Fig 2: Overexpression of miR-183 inhibits inflammation in CCI rats via the downregulation of the TXNIP-NLRP3 axis. a NLRP3 protein expression in the spinal cord of CCI rats determined using Western blot analysis, normalized to GAPDH (n = 10). b Expression of NLRP3 in Iba-1-labeled microglial cells of CCI rat spinal cord detected by immunofluorescence staining (n = 5) (scale bar = 50 µm). c NLRP3 protein expression in microglial cells treated with exogenous miR-183 mimicdetermined using Western blot analysis, normalized to GAPDH (n = 3). d Binding relationship between TXNIP and NLRP3 detected by Co-IP assay. e Protein expression of NLRP3, ASC, and caspase-1 in microglial cells after forced expression miR-183 and TXNIP measured using Western blot analysis, normalized to GAPDH. f Expression of IL-1ß in microglial cells after forced expression miR-183 and TXNIP measured using ELISA (n = 10). g Expression of NLRP3, ASC, and caspase-1 in spinal cord of CCI rats injected with lentivirus expressing miR-183 agomir or in combination with oe-TXNIP measured using Western blot analysis, normalized to GAPDH. h Expression of IL-1ß in spinal cord of CCI rats injected with lentivirus expressing miR-183 agomir or in combination with oe-TXNIP measured using ELISA (n = 10). *relative to sham-operated rats, microglial cells treated with mimic NC and #relative to microglial cells treated with miR-183 mimic or CCI rats injected with miR-183 agomir indicate p < 0.05. All measurement data were expressed as mean ± standard deviation. Unpaired t test was used to analyze data between the two groups, one-way ANOVA and Tukey’s post hoc test to analyze data among multiple groups, and repeated measures ANOVA and Bonferroni post hoc test to analyze data at different time points

Fig 3: TXNIP regulates the protein expression of spinal NLRP3 inflammasome in SNL-induced neuropathic pain. a Co-IP of TXNIP and NLRP3. Rabbit IgG is used as control for co-IP assays. *p < 0.05, **p < 0.01 versus the corresponding sham group by two-tailed unpaired Student’s t test; n = 5 per group. b Increased NLRP3 inflammasome complex after pSNL, including NLRP3, ASC, P-Caspase1, C-Caspase1, and mature IL-1ß, were reversed by knockdown of TXNIP with siRNA. Inflammasome complex expression was determined on day 7 after pSNL or at 24 h after 3-day injections of TXNIP siRNA or Scr, beginning on day 7 after pSNL. One-way ANOVA (expression versus the treated groups) followed by post hoc Tukey test, F (3, 8) = 76.56 for TXNIP, 106.9 for NLRP3, 79.01 for ASC, 191.3 for P-Caspase1, 26.68 for C-Caspase1, and 33.34 for mature IL-1ß, **p < 0.01, ***p < 0.001 versus sham group. #p < 0.05, ##p < 0.01, and ###p < 0.001 versus pSNL+Scr group. c Increased NLRP3 inflammasome were reversed by intrathecal injection of Lenti-miR-23a in pSNL mice. Inflammasome complex expressions were measured at 7 days after pSNL surgery or at 48 h after 3-day injections of Lenti-miR-23a or Lenti-vector, beginning on day 7 after pSNL. One-way ANOVA (expression versus the treated groups) followed by post hoc Tukey test, F (3, 8) = 20.79 for NLRP3, 96.78 for ASC, 29.35 for P-Caspase1, 106.1 for C-Caspase1, and 42.21 for mature IL-1ß, **p < 0.01, ***p < 0.001 versus sham group. #p < 0.05, ##p < 0.01, and ###p < 0.001 versus pSNL+Lenti-miR-23a group. d LV-miR-23a-induced expression of NLRP3 inflammasome was reversed by knockdown of TXNIP with siRNA in naïve mice. Content of inflammasome was examined at 48 h after 3-day injections of LV-miR-23a or at 24 h after 3-day injections of TXNIP siRNA or Scr (starting after the injections LV-miR-23a or vector). One-way ANOVA (expression versus the treated groups) followed by post hoc Tukey test, F (3, 8) = 106.7 for NLRP3, 138.4 for ASC, 54.04 for P-Caspase1, 133 for C-Caspase1, and 50.59 for mature IL-1ß, **p < 0.01, ***p < 0.001 versus vector group. #p < 0.05, ##p < 0.01, and ###p < 0.001 versus LV-miR-23a + Scr group. e Increased NLRP3 inflammasome induced by injection of Lenti-CXCR4 was reversed by injection of TXNIP siRNA in naïve mice. Inflammasome complex expressions were measured at 48 h after 3-day injections of Lenti-CXCR4 or at 24 h after 3-day injection of TXNIP siRNA or Scr (starting after injections of Lenti-CXCR4 or Lenti-vector). One-way ANOVA (expression versus the treated groups) followed by post hoc Tukey test, F (3, 8) = 18.84 for NLRP3, 48.48 for ASC, 84.25 for P-Caspase1, 8.63 for C-Caspase1, and 48.37 for mature IL-1ß, **p < 0.01, ***p < 0.001 versus Lenti-vector group. #p < 0.05, ##p < 0.01, and ###p < 0.001 versus Lenti-CXCR4 + Scr group. f NLRP3 inflammasome upregulated by injection of LV-miR-23a was reversed by injection of CXCR4 siRNA in naïve mice. One-way ANOVA (expression versus the treated groups) followed by post hoc Tukey test, F (3, 8) = 42.41 for NLRP3, 72.1 for ASC, 10.9 for P-Caspase1, 82.57 for C-Caspase1, and 290.7 for mature IL-1ß, **p < 0.01, ***p < 0.001 versus vector group. #p < 0.05, ##p < 0.01 versus LV-miR-23a + Scr group. Expression of inflammasome was measured at 48 h after 3-day injections of LV-miR-23a or at 6 h after injection of CXCR4 siRNA or Scr (beginning after injections of LV-miR-23a or Vector). Data are presented as mean ± SEM; n = 3 per group

