Icariin Promotes the Chondrocyte Proliferation and Stabilizes Extracellular Matrix by Regulating NEAT1/MiR-27a-3p/MAPK Signaling Pathway

Abstract

Background: Osteoarthritis (OA) is a frequently occurring degenerative joint disease. As the primary bioactive ingredient extracted from Epimedium, icariin (ICA) is reported to promote osteogenic differentiation and bone formation. Nonetheless, the specific mechanisms of ICA in treating OA warrant further elucidation. The current study is targeted at ascertaining the mechanism of ICA in modulating the phenotypes of chondrocytes with an in-vitro model. Methods: To construct an in-vitro model of OA, we used interleukin (IL)-1β to induce human chondrocytes (C-28/I2). Nuclear paraspeckle assembly transcript 1 (NEAT1) and miR-27a-3p expressions were examined through qRT-PCR. Cell viability was determined through Cell Counting Kit-8 (CCK-8) assay; flow cytometry was employed to analyze cell cycle progression; cell migration was examined by Transwell assay; enzyme-linked immunosorbent assay (ELISA) was conducted to measure the levels of inflammation-related factors (IL-8 and IL-6). The binding relationship of miR-27a-3p with NEAT1 was verified through dual-luciferase reporter gene assay; Western blot assay was conducted to measure the expressions of extracellular matrix-related proteins (ADAM metallopeptidase with thrombospondin type 1 motif 5 (ADAMTS-5), collagen II, matrix metalloproteinase 13 (MMP-13), and aggrecan) and mitogen-activated protein kinase (MAPK) signaling pathway-associated proteins (p-p38, p-Jun N-terminal kinase (JNK), and phosphorylated MAPK1 and MAPK2 (p-ERK1/2)). MiR-27a-3ps downstream target genes were predicted using the miRwalk, StarBase, miR-Database (DB), TargetScan databases, and the DAVID database was adopted to perform a Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis of the target genes. Results: ICA significantly boosted chondrocyte viability and migration, as well as suppressed the apoptosis, inflammatory response and extracellular matrix degradation of C-28/I2 cells. ICA down-regulated NEAT1 expression in chondrocytes. MiR-27a-3p was recognized as NEAT1s downstream target, and bioinformatics analysis implied that miR-27a-3ps potential target genes might be related to the MAPK signaling pathway activation. ICA could inhibit p-ERK1/2, p-p38, and p-JNK expressions through promoting miR-27a-3p expression. Conclusions: ICA protects chondrocyte via modulating the NEAT1/miR-27a-3p/MAPK axis, and our findings partly explain the mechanism of ICA in ameliorating OA.