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》

miR-99b-3p/Mmp13 axis regulates NLRP3 inflammasome-dependent microglial pyroptosis and alleviates neuropathic pain via the promotion of autophagy.

Gao X ，Gao LF ，Zhang ZY ，Jia S ，Meng CY ... -  《-》

Dental pulp mesenchymal stem cell-derived exosomes inhibit neuroinflammation and microglial pyroptosis in subarachnoid hemorrhage via the miRNA-197-3p/FOXO3 axis.

Subarachnoid hemorrhage (SAH) is a severe stroke subtype that lacks effective treatment. Exosomes derived from human dental pulp stem cells (DPSCs) are a promising acellular therapeutic strategy for neurological diseases. However, the therapeutic effects of DPSC-derived exosomes (DPSC-Exos) on SAH remain unknown. In this study, we investigated the therapeutic effects and mechanisms of action of DPSC-Exos in SAH. SAH was established using 120 male Sprague-Dawley rats. One hour after SAH induction, DPSC-Exos were administered via tail vein injection. To investigate the effect of DPSC-Exos, SAH grading, short-term and long-term neurobehavioral assessments, brain water content, western blot (WB), immunofluorescence staining, Nissl staining, and HE staining were performed. The role of miR-197-3p/FOXO3 in regulating pyroptosis was demonstrated through miRNA sequencing, bioinformatics analysis, and rescue experiments. The SAH model in vitro was established by stimulating BV2 cells with hemoglobin (Hb) and the underlying mechanism of DPSC-Exos was investigated through WB and Hoechst/PI staining. The expressions of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) were increased after SAH. DPSC-Exos alleviated brain edema and neuroinflammation by inhibiting the expression of FOXO3 and reducing NLRP3 inflammasome activation, leading to improved neurobehavioral functions at 24 h after SAH. In vitro, the expression of the NLRP3 inflammasome components (NLRP3 and caspase1-p20), GSDMD-N, and IL-18 was inhibited in BV2 cells pretreated with DPSC-Exos. Importantly, DPSC-Exos overexpressing miR-197-3p had a more obvious protective effect than those from NC-transfected DPSCs, while those from DPSCs transfected with the miR-197-3p inhibitor had a weaker protective effect. Functional studies indicated that miR-197-3p bound to the 3'-untranslated region of FOXO3, inhibiting its transcription. Furthermore, the overexpression of FOXO3 reversed the protective effects of miR-197-3p. DPSC-Exos inhibited activation of the NLRP3 inflammasome and related cytokine release via the miR-197-3p/FOXO3 pathway, alleviated neuroinflammation, and inhibited microglial pyroptosis. These findings suggest that using DPSC-Exos is a promising therapeutic strategy for SAH.

Liang X ，Miao Y ，Tong X ，Chen J ，Liu H ，He Z ，Liu A ，Hu Z ... -  《JOURNAL OF NANOBIOTECHNOLOGY》

Resveratrol protects BV2 mouse microglial cells against LPS-induced inflammatory injury by altering the miR-146a-5p/TRAF6/NF-κB axis.

Ge YT ，Zhong AQ ，Xu GF ，Lu Y ... -  《-》

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 ... -  《-》