Exosomes derived from Wharton's jelly of human umbilical cord mesenchymal stem cells reduce osteocyte apoptosis in glucocorticoid-induced osteonecrosis of the femoral head in rats via the miR-21-PTEN-AKT signalling pathway

Abstract

Purpose: Glucocorticoid-induced osteonecrosis of the femoral head (GIONFH) is a common disease after long-term or high-dose glucocorticoid use. The pathogenesis of GIONFH is still controversial, and abnormal bone metabolism caused by glucocorticoids may be one of the important factors. Exosomes, owing to their positive effect on bone repair, show promising therapeutic effects on bone-related diseases. In this study, we hypothesised that exosomes reduce osteocyte apoptosis in rat GIONFH via the miR-21-PTEN-AKT signalling pathway. Methods: To evaluate the effects of exosomes in GIONFH, a dexamethasone-treated or exosome-treated in vitro cell model and a methylprednisolone-treated in vivo rat model were set up. In vitro, a CCK-8 assay and 5-ethynyl-2'-deoxyuridine staining were performed to evaluate the proliferation of osteocytes. Further, a terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay, annexin V-fluorescein isothiocyanate-propidium iodide staining, and western blotting were conducted to evaluate the apoptosis of osteocytes. In vivo, we used micro-computed tomography and histological and immunohistochemical analyses to assess the effects of exosomes. Moreover, the mechanism of exosome action on osteocyte apoptosis through the miR-21-PTEN-AKT pathway was investigated by high-throughput RNA sequencing, fluorescence in situ hybridisation, luciferase reporter assays, and western blotting. Results: High-throughput RNA sequencing results showed that the AKT signalling pathway was up-regulated in the exosome group. Quantitative PCR and western blotting confirmed that the relative expression of genes in the AKT pathway was up-regulated. Western blotting revealed that AKT activated by exosomes inhibited osteocyte apoptosis. RNA fluorescence in situ hybridisation and luciferase reporter assays were performed to confirm the interaction between miR-21 and PTEN. According to the experiment in vivo, exosomes prevented GIONFH in a rat model as evidenced by micro-computed tomography scanning and histological and immunohistochemical analyses. Conclusions: Exosomes are effective at inhibiting osteocyte apoptosis (in MLO-Y4 cells) and at preventing rat GIONFH. These beneficial effects are mediated by the miR-21-PTEN-AKT signalling pathway.

Keywords: AKT signalling pathway; GIONFH; Wharton's jelly of human umbilical cord mesenchymal stem cells; apoptosis; exosomes.

Figure 1

(A) Morphology of hWJ-MSC-Exos identified by TEM. (B) Western blotting analysis of the surface biomarkers CD9, CD63, CD81, and TSG101. (C) Particle size distribution of hWJ-MSC-Exos as measured by DLS. (D) The cell proliferation ability of MLO-Y4 cells treated with Dex and with or without hWJ-MSC-Exos. (E) The TUNEL assay was performed to evaluate the apoptosis of MLO-Y4 cells. (F) The percentage of TUNEL-positive cells was calculated in ImageJ. (G) Western blotting was performed to detect the expression of apoptosis-related proteins including caspase 3, cleaved caspase 3, BCL-2, and BAX. (H) hWJ-MSC-Exos were labelled with PKH-26, and endocytosis of hWJ-MSC-Exos was observed under a confocal microscope. (I) The cell proliferation ability of MLO-Y4 cells was evaluated by the EdU assay.

Figure 2

The heat map (A), scatter plot (B), and volcano plot (C) of RNA-seq between groups 'Dex' and 'Dex with hWJ-MSC-Exos'. (D) Gene Ontology biological process classification of the gene sets from RNA-seq. (E) Significant Gene Ontology terms enriched in the plot figure. (F) Up-regulated signalling pathways and (G) down-regulated signalling pathways enriched in the set of differentially expressed genes.

Figure 3

(A-E) The expression of genes including CCND1, PIK3CA, PIK3R1, AKT1, and IGF-1 among the AKT signalling pathway gene sets was verified by RT-PCR. (F) Western blotting was performed to evaluate the protein amounts of AKT, p-AKT, caspase 3, cleaved caspase 3, and p-BAD. (G) The quantitative analysis of western blotting data in ImageJ. (H) Immunofluorescence staining was then performed to examine the amount of p-AKT in MLO-Y4 cells treated with Dex or exosomes. (I) Fluorescence intensity was quantified by means of ImageJ.

Figure 4

(A) Main reported miRNAs participating in apoptosis, bone-related diseases, and miRNAs present in exosomes; some miRNAs are multifunctional and are members of several groups, e.g. miR-21, miR-155, and miR-125b. (B) RT-PCR was performed to compare the level of miR-21 between hWJ-MSC-Exos and osteocyte exosomes. (C) RT-PCR was carried out to measure the level of miR-21 in osteocytes co-cultured with or without exosomes. (D) The binding site for miR-21 in PTEN mRNA. (E) The PTEN level when osteocytes were transfected with miR-21 mimics. (F) A luciferase reporter assay was performed to verify the interaction between miR-21 and PTEN. (G) FISH was conducted to identify the cell location of miR-21. (H) Western blotting was used to detect the interaction between miR-21 and PTEN. (I) Western blotting was conducted to evaluate the miR-21-PTEN-p-AKT axis. (J) The apoptosis of MLO-Y4 cells was assessed through annexin V-FITC/PI double staining with flow-cytometric analysis of the miR-21-PTEN-AKT signalling pathway.

Figure 5

(A) The GIONFH rat model was created by means of MPS, and exosomes were tested for the treatment of GIONFH. (B) Micro-CT scanning was performed to assess the bone volume of the femoral head in the different rat groups. (C) HE staining of the femoral heads in rats receiving different treatments. (D) Quantitative analyses of trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), bone volume per tissue volume (BV/TV), and trabecular number (Tb.N) in the different rat groups. (E) Immunohistochemical staining of p-AKT in samples from the different rat groups.