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Azithromycin Promotes the Osteogenic Differentiation of Human Periodontal Ligament Stem Cells after Stimulation with TNF-?.

ABSTRACT:

Background and objective

This study investigated the effects and underlying mechanisms of azithromycin (AZM) treatment on the osteogenic differentiation of human periodontal ligament stem cells (PDLSCs) after their stimulation with TNF-? in vitro. Methods. PDLSCs were isolated from periodontal ligaments from extracted teeth, and MTS assay was used to evaluate whether AZM and TNF-? had toxic effects on PDLSCs viability and proliferation. After stimulating PDLSCs with TNF-? and AZM, we analyzed alkaline phosphatase staining, alkaline phosphatase activity, and alizarin red staining to detect osteogenic differentiation. Real-time quantitative polymerase chain reaction (RT-qPCR) analysis was performed to detect the mRNA expression of osteogenic-related genes, including RUNX2, OCN, and BSP. Western blotting was used to measure the NF-?B signaling pathway proteins p65, phosphorylated p65, I?B-?, phosphorylated I?B-?, and ?-catenin as well as the apoptosis-related proteins caspase-8 and caspase-3. Annexin V assay was used to detect PDLSCs apoptosis.

Results

TNF-? stimulation of PDLSCs decreased alkaline phosphatase and alizarin red staining, alkaline phosphatase activity, and mRNA expression of RUNX2, OCN, and BSP in osteogenic-conditioned medium. AZM enhanced the osteogenic differentiation of PDLSCs that were stimulated with TNF-?. Western blot analysis showed that ?-catenin, phosphorated p65, and phosphorylated I?B-? protein expression decreased in PDLSCs treated with AZM. In addition, pretreatment of PDLSCs with AZM (10??g/ml, 20??g/ml) prevented TNF-?-induced apoptosis by decreasing caspase-8 and caspase-3 expression.

Conclusions

Our results showed that AZM promotes PDLSCs osteogenic differentiation in an inflammatory microenvironment by inhibiting the WNT and NF-?B signaling pathways and by suppressing TNF-?-induced apoptosis. This suggests that AZM has potential as a clinical therapeutic for periodontitis.

SUBMITTER: Meng T

PROVIDER: S-EPMC6234456 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Azithromycin Promotes the Osteogenic Differentiation of Human Periodontal Ligament Stem Cells after Stimulation with TNF-<i>α</i>.

Meng Tingting T Zhou Ying Y Li Jingkun J Hu Meilin M Li Xiaomeng X Wang Pingting P Jia Zhi Z Li Liyu L Liu Dayong D

Stem cells international 20181031

<h4>Background and objective</h4>This study investigated the effects and underlying mechanisms of azithromycin (AZM) treatment on the osteogenic differentiation of human periodontal ligament stem cells (PDLSCs) after their stimulation with TNF-<i>α in vitro. Methods</i>. PDLSCs were isolated from periodontal ligaments from extracted teeth, and MTS assay was used to evaluate whether AZM and TNF-<i>α</i> had toxic effects on PDLSCs viability and proliferation. After stimulating PDLSCs with TNF-<i> ...[more]

PMID: 30515223

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Project description:BACKGROUND:Long non-coding RNAs (lncRNAs) have been widely known to have an appreciable effect in physiology and pathology. In tooth regeneration, periodontal ligament stem cells (PDLSCs) are regarded as a key effector, whereas, how lncRNA acts in the osteogenic differentiation of PDLSCs have not been completely understood. This study aims to find out the relationship between lncRNA DANCR and the proliferation and osteogenic differentiation of PDLSCs. METHODS:Microarray was used to observe the different expression of lncRNAs in differentiated and undifferentiated PDLSCs. And then osteogenic-related lncRNA, DANCR was screened out. Its effects on proliferation and osteogenic differentiation was explored by constructing an overexpression and inhibition model. qRT-PCR was used to detect the mRNA expression of osteogenesis related genes. MTT assay was performed to assess the effects of DANCR on cell growth curve. To quantify the effects of DANCR on osteogenic differentiation of PDLSCs, ALP staining and alizarin red was performed in basic culture medium and osteogenic medium. Data were statistically processed. RESULTS:Compared with the undifferentiated PDLSCs, the alizarin red staining level was higher in differentiated PDLSCs. And the expressions of osteogenic differentiation marker genes Runt-related transcription factor 2 (Runx2), osteocalcin (OCN) and bone morphogenetic protein (BMP-2) were significantly increased in the differentiated PDLSCs. Furthermore, we noticed that comparing with control groups, the expression of lncRNA DANCR decreases markedly in osteogenically induced PDLSCs. DANCR promoted proliferation of PDLSCs, as evidenced by cell viability. Further investigation has proven that the downregulation of DANCR shows in the calcium sediment forming, alkaline phosphatase (ALP) activation and some osteogenic-related gene markers' upregulation including Runx2, OCN and BMP-2, which finally results in the osteogenic differentiation of PDLSCs following the transfection and induction. Conversely, DANCR upregulation was shown to repress the osteogenic differentiation potential of PDLSCs. CONCLUSIONS:The osteogenic differentiation of PDLSCs has proven to related to the down regulation of lncRNA DANCR. And this paper throws light on the effects of DANCR in the process of PDLSCs' osteogenic differentiation.