Go-sha-jinki-Gan (GJG) ameliorates allodynia in chronic constriction injury-model mice via suppression of TNF-α expression in the spinal cord

Abstract

Background: Alternative medicine is noted for its clinical effect and minimal invasiveness in the treatment of neuropathic pain. Go-sha-jinki-Gan, a traditional Japanese herbal medicine, has been used for meralgia and numbness in elderly patients. However, the exact mechanism of GJG is unclear. This study aimed to investigate the molecular mechanism of the analgesic effect of GJG in a chronic constriction injury model.

Results: GJG significantly reduced allodynia and hyperalgesia from the early phase (von Frey test, p<0.0001; cold-plate test, p<0.0001; hot-plate test p¼0.011; two-way repeated measures ANOVA). Immunohistochemistry and Western blot analysis revealed that GJG decreased the expression of Iba1 and tumor necrosis factor-a in the spinal cord. Double staining immunohistochemistry showed that most of the tumor necrosis factor-a was co-expressed in Iba1-positive cells at day 3 post-operation. GJG decreased the phosphorylation of p38 in the ipsilateral dorsal horn. Moreover, intrathecal injection of tumor necrosis factor-a opposed the anti-allodynic effect of GJG in the cold-plate test.

Conclusions: Our data suggest that GJG ameliorates allodynia in chronic constriction injury model mice via suppression of tumor necrosis factor-a expression derived from activated microglia. GJG is a promising drug for the treatment of neuropathic pain induced by neuro-inflammation.

Figure 1.

3D-HPLC analysis of GJG.

Figure 2.

GJG administration attenuated CCI-induced allodynia and hyperalgesia as assessed by behavioral tests. (a) von Frey test (mechanical allodynia). (b) cold-plate test (cold allodynia). (c) hot-plate test (thermal hyperalgesia). Data are presented as means ± SD. Data were analyzed using two-way repeated measures ANOVA with Tukey's HSD post hoc analyses (***p < 0.0001 and *p < 0.05 indicate significant differences between the GJG-treated CCI group and the control CCI group, n = 5–8).

Figure 3.

GJG inhibited the expression of Iba1, a microglial activation marker, in the ipsilateral dorsal horn. (a) Immunohistochemical analysis of Iba1 from day 1 to day 7 post-operation. Confocal images were taken at 40 × magnification. Magenta fluorescence indicates Iba1-positive cells. Scale bar = 100 µm. (b) Results of Western blotting analysis of Iba1 from day 1 to day 7 post-operation. GAPDH is shown as a loading control. (c) Quantification of Western blotting analysis of Iba1 in Sham, GJG-treated Sham, CCI, and GJG-treated CCI groups on day 3 post-operation. The left blot was densitometrically analyzed, and the ratio of Iba1 immunoreactivity to GAPDH immunoreactivity was determined. Each data point represents the mean ± SD. n = 3. Data were analyzed using Dunnett’s test (*p < 0.05 indicates a significant difference between the indicated groups).

Figure 4.

GJG inhibited the expression of TNF-α in the ipsilateral spinal cord. (a) Western blotting analysis of TNF-α in the lumbar ipsilateral spinal cord from day 1 to day 7 post-operation. The left panel shows TNF-α. GAPDH is shown at right as a loading control. (b) Quantification of Western blotting analysis of TNF-α in Sham, GJG-treated Sham, CCI, and GJG-treated CCI groups on day 1 (top panels) and day 3 (bottom panels) post-operation. The left blots were densitometrically analyzed, and the ratio of TNF-α immunoreactivity to GAPDH immunoreactivity was determined. Each data point represents the mean ± SD. n = 3. Data were analyzed using Dunnett’s test (*p < 0.05 indicates a significant difference between the indicated groups). (c) Western blotting analysis of phospho-p38 and p38 MAPK expression in the ipsilateral spinal cord from day 1 to day 7 post-operation. (d) Quantification of Western blotting analysis of phospho-p38 in Sham, GJG-treated Sham, CCI, and GJG-treated CCI groups on day 3 post-operation. The left blots were densitometrically analyzed, and the ratio of phosho-p38 immunoreactivity to p38 immunoreactivity was determined. Each data point represents the mean ± SD. n = 3. Data were analyzed using Dunnett’s test (**p < 0.001 and *p < 0.05 indicates a significant difference between the indicated groups.).

Figure 5.

GJG inhibited the expression of TNF-α in Iba1-positive microglia in the ipsilateral dorsal horn. (a) Immunohistochemical images of Iba1 and TNF-α in the ipsilateral dorsal horn of the lumbar spinal cord on day 3 post-operation. Left panels show Iba1-positive cells (deep red). Middle panels show TNF-α-positive cells (green). Right panels showed merged images of Iba1 (deep red) and TNF-α (green). Merged images of double-stained cells indicated that TNF-α was localized in Iba1-positive cells (yellow). The white arrowheads indicate Iba1/TNF-α double-positive cells. Confocal images were taken at 40 × magnification. Scale bar = 100 µm. (b) Histogram of the mean percentage value of the number of Iba1/TNF-α-positive cells in the superficial laminae of the ipsilateral dorsal horn in each mouse group. The average numbers of Iba1-positive cells/10⁴µm² were 14.7 ± 7.2 in Sham, 27.4 ± 3.7 in CCI and 22.2 ± 4.5 in CCI + GJG. The average numbers of both Iba1-positive and TNF-α-positive cells/10⁴µm² were 3.9 ± 1.6 in Sham, 19.9 ± 4.2 in CCI and 9.7 ± 1.8 in CCI + GJG. Each data point represents the mean ± SD. Data were analyzed using one-way repeated measures ANOVA with Tukey's HSD post hoc analyses (***p < 0.0001 and **p < 0.01 indicates a significant difference between the Sham group versus the CCI group and between the CCI group versus the GJG-treated CCI group; n = 3).

Figure 6.

Intrathecal (i.t.) injection of TNF-α reversed the effects of GJG on cold allodynia. GJG was administered for two weeks to CCI model mice with i.t. TNF-α (10 pg) or with i.t. PBS. Pain behavior was assessed with the von Frey test (a) and the cold-plate test (b). The area under the effect-time curves (AUC) and significant differences between the AUC values for differently treated CCI mice in the cold-plate test were determined (c). Synergistic interaction between TNF-α and GJG in CCI mice in the cold-plate test (d). Data are presented as means ± SD. Data were analyzed using two-way repeated measures ANOVA or one-way repeated measures ANOVA (***p < 0.0001 and *p < 0.05 indicate a significant difference between the indicated groups. n = 4).