The Study On Psoralen Stimulating Osteoblast Proliferation By Activating NF-kB-MAPK Signaling

ObjectiveOsteoporosis is a kind of systemically skeletal disease, which can make body bones more fragile and thus increase the incidence of fracture. About 50%postmenopausal women may suffer from osteoporosis due to postmenopausal estrogen deficiency. Nowadays medication, which aimed to increase bone formation or decrease bone resorption, is mainly used in clinic to reduce fractures caused by osteoporosis that have some side-effects at the sametime. People have been looking for natural alternatives which have the advantage of selective estrogen receptor modulators (SERMs), but brought less adverse effects compared to traditional medication. Recent studies showed that phytoestrogen is a kind of ideal natural selective estrogen receptor modulators for the treatment of osteoporosis. In Chinese herbal medicine, Psoralen, the main substance of Psoralea corylifolia, which has been an identity as a phytoestrogen, is used for osteoporosis treatment. Several studies have showed the effect of Psoralen in bone formation. However, the pathways and underlying molecular mechanisms participate in Psoralen-induced osteoblasts formation are unclear. The objective of this study was to identify the effects of Psoralen on osteoblasts and the signaling way which may involve by treating hFOBl.19(osteoblasts) with Psoralen in vitro.Methods(1) Effects of Psoralen on hFOBl.19 cells proliferation:psoralen in different concentration were added to hFOBl.19 cell medium. The cells were collected after 24h, CCK8 assay was used to detected its activity, while the qPCR and Western blotting assay were used to testing the expression level of GLUT3, then to determine the concentration and the time it takes to treat the hFOBl.19 cells.(2) Research on psoralen to MAPK signaling pathway:cells were divided into five groups, including normal group, psoralen group (psoralen added to medium),p38 signaling pathway inhibitor SB203580 group (psoralen and p38 signaling pathway inhibitor SB203580 added to medium), ERK signaling pathway inhibitor SCH772984 group (psoralen and ERK signaling pathway inhibitor SCH772984 added to medium) and JNK pathway inhibitor SP600125 group (psoralen and JNK signaling pathway inhibitor SP600125 added to medium). Cultured cells were collected after 48h. Cell proliferation was detected by CCK8 assay, then extracted proteins, to test the p38, ERK1/2, JNK protein phosphorylation level and p65, GLUT3 expression levels by Western blotting method.(3) Study of psoralen on NF-kB signaling pathway:cells were divided into three groups, including normal group, psoralen group (psoralen added in medium), NF-kB signaling pathway inhibitor group (psoralen and NF-kB signaling pathway inhibition added medium. Cultured cells were collected after 48h. Cell proliferation was detected by CCK8 assay, then extracted proteins, to test the p65, GLUT3 expression levels by Western blotting method.Results(1) Psoralen can promote hFOBl.19 cells proliferation:CCK-8 assay told that hFOBl.19 cells had showed obvious dependence when psoralen at a concentration of 15μM, GLUT3 expressed most obvious at this concentration by qPCR and Western blotting analysis, which was consistent with CCK-8 detection. The best time tips that hFOBl.19 cells proliferation most obvious, the study showed through CCK-8 assay that hFOBl.19 cells proliferate significantly in the concentration of 15μM within 48h, the number of cells began to decrease after 72h, while the GLUT3 express most obviously when psoralen treated hFOBl.19 cells for 48h through qPCR and Western blotting detection, which consistent with CCK-8 test (P< 0.05)(2) Psoralen can activate MAPK signaling pathways:In the study,15μM of psoralen was added to hOBl.19 cells medium, then ERK, P38, JNK and the protein GLUT3 were extracted after these cells medium treated by SCH772984、 SB203580 and SP600125. The Western blotting analysis told that ERK, P38 and JNK protein levels in three groups had shown no significant difference. The ERK, P38, phosphorylation of p65 and JNK protein GLUT3 protein levels were significantly reduced as the addition of inhibitor compared to psoralen plus separate groups. CCK-8 assay had shown that MAPK inhibitors significantly inhibited psoralen-induced proliferation of osteoblasts (P<0.05)(3) Psoralen can activate NF-kB signaling pathways:15μM of psoralen was added to hOBl.19 cells medium, then p65 and the protein GLUT3 were extracted after these cells treated by NF-kB inhibitor (PDTC). The Western blotting analysis told that the p65 and the protein GLUT3 levels were significantly reduced as the addition of NF-kB inhibitor (PDTC) compared to psoralen plus separate groups. CCK-8 assay had shown that NF-kB inhibitor (PDTC) can decline the proliferation effects of psoralen to hOBl.19 cells (P<0.05)ConclusionPsoralen can stimulate osteoblast proliferation by activation of NF-κ B-MAPK signaling. It can be an ideal choice as selective estrogen receptor modulators (SERMs), which has broad prospects to the treatment of osteoporosis.