Fig 2: rDKK3 promoted the switch of microglia from M1 type to M2 type in neuropathic pain rats via Kremen-1 and DVL-1. a–c Western blot and immunofluorescence results showed that rDKK3 ameliorated the up-regulated protein expression level of Iba-1 in the spinal cord and suppressed the activated microglia caused by neuropathic pain, while treatment with Kremen-1 siRNA or DVL-1 siRNA abolished the effect of rDKK3 on microglia activation (****p < 0.0001 compared with Sham group, ####p < 0.0001 compared with SNI + Vehicle group, &&&&p < 0.0001 compared with SNI + rDKK3 group, n = 6 in each group). d–f Western blot results indicated that treatment with rDKK3 attenuated the increased protein expression level of CD16, CD86 and iNOS in the spinal cord induced by neuropathic pain, while Kremen-1 siRNA or DVL-1 siRNA abolished the effect of rDKK3 on M1 type biomarkers (**p < 0.01, ***p < 0.001, ****p < 0.0001 compared with Sham group, ##p < 0.01, ###p < 0.001, ####p < 0.0001 compared with SNI + Vehicle group, &&p < 0.01, &&&p < 0.001, &&&&p < 0.0001 compared with SNI + rDKK3 group, n = 6 in each group). g–i Western blot results indicated that application with rDKK3 up-regulated the decreased protein expression level of Arg1, CD206 and IL-10 in the spinal cord caused by neuropathic pain, while administration with Kremen-1 siRNA or DVL-1 siRNA attenuated the effect of rDKK3 on M2 type biomarkers (**p < 0.01, ****p < 0.0001 compared with Sham group, #p < 0.05, ####p < 0.0001 compared with SNI + Vehicle group, &&p < 0.01, &&&&p < 0.0001 compared with SNI + rDKK3 group, n = 6 in each group)

Fig 3: LGK-974 regulates functional markers for macrophage via blocking Wnt/β-catenin signaling. 16HBE, A549 or H1299 cells were co-cultured with LGK-974 and/or Wnt3a/5aA for 24 h. Then, total RNA was isolated and the expression levels of functional markers for MR, IL-10 and Arg1 (A), and functional markers for M1-macrophage (TNF-α, IL-12 and iNOS) (B) were measured by performing reverse transcription-quantitative PCR. Arg1 protein level was detected by western blotting and IL-10 section in supernatant was measured by performing ELISA (A, right panel). TNF-α and IL-12 section in supernatant was measured by performing ELISA (B, right panel). *P<0.05 vs. mock group. MR, mannose receptor; iNOS, inducible nitric oxide synthase; Arg1, argininase-1.

Fig 4: rDKK3 promoted the switch of microglia from M1 type to M2 type in rats with neuropathic pain. a–c Western blot and immunofluorescence results showed that rDKK3 ameliorated the up-regulated protein expression level of Iba-1 in the spinal cord and suppressed the activated microglia caused by neuropathic pain (****p < 0.0001 compared with Sham + Vehicle group, ####p < 0.0001 compared with SNI + Vehicle group, n = 6 in each group). d–f Western blot results indicated that treatment with rDKK3 attenuated the increased protein expression level of CD16, CD86 and iNOS in the spinal cord induced by neuropathic pain (****p < 0.0001 compared with Sham + Vehicle group, ####p < 0.0001 compared with SNI + Vehicle group, n = 6 in each group). g–i Western blot result indicated that application with rDKK3 up-regulated the decreased protein expression level of Arg1, CD206 and IL-10 in the spinal cord caused by neuropathic pain (*p < 0.05, **p < 0.01, ***p < 0.001 compared with Sham + Vehicle group, #p < 0.05, ###p < 0.001, ####p < 0.0001 compared with SNI + Vehicle group, n = 6 in each group)

Fig 5: EST12 induces classical M1 activation relying on RACK1-JNK signaling pathway. (A-D) BMDMS isolated from WT, RACK1 KO C57BL/6 mice, respectively, were stimulated with EST12 protein (2 µM) for the indicated time, the expression of iNOS and Arg1 was determined by Western blot (A); NO in the supernatant was determined as described above (B); the secreted IL-6 (C) and TNF-a (D) levels were determined by ELISA. (E-H) RAW264.7 cells were pretreated with JNK inhibitor (10 µM), and then stimulated with EST12 protein (2 µM) for the indicated time, the expression of iNOS and Arg1 was determined by Western blot (E); NO in the supernatant was determined as described above (F); the expression of IL-6 (G) and TNF-a (H) were determined by ELISA. Two-way ANOVA with Tukey’s multiple comparison test was used for (B-D) and (F-H). The data are expressed as the mean ± SEM of three independent experiments. p > 0.05, not significant (ns), ***p < 0.001.