ABSTRACT
Objective @#To study the effect of light-emitting diode (LED) red light on the osteogenic/odontogenic differentiation of human dental pulp stem cells (hDPSCs) and its mechanism were discussed. @*Methods@#This study has been reviewed and approved by the Ethics Committee. hDPSCs were cultured by tissue block enzyme digestion. The proliferative capacity of hDPSCs was detected by the CCK-8 at days 1, 3, 5 and 7 under stimulation with 0, 1, 5 and 10 μg/mL lipopolysaccharide (LPS), and the LPS stimulatory concentration was screened. The CG group (mineralization induction), LPS+CG group, and LPS+CG+ (2, 4, 6, 8, and 10 J/cm2) LED red light groups were set. On day 7, alkaline phosphatase (ALP) staining and ALP activity were determined. Relative expression levels of the ALP, osterix (OSX), dentin matrix protein-1 (DMP-1) and dentin sialophosphoprotein (DSPP) genes were measured by qRT-PCR. On day 21, alizarin red staining and calcium nodule quantitative determination were performed to screen the best light energy. The LPS+CG group and LPS+CG+LED group (optimal energy) were set up, and the secretion and expression levels of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were detected by ELISAs on days 1, 3, 5 and 7. The relative expression levels of the extracellular regulated protein kinases 1/2 (ERK1/2), p38, c-Jun N-terminal kinase (JNK), and extracellular regulated protein kinases 5 (ERK5) proteins and their phosphorylated proteins in the MAPK signaling pathway were detected by Western blots. After the pathway was blocked, the relative expression levels of the ALP, OSX, DMP-1, and DSPP proteins after LED red light irradiation on day 7 were detected by Western blots.@*Results@# CCK-8 assays showed that the proliferation of hDPSCs induced by 10 μg/mL LPS was lower than that of the 0, 1, and 5 μg/mL groups on the 5th and 7th days (P<0.05), and 10 μg/mL was selected as the LPS stimulatory concentration in the follow-up experiment. ALP staining, ALP activity, gene expression levels of ALP, OSX, DMP-1 and DSPP and calcium nodule quantification in the LPS+CG+4 J/cm2 group were higher than those in the other treatment groups (P<0.05). 4 J/cm2 LED red light had the strongest ability to promote osteogenic/odontogenic differentiation and was used as the LED light energy density in subsequent experiments. ELISA showed that the secretion and expression levels of TNF-α and IL-1β in the LPS+CG+LED group were lower than those in the LPS+CG group on the 5th and 7th days (P<0.05). Western blot analysis showed that 4 J/cm2 LED red light promoted the expression levels of the p-ERK1/2, p-p38, p-JNK and p-ERK5 proteins. After the MAPK pathway was blocked, the expression levels of the ALP, OSX, DMP-1, and DSPP proteins in the LPS+CG+LED+U0126 (ERK1/2 inhibitor), SP600125 (JNK inhibitor), and BIX02189 (ERK5 inhibitor) groups were lower than those in the LPS+CG+LED group (P<0.001). The protein expression levels of ALP, OSX and DMP-1 in the LPS+CG+LED+SB203580 (p38 inhibitor) group were not significantly different from those in the LPS+CG+LED group (P>0.05).@*Conclusion@#In inflammatory conditions, LED red light promotes osteogenic/odontogenic differentiation of hDPSCs. This effect may be attributed to enhancement of the ERK1/2, JNK, and ERK5 signaling pathways, which reduces the production of the inflammatory cytokines TNF-α and IL-1β.