Abstract

Osteoarthritis (OA) is a progressive degeneration of articular cartilage with involvement of synovial membrane, and subchondral bone. Recently, cell-based therapies, including the application of stem cells such as mesenchymal stem cells (MSCs), have been introduced for restoration of the articular cartilage. Toll-like receptors (TLRs) were reported to participate in OA progression and MSC chondrogenesis. Here, the role and molecular mechanism of toll like receptor 4 (TLR4) in chondrogenic differentiation of synovium-derived MSCs (SMSCs) were investigated. Molecular markers (CD44, CD90, CD45 and CD14) on SMSC surfaces were identified by flow cytometry. Multi-potential differentiation capacities of SMSCs for chondrogenesis, adipogenesis and osteogenesis were examined by Alcian blue, oil red O and Alizarin red staining, respectively. TLR4 and miR-145-5p levels in SMSCs were assessed using RT-qPCR. The protein expression of TGFB3, Col II, SOX9 and Aggrecan in SMSCs was tested by western blotting. Cytokine secretions were analyzed with ELISA for IL-1β and IL-6. Intracellular NAD⁺ content and NAD⁺/NADH ratio were assessed. The interaction between miR-145-5p and TLR4 was confirmed by RNA pulldown and luciferase reporter assays. In this study, SMSCs were identified to have immunophenotypic characteristics of MSCs. TLR4 knockdown inhibited chondrogenic and osteogenic differentiation of SMSCs. Mechanistically, TLR4 was targeted by miR-145-5p in SMSCs. Moreover, TLR4 elevation offset the inhibitory impact of miR-145-5p upregulation on chondrogenic differentiation of SMSCs. Overall, miR-145-5p restrains chondrogenesis of SMSCs by suppressing TLR4.

摘要

骨关节炎（OA）是关节软骨的进行性退化，涉及滑膜和软骨下骨。最近，已经引入了基于细胞的疗法，包括应用间充质干细胞 (MSCs) 等干细胞来修复关节软骨。据报道，Toll 样受体 (TLR) 参与 OA 进展和 MSC 软骨形成。在这里，研究了 toll 样受体 4 (TLR4) 在滑膜来源的 MSCs (SMSCs) 软骨分化中的作用和分子机制。通过流式细胞术鉴定 SMSC 表面上的分子标记（CD44、CD90、CD45 和 CD14）。分别通过阿尔新蓝、油红 O 和茜素红染色检测 SMSCs 对软骨形成、脂肪形成和成骨形成的多潜能分化能力。使用 RT-qPCR 评估 SMSC 中的 TLR4 和 miR-145-5p 水平。通过蛋白质印迹检测 SMSCs 中 TGFB3、Col II、SOX9 和 Aggrecan 的蛋白表达。用ELISA分析细胞因子分泌物的IL-1β和IL-6。细胞内NAD⁺含量和 NAD ⁺ /NADH 比率进行了评估。miR-145-5p 和 TLR4 之间的相互作用通过 RNA 下拉和荧光素酶报告基因测定得到证实。在这项研究中，SMSCs 被鉴定为具有 MSCs 的免疫表型特征。TLR4 敲低抑制了 SMSCs 的软骨形成和成骨分化。机制上，TLR4 被 SMSC 中的 miR-145-5p 靶向。此外，TLR4 升高抵消了 miR-145-5p 上调对 SMSCs 软骨分化的抑制作用。总体而言，miR-145-5p 通过抑制 TLR4 来抑制 SMSC 的软骨形成。