Fig 4: RU486 suppresses TXNIP transcription and improves astrocytic glucose uptake and glycolysis. (A) RU486 (1 µM) inhibited luciferase activity of A9433 cells obviously, n = 6 cell cultures/group; P CortxCort + RU486 < 0.0001. (B,C) RU486 (1 µM) reduced TXNIP mRNA [(B), n = 5 cell cultures/group; P CortxCort + RU486 = 0.0017)] and protein [(C), n = 6 cell cultures/group; P CortxCort + RU486 = 0.0007)] levels of primary astrocytes. (D–I) RU486 (1 µM) increased GLUT1 PM translocation [(D), n = 6 cell cultures/group; Total: P CortxCort + RU486 = 0.0007; PM: P CortxCort + RU486 = 0.0094; CYT: P CortxCort + RU486 = 0.0188)], 2-NBDG uptake [(E), scale bar = 100 µm; (F), n = 6 cell cultures/group; P CortxCort + RU486 = 0.0001)], glucose uptake [(G), n = 6 cell cultures/group; P CortxCort + RU486 = 0.0257)], glycolytic rate [(H), n = 6 cell cultures/group)] and lactate release [(I), n = 6 cell cultures/group; P CortxCort + RU486 = 0.0095)] of in vitro cultured primary astrocytes. All data were expressed as mean ± SEM, *p < 0.05, **p < 0. 01, ***p < 0.001 vs. Ctrl (control) group, # p < 0.05, ## p < 0.01, ### p < 0.001 vs. Cort (corticosterone) group (Normality of data was checked by Shapiro-Wilk test. Data with or without normal distribution were analyzed by one-way ANOVA or Kruskal-Wallis test followed by Dunnett’s post-hoc test for multiple comparisons).

Fig 5: The schematic of miR-23a targeting CXCR4 regulates neuropathic pain via TXNIP/NLRP3 inflammasome in spinal glial cells of mice. In pSNL-induced chronic neuropathic pain, spinal miR-23a expression was significantly reduced, which increased the expression of spinal CXCR4, and subsequently the expression of TXNIP and NLRP3 inflammasome including NLRP3, ASC, Caspase-1, and IL-1ß