Fig 2: DRG-Exo are involved in the development of NP through the delivery of miR-16-5p. a Microarray analysis of differentially enriched miRNAs in BV2 cells after DRG-Exo treatment. b miR-16-5p expression in the spinal cord of SNL and DRG-Exo-treated mice using RT-qPCR. c RT-qPCR detection of miR-16-5p enrichment in the sham-operated, SNL-treated, and naive mice-derived DRG-Exo. d miR-16-5p expression in DRG neurons upon miR-16-5pKD or miR-NCKD using RT-qPCR. e miR-16-5p expression in DRG-Exo-KD or DRG-Exo-NC using RT-qPCR. Assessment of mechanical allodynia response in mice treated with DRG-Exo-KD or DRG-Exo-NC using 0.07 g (f) or 0.4 g (g) von Frey filaments at different time points before and after SNL surgery, respectively. h The responses of mice treated with DRG-Exo-KD or DRG-Exo-NC to thermal hyperalgesia were evaluated at different time points before and after SNL surgery. i The responses of mice treated with DRG-Exo-KD or DRG-Exo-NC to cold hyperalgesia were assessed at different time points before and after SNL surgery, respectively. j Inflammatory infiltration in the spinal cord (L4) of the mice treated with DRG-Exo-KD or DRG-Exo-NC was observed using HE staining. k The mRNA expression of IL-6 and IL-1β in the spinal cord by RT-qPCR. l Expression of iba1, a marker of microglia activation in the spinal cord by immunofluorescence. m Expression of miR-16-5p in the spinal cord by RT-qPCR. Values are expressed as mean ± SD. **p < 0.01 vs the sham group; ##p < 0.01 vs the SNL + PBS group; @@p < 0.01 vs the supernatant group; $$p < 0.01 vs the miR-NC.KD group; &&p < 0.01 vs the DRG-Exo-NC group by unpaired t-test (d, e, l, m), one-way (b) or two-way ANOVA (c, f, g, h, i, k)

Fig 3: miR-16-5p interacts with HECTD1 to promote microglial activation. a The intersection of predicted downstream targets of miR-16-5p in Starbase, Targetscan, miRWalk, miRBD, and DIANA TOOLS databases. b Detection of HECTD1 expression in the spinal cord of mice by RT-qPCR. c Binding sites of miR-16-5p and HECTD1 predicted by the Starbase database and the luciferase activity in 293T cells with WT and MUT HECTD1 binding sites using dual-luciferase assays. d The expression of HECTD1 mRNA in biotin-coupled miR-16-5p pull-down complexes using an RNA pull-down assay. e Detection of HECTD1 mRNA expression in LPS- and DRG-Exo-treated BV2 cells. f Co-localization of HECTD1 and iba1 in LPS and DRG-Exo-treated BV2 cells using immunofluorescence staining. g Detection of HECTD1 mRNA expression after knockdown of HECTD1 in BV2 cells using RT-qPCR. h Iba1 and iNOS protein expression in BV2 cells after treatment with DRG-Exo-KD and HECTD1 knockdown. i IL-6 and IL-1β levels in BV2 cells after treatment with DRG-Exo-KD and HECTD1 knockdown using ELISA. j Cell migration ability in BV2 cells after treatment with DRG-Exo-KD and HECTD1 knockdown detected by cell scratch assay. Values are expressed as mean ± SD and analyzed using unpaired t-test or one-way/two-way ANOVA. *p < 0.05, **p < 0.01 vs the sham, miR-NCKD, NC-Bio, control, sh-NC, or DRG-Exo-NC group; ##p < 0.01 vs the SNL + PBS, LPS + PBS, or DRG-Exo + sh-NC group; @@p < 0.01 vs the DRG-Exo-NC group by unpaired t-test (d), one-way (b, e, g, i, j) or two-way ANOVA (c, h). Cell experiments were independently repeated three times

Fig 4: DRG-Exo promote neuroinflammation in SNL-induced mice by activating microglial activation. a Expression of iba1, a marker of microglial activation in the spinal cord of mice by immunofluorescence. b Expression of IL-6 and IL-1β in the spinal cord of mice by RT-qPCR. c The uptake of DRG-Exo by BV2 cells. d Activation marker iba1 and pro-inflammatory marker iNOS expression in LPS and DRG-Exo-treated BV2 cells. e The levels of IL-6 and IL-1β in LPS and DRG-Exo-treated BV2 cells using ELISA. f The migration capacity of BV2 cells after LPS and DRG-Exo treatment was measured using cell scratch assay. Values are expressed as mean ± SD. *p < 0.05, **p < 0.01 vs the control group and ##p < 0.01 vs the SNL + PBS or LPS + PBS group by one-way (a, e, f) or two-way ANOVA (b, d). The cell experiment was independently repeated three times

Fig 5: Binge alcohol exposure caused more intestinal disintegration and increased serum FITC-D4 and endotoxin (LPS) levels in Aldh2-KO mice compared to WT mice. (A) Representative H&E-stained histology images showed more ruptured intestinal villi structures in alcohol-exposed Aldh2-KO mice than corresponding WT mice, (B) despite the similar levels of serum EtOH concentrations between both mouse strains 1 h after alcohol exposure. (C–F) Binge alcohol exposure significantly increased the levels of (C) serum FITC-D4, (D) endotoxin (LPS), (E) IL-6, and (F) TNF-α in Aldh2-KO mice compared to the WT counterparts (samples from n = 3~5/group). Data were analyzed by two-way ANOVA, where ** p < 0.01, *** p < 0.001, and **** p < 0.0001.