Abstract
Chronic morphine-induced antinociceptive tolerance is a major unresolved issue in clinical practices, which is associated with microglia activation in the spinal cord. E3 ubiquitin ligase Pellino1 (Peli1) is known to be an important microglia-specific regulator. However, it is unclear whether Peli1 is involved in morphine tolerance. Here, we found that Peli1 levels in the spinal cord were significantly elevated in morphine tolerance mouse model. Notably, Peli1 was expressed in a great majority of microglia in the spinal dorsal horn, while downregulation of spinal Peli1 attenuated the development of morphine tolerance and associated hyperalgesia. Our biochemical data revealed that morphine tolerance-induced increase in Peli1 was accompanied by spinal microglia activation, activation of mitogen-activated protein kinase (MAPK) signaling, and production of proinflammatory cytokines. Peli1 additionally was found to promote K63-linked ubiquitination of tumor necrosis factor receptor-associated factor 6 (TRAF6) in the spinal cord after repeated morphine treatment. Furthermore, knocking down Peli1 in cultured BV2 microglial cells significantly attenuated inflammatory reactions in response to morphine challenge. Therefore, we conclude that the upregulation of Peli1 in the spinal cord plays a curial role in the development of morphine tolerance via Peli1-dependent mobilization of spinal microglia, activation of MAPK signaling, and production of proinflammatory cytokines. Modulation of Peli1 may be a potential strategy for the prevention of morphine tolerance.
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Acknowledgments
This study was supported by the National Natural Science Foundation of China (81600967 (Chen Lu), 81703493 (Tianya Liu)), the National Science Foundation for Young Scientists of Jiangsu Province (BK20150214 (Chen Lu), BK20170258 (Tianya Liu)), and Jiangsu Students for innovation and entrepreneurship training program (201610313065X (Chen Lu)).
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In this paper, LJW, CY, and YYX carried out cell experiments, animal surgery, and molecular biology studies. TYL and YZ performed the experimental data analysis. TYL and BW contributed reagents or analytic tools. CL and MA revised the manuscript. CL and JLC designed, supervised the study, and drafted the manuscript.
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10571_2020_797_MOESM1_ESM.docx
Fig. S1 The expression of GFP in the spinal dorsal horn. Representative immunofluorescence images showing the labeling of astrocytes (GFAP, red), microglial cells (Iba1, red), and neurons (NeuN, red) with GFP (green) in the spinal dorsal horn 7 days after the intrathecal injection. Scale bar represents 50 μm. n=3 per group. (DOCX 1334 kb)
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Fig. S2 Downregulation of spinal Peli1 does not reverse the established morphine tolerance. Morphine was administered subcutaneously twice daily for 11 days. Peli1 siRNA (siPeli1) or negative siRNA (siNeg) was intrathecally injected once daily from days 9 to 11 when morphine tolerance had fully developed. Each intrathecal injection is indicated by arrows. a Effectiveness of Peli1 siRNA or negative siRNA on expression of Peli1 were determined using western blot. Tissues were taken 4 hours after the last siRNA injection. n=4 per group. b MPE was determined using tail immersion test before and on day 8, 9, 10, 11 of morphine treatment. n=5 per group. c-d The paw withdrawal latency (c) and paw withdrawal threshold (d) were examined before and on day 11 of morphine treatment. n=5 per group. Results are expressed as the Mean ± SEM. *p<0.05 compared with indicated group. ns, statistically not significant. (DOCX 181 kb)
10571_2020_797_MOESM3_ESM.docx
Fig. S3 The effect of Peli1 on phosphorylated NF-B p65 levels after chronic morphine treatment in the spinal cord. Knockdown of spinal Peli1 did not attenuate morphine tolerance-induced increase in the phosphorylation of NF-B p65 (n=4 per group). Results are expressed as the Mean ± SEM. *p<0.05 compared with indicated group. ns, statistically not significant. (DOCX 108 kb)
10571_2020_797_MOESM4_ESM.docx
Fig. S4 Peli1 is involved in morphine-induced inflammatory reactions and microglia activation in BV2 cells. a BV2 cells were treated with or without 50 µM, 100 µM and 200 µM morphine for 24 h. Levels of Peli1 mRNA was determined using qPCR (n=4 per group). b-h BV2 cells were transfected with 75 µM Peli1 siRNA (siPeli1) or negative siRNA (siNeg) 24 h followed by the addition of morphine (200 µM) for 24 h. The expression of Peli1 mRNA (b, n=4 per group) and Peli1 protein (c, n=4 per group) were determined. Quantitative Western blot analysis showing the effect of Peli1 on P-NF-B p65 (d, n=4 per group) in BV2 microglial culture upon morphine treatment. e TNF-α (n=3-6 per group), IL-6 (n=4-6 per group), and IL-1β (n=4-6 per group) mRNA levels in BV2 cells were determined using qPCR. f After morphine stimulation, transfected BV2 cells were fixed and stained with DAPI (blue) and anti-Iba1 (red). Scale bar represents 20 m. n=3 per group. g qPCR analysis showing the inhibitory effect of Peli1 siRNA on Iba1 mRNA levels in BV2 cells after morphine treatment. n=4-5 per group. h Transfection of Peli1 siRNA inhibits the migration of BV2 cells into the scratched area. n=5 per group. Representative images were taken before and after scratch (4× magnification). Dashed line indicates the width of gap. Results are expressed as the Mean ± SEM. *p<0.05 compared with indicated group. ns, statistically not significant. (DOCX 1068 kb)
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Wang, L., Yin, C., Xu, X. et al. Pellino1 Contributes to Morphine Tolerance by Microglia Activation via MAPK Signaling in the Spinal Cord of Mice. Cell Mol Neurobiol 40, 1117–1131 (2020). https://doi.org/10.1007/s10571-020-00797-3
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DOI: https://doi.org/10.1007/s10571-020-00797-3