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.

PTEN inhibitor VO-OHpic attenuates GC-associated endothelial progenitor cell dysfunction and osteonecrosis of the femoral head via activating Nrf2 signaling and inhibiting mitochondrial apoptosis pathway

“…The extrinsic death receptor pathway and the intrinsic mitochondrial pathway are two major systems that could activate the expression of caspases and subsequent cell apoptosis [44]. Previous studies have reported the proapoptosis effects of excess GC on osteoblasts [45], osteocytes [46], and endothelial cells [47]. In the present study, we firstly reported that excess GC could induce EPC apoptosis through activating mitochondrial pathway.…”

Exosomal miR-186 Derived from BMSCs Promote Osteogenesis Through Hippo Signaling Pathway in Postmenopausal Osteoporosis

“…Yang et al [32] performed RNA-seq and found miR-1263 prevented BMSC apoptosis in disuse osteoporosis. In a study conducted by Kuang et al, exosomes derived from human umbilical cord mesenchymal stem cells transferred miR-21 to regulate osteogenesis in GIONFH [12]. In our study, we extracted BMSCs from OVX or normal rats, and we performed miRNA-seq between BMSCs from OVX and BMSCs from OVX + exosomes groups; differentially expressed miRNAs were explored.…”

“…After rinsing, cells were xed with 4% paraformaldehyde. Mineralized nodules were observed by staining with a 1% alizarin red solution (Cyagen, Suzhou, China) following manufacturer instructions [12]. ALP staining was used to investigate the osteogenesis of BMSCs after induction [18].…”

“…Xiao et al reported that cardiac progenitor cell-derived exosomal miR-21 can decrease oxidative stress to protect myocardium by targeting PDCD4 [10]. Kuang et al showed that exosomes derived from Wharton's jelly of human umbilical cord mesenchymal stem cells can transfer miR-21-5p and signi cantly reduce osteocyte apoptosis for the treatment of glucocorticoid-induced osteonecrosis of femoral heads in rats by activating the AKT signaling pathway [12]. Liu et al reported that stem cell-derived exosomes can promote cartilage regeneration [13].…”

Mesenchymal Stem Cell-Derived Extracellular Vesicles: Challenges in Clinical Applications

“…The DiD-labeled MSC-EV distributed maximally in the liver and spleen, lesser in the bone marrow of the spine, femur, and tibia, and were undetectable in the lung, heart, and kidney (Wen et al, 2019). PKH-26A, a lipophilic dye that integrates into cell membranes, is commonly used to label MSC-EV (Bucan et al, 2019;Karaoz et al, 2019;Kuang et al, 2019). Bucan et al (2019) evaluated the effects of rat adipose-derived MSC-EV (rAMSC-EV) on sciatic nerve regeneration and neurite growth.…”