Exosomes-Shuttled lncRNA SNHG7 by Bone Marrow Mesenchymal Stem Cells Alleviates Osteoarthritis Through Targeting miR-485-5p/FSP1 Axis-Mediated Chondrocytes Ferroptosis and Inflammation

Abstract

Background: Osteoarthritis (OA), a degenerative joint disorder, is a major reason of disability in adults. Accumulating evidences have proved that bone marrow mesenchymal stem cells (BMSCs)-carried exosomes play a significant therapeutic effect on OA. However, the precise regulatory network remains unknown.

Methods: OA and normal cartilage samples were acquired from patients, and chondrocytes were exposed to IL-1β to conduct a cellular OA model. Exosomes prepared from BMSCs were identified using nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). Cell viability was determined with CCK-8 assay. Inflammatory injury was assessed by LDH and inflammatory factors (TNF-α and IL-6) using corresponding ELISA kits, respectively. Ferroptosis was evaluated by GSH, MDA and iron levels using corresponding kits, and ROS level with DCFH-DA. The expressions of genes/proteins were determined with RT-qPCR/western bolt. RNA immunoprecipitation and luciferase activity assay were conducted for testing the interactions of small nucleolar RNA host gene 7 (SNHG7)/ferroptosis suppressor protein 1 (FSP1) and miR-485-5p.

Results: The expressions of SNHG7 and FSP1 were both reduced in IL-1β-induced chondrocytes and OA cartilage tissues, and there was a positive correlation between them in clinical level. Moreover, SNHG7 was enriched in BMSCs-derived exosomes (BMSCs-Exos) and could be internalized by chondrocytes. Functional analysis illustrated that BMSCs-Exos administration repressed inflammatory injury, oxidative stress and ferroptosis in IL-1β-induced chondrocytes, while these changes were reinforced when SNHG7 was overexpressed in BMSCs-Exos. Notably, FSP1 silencing in chondrocytes abolished the beneficial effects mediated by exosomal SNHG7.

Conclusions: Exosomal SNHG7 released from BMSCs inhibited inflammation and ferroptosis in IL-1β-induced chondrocytes through miR-485-5p/FSP1 axis. This work suggested that BMSCs-derived exosomal SNHG7 would be a prospective target for OA treatment.

Keywords: Bone marrow mesenchymal stem cells; Exosomes; Ferroptosis; Inflammation; MiR-485-5p/FSP1; Osteoarthritis; SNHG7.

Fig. 1

The abnormal expression of SNHG7 and FSP1 in OA patients and chondrocytes. OA and normal cartilage tissues were collected from OA patients (n = 29) and patients without OA or RA (n = 12). A–B The levels of SNHG7 RNA and FSP mRNA were detected by RT-qPCR. c Western blot detection of FSP1 protein. D Pearson analysis for evaluating the correlation of SNHG7 and FSP1. Chondrocytes were treated by IL-1β (10 ng/mL) for 24 h. E–G The RNA level of SNHG7 was detected by RT-qPCR. F–G FSP1 mRNA and protein were detected by RT-qPCR and western blot, respectively. Data are means ± SD. *p < 0.05, **p < 0.01

Fig. 2

SNHG7 was enriched in BMSCs-Exos and could be absorbed by chondrocytes. A TEM image of exosomes isolated from BMSCs. B The size distribution of exosomes determined by NTA. C Western blot for examining exosomes-specific proteins (CD9, CD63 and CD81). D SNHG7 was detected by agarose gel electrophoresis after amplification by specific primer. E, F BMSCs were transfected with pcDNA3.1 vectors or pcDNA3.1-SNHG7 vectors, respectively. E–F RT-qPCR was performed for determining the RNA level of SNHG7 in BMSCs and BMSCs-Exos. G Chondrocytes was co-cultured with PKH67-labed BMSCs-Exos, the internalization efficiency was determined by immunofluorescence. Exosomes derived from BMSCs-transfected with pcDNA3.1 vectors or pcDNA3.1-SNHG7 vectors (Exos^−vector, Exos^−SNHG7) were added into IL-1β-treated chondrocytes. H–I The levels of SNHG7 RNA and FSP mRNA were detected by RT-qPCR. Data are means ± SD. *p < 0.05, **p < 0.01

