Wnt3a/YTHDF1 Regulated Oxaliplatin-Induced Neuropathic Pain Via TNF-α/IL-18 Expression in the Spinal Cord

Abstract

Oxaliplatin is widely used in cancer treatment, however, many patients will suffer from neuropathic pain (NP) induced by it at the same time. Therefore exploring the mechanism and founding novel target for this problem are needed. In this study, YTHDF1 showed upregulation in oxaliplatin treated mice. As m6A is known as conserved and it widely functions in numerous physiological and pathological processes. Therefore, we focused on exploring the molecular mechanism of whether and how YTHDF1 functions in NP induced by oxaliplatin. IHC and western blotting were conducted to measure proteins. Intrathecal injection for corresponding siRNAs in C57/BL6 mice or spinal microinjection for virus in YTHDF1^flox/flox mice were applied to specially knockdown the expression of molecular. Von Frey, acetone test and ethyl chloride (EC) test were applied to evaluate NP behavior. YTHDF1, Wnt3a, TNF-α and IL-18 were increased in oxaliplatin treated mice, restricted the molecular mentioned above respectively can significantly attenuate oxaliplatin-induced NP, including the mechanical allodynia and cold allodynia. Silencing YTHDF1 and inhibiting Wnt3a and Wnt signaling pathways can reduce the enhancement of TNF-α and IL-18, and the decreasing of the upregulation of YTHDF1 can be found when inhibiting Wnt3a and Wnts signaling pathways in oxaliplatin treated mice. Our study indicated a novel pathway that can contribute to oxaliplatin-induced NP, the Wnt3a/YTHDF1 to cytokine pathway, which upregulating YTHDF1 functioned as the downstream of Wnt3a signal and promoted the translation of TNF-α and IL-18 in oxaliplatin treated mice.

Keywords: IL18; Neuropathic pain; TNF alpha; Wnt; YTHDF1.

Fig. 1

Arrangement for medicine treatment. a Arrangement for oxaliplatin administration. b Arrangement for Wnt signaling pathway inhibitors (IWP-2 and IWR-1-endo) treatment. c Arrangement for siRNA administration. d. Arrangement for virus microinjection

Figure2

a–c Behavioral tests for oxaliplatin treated mice. [Von Frey test (a), F_{12, 144} = 5.401, P < 0.001; ***P < 0.001 vs. the corresponding days vehicle group, two-ways repeated measured ANOVA, n = 10; Acetone test (b), F_{12, 144} = 7.737, P < 0.001; ***P < 0.001 vs. the corresponding days vehicle group, two-ways repeated measured ANOVA, n = 10; Ethyl chloride (EC) test (c); F_{12, 144} = 4.208, P < 0.001; *P < 0.05, ***P < 0.001 vs. the corresponding days vehicle group, two-ways repeated measured ANOVA, n = 10]. d Immunofluorescent image showed that YTHDF1 was mainly co-stain with Neun. Scale bar, 100 μm; (n = 3). The data are shown as the mean ± sem

Fig. 3

The role of YTHDF1 in oxaliplatin-induced NP. a The expression of YTHDF1 in oxaliplatin-treated mice (*P < 0.05, **P < 0.01, ***P < 0.001 vs. the control group, one-way ANOVA, n = 6). b Immunofluorescent image testified that the protein of YTHDF1 on day 7 in oxaliplatin-treated rodents were obviously higher than the vehicle group. Scale bar, 100 μm; (n = 3). c YTHDF1 expression was inhibited by YTHDF1 siRNA in naïve mice or and AAV-CMV-Cre-EGFP in YTHDF1^flox/flox mice (**P < 0.01 vs. the siRNA NC group, **P < 0.01 vs. the AAV group, one-way ANOVA, n = 6). d–f Behavioral tests for YTHDF1 siRNA injection in oxaliplatin treated mice [von Frey (d), F_{20, 216} = 6.304, P < 0.001; *P < 0.05, ***P < 0.001 vs. corresponding days scramble group, two-ways repeated measured ANOVA, n = 10; Acetone test (e), F_{20, 216} = 12.447, P < 0.001; **P < 0.01, ***P < 0.001 vs. corresponding days scramble group, two-ways repeated measured ANOVA, n = 10; EC test (f), F_{20, 216} = 12.447, P < 0.001; **P < 0.01, ***P < 0.001 vs. corresponding days scramble group, two-ways repeated measured ANOVA, n = 10]. g–i Behavioral tests for AAV-CMV-Cre-EGFP injection in oxaliplatin treated YTHDF1^flox/flox mice [von Frey (g), F_{20, 216} = 5.648, P < 0.001; **P < 0.01 vs. the corresponding days scramble group, two-ways repeated measured ANOVA, n = 10; Acetone test (h), F_{20, 216} = 13.334, P < 0.001; **P < 0.01 vs. the corresponding days scramble group, two-ways repeated measured ANOVA, n = 10; EC test (i), F_{20, 216} = 3.553, P < 0.001; **P < 0.01 vs. the corresponding days scramble group, two-ways repeated measured ANOVA, n = 10]. The data are shown as the mean ± sem

