Human recombinant CIRP peptides were purchased from Abcam. NF-κB inhibitor BAY 11-7082 and cell counting kit-8 (CCK-8) was purchased from Beyotime Institute of Biotechnology. TLR4 inhibitor TAK-242 was purchased from Shanghai Xianding Biotechnology Co., Ltd. Primary antibodies directed against inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), MMP1, MMP3, MMP13, Lamin B1, CD63, CD9, 130 kDa cis-Golgi matrix protein 1 (GM130) and GAPDH were purchased from Proteintech Group, Inc., ADAM metallopeptidase with thrombospondin type 1 motif 5 (ADAMTS5) was purchased from ABclonal. Human Reactive Inflammasome Antibody Sampler Kit II, CIRP, IκBα and p65 were purchased from Cell Signaling Technology, Inc. Collagen II was purchased from Beyotime Institute of Biotechnology. Anti-rabbit IgG, HRP-linked antibody and Anti-mouse IgG, HRP-linked antibody were purchased from Beyotime Institute of Biotechnology.

The collection of cartilage tissues from patients was approved by the Ethics Committee of Shenzhen Second People's Hospital (2023-157-01YJ). Samples used in this study were collected after all patients received detailed explanation of the research and submitted written informed consents. Human articular cartilage tissues were obtained from volunteer donors who had undergo joint replacement or joint surgery (December 2023-September 2024). Normal chondrocytes were obtained from the articular cartilage of patients who underwent joint replacement surgery due to femoral neck fractures. Notably, these patients did not have a diagnosis of OA. The control group comprised 11 patients (5 males and 6 females), all aged from 60-74 years old. By contrast, OA chondrocytes were sourced from the articular cartilage of individuals diagnosed with OA, comprising 11 patients (3 males and 8 females), also aged from 60 to 81 years old. Washing the cartilage samples 3 times with phosphate buffer saline (PBS) containing penicillin and streptomycin and then cutting them into small fragments (1-3 mm³). The minced samples were placed in 1 mg/ml collagenase II (MilliporeSigma) for digestion at 37°C for 16 h in a shaker, digesting the samples multiple times until collagen was completely dissolved. After digestion, the suspension was filtered with a 100-mm nylon cell strainer to remove matrix debris and the cells collected via centrifugation at 108 × g for 10 min at 24°C. After centrifugation, cells were resuspended and subcultures with chondrocyte growth medium (DMEM/F12; 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 100 IU/ml penicillin and 100 μg/ml streptomycin (Gibco; Thermo Fisher Scientific, Inc.). The cells were cultured in an incubator with 5% CO₂ atmosphere at 37°C. The medium was changed when the cell was attached and the cells were passaged at 80% confluence. Only the passage 1(P1) and passage 2 (P2) chondrocytes were used for the experiments. Toluidine blue staining was used to assess chondrocyte morphology. Briefly, the samples were stained with toluidine blue solution for 5 min at room temperature, followed by the addition of an equal volume of distilled water and mixed thoroughly. After allowing the mixture to stand for 10 min at room temperature, images were captured using an Olympus microscope (Olympus Corporation).

Human primary chondrocytes cells were seeded in 96-well cell culture plates at a density of 1×10⁴/well and cultured overnight. The concentration range of BAY 11-7082 and TAK-242 used in this assay was based previous studies (37,38). The concentration range of CIRP used in this assay was from 0-8 μg/ml. After incubation with CIRP for 48 h at 37°C, the cell viability was measured using CCK-8 following the standard protocol. Briefly, the culture medium and CCK-8 solution were mixed at a ratio of 10:1 and 100 μl mixture was added into each well. After 1 h incubation at 37°C in a cell culture incubator, the absorbance at 450 nm was detected using a microplate reader.

