Neuronal CRMP2 phosphorylation inhibition by the flavonoid, naringenin, contributes to the reversal of spinal sensitization and arthritic pain improvement

Abstract

Background: Rheumatoid arthritis patients usually suffer from arthritic chronic pain. However, due to an incomplete understanding of the mechanisms underlying autoimmune disorders, the management of arthritic pain is unsatisfactory. Here, we investigated the analgesic effect and underlying mechanism of the natural flavonoid naringenin (NAR) in collagen-induced arthritis (CIA) pain.

Methods: NAR was injected (i.p.) once per day for 42 days after initial immunization, and rats were sacrificed on the 28th (the 21st day after final immunization, PID 21) and 42nd days (PID 35). The inflammatory factors, central sensitization indicators, and CRMP2 phosphorylation, as well as the anti-rheumatoid activity and analgesic effect of NAR, were further investigated.

Results: We found that NAR decreased the arthritis score and paw swelling, as well as the mechanical and thermal pain. The immunofluorescence results also showed a dose dependent effect of NAR on reducing the expressions of spinal cFos, IBA-1, and GFAP on the 28th (PID 21) and 42nd day (PID 35). NAR decreased the phosphorylation of CRMP2 S522 and the expression of the kinase CDK5 in the spinal dorsal horn, but pCRMP2 Y479 was unchanged. In addition, CRMP2 was co-localized with NEUN, but not IBA-1 or GFAP, indicating the involvement of neural CRMP2 phosphorylation in CIA-related pain. Finally, CRMP2 S522 phosphorylation selective inhibitor (S)-lacosamide also alleviated arthritic pain.

Conclusions: Taken together, our results demonstrate that NAR alleviates inflammation and chronic pain in CIA model, which might be related to its inhibition of neuronal CRMP2 S522 phosphorylation, potentially mitigating the central sensitization. Our study provide evidence for the potential use of NAR as non-opioid-dependent analgesia in arthritic pain.

Keywords: CRMP2; Central sensitization; Chronic pain; Naringenin; Rheumatoid arthritis.

Fig. 1

Collagen treatment successfully induced RA, and NAR treatment alleviated the degree of arthritic score and pain in CIA rats. A Experimental design and treatment strategies. B Arthritic score in CIA rats. C CIA rats toe volume. D Mechanical pain threshold in CIA rats. E Thermal withdrawal latency in CIA rats. *P < 0.05 vs. naive group, **P < 0.01 vs. naive group. ^#P < 0.05 vs. CIA model group, ^##P < 0.01 vs. CIA model group, by repeated-measures two-way ANOVA followed by post hoc Dunnett’s multiple comparisons test (Rats from PID0 to PID21 are n = 12/group. From PID28 to PID35 are n = 6/group). PID: days post the second immunization. In this and subsequent figures, the experimenters were blinded to the treatment condition(s)

Fig. 2

Naringenin decreased the expression of cFos in the spinal cord dorsal horn in CIA rats. A Representative micrographs of the expression of cFos in the spinal cord dorsal horn on days 28 (PID 21) and 42 (PID 35). B, C Expression of cFos in the spinal cord dorsal horn in CIA and NAR groups on the day 28 (PID 21) and day 42 (PID 35). *P < 0.05 vs. naive group, ** P < 0.01 vs. naive group, ^#P < 0.05 vs. CIA model group, by repeated-measures one-way ANOVA followed by post hoc Dunnett’s multiple comparisons test. The dotted white lines lineate the edge of the section. The second column is a higher magnification of the white box indicated in the first column images. All scale bars are 50 μm. The relative mean fluorescence intensity of cFos were quantified using the Image J software. The data are presented as means ± SEM (n = 5 rats per group)

Fig. 3

Naringenin decreased the expression of IBA-1 in the spinal cord dorsal horn in CIA rats. A The expression of IBA-1 in the spinal cord dorsal horn on days 28 (PID 21) and 42 (PID 35) were examined by immunofluorescence. B, C Expression of IBA-1 in the spinal cord dorsal horn in CIA and NAR groups on the day 28 (PID 21) and day 42 (PID 35). D, E Representative Western blots showing the expression of IBA-1 in the spinal cord dorsal horn in CIA and NAR groups on days 28 (PID 21) and 42 (PID 35). *P < 0.05, **P < 0.01 vs. naive group, ^#P < 0.05, ^##P < 0.01 vs. CIA model group, by repeated-measures one-way ANOVA followed by post hoc Dunnett’s multiple comparisons test. All scale bars are 50 μm. The relative mean fluorescence intensity of IBA-1 was quantified using the Image J software. The data are presented as means ± SEM (n = 5 rats per group)

