淫羊藿苷促进成骨细胞增殖分化的非核效应信号通路.

BACKGROUND: Estrogen deficiency can inhibit osteoclast proliferation and differentiation. Epimedium is a phytoestrogen that acts as a traditional kidney tonic herb, and its active ingredient, icariin, can produce some effects of estrogen. OBJECTIVE: To investigate the possible signaling pathways involved in the non-nuclear effects of icariin in promoting osteoblast proliferation and differentiation, as well as the possible mechanisms of phosphorylated protein cascade regulation in the signaling pathway by phosphorylated proteomics techniques. METHODS: Primary osteoblasts were obtained from newborn Sprague-Dawley rats, cultured, and identified. The effect of icariin on osteoblast activity was measured by cell counting kit-8 method. The primary osteoblasts were passed to the third generation. After 24 hours, passaged cells were then divided into six groups: bovine serum albumin intervention group, PBS intervention group, icariin intervention group, icariin-bovine serum albumin coupling intervention group, ICI182780+icariin-bovine serum albumin coupling intervention group, and PD98059+icariin-bovine serum albumin coupling intervention group. The cells of each group were collected at 0, 5, 15 and 25 minutes and used for protein extraction, enzymatic digestion, peptide labeling and phosphorylated peptide enrichment. Phosphoproteomic analysis was performed using liquid chromatography-mass spectrometry technology RESULTS AND CONCLUSION: 10 μg/L Icariin significantly promoted the proliferation and differentiation of osteoblasts. Icariin-bovine serum albumin coupling was able to induce phosphorylation of ERK in osteoblasts without membrane permeation, confirming the non-nuclear effect. Compared with the icariinbovine serum albumin coupling intervention group, there were 971 differentially expressed proteins (499 up-regulated and 472 down-regulated) in the icariin intervention group, 974 (509 up-regulated and 465 down-regulated) in the bovine serum albumin intervention group, 836 (377 up-regulated and 459 downregulated) in the PBS intervention group, 231 (126 up-regulated and 105 down-regulated) in the ICI182780+icariin-bovine serum albumin coupling intervention group, and 150 (65 up-regulated and 85 down-regulated ) in the PD98059+icariin-bovine serum albumin coupling intervention group. Among them, 65 and 62 significantly expressed proteins were down-regulated (Fc value < 0.8) in the ICI182780+icariin-bovine serum albumin coupling intervention group and PD98059+icariin-bovine serum albumin coupling intervention group, respectively, most of which were involved in important biological processes, such as mRNA processing and stability regulation, RNA splicing, protein phosphorylation, and mitosis, as well as in signaling pathways analyzed by KEGG library, such as MAPK, protein ubiquitination, mTOR, and PI3K/Akt pathways. To conclude, icariin promotes osteoblast proliferation and differentiation through non-nuclear effects in relation to MAPK, protein ubiquitination, mTOR, and PI3K/Akt signaling pathways. [ABSTRACT FROM AUTHOR]