Total RNA was extracted from human chondrocytes using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. In brief, the cells were first lysed in TRIzol^® to release the RNA, and then chloroform was added to facilitate phase separation. After mixing and centrifugation at 12,000 × g for 15 min at 4°C, the upper aqueous phase containing the RNA was transferred to a new tube. Isopropanol was subsequently added to precipitate the RNA. The RNA pellet was washed with 75% ethanol, dried and finally resuspended in RNase-free water. Then cDNA was synthesized using PrimeScript RT Reagent Kit with gDNA Eraser (Takara Biotechnology Co., Ltd.) according to the manufacturer's instructions. RT-qPCR was conducted with SYBR Green Supermix (Beyotime Institute of Biotechnology) using aViiA 7 Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The PCR cycling conditions consisted of an initial denaturation at 95°C for 10 min, followed by 35 cycles of denaturation at 95°C for 15 sec, annealing at 60°C for 1 min, and extension at 72°C for 30 sec. Transcript levels were normalized to GAPDH (for mRNA) or the small U6 RNA (for miRNA). The specific primers used for these analyses are listed in Table I. Gene expression was calculated using the 2^−ΔΔCq method (39) and each experiment was performed in triplicate.

The whole cell proteins were extracted from chondrocytes using lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl and 0.1% sodium dodecyl sulfate) with protease inhibitors supplement. Separate extraction of nuclear and cytoplasmic proteins was conducted using a Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Institute of Biotechnology). The determination of protein concentration was conducted using the BCA protein concentration detection kit (Thermo Fisher Scientific, Inc.). Denatured protein solution (40 μg) was subjected to 12% SDS-PAGE followed by immunoblot analysis. The sample was then transferred to a PVDF membrane (Merck KGaA) that had been incubated in methanol for 1 min at room temperature. After removing the methanol, the membrane was equilibrated in transfer buffer until it was ready for use. Following a transfer at 100 V for 1 h at 4°C, the membrane was blocked with 5% skimmed milk for 1 h at room temperature before being incubated with primary antibodies against GADPH (1:50,000; cat. no. 60004-1-Ig; ProteinTech Group, Inc.), MMP1 (1:1,000, 10371-2-AP, ProteinTech Group, Inc.), MMP3 (1:5,000; cat. no. 66338-1-Ig; ProteinTech Group, Inc.), MMP13 (1:1,000; cat. no. 18165-1-AP; ProteinTech Group, Inc.), a disintegrin and metalloproteinase with thrombospondin motif-5 (Adamts5; 1:500; cat. no. A2836; ABclonal Biotech Co., Ltd.), iNOS (1:500; cat. no. 18985-1-AP; ProteinTech Group, Inc.), cyclooxygenase-2 (COX-2; 1:500; cat. no. 12375-1-AP; ProteinTech Group, Inc.), lamin B1 (1:5,000; cat. no. 12987-1-AP; ProteinTech Group, Inc.), CD63 (1:500; cat. no. 32151-1-AP, ProteinTech Group, Inc.), CD9 (1:500; cat. no. 84801-13-RR, ProteinTech Group, Inc.), GM130 (1:500; cat. no. 11308-1-AP, ProteinTech Group, Inc.), Human Reactive Inflammasome Antibody Sampler Kit II (1:1,000; cat. no. 25620T; Cell Signaling Technology Inc.), CIRP (1:1,000; cat. no. 68522; Cell Signaling Technology Inc.), p65 (1:1,000; cat. no. 8242; Cell Signaling Technology Inc.), IκBα (1:1,000, cat. no. 4814; Cell Signaling Technology Inc.), and collagen II (1:500; cat. no. AF6528, Beyotime Institute of Biotechnology) overnight at 4°C. Subsequently, the membranes were incubated with a HRP-conjugated goat anti-rabbit (1:1,000; cat. no. A0208; Beyotime Institute of Biotechnology Inc.) or goat anti-mouse IgG (1:1,000; cat. no. A0216; Beyotime Institute of Biotechnology Inc.) secondary antibody for 2 h at room temperature. ECL reagent (Thermo Fisher Scientific, Inc.) was used for development according to the manufacturer's protocol. The semi-quantitatively analysis of immunoreactive bands was performed using ImageJ software 1.53e (National Institutes of Health).

