Yes-associated protein-mediated melatonin regulates the function of periodontal ligament stem cells under oxidative stress conditions

Figure 1 Characterization of periodontal ligament stem cells. A: Periodontal ligament stem cells (PDLSCs) were cultured (scale bar = 200 μm); B: PDLSCs expressed CD105, CD73, CD29, CD44 and CD90 positively and expressed CD34, CD31 and CD45 negatively; C: Alkaline phosphatase staining of PDLSCs; D: Alizarin Red staining of PDLSCs; E: Oil Red O staining of PDLSCs. Data are presented as mean ± SD, n = 3.

Figure 2 Hydrogen peroxide-induced oxidative stress in periodontal ligament stem cells. A: Hydrogen peroxide (H₂O₂) affected the growth state and cell morphology of periodontal ligament stem cells (PDLSCs); B: H₂O₂ promoted apoptosis of PDLSCs; C: H₂O₂ increased the reactive oxygen species fluorescence intensity of PDLSCs; D: H₂O₂ increased the proportion of reactive oxygen species-positive cells in PDLSCs. Data are presented as mean ± SD, n = 3. ^aP < 0.05; ^bP < 0.01; ^dP < 0.0001. NC: Negative control; ROS: Reactive oxygen species; H₂O₂: Hydrogen peroxide.

Figure 3 Hydrogen peroxide inhibited stemness and promoted senescence in periodontal ligament stem cells. A: Hydrogen peroxide (H₂O₂) inhibited the expression level of stemness-related proteins C-MYC, OCT-4, and NANOG in periodontal ligament stem cells (PDLSCs); B: H₂O₂ suppressed the expression levels of stemness-related genes C-MYC, OCT-4, and NANOG in PDLSCs; C: Alkaline phosphatase staining and alkaline phosphatase activity showed that the osteogenic differentiation ability of the H₂O₂-induced PDLSCs was weaker than that of the control group; D: Alizarin Red staining and quantitative analysis showed that the osteogenic differentiation ability of the H₂O₂-induced PDLSCs was weaker than that of the control group; E: Senescence-associated β-galactosidase staining indicated significantly more senescence-associated β-galactosidase-positive cells in H₂O₂-induced PDLSCs compared with the control group; F: Western blot results showed that H₂O₂ inhibited the expression of p-RB but increased the expression of p21 in PDLSCs. Data are presented as mean ± SD, n = 3. ^aP < 0.05; ^bP < 0.01; ^cP < 0.001; ^dP < 0.0001. NC: Negative control; H₂O₂: Hydrogen peroxide; ALP: Alkaline phosphatase; SA β-gal: Senescence-associated β-galactosidase.

Figure 4 Melatonin improved proliferative capacity and reduced oxidative stress levels in hydrogen peroxide-induced periodontal ligament stem cells. A: Melatonin (MT) restored the growth state and enhanced the proliferative capacity of hydrogen peroxide (H₂O₂)-induced periodontal ligament stem cells (PDLSCs); B: MT reduced the proportion of G0/G1 phase and increased the proportion of G2/M phase in H₂O₂-induced PDLSCs; C: MT decreased the reactive oxygen species fluorescence intensity of H₂O₂-induced PDLSCs; D: MT decreased the proportion of reactive oxygen species-positive cells in H₂O₂-induced PDLSCs. Data are presented as mean ± SD, n = 3. ^aP < 0.05; ^bP < 0.01; ^dP < 0.0001. NS: No significance; H₂O₂: Hydrogen peroxide; MT: Melatonin; NC: Negative control.

Figure 5 Melatonin increased stemness and delayed senescence in hydrogen peroxide-induced periodontal ligament stem cells. A: Melatonin (MT) restored the expression levels of stemness-related proteins C-MYC, OCT-4, and NANOG in hydrogen peroxide (H₂O₂)-induced periodontal ligament stem cells (PDLSCs); B: Alkaline phosphatase staining and alkaline phosphatase activity showed that MT enhanced the osteogenic differentiation ability of the H₂O₂-induced PDLSCs; C: Alizarin Red staining and quantitative analysis showed that MT enhanced the osteogenic differentiation ability of the H₂O₂-induced PDLSCs; D: Senescence-associated β-galactosidase staining indicated MT decreased senescence-associated β-galactosidase-positive cells in H₂O₂-induced PDLSCs; E: Western blot results showed that MT increased the expression of p-RB but inhibited the expression of p21 in H₂O₂-induced PDLSCs. Data are presented as mean ± SD, n = 3. ^aP < 0.05; ^bP < 0.01; ^cP < 0.001; ^dP < 0.0001. H₂O₂: Hydrogen peroxide; ALP: Alkaline phosphatase; ARS: Alizarin Red staining; MT: Melatonin; SA β-gal: Senescence-associated β-galactosidase.

Figure 6 Yes-associated protein mediated melatonin to enhance stemness and reduce senescence in hydrogen peroxide-induced periodontal ligament stem cells. A: Hydrogen peroxide (H₂O₂) reduced the mRNA expression level of Yes-associated protein (YAP) in periodontal ligament stem cells (PDLSCs); B: H₂O₂ reduced the protein expression level of YAP in PDLSCs; C: Melatonin (MT) increased the protein expression level of YAP in H₂O₂-induced PDLSCs; D: Verteporfin reduced the protein expression level of YAP in MT-increased PDLSCs; E: Verteporfin decreased the protein expression levels of stemness-related proteins C-MYC, OCT-4 and NANOG in MT-increased PDLSCs; F: Alkaline phosphatase staining and alkaline phosphatase activity showed that verteporfin reduced the osteogenic differentiation ability in MT-increased PDLSCs; G: Alizarin Red staining and quantitative analysis showed that verteporfin reduced the osteogenic differentiation ability in MT-increased PDLSCs; H: Senescence-associated β-galactosidase staining indicated verteporfin increased senescence-associated β-galactosidase-positive cells in MT-increased PDLSCs; I: Western blot results showed that verteporfin decreased the expression of p-RB but increased the expression of p21 in MT-treated PDLSCs. Data are presented as mean ± SD, n = 3. ^aP < 0.05; ^bP < 0.01; ^cP < 0.001; ^dP < 0.0001. H₂O₂: Hydrogen peroxide; MT: Melatonin; YAP: Yes-associated protein; VP: Verteporfin; ALP: Alkaline phosphatase; ARS: Alizarin Red staining.