Effects Of Trichostatin A On Imflammatory Responses Of Human Gingival Stem Cells And The Underlying Mechanism

Objective:This study was to investigate the effects of histone deacetylation inhibitor--Trichostatin A (TSA) on inflammatory response in gingival stem cells. The underlying mechanisms by which TSA affect proinflammatory cytokines production was also explored.Methods:1. Gingival tissue samples from health and periodontitis patients were obtained from clinic following routine and tissues were dissociated in collagenase I and Dispase. DMEM medium was used to culture cells were. Flow cytometric analysis were tested the surface markers of stem cell including OCT-4, SSEA-4, CD146 and CD90. Adipogenesis and osteogenesis differentiation were detected the multiple different potentials of GMSCs were subsequently observed by Alizarin Red staining, ALP activity and oil red O staining.2. The effects of histone deacetylase inhibitors TSA on the inflammatory cytoKines production in MSCs were isloated from health and periodontitis patients’gingival tisssues.The mRNA levels of histone deacetylase Ⅰ, Ⅲ, Ⅳ, interleuKin 6 and interleuKin 8 were detected by RT-PCR in N-GMSCs and I-GMSCs. MTT and Annexin V analysis were used to detect the effect of TSA on the proliferation and apoptosis of GMSCs. Flow cytometric CBA assay and RT-PCR were performed to detect the concentration of interleuKin 6 and interleuKin 8 in suprenatants of cultured GMSCs with TSA stimulation.3. NF-κB signaling pathwayGMSCs were stimulated by LPS and TSA for 0,15,30,45,60 min, extracted the nucleus protein to detect the DNA binding activity of p65 by NF-κB (p65) transcription factor assay kit. I-κBα, phosphorated-I-κBα, p65 and phosphorated-p65 were detected by western-blot.Results:1. Flow cytometric analysis showed GMSC expressed were positive with anti-SSEA-4, OCT-4, CD90 and CD146 which were considered as biomarkers of MSC or embryonic stem cells. After 7 days or 3 weeks of cultuer with osteogenic medium and 4 weeks of culture with an adipogenic medium, the GMSCs had the potential to osteogenic and adipogenic differentiation.2. The mRNA expressions of HDAC Ⅰ, Ⅲ, Ⅳ, IL-6 and IL-8 in inflamed gingival tissue were higher than that in healthy tissue. RT-PCR and flow cytometry CBA were used to detect the expression of IL-6 and IL-8 when treated with LPS and TSA, we found that TSA could control the expression of IL-6 and IL-8 both in mRNA and protein levels.3. NF-κB (p65) transcription factor assay kit showed that LPS could enhance the DNA binding activity of p65, however TSA could inhibit p65 DNA binding activity. Western-blot showed that cytoplasmic protein I-κB and nuclear protein p65 were phosphorylated after LPS stimulation. However, after LPS and 100 nM TSA treated, the expression of pI-KBa and p-p65 in GMSC were down-ergulated.Conclusions:1. Mesenchymal stem cells could be isolated from inflammted and healthy gingival tissues. N-GMSC and I-GMSC were identified by the biomarkers of stem cells, and multiple differentiation capability.2. TSA could inhibit inflammatory cytokines production in gingival mesenchymal stem cells were stimulated by LPS in vitro.3. Mechanically, TSA reperss inflammatory cytokins production in GMSCs by inhibiting NF-κB signaling pathway. Our results suggest that TSA might be a promise pharmaceutical in treating periodontitis.