Huc-MSCs-derived exosomes attenuate neuropathic pain by inhibiting activation of the TLR2/MyD88/NF-κB signaling pathway in the spinal microglia by targeting Rsad2.

Mesenchymal stem cells (MSCs)-derived exosomes have shown promise as a cell-free therapeutic strategy for neuropathic pain. This study was conducted to explore the potential mechanisms underlying the analgesic effects of MSC-derived exosomes in treating neuropathic pain. Human umbilical cord MSCs (huc-MSCs)-derived exosomes were isolated and identified. BV-2 microglia were stimulated with lipopolysaccharide (LPS) in the presence or absence of exosomes. Differentially expressed proteins were identified by tandem mass tag (TMT)-based proteomic analysis. The analgesic effects of huc-MSCs-derived exosomes were evaluated in a rat model of chronic constriction injury (CCI). The underlying mechanism was investigated by flow cytometry, RT-qPCR, Western blotting, immunofluorescent staining, and small interfering RNA transfection. In vitro, huc-MSCs-derived exosomes suppressed LPS-induced microglial activation and inhibited activation of the TLR2/MyD88/NF-κB signaling pathway. Based on the proteomic analysis, Rsad2 was identified and confirmed to be down-regulated by huc-MSCs-derived exosomes. Importantly, knockdown of Rsad2 also inhibited microglial activation and restrained activation of the TLR2/MyD88/NF-κB signaling pathway. In vivo, intrathecal injection of exosomes ameliorated CCI-induced mechanical allodynia, down-regulated Rsad2 expression and restrained TLR2/MyD88/NF-κB signaling activation in the spinal microglia. Huc-MSCs-derived exosomes exerted analgesic effects on neuropathic pain by inhibiting activation of the TLR2/MyD88/NF-κB signaling pathway in the spinal microglia. The mechanism underlying these antinociceptive effects involved exosome-mediated interference with Rsad2 expression, thereby inhibiting microglial activation.

Gao X ，Gao LF ，Zhang YN ，Kong XQ ，Jia S ，Meng CY ... -  《-》

Anti-inflammatory protein TSG-6 secreted by bone marrow mesenchymal stem cells attenuates neuropathic pain by inhibiting the TLR2/MyD88/NF-κB signaling pathway in spinal microglia.

Yang H ，Wu L ，Deng H ，Chen Y ，Zhou H ，Liu M ，Wang S ，Zheng L ，Zhu L ，Lv X ... -  《Journal of Neuroinflammation》

Exosomal GDNF from Bone Marrow Mesenchymal Stem Cells Moderates Neuropathic Pain in a Rat Model of Chronic Constriction Injury.

Both of exosomes derived from mesenchymal stem cells (MSCs) and glial cell line-derived neurotrophic factor (GDNF) show potential for the treatment of neuropathic pain. Here, the analgesic effects of exosomes derived from bone marrow MSCs (BMSCs) were investigated. BMSCs-derived exosomes were isolated and characterized. Chronic constriction injury (CCI) was constructed to induce neuropathic pain in rats, which were then treated with exosomes. Pain behaviors were evaluated by measuring paw withdrawal thresholds and latency. The changes of key proteins, including cytokines, were explored using Western blot and ELISA. Administration of BMSCs-derived exosomes alleviated neuropathic pain, as demonstrated by the decrease of thermal hyperalgesia and mechanical allodynia, as well as the reduced secretion of pro-inflammatory cytokines in CCI rats. These effects were comparable to the treatment of GDNF alone. Mechanically, the exosomes suppressed the CCI-induced activation of TLR2/MyD88/NF-κB signaling pathway, while GDNF knockdown impaired their analgesic effects on CCI rat. BMSCs-derived exosomes may alleviate CCI-induced neuropathic pain and inflammation in rats by transporting GDNF.

Zhang X ，Liu H ，Xiu X ，Cheng J ，Li T ，Wang P ，Men L ，Qiu J ，Jin Y ，Zhao J ... -  《-》

Huc-MSCs-derived exosomes attenuate inflammatory pain by regulating microglia pyroptosis and autophagy via the miR-146a-5p/TRAF6 axis.

