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

(2022) 20:324 Hua et al. Journal of Nanobiotechnology https://doi.org/10.1186/s12951-022-01522-6 Journal of Nanobiotechnology Open Access RESEARCH Huc‑MSCs‑derived exosomes attenuate inflammatory pain by regulating microglia pyroptosis and autophagy via the miR‑146a‑5p/ TRAF6 axis Tong Hua1†, Mei Yang1†, Honghao Song1†, Erliang Kong1, Mengqiu Deng1, Yongchang Li1, Jian Li1, Zhixiao Liu2, Hailong Fu1*, Yue Wang3* and Hongbin Yuan1* Abstract Background: 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. Methods: C57BL/6J male mice were used to establish a CFA-induced inflammatory pain model, and huc-MSCsderived 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 YoPro-1 dye staining were performed to investigate the related mechanisms. Results: 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-MSCsderived exosomes on BV2 cell pyroptosis. Importantly, huc-MSCs-derived exosomes transfected with miR-146a-5p † Tong Hua, Mei Yang and Honghao Song contributed equally to this work *Correspondence: ; ; 1 Department of Anesthesiology, Changzheng Hospital, Naval Medical University, Shanghai 200003, China 3 Stem Cell and Regeneration Medicine Institute, Research Center of Translational Medicine, Naval Medical University, Shanghai 200433, China Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Hua et al. Journal of Nanobiotechnology (2022) 20:324 Page 2 of 18 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. Conclusion: Huc-MSCs-derived exosomes attenuated inflammatory pain via miR-146a-5p/TRAF6, which increased the level of autophagy and inhibited pyroptosis. Keywords: Huc-MSCs-derived exosomes, Inflammatory pain, Microglia, Pyroptosis, Autophagy Graphical Abstract Background Chronic pain, such as chronic inflammatory pain, is one of the most common and complex health problems, which causes insomnia, depression and increases suicide occurrence [1]. In addition, chronic pain brings about huge social and economic burdens [2]. However, the mechanism of chronic pain is still not completely understood, leading to a lack of effective treatments. Available evidence in the past few years suggests that neuroinflammation plays a predominant role in the pathological process of chronic pain [3, 4]. Opioids and nonsteroidal anti-inflammatory drugs (NSAIDs) are most commonly used to alleviate chronic pain resulting from neuroinflammation [5]. However, continuously using opioids increases the risk of serious adverse effects, such as drug addiction, opioid tolerance, and opioid-induced hyperalgesia [6]. On the other hand, opioids and NSAIDs cannot completely inhibit the generation of chronic pain. Thus, it is imperative to pay more attention to exploring the mechanisms of chronic inflammatory pain, and develop an effective therapeutic strategy. Microglia play a double-edged sword role in protecting against stimulation resulting from tissue damage or pathogen infection. An excessive inflammatory response, especially secreting abundant proinflammatory cytokines, eventually leads to neuroinflammation [7]. Microglia pyroptosis is a newly discovered inflammatory cell death via inflammasomes associated with immune and inflammation-related disorders in the central nervous system, covering spinal cord injury and depression [8–10]. NLRP3 inflammasome is one classic member of inflammasomes which is the most studied molecules in chronic pain [11, 12]. The main components of NLRP3 inflammasome include pattern recognition receptor NLRP3, adaptor protein apoptosis-associated speck-like protein (ASC) and pro-caspase-1 enzyme [13]. Once NLRP3 inflammasome is activated by a diversity of stimuli and ligands, the assembly of NLRP3 with ASC into the inflammasome complex is triggered. The activation of NLRP3 and caspase-1 cleaves pro-IL-1β and pro-IL-18, ultimately increasing the secretion of mature IL-1β and IL-18 [13]. Moreover, NLRP3 inflammasome Hua et al. Journal of Nanobiotechnology (2022) 20:324 is closely related to pyroptosis, via cleaving GSDMD into its active form, GSDMD-N, which forms pores in the cell membranes [14]. Autophagy is an evolutionarily conserved biological process that eliminates misfolded proteins or ligands and influences immune infla (...truncated)