Fig. 3

SNHG7 delivered by BMSCs-Exos alleviated IL-1β-triggered inflammatory injury in chondrocytes via upregulation of FSP1. A Chondrocytes were transfected with shNC or shFSP1, respectively. FSP1 mRNA was detected by RT-qPCR. Chondrocytes divided into following group: control, IL-1β, IL-1β + Exos^−vector, IL-1β + Exos^−SNHG7, IL-1β + Exos^−SNHG7 + shNC, IL-1β + Exos.^−SNHG7 + shFSP1. B FSP1 mRNA was detected by RT-qPCR. C Cell viability detection by CCK-8. D LDH release detected by corresponding kit. E–F TNF-α and IL-6 levels were tested by ELISA. Data are means ± SD. *p < 0.05, **p < 0.01

Fig. 4

Exosomal SNHG7 secreted by BMSCs inhibited IL-1β-triggered ferroptosis by elevating FSP1. Chondrocytes were divided into following groups: control, IL-1β, IL-1β + Exos^−vector, IL-1β + Exos^−SNHG7, IL-1β + Exos^−SNHG7 + shNC, IL-1β + Exos^−SNHG7 + shFSP1. A, B ELISA assay for measuring the levels of GSH activity and MDA level. C DCFH-DA staining for measuring ROS level. D Fe.²⁺ was determined by commercial kit. E Western blot for detecting GPX4 and PTGS2 protein levels. Data are means ± SD. *p < 0.05, **p < 0.01

Fig. 5

SNHG7 upregulated FSP1 expression by sponging miR-485-5p. A Image of predicted miRNAs which both interacted with SNHG7 and FSP1 3′-UTR. Chondrocytes were transfected with pcDNA3.1 vectors or pcDNA3.1-SNHG7 vectors, respectively. B The RNA level of SNHG7 was detected by RT-qPCR. C RT-qPCR for testing the levels of miRNAs including hsa-miR-485-5p, hsa-miR-224-5p, hsa-miR-146a-5p, hsa-miR-186-5p, hsa-miR-146b-5p, hsa-miR-214-5p and hsa-miR-543. D RT-qPCR was used to detect miRNAs levels in chondrocytes with/without IL-1β treatment. E Image of the predicted binding sites between SNHG7 and miR-485-5p. f, G The binding relationship between SNHG7 and miR-485-5p was validated by Dual luciferase reporter assay (F) and RIP (G). H Image of predicted binding site between FSP1 3′-UTR and miR-485-5p. I Dual luciferase reporter assay for detecting the relationship of SNHG7 and miR-485-5p. J Chondrocytes were transfected with mimics NC or miR-485-5p mimics, then miR-485-5p level was detected by RT-qPCR. K Chondrocytes were transfected with pcDNA3.1-SNHG7 vectors or co-transfected with pcDNA3.1-SNHG7 vectors plus miR-485-5p mimics. FSP1 mRNA was detected by RT-qPCR. Data are means ± SD. *p < 0.05, **p < 0.01

Fig. 6

MiR-485-5p overexpression reversed the role of exosomal SNHG7 in IL-1β-stimulated inflammatory injury. Chondrocytes were divided into following groups: IL-1β, IL-1β + Exos^−vector, IL-1β + Exos^−SNHG7, IL-1β + Exos.^−SNHG7 + miR-485-5p mimics. A Cell viability detection by CCK-8. B LDH release detected by corresponding kit. C–D TNF-α and IL-6 levels were tested by ELISA. Data are means ± SD. *p < 0.05, **p < 0.01

Fig. 7

MiR-485-5p overexpression diminished the protective role of exosomal SNHG7 in IL-1β-induced ferroptosis. Chondrocytes were divided into following groups: IL-1β, IL-1β + Exos^−vector, IL-1β + Exos^−SNHG7, IL-1β + Exos^−SNHG7 + miR-485-5p mimics. A, B ELISA assay for measuring the levels of GSH activity and MDA level. C DCFH-DA staining for measuring ROS level. D Fe.²⁺ was determined by commercial kit. E Western blot for detecting GPX4 and PTGS2 protein levels. Data are means ± SD. *p < 0.05, **p < 0.01