Fig. 4

The role of Wnt3a in oxaliplatin-induced NP. a The level of Wnt3a in oxaliplatin-treated rodents (*P < 0.05, **P < 0.01, vs. the control group, one-way ANOVA, n = 6). b Immunofluorescent image testified that the expression of Wnt3a on day 7 in oxaliplatin-treated mice were significantly higher than the vehicle group. Scale bar, 100 μm; (n = 3). c Western blotting assay showed both IWP-2 (20 μM) and IWR-1-endo (5 μM) treatment can significantly inhibited the expression of Wnt3a (**P < 0.01 vs. the DMSO group, one-way ANOVA, n = 6). d–f Behavioral tests for IWP-2 injection in oxaliplatin treated mice [von Frey (d), F_{8, 108} = 2.357, P = 0.022; ***P < 0.001 vs. the corresponding days DMSO group, two-ways repeated measured ANOVA, n = 10; Acetone test (e), F_{8, 108} = 3.792, P < 0.01; ***P < 0.001 vs. the corresponding days DMSO group, two-ways repeated measured ANOVA, n = 10; EC test (f), F_{8, 108} = 2.248, P = 0.029; ***P < 0.001 vs. the corresponding days DMSO group, two-ways repeated measured ANOVA, n = 10. g–i Behavioral tests for IWR-1-endo injection in oxaliplatin treated mice [von Frey (g), F_{8, 108} = 3.738, P < 0.01; ***P < 0.001 vs. the corresponding days DMSO group, two-ways repeated measured ANOVA, n = 10; Acetone test (h), F_{8, 108} = 5.208, P < 0.001; *P < 0.05, ***P < 0.001 vs. the corresponding days DMSO group, two-ways repeated measured ANOVA, n = 10; EC test (i), F_{8, 108} = 4.812, P < 0.001; **P < 0.01, ***P < 0.001 vs. the corresponding days DMSO group, two-ways repeated measured ANOVA, n = 10; The data are shown as the mean ± sem

Fig. 5

Wnt3a/YTHDF1 pathway functioned in oxaliplatin-induced NP by regulating TNF-α and IL-18. a The expression of TNF-α in oxaliplatin-treated mice (**P < 0.01, ***P < 0.001 vs. the control group, one-way ANOVA, n = 6). b Immunofluorescent image testified that the protein levels of TNF-α on day 7 in oxaliplatin-treated mice were significantly higher than the vehicle group. Scale bar, 100 μm; (n = 3). c The IL-18 in oxaliplatin-treated mice(***P < 0.001 vs. the control group, one-way ANOVA, n = 6). d Immunofluorescent image testified that the protein levels of IL-18 on day 7 in oxaliplatin-treated mice were significantly higher than the vehicle group. Scale bar, 100 μm; (n = 3). e Western blotting assay showed IWP-2 (20 μM) and IWR-1-endo (5 μM) treatment can significantly inhibited the expression of YTHDF1(**P < 0.01, ***P < 0.001 vs. the Oxa + DMSO group, one-way ANOVA, n = 6). f Western blotting assay showed IWP-2 (20 μM) and IWR-1-endo (5 μM) treatment can significantly inhibited the expression of TNF-α (**P < 0.01 vs. the Oxa + DMSO group, one-way ANOVA, n = 6). g Western blotting assay showed IWP-2 (20 μM) and IWR-1-endo (5 μM) treatment can significantly inhibited the expression of IL-18 (**P < 0.01 vs. the Oxa + DMSO group, one-way ANOVA, n = 6). h The protein level of TNF-α was downregulated after AAV-CMV-Cre-EGFP treatment (*P < 0.05 vs. the Oxa group, **P < 0.01 vs. the Oxa + AAV-CMV-EGFP group, one-way ANOVA, n = 6). i The protein level of TNF-α was downregulated after YTHDF1 siRNA treatment (**P < 0.01 vs. the Oxa group, *P < 0.05 vs. the Oxa + siRNA NC group, one-way ANOVA, n = 6). j IL-18 was downregulated after AAV-CMV-Cre-EGFP treatment (**P < 0.01 vs. the Oxa group, *P < 0.05 vs. the Oxa + AAV-CMV-EGFP group, one-way ANOVA, n = 6). k The level of IL-18 was downregulated after YTHDF1 siRNA treatment (**P < 0.01 vs. the Oxa group, **P < 0.01 vs. the Oxa + siRNA NC group, one-way ANOVA, two-tailed, n = 6). The data are shown as the mean ± sem

Fig. 6

Behavioral tests for TNF-α siRNA and IL-18 siRNA injection in oxaliplatin treated mice. a von Frey (F_{8, 108} = 2.364, P < 0.05; #P < 0.05, ##P < 0.01, **P < 0.01, ***P < 0.001, vs. corresponding days siRNA NC group, two-ways repeated measured ANOVA, n = 10). b Acetone test (F_{8, 108} = 2.039, P < 0.05; *P < 0.05, #P < 0.05,**P < 0.01, ##P < 0.01 vs. corresponding days siRNA NC group, two-ways repeated measured ANOVA, n = 10). c EC test (F_{8, 108} = 2.051, P < 0.05; *P < 0.05, #P < 0.05, ##P < 0.01, ***P < 0.001 vs. corresponding days siRNA NC group, two-ways repeated measured ANOVA, n = 10)