For immunofluorescence, cells were treated with CIRP for 24 h. The cells were fixed with 4% paraformaldehyde for 15 min at room temperature. Fixed samples were permeabilized with 0.3% Triton X-100 for 10 min at room temperature and then blocked with 5% BSA for 30 min. Chondrocytes were incubated with p65 (1:50) primary antibodies overnight at 4°C. Afterwards, they were washed with PBS 3 times and incubated with Cy3-conjugated secondary antibodies for 1 h at room temperature in the dark. Finally, the cells were incubated with DAPI for nucleuses stain at room temperature for 10 min. A fluorescence microscope was used to acquire the images at a magnification of 400×. Collagen II (1:50) and MMP13 (1:50) primary antibody were used to detect its expression in human chondrocytes using the same immunofluorescence protocol, and a fluorescence microscope was used to acquire the images at a magnification of 100×. The cell density seeded onto the slide was 1×10⁵ cells per well.

Exosomes were harvested through a sequential centrifugation method. Cells were cultured in serum-free medium for 48 h. The medium was collected and centrifuged sequentially at 300 × g for 10 min, at 2,000 × g for 15 min and at 10,000 × g for 30 min at 4°C. The supernatant was filtered using a 0.22 mm filter (Beyotime Institute of Biotechnology) to remove remaining cells and debris and then centrifuged at 120,000 × g for 70 min at 4°C to harvest exosomes. PBS was used to re-suspend the purified exosomes. Exosomes were kept either at −80°C for long term preservation or at −20°C for short term preservation.

Exosomes were entrusted to Wuhan Servicebio Technology Co., Ltd. for transmission electron microscopy. In brief, fix the exosomes with 1 ml of 2% paraformaldehyde for 5 min at room temperature. Then, 10 μl of exosomes was applied to carbon-coated copper grids for 1 min, then washed with double-distilled water and dried using filter paper. Afterwards, ~15 μl of 2% uranyl acetate staining solution was added for 1 min at room temperature. The grids were examined using a Hitachi HT7700 transmission electron microscope (Hitachi High-Technologies Corporation). The particle size and particle concentration were determined by a Nano sight Tracking Analysis (NTA) system NanoSight NS300 (Malvern Panalytical Instruments Corp.). Briefly, exosomes were re-suspended in PBS at a concentration of ~3 μg protein content/ml. Then the solution was further diluted by 100 to 500 hundred folds for analysis. A 1 min video was recorded by the NTA software (NTA 3.4-Sample Assistant Build 3.4.003-SA, Malvern Panalytical Ltd.) for further analysis. Western blotting was used to detect the exosome marker protein expression.

To predict the miRNAs that could potentially regulate CIRP expression, the miRWalk (http://mirwalk.umm.uni-heidelberg.de/), TargetScan (http://www.targetscan.org/), StarBase (http://starbase.sysu.edu.cn/), miRDIP (http://ophid.utoronto.ca/mirDIP/) and a chondrogenesis-related microRNA databases (GSE135588) were used to find CIRP targeting miRNAs. A Venn diagram (http://bioinformatics.psb.ugent.be/cgi-bin/liste/Venn/calculate_venn.htpl) was created based on comparisons of these miRNAs.

To study the interaction between miR-145-5p and the 3' untranslated region (UTR) of the CIRP gene, plasmid vectors containing the wild-type CIRP 3' UTR that included the predicted binding sites for miR-145-5p were constructed. These plasmids were transfected into 293 cells using GP-transfect-Mate (GenePharma, Inc.). To evaluate transfection efficiency, a Renilla luciferase vector was co-transfected in each experiment. After transfecting the cells, incubate them in a 37°C incubator for 24 h, the luciferase activity was measured in relative light units, with the firefly luciferase activity normalized against the Renilla luciferase activity using Dual-Luciferase Reporter Assay System (Promega Corporation). In brief, 250 μl of 1X cell lysis buffer was added to each well, and 50 μl of the lysate transferred to a microplate, setting up three replicates. Then, 10 μl of firefly luciferase reaction solution was added, gently mixed, and the firefly luciferase activity measured within 30 min. Finally, 30 μl of Renilla luciferase reaction solution was added, mixed immediately and the Renilla luciferase activity measured.