Fig. 4

Naringenin decreased the expression of GFAP in the spinal cord dorsal horn in CIA rats. A The expression of GFAP in the spinal cord dorsal horn on the day 28 (PID 21) and day 42 (PID 35) were examined by immunofluorescence. B, C Representative Western blots showing expression of GFAP in the spinal cord dorsal horn in CIA and NAR groups on days 28 (PID 21) and 42 (PID 35). D, E Representative Western blots showing expression of GFAP in the spinal cord dorsal horn in CIA and NAR groups on the day 28 (PID 21) and day 42 (PID 35). *P < 0.05 vs. naive group, ** P < 0.01 vs. naive group, ^#P < 0.05 vs. CIA model group, by repeated-measures one-way ANOVA followed by post hoc Dunnett’s multiple comparisons test. All scale bars are 50 μm. The relative mean fluorescence intensity of GFAP were quantified using the Image J software. The data are presented as means ± SEM (n = 5 rats per group)

Fig. 5

Naringenin reduces phosphorylation of CRMP2 S522 in spinal cord dorsal horn of CIA rats on day 28 (PID 21) following the first day of initial immunization. A, B The expression levels of CRMP2 and pCRMP2 S522 in the spinal cord dorsal horn on day 28 (PID 21) were examined by immunofluorescence. Representative Western blots showing expression of CRMP2 (C), CDK5 (D), pCRMP2 S522 (E), and pCRMP2 Y479 (F) in the spinal cord dorsal horn in CIA and NAR groups on day 28 (PID 21). G Representative blots are shown. *P < 0.05 vs. naive group, **P < 0.01 vs. naive group, ^#P < 0.05 vs. CIA model group, ^##P < 0.01 vs. CIA model group, by repeated-measures one-way ANOVA followed by post hoc Dunnett’s multiple comparisons test. The data are presented as means ± SEM (n = 5 rats per group). All scale bars are 50 μm. The relative mean fluorescence intensity of pCRMP2 S522 were quantified using the Image J software. The data are presented as means ± SEM (n = 5 rats per group)

Fig. 6

Naringenin reduces phosphorylation of CRMP2 in spinal cord dorsal horn of CIA rats on day 42 (PID 35) following the first day of initial immunization. A, B The expression levels of CRMP2 and pCRMP2 S522 in the spinal cord dorsal horn on day 42 (PID 35) were examined by immunofluorescence. Representative Western blots showing expression of CRMP2 (C), CDK5 (D), pCRMP2 S522 (E), and pCRMP2 Y479 (F) in the spinal cord dorsal horn in CIA and NAR groups on day 42 (PID 35). G Representative blots are shown. *P < 0.05 vs. naive group, **P < 0.01 vs. naive group, ^#P < 0.05 vs. CIA model group, ^##P < 0.01 vs. CIA model group, by repeated-measures one-way ANOVA followed by post hoc Dunnett’s multiple comparisons test. The data are presented as means ± SEM (n = 5 rats per group). All scale bars are 50 μm. The relative mean fluorescence intensity of pCRMP2 S522 were quantified using the Image J software. The data are presented as means ± SEM (n = 5 rats per group)

Fig. 7

The CRMP2 S522 phosphorylation inhibitor (S)-LCM significantly reverses CIA-induced pain like behaviors. A Experimental design and treatment strategies. Paw withdrawal thresholds (B) and paw withdrawal latencies (C) of DBA1 mice were measured at the indicated days post initial day of immunization. (S)-LCM was given intraperitoneally at 20 mg/kg and behaviors were assessed 1 hour later. **P < 0.01 vs. naive group, ^##P < 0.01 vs. CIA model group, by repeated-measures two-way ANOVA followed by post hoc Dunnett’s multiple comparisons test. The data are presented as means ± SEM (n = 8 per group). PID: Days post the second immunization