined;according to the results,the experiment was divided into three groups: control group(containing only the Bone culture medium),TNF-α group(osteogenic medium+10ng/ml TNF-α),TNF-α+ Mela group(osteogenic medium+10ng/ml TNF-α+100μM melatonin);(3)Alkaline phosphatase staining and activity analysis,alizarin red staining and semi-quantitative analysis,The expression of osteogenic genes and proteins(OPN and RUNX2)was detected by RT-q PCR and western blot,and to observe the effect of melatonin on the osteogenic differentiation ability of h PDLSCs under inflammatory microenvironment;(4)ROS levels in cells incubated with DCFH-DA fluorescent probe were observed by fluorescence microscopy and flow cytometry,and oxidase and antioxidant enzyme-related genes and proteins(NOX1 and CAT)expression levels were detected by RT-q PCR and Western blotting,to observe the changes of melatonin on oxidative stress of h PDLSCs under inflammatory microenvironment;(5)The expression of NF-κB signaling pathway-related proteins P65/P-P65 and IκBα in h PDLSCs under inflammatory microenvironment was detected by western blotting.Results:(1)The results of flow cytometry showed that the expression rates of the surface markers CD90 and CD146 of the cells extracted in this experiment were 99.6% and63.7%,while the expression rate of CD45 was only 0.27%.In addition,h PDLSCs have the ability to differentiate into osseous and adipocytes,consistenting with the typical characteristics of mesenchymal stem cells.Combined with the material obtained,it indicates that the cells extracted in this experiment are human periodontal ligament stem cells;(2)100 μM melatonin can restore the alkaline phosphatase activity of h PDLSCs in the inflammatory microenvironment to the greatest extent,so this concentration was selected for subsequent experiments;(3)After TNF-αstimulated h PDLSCs,the staining degree and activity of alkaline phosphatase were down-regulated.Alizarin red staining and semi-quantitative analysis showed that the formation of mineralization was reduced.At the same time,the gene and protein expression levels of OPN and RUNX2 were reduced.Melatonin could partially restore the osteogenic potential of h PDLSCs inhibited by TNF-α(P<0.05);(4)Melatonin can reduce the level of ROS in the inflammatory microenvironment,down-regulate the expression of oxidase NOX1 and up-regulate the expression of antioxidant enzyme CAT(P<0.05);(5)Western blotting results showed that p65 in the TNF-α group The phosphorylation level of protein was up-regulated and the protein level of IκBα was down-regulated,while the phosphorylation level of p65 protein was down-regulated and the protein level of IκBα was up-regulated in TNF-α + Mela group(P<0.05).Conclusion:Melatonin can inhibit TNF-α-induced ROS production during the osteogenic differentiation of h PDLSCs,and reverse the TNF-α-suppressed osteogenic differentiation of h PDLSCs in vitro,which is accompanied by activation and inactivation of the NF-κB signaling pathway.Therefore,these findings provide more evidence that melatonin can be used as a drug candidate for the treatment of periodontitis.