Electroporation method was used to load miR-145-5p mimics into exosomes. Exosomes at a final concentration of 100 μg/μl protein (measured by BCA) were mixed with electroporation buffer (in a 1:1 ratio) and 100 pmol of synthetic miR-145-5p mimics (sense 5'-GUCCAGUUUUCCCAGGAAUCCCU-3', antisense 5'-GGAUUCCUGGGAAAACUGGACUU-3') or negative control (mimics NC, sense 5'-UUCUCCGAACGUGUCACGUTT-3', antisense 5'-ACGUGACACGUUCGGAGAATT-3') and electroporated at 0.400 kV using an electroporation instrument. After the loading of miR-145-5p mimics, the exosome samples were diluted with PBS and centrifuged at 10,000 × g for 30 min and 100,000 × g for 70 min at 4°C to remove unloaded miR-145-5p mimics.

All procedures were approved by the Animal Research Committee of Southwest Medical University (approval no. 20220307-004). A total of 20 six-week-old male C57BL/6J mice were randomized into four groups: normal group, OA group, miR-NC-Exos OA group and miR-145-Exos OA group, with each group comprising five mice. The OA model was built on the instability of the medial meniscus (DMM) caused by surgical therapy (40). The normal and OA group were given articular cavity injections of PBS. MiR-NC-Exos OA group were given articular cavity injections of 100 μg exosomes from cells overexpressed miR-NC mimics and miR-145-Exos OA group were given articular cavity injections of 100 μg exosomes from cells overexpressed miR-145 mimics. The injections were carried out twice a week for four consecutive weeks. All animals were sacrificed via cervical dislocation while under anesthesia induced by an intraperitoneal injection of pentobarbital sodium (50 mg/kg). Mortality was confirmed through the observation of respiratory arrest. a total of 10 cartilage samples were collected for safranin-O staining, hematoxylin and eosin staining and immunohistochemical (IHC) staining.

The calcification of the knee joint was removed with a 10% EDTA solution after it had been fixed with 4% paraformaldehyde (24 h; 4°C). The tissue was then dehydrated by immersing it in 70% ethanol for 30 min, followed by 80% ethanol for 30 min, 95% ethanol for 30 min, and finally 100% ethanol for 1 h to ensure complete dehydration. After dehydration, the tissue was cleared and placed in melted paraffin wax at approximately 60°C for 3 h, which was then transferred into a mold filled with fresh paraffin wax to cool and solidify. The paraffin blocks were sliced into frontal serial slices with a thickness of 5 mm. Hematoxylin and eosin (H&E) staining was performed at room temperature, with hematoxylin applied for 10 min, and eosin applied for 3 min. Safranin O-fast green (S-O) staining was also performed at room temperature, with safranin O staining lasting 5 min, and fast green staining for 15 sec. And a light microscope was used to identify the degree of cartilage degeneration.

MMP3 and collagen II expression levels were detected in the cartilage by immunohistochemistry. Briefly, the slides were dewaxed and rehydrated. The slide was sealed with 5% BSA for 30 min after a 20 min incubation with 0.4% pepsin (Sangon Biotech Co., Ltd.) in 5 mM HCl to repair the antigen. Then, with primary antibodies specific to MMP3 (1:500) and collagen II (1:50) overnight (at 4°C), incubation of the slides was performed. After 50 min (room temperature) of incubation, the slides were subjected to incubation with HRP-conjugated goat anti-rabbit (1:50; cat. no. A0208; Beyotime Institute of Biotechnology, Inc.) and images were captured with an Olympus BX51 microscope (Olympus Corporation).

RNA-seq was conducted and analyzed by Genedenov Co., Ltd. Briefly, total RNA extraction from the cells was performed using TRIzol^® Reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and genomic DNA removal was performed with DNase I (Takara Biotechnology Co., Ltd.). After preparation of the RNA-seq transcriptome library, the Illumina HiSeq xten/NovaSeq 6000 sequencer (Illumina, Inc.) was used to perform RNA-seq. Gene Ontology (GO) functional enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were conducted using omicshare (https://www.omicshare.com). The RNA-seq dataset has been deposited in the BioProject database under the accession number PRJNA1302182 and the URL is https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1302182.

