Exosomes derived from human platelet-rich plasma prevent apoptosis induced by glucocorticoid-associated endoplasmic reticulum stress in rat osteonecrosis of the femoral head via the Akt/Bad/Bcl-2 signal pathway

Abstract

An excess of glucocorticoids (GCs) is reported to be one of the most common causes of osteonecrosis of the femoral head (ONFH). In addition, GCs can induce bone cell apoptosis through modulating endoplasmic reticulum (ER) stress. Among the three main signal pathways in ER stress, the PERK (protein kinase RNA-like ER kinase)/CHOP (CCAAT-enhancer-binding protein homologous protein) pathway has been considered to be closely associated with apoptosis. Platelet-rich plasma (PRP) has been referred to as a concentration of growth factors and the exosomes derived from PRP (PRP-Exos) have a similar effect to their parent material. The enriched growth factors can be encapsulated into PRP-Exos and activate Akt and Erk pathways to promote angiogenesis. Activation of the Akt pathway may promote the expression of anti-apoptotic proteins like Bcl-2, while CHOP can inhibit B-cell lymphoma 2 (Bcl-2) expression to increase the level of cleaved caspase-3 and lead to cell death. Consequently, we hypothesized that PRP-Exos prevent apoptosis induced by glucocorticoid-associated ER stress in rat ONFH via the Akt/Bad/Bcl-2 signal pathway. To verify this hypothesis, a dexamethasone (DEX)-treated in vitro cell model and methylprednisolone (MPS)-treated in vivo rat model were adopted. Characterization of PRP-Exos, and effects of PRP-Exos on proliferation, apoptosis, angiogenesis, and osteogenesis of cells treated with GCs in vitro and in vivo were examined. Furthermore, the mechanism by which PRP-Exos rescue the GC-induced apoptosis through the Akt/Bad/Bcl-2 pathway was also investigated. The results indicate that PRP-Exos have the capability to prevent GC-induced apoptosis in a rat model of ONFH by promoting Bcl-2 expression via the Akt/Bad/Bcl-2 signal pathway under ER stress.

Keywords: apoptosis.; endoplasmic reticulum stress; exosomes; glucocorticoids; osteonecrosis of the femoral head; platelet-rich plasma.

Figure 1

Characterization of exosomes derived from PRP. (A) Particle size distribution of PRP-Exos measured by DLS. (B) Morphology of PRP-Exos examined by transmission electron microscopy. Scale bar: 100 nm. (C) Western blotting analysis of the surface biomarkers TSG101, CD9, CD63, and CD81 on PRP-Exos, the source marker CD41 and the encapsulated proteins calnexin, PDGFBB, TGF-β, bFGF, and VEGF, as compared with platelet lysate (PL).

Figure 2

Effects of PRP-Exos on cell proliferation in vitro and in vivo.(A) The cell viability of HMEC-1, BMSC and MC3T3-E1 cells treated with DEX together with or without PRP-Exos, examined by CCK-8 analysis. (B) The cell proliferation of HMEC-1, BMSC and MC3T3-E1 cells treated with DEX together with or without PRP-Exos was confirmed by EdU kit assay. (C) The effects of PRP-Exos on cellular proliferation in the femoral heads of rats in the short-term subgroup were evaluated by Ki67 immunostaining in vivo. Scale bar: 50 μm.

Figure 3

Effects of PRP-Exos on GC-induced apoptosis in vitro and in vivo.(A-C) In vitro, the apoptosis of HMEC-1, BMSC and MC3T3-E1 cells induced by DEX together with or without PRP-Exos were assessed through Annexin V-FITC/PI double staining with flow cytometric analysis. (D) Western blotting was used to examine the cleaved caspase-3 expression in HMEC-1, BMSC and MC3T3-E1 cells exposed to DEX and co-treated with or without PRP-Exos. (E) In vivo, the effects of PRP-Exos on the rat model in the short-term subgroup were evaluated by TUNEL assay. Scale bar: 50 μm.

Figure 4

Effects of PRP-Exos on angiogenesis of cells or tissue treated with GCs in vitro and in vivo. (A) The angiogenesis of HMEC-1 cells induced by DEX together with or without PPR-Exos was evaluated by tube formation assay. Scale bar: 50 μm. (B) After 3 h induction by DEX together with or without PRP-Exos, the protein expression of Erk, p-Erk, Akt and p-Akt was examined by western blotting. (C) After induction by DEX together with or without PRP-Exos for 24 h, the protein expression of VEGF-A was examined by western blotting. (D) In vivo, the blood supply of the rat model in the long-term subgroup treated with PRP-Exos was examined using MicroFil.

Figure 5

Effects of PRP-Exos on osteogenesis of BMSCs and MC3T3-E1 cells treated with DEX in vitro. Osteogenesis was induced together with or without DEX or PRP-Exos in BMSCs and MC3T3-E1 cells. (A) After 21 days, the formation of calcium mineral deposits was measured by Alizarin red staining. Scale bar: 50 μm. (B) After 7 days, the protein expression levels of Runx2, type I collagen and β-catenin were examined by western blotting. (C) After 7 days, the ALP expression level and (D), after 14 days, the OCN expression level, were examined by ELISA.

Figure 6

The osteogenesis-promoting effects of PRP-Exos on the rat model of GC-induced ONFH. (A) Reconstructed coronal, transverse and sagittal images of bones within the normal, MPS and PRP-Exos groups. (B) 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 treatment groups. (C) HE staining of the femoral heads in rats receiving different treatments. Scale bar: 100 μm. (D) Immunohistochemical staining of type I collagen in samples from the different treatment groups. Scale bar: 100 μm.

Figure 7

PRP-Exos rescued cells from GC-induced apoptosis through the Akt/Bcl-2 pathway. (A) In the different treatment groups, the p-PERK, PERK, CHOP, Bcl-2 and cleaved-caspase-3 protein expression levels were examined by western blotting. (B) In the different treatment groups, the p-Akt, Akt, P-Bad, Bad, Bcl-2 and cleaved-caspase-3 protein expression levels were examined by western blotting. (C) A brief schematic diagram showing the underlying mechanism of PRP-Exos prevention of GC-induced ONFH.