Fig 2: δ-opioid receptor (DOR) knockdown and overexpression induced opposite effects on microglia-mediated inflammatory events. (A) Effects of DOR knockdown and overexpression on inflammatory cytokines under LPS insult. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. NC; Δp < 0.05, ΔΔp < 0.01, ΔΔΔp < 0.001 vs. NC-LPS. Note that DOR knockdown aggravated the inflammatory situation induced by LPS, and it also promoted the anti-inflammatory events to fight against LPS injury. DOR overexpression largely downregulated LPS-induced generations of CD86, iNOS and TNF-α, while showed an inappreciable effect on CD206, IL-10 and TGF-β. (B) Effects of DOR knockdown or overexpression on inflammatory cytokines under hypoxic stress. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. NC; Δp < 0.05, ΔΔp < 0.01, ΔΔΔp < 0.001, ΔΔΔΔp < 0.0001 vs. NC-Hypoxia. Note that DOR knockdown in BV2 cells significantly increased both the inflammatory cytokines and anti-inflammatory cytokines level under hypoxic stress. DOR overexpression in BV2 cells suppressed the inflammatory events, and downregulated M2 phenotype marker CD206 as well.

Fig 3: δ-opioid receptor (DOR) regulation of inflammatory cytokines and anti-inflammatory cytokines under LPS, IL4, or hypoxic condition. The mRNA levels of CD86, iNOS, TNF-α, CD206, IL10, and TGF-β were measured by RT-PCR in BV2 cells under LPS/hypoxic insults (N ≥ 3 in each group). (A) Effects of DOR activation on inflammatory events under LPS insult. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. C; Δp < 0.05, ΔΔp < 0.01 vs. L. Note that 10–20 μM of UFP-512 significantly inhibited the M1 type shift of BV2 cells under LPS insult, as well as downregulating iNOS and TNF. (B) DOR-mediated suppressions of anti-inflammatory events in IL4-treated BV2 cells. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. C; Δp < 0.05, ΔΔp < 0.01 vs. I. Note that IL4 caused a significant increase in M2 phenotype marker CD206 and anti-inflammatory cytokines IL-10 and TGF- β. Following the increase of UFP-512 concentration, the IL4- mediated anti-inflammatory events were attenuated. (C) Effects of DOR activation on inflammatory events under hypoxic stress. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. C; Δp < 0.05, ΔΔp < 0.01 vs. H. Note that the hypoxia-induced upregulation of CD86, iNOS and TNF-α were inhibited by DOR activation with UFP-512, while the addition of naltrindole reversed these effects.

Fig 4: δ-opioid receptor (DOR) expression and distribution in BV2 cells. The experiments were conducted on BV2 cells (N = 3 in each group). C: normal control. L: LPS. I: IL4. H: 1% O2 hypoxia. (A) Representative immunofluorescent images of BV2 cells exposed to normal, LPS, IL4, or hypoxic environment. The merge images merged Iba1 (green), DOR (red), and Hoechst (blue) images in each group. Note that the immunofluorescence staining showed that DOR maintained a mild expression level under normal condition and LPS insult, but the red fluorescence labeling DOR significantly enhanced after 24 h of IL4 treatments. Hypoxia induced both high-expressed DOR and low-expressed DOR BV2 cells. (B) Differential MHCII expression in BV2 cells under physiological, LPS, IL4, or hypoxic condition. *p < 0.05, **p < 0.01 vs. C. Note that LPS significantly upregulated MHCII expression, while hypoxic insult also induced a slight increase in MHCII protein in BV2 cells. (C) Differential Arginase expression in BV2 cells under physiological, LPS, IL4, or hypoxic condition. ****p < 0.0001 vs. C. Note that arginase is difficult to detect under normal condition or LPS insult. IL4 caused a sharp increase of arginase, and hypoxia also led to an upregulation of Arginase protein. (D) Alterations of DOR density in BV2 cells under physiological, LPS, IL4, or hypoxic condition. **p < 0.01 vs. C. Note that DOR density kept a low level under normal condition and LPS insult, but significantly increased after exposure to IL4 or hypoxia.

Fig 5: Differential changes in striatal and hippocampal mRNA expression after a month of forced abstinence from oxycodone SA. a Increased Oprm1 receptor mRNA levels in the striatum of LgA-H rats. b Striatal Oprd1 mRNA levels are increased in ShA rats. c Striatal Oprk1 expression showed no significant changes. d Decreased hippocampal Oprm1 receptor mRNA levels in LgA rats. e Hippocampal Oprd1 mRNA levels show no significant changes. f Hippocampal Oprk1 mRNA levels are decreased in all oxycodone groups. The values in the bar graphs represent means ± SEM (n = 5–12 animals per group). Note the differences in scales on the Y-axis. Key to statistics: *, **, *** = p < 0.05, 0.01, 0.001, respectively, in comparison to saline rats; ##p < 0.01 in comparison to ShA rats; $p < 0.05 in comparison to LgA-L