The data were expressed as the mean ± SD and all experiments were repeated three times with independent cultures. Prior to statistical analysis, the normality of the data distribution was verified using the Shapiro-Wilk test. The statistical significance of differences for the mean values between two groups was determined with an unpaired two-tailed Student's t-test and multiple groups compared using one-way analysis of variance (ANOVA) with Bonferroni post hoc test. P<0.05 was considered to indicate a statistically significant difference.

To assess the effect of CIRP on OA progression, chondrocytes were first collected from patients with fractures or OA undergoing joint replacement surgery to evaluate its expression. As shown in Fig. 1A, the extracted human chondrocytes were stained with toluidine blue, confirming that the isolated cells were indeed human chondrocytes and suitable for further study. In Fig. 1B, qPCR analysis revealed that the expression of CIRP was markedly increased, while collagen Ⅱ levels decreased in OA chondrocytes compared with normal chondrocytes. Additionally, the expression of MMP1, MMP3, MMP13 and ADAMTS5 were also overexpressed in OA chondrocytes. The present study then analyzed the protein levels of these genes and the data presented in Fig. 1C and D supported the mRNA findings. These results indicated that CIRP is overexpression in OA chondrocytes and may contribute to the progression of OA.

Recent studies revealed that CIRP can be released into extracellular space by various cell types, including cardiomyocytes (41-43). The exosomes released by OA chondrocytes were next collected and analyzed using TEM (Fig. 2A). The observations indicated that the exosomes derived from chondrocytes were round or oval-shaped with uneven sizes and ranging in diameter from 150-200 nm (Fig. 2B). Western blotting revealed positive expression of CD63 and CD9, along with negative expression of GM130 in the exosomes (Fig. 2C). It was then examined whether CIRP was present in chondrocyte-derived exosomes. The western blotting results showed that chondrocyte exosomes indeed contained CIRP and its expression was markedly increased in OA chondrocytes compared with normal chondrocytes (Fig. 2D and E). Moreover, treatment of chondrocytes with 10 ng/ml IL-1β also resulted in an increase in CIRP expression (Fig. 2F-H). Furthermore, the exosome release inhibitor GW4869 suppressed the secretion of CIRP in human chondrocytes (Fig. 2I). These results indicate that CIRP is overexpressed and released in an exosomal manner in OA chondrocytes, which may contribute to the progression of OA.

From the aforementioned results, it was observed that the expression of CIRP was upregulated in human OA chondrocytes. This upregulation was associated with increased expression of inflammatory cytokines and MMPs, suggesting that CIRP may play a role in progression of OA. OA progression is strongly associated with inflammatory responses and ECM degradation in chondrocytes. To study the role of CIRP in OA, chondrocytes were treated with CIRP concentrations ranging from 0-8 μg/ml. It was found that 4 μg/ml CIRP did not affect cell viability, while 8 μg/ml CIRP did (data not shown). Therefore, the present study chose to use 4 μg/ml CIRP to treat chondrocytes and analyze its effect on inflammatory cytokines expression. The results demonstrated that CIRP treatment increased the expression of IL-6, IL-1β and TNFα (Fig. 3A). Additionally, the expression of iNOS and COX-2, two crucial inflammatory response factors in OA, was also markedly upregulated following CIRP treatment (Fig. 3A and B). The present study then examined the effect of CIRP on the expression of extracellular matrix proteins. As shown in Fig. 3C, treatment with 4 μg/ml CIRP markedly decreased collagen Ⅱ expression and increased the expression of MMP1, MMP3, MMP13 and ADAMTS5 at mRNA level. The western blotting results in Fig. 3D also showed that CIRP induced the upregulation of MMP1, MMP3, MMP13 and ADAMTS5, along with the downregulation of collagen II, with IL-1β treatment serving as a positive control. Furthermore, Immunofluorescence results indicated that collagen Ⅱ expression was markedly reduced, while MMP13 expression was induced by CIRP treatment (Fig. 3E).

