Granulocyte Colony Stimulating Factor (GCSF) Can Attenuate Neuropathic Pain by Suppressing Monocyte Chemoattractant Protein-1 (MCP-1) Expression, through Upregulating the Early MicroRNA-122 Expression in the Dorsal Root Ganglia

Abstract

Our previous animal studies and several human clinical trials have shown that granulocyte-colony stimulating factor (GCSF) can attenuate neuropathic pain through various mechanisms. GCSF itself is also a multipotent cytokine that can modulate microribonucleic acid (microRNA) expression profiles in vitro. In this study, we used the NanoString nCounter analysis system to screen the expression of different rodent microRNAs at early stage after nerve injury and studied the expression of related cytokines/chemokines in the dorsal root ganglia (DRGs) of rats that underwent chronic constriction injury (CCI) to explore the underlying mechanisms of the analgesic effects of GCSF. We found that microRNA-122 expression was downregulated by CCI; in contrast, GCSF treatment significantly upregulated microRNA-122 expression in the DRGs of CCI rats on the 1st day after nerve injury. We further studied the expression of different cytokines/chemokines (IL-1β, IL-6, and monocyte chemoattractant protein-1 (MCP-1)) that were modulated by microRNA-122. MCP-1 has been reported to participate in neuropathic pain development, and its expression on the DRGs of vehicle-treated CCI rats was significantly higher than that on the DRGs of sham-operated rats; in contrast, GCSF-treated rats exhibited significantly lower MCP-1 expression in the DRG than vehicle-treated rats on the 7th day after nerve injury. An early GCSF treatment can suppress MCP-1 expressions, through upregulating microRNA-122 expressions in the DRGs of CCI rats at an earlier stage, thus indirectly attenuating neuropathic pain development.

Keywords: chronic constriction injury; dorsal root ganglia; granulocyte colony stimulating factor; microRNA-122; monocyte chemoattractant protein-1; neuropathic pain; rats.

Figure 1

The time sequence of the analgesic effects of granulocyte colony stimulating factor (GCSF) on neuropathic pain. Compared to vehicle-treated chronic constriction injury (CCI) rats, the sequential effects of GCSF on GCSF-treated CCI rats increased opioid containing polymorphonuclear cells (PMNs) at injury site from 12 to 48 h and increased mu opioid receptor (MOR) levels on days 1–3 at the nerve ligature site after nerve injury; decreased IL-6 and TNF-α levels on days 2–6, and days 3–6, respectively, in the dorsal root ganglia (DRGs) after nerve injury; decreased IL-6 but increased IL-4 levels from days 1–7 in the spinal dorsal horns (SDHs) after nerve injury; and suppressed microglia and p-p38 activation on days 3–6 and on day 3, respectively, in the spinal dorsal horn after nerve injury [1,2]. (↑: increase, ↓: decrease).

Figure 2

Early GCSF treatment alleviated mechanical allodynia in CCI rats on the 1st and 7th days after nerve injury. The paw withdrawal thresholds of the vehicle-treated CCI rats were significantly lower than those of the sham-operated controls on the 1st and 7th days after nerve injury, as determined by von Frey filaments (on the 1st and 7th days after nerve injury). In contrast, the GCSF-treated CCI rats exhibited significantly attenuated mechanical allodynia compared to that of the vehicle-treated CCI rats on the 1st and 7th days after nerve injury (two-way ANOVA, post hoc Tukey’s test; n = 9 in each group; ## p < 0.01: vehicle-treated rats compared to sham-operated controls; ** p < 0.01: GCSF-treated CCI rats compared to vehicle-treated CCI rats).

Figure 3

GCSF treatment upregulated the levels of microRNA-122 in the DRGs of CCI rats on the 1st day after nerve injury. MicroRNA-122 levels in the DRGs of the vehicle-treated CCI rats were significantly decreased compared to those in the DRGs of the sham-operated controls on the 1st day after nerve injury. In contrast, the microRNA levels in the DRGs of the GCSF-treated CCI rats were significantly higher than those in DRGs of the vehicle-treated CCI rats on the 1st day after nerve injury (A). There was no significant difference in microRNA-122 levels between sham-operated, vehicle-treated CCI, and GCSF-treated CCI rats on the 7th day after nerve injury (B). The other 419 screened microRNAs did not show a similar trend. The data are shown as the means ± SEMs (Day 1: n = 3 in each group; Day 7: n = 2 in each group; # p < 0.05: vehicle-treated rats compared to sham-operated rats; * p < 0.05: GCSF-treated CCI rats compared to vehicle-treated CCI rats, unpaired t-test between each group by the default setting of NanoString nSolver software 3.0).

