Icariin promotes osteoblast proliferation and differentiation through a non-nuclear signaling pathway

doi:10.12307/2023.495

Abstract

Abstract: 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 icariin-bovine 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 down-regulated) 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.

Key words: icariin, osteoblast, proliferation, differentiation, non-nuclear effect, signaling pathway

CLC Number:

Mei Jie, He Qiang, Sun Xin, Yin Hong, Qian Weiqing. Icariin promotes osteoblast proliferation and differentiation through a non-nuclear signaling pathway[J]. Chinese Journal of Tissue Engineering Research, 2023, 27(20): 3129-3135.

Figures/Tables 6

对鉴定到的磷酸化位点对应的蛋白，基于GO数据库(http://www.geneontology.org/)提取各注释信息，挖掘蛋白功能。选取GO富集分析Top30(筛选3种分类中对应蛋白数目大于1的GO条目，按照每个条目对应的-log10P值由大到小排序的各10条)，见图5A，图中X坐标为GO条目名称，Y坐标为对应条目的蛋白数量和其百分比。这些蛋白主要在7种生物学过程中发挥作用，包括mRNA加工、RNA剪接、核小体组装、翻译起始、翻译的正负调控、mRNA稳定性调节、细胞对热量的反应等；8种细胞组分被包含其中，包括细胞核、核仁、肌动蛋白细胞骨架、催化步骤2剪接体、一种含boxC/DsnoRNP的核糖核蛋白复合物、伴侣复合体、含蛋白质复合物、黏附连接等；与14类分子功能紧密相关，包括RNA结合、mRNA结合、激酶结合、核小体DNA结合、组蛋白甲基转移酶结合、dATP结合、ATP结合、蛋白质结构域特异性结合、肌动蛋白丝结合、组蛋白结合、相同蛋白质结合、染色质结合、支架蛋白结合、GTP酶结合。 KEGG是系统分析蛋白质在细胞中代谢途径的主要公共数据库。利用KEGG数据库(http://www.genome.jp/kegg/)对蛋白进行Pathway分析(结合KEGG注释结果)，并用超几何分布检验的方法计算每个Pathway条目中蛋白富集的显著性并用P值来表示。KEGG富集分析Top20(按照每个条目对应的-log10P值由大到小排序)气泡图见图5B，图中X轴Enrichment Score为富集分值，Y轴为top20的pathway通路信息。气泡越大的条目包含的差异位点对应蛋白数目越多，气泡颜色由红-绿-蓝-紫变化，其富集P值越小，显著程度越大。结果显示：淫羊藿苷-牛血清白蛋白组和牛血清白蛋白组中差异蛋白多集中在RNA转运、剪切体等信号通路，且差异程度较大。淫羊藿苷-牛血清白蛋白组和PBS组中差异蛋白多集中在RNA转运、剪切体等信号通路，且差异程度较大。ICI182780+淫羊藿苷-牛血清白蛋白组和淫羊藿苷-牛血清白蛋白组中差异蛋白多集中在自噬、mTOR等信号通路，且差异程度较大。淫羊藿苷-牛血清白蛋白组和淫羊藿苷组中差异蛋白多集中在RNA转运、剪切体、紧密连接等信号通路，且差异程度较大。PD98059+淫羊藿苷-牛血清白蛋白组和淫羊藿苷-牛血清白蛋白组中差异蛋白多集中在间隙连接、癌症蛋白聚糖等信号通路，其中差异程度较大的是间隙连接信号通路。淫羊藿苷-牛血清白蛋白组和淫羊藿苷组中差异蛋白多集中在RNA转运、黏附斑等信号通路，且差异程度较大。"

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