Next, chondrocytes were treated with 4 μg/ml CIRP and cells were collected for RNA-seq. Fig. 4A shows the genes affected by CIRP treatment. KEGG signaling pathway enrichment analysis indicated that the differentially expressed genes were highly enriched in functions related to inflammatory processes, including TNF-α signaling, cytokine-cytokine receptor interaction, NOD-like receptor signaling and Toll-like receptor signaling pathways (Fig. 4B). The activation of NF-κB signaling pathway plays crucial roles in the inflammatory response and cartilage degradation associated with OA. Studies have reported that CIRP can activate the NF-κB signaling pathway via TLR4/MD2. The present study found that 4 μg/ml CIRP increased the expression of p65 in the nucleus and promoted the degradation of IκBα (Fig. 4C and D). It was observed that CIRP markedly induced the nuclear translocation of p65 in chondrocytes (Fig. 4G). These results demonstrate that CIRP induces NF-κB activation in human chondrocytes. The present study also analyzed the activation of NF-κB downstream NLRP3 inflammasome signaling pathway and the results showed that CIRP induced the upregulation of NLRP3, ASC and cleaved-caspase1 (Fig. 4E and F).

It has been reported that extracellular CIRP stimulates inflammatory responses by activating its receptors, including TLR4, IL-6R and TREM-1 (41,44,45). Based on the RNA-seq analysis, the present study sought to determine whether inhibition of TLR4/NF-κB signaling pathway would eliminate CIRP's function. The inhibitors BAY 11-7082 and TAK-242 were used to block the TLR4/NF-κB signaling pathway. Fig. 5A shows their toxicity on human chondrocytes. Next, chondrocytes were pretreated with 5 μM BAY 11-7082 or TAK-242 and then treated with 4 μg/ml CIRP to analyze the effects of NF-κB signaling pathway inhibition on CIRP. The results in Fig. 5B and C indicate that treatment with 5 μM BAY 11-7082 and TAK-242 inhibited the activation of the NF-κB signaling pathway. Furthermore, these two inhibitors suppressed CIRP-induced activation of the NLRP3 signaling pathway (Fig. 5D and E). Additionally, these two inhibitors suppressed CIRP-induced downregulation of collagen Ⅱ and upregulation of MMP1, MMP3, MMP13 and ADAMTS5 (Fig. 5F and G). These results indicate that CIRP-induced inflammatory responses and ECM degradation in human chondrocytes are dependent on the TLR4/NF-κB/NLRP3 signaling pathway.

Subsequently, miRWalk, TargetScan, StarBase, miRDIP and a chondrogenesis-related microRNA databases were applied to predict the miRNAs that could potentially regulate CIRP expression. A Venn diagram was created based on comparisons of these miRNAs (Fig. 6A), which revealed six candidate miRNAs of interest. Among them, has-miR-145-5p has been reported to play an important role in chondrogenesis (34-36). Therefore, it was hypothesized that miR-145-5p could influence OA progression by targeting CIRP. To validate this hypothesis, online tools indicated the presence of specific binding sites between CIRP mRNA and miR-145-5p (Fig. 6B), suggesting that CIRP might be a downstream target gene of miR-145-5p. Moreover, a dual luciferase reporter gene assay showed that, compared with the NC-mimic control, luciferase activity decreased after the introduction of miR-145-5p mimic and the CIRP-WT reporter plasmid (Fig. 6C). Next, the expression of miR-145-5p in OA human chondrocytes and IL-1β treated human chondrocytes was analyzed by qPCR. The results revealed that the miR-145-5p expression was reduced in OA chondrocytes compared with normal chondrocytes. Moreover, chondrocytes treated with IL-1β exhibited lower miR-145-5p expression compared with untreated chondrocytes (Fig. 6D and E). Subsequently, the expression of CIRP in human chondrocytes treated with miR-145-5p mimic or miR-145-5p inhibitor was assessed. The results revealed that miR-145-5p mimic led to increased miR-145-5p expression (Fig. S1A) and decreased CIRP expression (Fig. 6F, H and I), whereas the miR-145-5p inhibitor decreased miR-145-5p (Fig. S1B) induced the opposite effects (Fig. 6G, J and K). Taken together, these results indicated that miR-145-5p is downregulated in OA and specifically binds to and suppresses CIRP expression.

