Erythropoietin/erythropoietin receptor signaling pathway not only participates in bone marrow hematopoiesis, but also regulates the metabolic response of non-hematopoietic tissues, such as brain, heart, skeletal muscle, and adipose tissue. Simultaneously, it can accelerate the mineralization process of periodontal ligament stem cells and reduce oxidative stress damage. However, the mechanism of action on osteogenic differentiation of periodontal ligament stem cells is still unclear.

To investigate the effect and action mechanism of erythropoietin/erythropoietin receptor signaling pathway on osteogenic differentiation of periodontal ligament stem cells.

Enzyme digestion method was used to isolate and culture periodontal ligament stem cells from periodontal disease patients and healthy people. The mRNA and protein levels of erythropoietin receptor in two kinds of periodontal ligament stem cells were detected by qRT-PCR and western blot assay. Erythropoietin receptor expression was silenced by small interfering RNA (siRNA) or activated by erythropoietin. qRT-PCR and western blot assay were used to detect the expression of erythropoietin receptor, levels of osteogenic marker genes Runt-related transcription factor 2 (Runx2), osteocalcin, osteopontin, and bone sialoprotein. Alkaline phosphatase staining and alizarin red staining were applied to measure osteogenic differentiation ability of periodontal ligament stem cells. The phosphorylation of signal transducer and activator of transcription 5 (STAT5) was detected by western blot assay.

(1) The results of qRT-PCR and western blot assay showed that the mRNA and protein levels of erythropoietin receptor in periodontal ligament stem cells in the disease group were significantly lower than those in periodontal ligament stem cells in the healthy group. (2) Alkaline phosphatase staining and alizarin red staining showed that knocking down the erythropoietin receptor can inhibit the osteogenic differentiation ability of periodontal ligament stem cells. qRT-PCR results showed that compared with the control group, knockdown of the erythropoietin receptor group significantly reduced expression levels of Runt-related transcription factor 2, osteocalcin, osteopontin, and bone sialoprotein (P < 0.05). (3) qRT-PCR results showed that after erythropoietin treatment, the expression of erythropoietin receptor in periodontal ligament stem cells recovered. Silencing erythropoietin receptor and then administration of erythropoietin treatment reversed the expression level of erythropoietin receptor. Erythropoietin treatment increased the osteogenic differentiation ability of periodontal ligament stem cells in the disease group and the expression level of the osteogenic marker gene Runt-related transcription factor 2 (P < 0.05). Silencing the expression of STAT5 inhibited this effect of erythropoietin. (4) Western blot assay results showed that with the extension of erythropoietin treatment time, the phosphorylation level of STAT5 increased in periodontal ligament stem cells in the disease group (P < 0.05). The above results indicate that erythropoietin restores the osteogenic differentiation ability of pathological periodontal ligament stem cells by inducing the phosphorylation of STAT5.