miRNA-382-5p Carried by Extracellular Vesicles in Osteoarthritis Reduces Cell Viability and Proliferation, and Promotes Cell Apoptosis by Targeting PTEN.

The objective of the study was to identify extracellular vesicle (EV) microRNAs (miRNAs) that play important roles in knee osteoarthritis (OA). Models of knee OA were surgically induced in nine male Sprague-Dawley rats. Tissue samples were collected at 0 weeks (Control), 6 weeks (6 weeks), and 12 weeks (12 weeks). The EVs were isolated and analyzed for size. Various biomarkers, including recombinant tetraspanin 30 cluster of differentiation (CD)63 and CD9 were detected. An Agilent array was used to screen for differentially expressed (DE) miRNAs. The levels of DE miRNAs and their target mRNAs were evaluated by quantitative reverse transcription-polymerase chain reaction and western blotting. The viability, proliferation, and apoptosis of lipopolysaccharide (LPS)-induced human synovial cells (HSCs) were examined by using Cell Counting Kit-8, EdU (5-ethynyl-2'-deoxyuridine), and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) assays, respectively. The OA model rats had significantly increased levels of inflammatory activity, damaged cells, and rough articular cartilage when compared with rats in the control group. The EVs from the model rats appeared as round vesicle-like structures with a mean diameter of ∼145 nm. Five miRNAs that showed gradual increases in the model rats were selected for further analysis; those miRNAs included miR-127-3p, miR-132-3p, miR-141-3p, miR-345-5p, and miR-382-5p. miR-382-5p was found to reduce the viability and proliferation and promote the apoptosis of LPS-induced HSCs. Moreover, phosphatase and tensin homolog deleted on chromosome 10 (PTEN) was negatively regulated by miR-382-5p. Our findings revealed that EVs produced by the OA rats contained miR-382-5p, which might reduce cell viability and proliferation, and promote cell apoptosis by targeting PTEN.

The silencing of miR-199a-5p protects the articular cartilage through MAPK4 in osteoarthritis.

Background: Osteoarthritis (OA) is the most common joint disorder, and places a heavy burden on individuals and society. As conventional therapies, such as surgery, rarely cure the disorder, targeted therapies represent a promising alternative. This research sought to explore the potential effect of miR-199a-5p on the development of OA. Methods: Based on the OA rat model, the serum was collected at 6 and 12 weeks, and microRNA (miRNA) sequencing was performed. A bioinformatics analysis was conducted to examine the differentially expressed micro ribonucleic acids, and qRT-PCR (real-time quantitative PCR) was conducted to determine their expression in the joint tissues of rats with OA. Rats articular chondrocytes were collected and treated with a miR-199a-5p antagomir or agomir. Afterwards, cell viability, autophagy was determinated. Dual luciferase was used to verify that miR-199a-5p targets the regulation of mitogen-stimulated protein kinase 4 (MAPK4). Subsequently, in chondrocytes, MAPK was knockdown to rescue the effect of miR-199a-5p inhibition, and cell viability and autophagy were examined. Finally, the OA model was treated with miR-199a-5p antagomir to detect joint pathology, cartilage tissue and inflammatory factor and autophagy was measured. Results: MiR-199a-5p was greatly upregulated in OA, and miRNA was found to be differentially expressed in OA tissues. MAPK4 was identified to be a target gene of miR-199-5p. Inhibiting miR-199a-5p not only decreased the survival of chondrocytes and induced apoptosis, but also relieved inflammation and decreased the content of pro-inflammatory cytokines. Further, the silencing of miR-199a-5p protected the articular cartilage and improved gait abnormalities, but this effect was abrogated by the silencing of MAPK4. Conclusions: The silencing of miR-199a-5p appears to improve gait abnormalities, promote the survival of chondrocytes, and improve the condition of OA. Our findings may lead to the development of miR-199a-5p-based targeted therapy for OA.

Quercetin Alleviates Osteoarthritis Progression in Rats by Suppressing Inflammation and Apoptosis via Inhibition of IRAK1/NLRP3 Signaling.

INTRODUCTION: Quercetin was recently reported to help protect against osteoarthritis (OA) progression, but the molecular mechanism for that protective affect remains unclear. METHODS: Here, OA model rats were intraperitoneally injected with quercetin, and the severity of cartilage damage in the rats was evaluated by H&E, Safranin O, and Toluidine blue, as well as by using the Osteoarthritis Research Society International (OARSI) Scoring System. Additionally, rat chondrocytes were treated with quercetin and then stimulated with IL-1ß. The levels of pro-inflammatory cytokines (IL-1ß, IL-18, and TNF-α) were detected by ELISA.Cell apoptosis was evaluated by flow cytometry and Hoechst staining. ROS levels were measured using a DCFH-DA probe. Protein expression was evaluated by Western blotting, immunohistochemical staining, and immunofluorescence. RESULTS: Our data showed that quercetin attenuated the degeneration and erosion of articular cartilage, suppressed inflammation and apoptosis, and downregulated the levels of IRAK1, NLRP3, and caspase-3 expression. In vitro data showed that overexpression of NLRP3 could reverse the suppressive effect of quercetin on IL-1ß-induced rat chondrocyte injuries. Importantly, rescue experiments confirmed that quercetin inhibited IL-1ß-induced rat chondrocyte injuries in vitro by suppressing the IRAK1/NLRP3 signaling pathway. CONCLUSION: Our study indicated that quercetin inhibits IL-1ß-induced inflammation and cartilage degradation by suppressing the IRAK1/NLRP3 signaling pathway.