Subsequently, to investigate the effect of miR-145-5p on human chondrocytes, the cells were transfected with miR-145-5p mimic, followed by overexpression of CIRP to induce the inflammation and ECM degradation in chondrocytes. qPCR and western blotting analysis were conducted to evaluate the mRNA and protein expression of ECM-related proteins (collagen Ⅱ, ADAMTS5 and MMPs) as well as pro-inflammatory factors (iNOS, COX-2, IL-6, TNF-α and IL-1β). As shown in Fig. 7A, B and E, the expression of pro-inflammatory factors was markedly increased after CIRP overexpression but was reduced by miR-145-5p mimic treatment. In Fig. 7C-E, the expression of collagen Ⅱ was markedly decreased following CIRP overexpression but was increased by miR-145-5p mimic treatment, with the expression of other ECM-related proteins showing inverse results. These findings indicated that miR-145-5p overexpression markedly increased collagen Ⅱ expression, decreased the expression of MMP1, MMP3, MMP13 and ADAMTS5 and reduced inflammatory response in human chondrocytes. Moreover, the damage induced by CIRP overexpression in human chondrocytes was alleviated by overexpressed miR-145-5p overexpression. These results indicate that miR-145-5p plays a key role in maintaining the function of chondrocytes in vitro.

The present study investigated the role of miR-145-5p in IL-1β-induced activation of the CIRP/NF-kB/NLRP3 signaling pathway. It was found that treatment with 10 ng/ml IL-1β increased the expression of p65 in the nucleus and promoted the degradation of IκBα in human chondrocytes (Fig. 8A and B). Additionally, IL-1β induced the nuclear translocation of p65 (Fig. 8C). However, the activation of the NF-κB signaling pathway was suppressed by treatment with miR-145-5p mimics (Fig. 8A-C). Moreover, IL-1β-induced expression of CIRP and activated the downstream NLRP3 inflammasome signaling pathway, both of which were suppressed by treatment with miR-145-5p mimics (Fig. 8D and E). These results indicated that miR-145-5p exerts a protective function in chondrocytes by inhibiting the CIRP/NF-κB/NLRP3 signaling pathway.

Finally, the effect of miR-145-5p on mice with OA was evaluated in vivo. The mice were divided into four groups: One sham group and three DMM groups (DMM group, DMM injected with miR-NC exosomes group and DMM injected with miR-145-5p mimic exosomes group). hematoxylin and eosin staining was conducted to observe the pathological changes in the articular cartilage of the knee joint and S/O staining was used to assess articular cartilage degeneration. The S/O staining revealed that the articular cartilage in the sham control group exhibited a normal, red-dyed area and a smooth surface. By contrast, the thickness of the articular cartilage was markedly reduced in both the DMM group and the DMM injected with miR-NC exosomes group compared with the sham control group. Notably, the DMM injected with miR-145-5p mimic exosomes group showed a thicker red-dyed area and a smoother surface of articular cartilage compared with the DMM injected with miR-NC exosomes group (Fig. 9A), suggesting that miR-145-5p plays a role in attenuating cartilage matrix degradation. Additionally, immunohistochemical assays for MMP3 and collagen Ⅱ were also performed in the OA model. In the DMM group and the DMM injected with miR-NC exosomes group, the expression of MMP3 was markedly increased compared with the sham group, while the DMM injected with miR-145-5p mimic exosomes group exhibited decreased expression of MMP3 (Fig. 9B). Similarly, the expression of collagen Ⅱ was markedly reduced in the DMM group and the DMM injected with miR-NC group compared with the sham group, while the DMM injected with miR-145-5p mimic exosomes group exhibited elevated expression levels (Fig. 9B). Therefore, these results indicate that, as demonstrated by the in vivo experiments, overexpressing of miR-145-5p can hinder OA-related damage.