Chronic inflammatory pain significantly reduces the quality of life and lacks effective interventions. In recent years, human umbilical cord mesenchymal stem cells (huc-MSCs)-derived exosomes have been used to relieve neuropathic pain and other inflammatory diseases as a promising cell-free therapeutic strategy. However, the therapeutic value of huc-MSCs-derived exosomes in complete Freund's adjuvant (CFA)-induced inflammatory pain remains to be confirmed. In this study, we investigated the therapeutic effect and related mechanisms of huc-MSCs-derived exosomes in a chronic inflammatory pain model. C57BL/6J male mice were used to establish a CFA-induced inflammatory pain model, and huc-MSCs-derived exosomes were intrathecally injected for 4 consecutive days. BV2 microglia cells were stimulated with lipopolysaccharide (LPS) plus adenosine triphosphate (ATP) to investigate the effect of huc-MSCs-derived exosomes on pyroptosis and autophagy. Bioinformatic analysis and rescue experiments were used to demonstrate the role of miR-146a-5p/ TRAF6 in regulating pyroptosis and autophagy. Western blotting, RT-qPCR, small interfering RNA and Yo-Pro-1 dye staining were performed to investigate the related mechanisms. Huc-MSCs-derived exosomes alleviated mechanical allodynia and thermal hyperalgesia in CFA-induced inflammatory pain. Furthermore, huc-MSCs-derived exosomes attenuated neuroinflammation by increasing the expression of autophagy-related proteins (LC3-II and beclin1) and inhibiting the activation of NLRP3 inflammasomes in the spinal cord dorsal horn. In vitro, NLRP3 inflammasome components (NLRP3, caspase1-p20, ASC) and gasdermin D (GSDMD-F, GSDMD-N) were inhibited in BV2 cells pretreated with huc-MSCs-derived exosomes. Western blot and Yo-Pro-1 dye staining demonstrated that 3-MA, an autophagy inhibitor, weakened the protective effect of huc-MSCs-derived exosomes on BV2 cell pyroptosis. Importantly, huc-MSCs-derived exosomes transfected with miR-146a-5p mimic promoted autophagy and inhibited BV2 cell pyroptosis. TRAF6, as a target gene of miR-146a-5p, was knocked down via small-interfering RNA, which increased pyroptosis and inhibited autophagy. Huc-MSCs-derived exosomes attenuated inflammatory pain via miR-146a-5p/TRAF6, which increased the level of autophagy and inhibited pyroptosis.

Hua T ，Yang M ，Song H ，Kong E ，Deng M ，Li Y ，Li J ，Liu Z ，Fu H ，Wang Y ，Yuan H ... -  《JOURNAL OF NANOBIOTECHNOLOGY》

Human umbilical cord mesenchymal stem cell-derived exosomes attenuate neuroinflammation and oxidative stress through the NRF2/NF-κB/NLRP3 pathway.

We investigated whether human umbilical cord mesenchymal stem cell (hUC-MSC)-derived exosomes bear therapeutic potential against lipopolysaccharide (LPS)-induced neuroinflammation. Exosomes were isolated from hUC-MSC supernatant by ultra-high-speed centrifugation and characterized by transmission electron microscopy and western blotting. Inflammatory responses were induced by LPS in BV-2 cells, primary microglial cultures, and C57BL/6J mice. H2 O2 was also used to induce inflammation and oxidative stress in BV-2 cells. The effects of hUC-MSC-derived exosomes on inflammatory cytokine expression, oxidative stress, and microglia polarization were studied by immunofluorescence and western blotting. Treatment with hUC-MSC-derived exosomes significantly decreased the LPS- or H2 O2 -induced oxidative stress and expression of pro-inflammatory cytokines (IL-6 and TNF-α) in vitro, while promoting an anti-inflammatory (classical M2) phenotype in an LPS-treated mouse model. Mechanistically, the exosomes increased the NRF2 levels and inhibited the LPS-induced NF-κB p65 phosphorylation and NLRP3 inflammasome activation. In contrast, the reactive oxygen species scavenger NAC and NF-κB inhibitor BAY 11-7082 also inhibited the LPS-induced NLRP3 inflammasome activation and switched to the classical M2 phenotype. Treatment with the NRF2 inhibitor ML385 abolished the anti-inflammatory and anti-oxidative effects of the exosomes. hUC-MSC-derived exosomes ameliorated LPS/H2 O2 -induced neuroinflammation and oxidative stress by inhibiting the microglial NRF2/NF-κB/NLRP3 signaling pathway.

Che J ，Wang H ，Dong J ，Wu Y ，Zhang H ，Fu L ，Zhang J ... -  《-》