The cartilage lesions mainly affect the superficial layer in EOA, and are gradually worsened and deepened with the progress of OA, suggesting that it is of great clinical significance to begin intervention in the early-stage of OA. In this study, we firstly verified that the intra-articularly injected miR-140-5p could rapidly enter the cartilage and reach the chondrocyte cytoplasm; then, we found that the earlier a single IAJ of miR-140-5p, the better the therapeutic effect, and multiple IAJs of miR-140-5p exhibit better therapeutic effect than single IAJ on EOA; finally, the bioinformatics analysis predicted the potential targets of miR-140-5p and the possible mechanisms by which miR-140-5p participates in OA pathogenesis. These results suggest that IAJs of miR-140-5p is an effective and promising strategy for the treatment of EOA, and also provide support for further exploration of miR-based OA therapeutics.
Intra-articular treatments have emerged in recent years and may offer safe therapeutics that have few extra-articular adverse effects [13–16], and we firstly proved in this study that the intra-articularly injected miR-140-5p could rapidly enter the cartilage and reach the chondrocyte cytoplasm, while no extra-articular uptake, and exhibit a protective role in OA. Based on these results, we further discovered that the potential target genes of rno-miR-140-5p were enriched in various biological processes and mainly located within 10 pathways, implying that the potential mechanisms by which rno-miR-140 attenuates OA progression may be the regulation of these pathways.
The PI3K-Akt, Notch, Wnt and MAPK signaling pathways are well known to be involved in OA pathogenesis, and regulate a variety of biological processes, including inflammation, cell growth, survival and metabolism [17–19]. For examples, activation of the PI3K-AKT pathway in chondrocytes accelerates cartilage degradation, while inhibition of the PI3K-AKT pathway attenuates cartilage degradation and inflammatory responses [20, 21]. Notch pathway plays a critical role in cell fate via regulating differentiation and apoptosis , and is activated in OA . Meanwhile, these pathways are involved in a wide variety of cellular processes, and there is also close interactions between them. For examples, Guo et al. reported that Notch2 negatively regulates cell invasion by inhibiting the PI3K-AKT signaling pathway in gastric cancer , and Villegas et al. found that PI3K-AKT cooperates with oncogenic Notch by inducing nitric oxide-dependent inflammation . However, few studies have reported the interactions between these pathways in chondrocytes and OA pathogenesis.
Although the central role of cartilage degeneration in OA has been recognized, OA is a total joint disease, while not just a dysfunction of cartilage. Although we found in this study that the intra-articularly injected miR-140-5p has no extra-articular distribution, it is substantially uptaken by the intra-articular non-cartilage tissues such as synovium and meniscus. Peng et al. reported that synovial fibroblasts (SFs) also respond to miR-140-5p which could inhibits the proliferation and migration of SFs and promotes apoptosis of SFs in autoimmune arthritis mice . Li et al. reported that miR-140 plays an important role in fracture healing , and Genemaras et al. found that miR-140-5p can be detected in meniscal cells . However, few studies reported the specific effect of miR-140-5p on osteoblasts, osteoclasts or meniscus cells. Therefore, there is still a need to further investigate the effects of miR-140-5p on intra-articular cells and tissues other than chondrocytes and cartilage.
Previous studies have confirmed that drugs or genes injected directly into the joint cavity will be cleared rapidly due to the action of lymphatic vessels and synovial blood vessels, and non-targeted aggregation further reduces the bioavailability, thus multiple, high-dose injections which might make the administration more cumbersome and increase the risk of infection are often required . In order to enhance the cartilage-targeting delivery efficiency and minimize dosage requirements, construction of carriers with good safety, precise cartilage-targeting and high delivery efficiency is essential. A number of delivery vectors for miRs have been reported, such as adenoviruses, collagen scaffolds and hydrogels [30, 31], and biochemical modifications that can selectively bind to cartilage or play specific biological functions may further improve the vectors. For example, Li et al. reported that N-Cadherin peptidomimetic modified self-assembling polypeptide nanomaterials enhances the chondrogenic differentiation of mesenchymal stem cells (MSCs) .
The cartilage matrix is dense and highly negatively charged (mainly from aggrecans), and is a major obstacle to the entry of drugs or genes into cartilage. Previous studies have reported that non-specific electrostatic interaction between cationic carrier and negatively charged extra-cellular matrix (ECM) is beneficial to the rapid penetration of drugs or genes into cartilage, and the high negative charge state of the ECM will greatly increase the residence time of the cationic carrier, thereby transforming the cartilage from the barrier into a reservoir . Therefore, if a functionalized vector was modified by appropriate cationic groups or molecules, it can not only serve as a carrier for cartilage-targeting and efficiently delivery of the negatively charged miRs, but also synergistically enhance the repair of cartilage lesions, possessing good application prospects .