Figure 4

GCSF treatment decreased MCP-1 expression in the DRGs of CCI rats on the 7th day after nerve injury. The vehicle-treated CCI rats exhibited significantly higher MCP-1 levels in the DRGs than those exhibited by the sham-operated rats on the 1st and 7th days after nerve injury (A,B). In contrast, the GCSF-treated rats exhibited significantly lower MCP-1 levels in the DRGs than the vehicle-treated rats on the 7th day after nerve injury (B). The data are shown as the means ± SEMs (one-way ANOVA, post hoc Tukey’s test or Kruskal–Wallis, post hoc Mann–Whitney rank-sum test, if appropriate; Day 1 and 7: n = 4 per group; ## p < 0.01: vehicle-treated rats compared to sham-operated controls; * p < 0.05: GCSF-treated CCI rats compared to vehicle-treated CCI rats).

Figure 5

Proinflammatory cytokine IL-6 expression in the DRGs of sham-operated rats, vehicle-treated CCI rats, and GCSF-treated CCI rats. The vehicle-treated CCI rats exhibited significantly higher IL-6 levels in the DRGs than the sham-operated rats on the 7th day after nerve injury (B). The GCSF-treated CCI rats exhibited lower IL-6 levels in the DRGs than the vehicle-treated CCI rats on the 1st and 7th days after nerve injury (A,B). However, the difference between the groups did not reach statistical significance (one-way ANOVA, post hoc Tukey’s test or Kruskal–Wallis, post hoc Mann–Whitney rank-sum test, if appropriate; Day 1 and 7: n = 8 per group; # p < 0.05: vehicle-treated rats compared to sham-operated controls).

Figure 6

Pro-inflammatory cytokine IL-1β expression in the DRGs of sham-operated rats, vehicle-treated CCI rats, and GCSF-treated CCI rats. There was a tendency for GCSF-treated CCI rats to exhibit lower IL-1β levels in the DRGs than vehicle-treated CCI rats on the 1st and 7th days after nerve injury (A,B). There was also a tendency for vehicle-treated CCI rats to exhibit higher IL-1β levels in the DRGs than sham-operated rats on the 7th day after nerve injury (B). However, the differences did not reach statistical significance (one-way ANOVA, post hoc Tukey’s test or Kruskal–Wallis, post hoc Mann–Whitney rank-sum test, if appropriate; Day 1 and 7: n = 4 per group).

Figure 7

GCSF treatment decreased the number of MCP-1 + CGRP-positive neurons in the DRGs of CCI rats (B,E). Immunohistochemical studies revealed that many MCP-1-positive neurons (red) in the DRGs were co-stained with IB4 (green (A), a marker of small-diameter, C-fiber nonpeptidergic sensory neurons), CGRP (green (B), a marker of small- to medium-diameter peptidergic sensory neurons), and NF200 (green (C), a marker of large-diameter myelinated sensory neurons) (Figure 7A–C). Significantly fewer MCP-1 + CGRP-positive neurons were observed in the DRGs of the GCSF-treated CCI rats than in the DRGs of the vehicle-treated CCI rats on the 7th day after nerve injury (E). There was a tendency for the GCSF-treated CCI rats to exhibit fewer MCP-1+ IB4-positive neurons in the DRGs than the vehicle-treated CCI rats and sham-operated rats. However, the differences were not statistically significant (D). In contrast, there were no significant differences in the number of MCP-1 + NF200-positive neurons in the DRGs between the different groups (F) (one-way ANOVA, post hoc Tukey’s test or Kruskal–Wallis, post hoc Mann–Whitney rank-sum test, if appropriate; Day 7: n = 4 per group). Scale bars = 50 μm. The arrows indicate MCP-1-positive/IB4-positive, MCP-1-positive/CGRP-positive, and MCP-1-positive/NF200-positive neurons. * p < 0.05.

Figure 7

GCSF treatment decreased the number of MCP-1 + CGRP-positive neurons in the DRGs of CCI rats (B,E). Immunohistochemical studies revealed that many MCP-1-positive neurons (red) in the DRGs were co-stained with IB4 (green (A), a marker of small-diameter, C-fiber nonpeptidergic sensory neurons), CGRP (green (B), a marker of small- to medium-diameter peptidergic sensory neurons), and NF200 (green (C), a marker of large-diameter myelinated sensory neurons) (Figure 7A–C). Significantly fewer MCP-1 + CGRP-positive neurons were observed in the DRGs of the GCSF-treated CCI rats than in the DRGs of the vehicle-treated CCI rats on the 7th day after nerve injury (E). There was a tendency for the GCSF-treated CCI rats to exhibit fewer MCP-1+ IB4-positive neurons in the DRGs than the vehicle-treated CCI rats and sham-operated rats. However, the differences were not statistically significant (D). In contrast, there were no significant differences in the number of MCP-1 + NF200-positive neurons in the DRGs between the different groups (F) (one-way ANOVA, post hoc Tukey’s test or Kruskal–Wallis, post hoc Mann–Whitney rank-sum test, if appropriate; Day 7: n = 4 per group). Scale bars = 50 μm. The arrows indicate MCP-1-positive/IB4-positive, MCP-1-positive/CGRP-positive, and MCP-1-positive/NF200-positive neurons. * p < 0.05.

Figure 8

GCSF treatment downregulated MCP-1 expression in the DRGs of CCI rats, through upregulating microRNA-122 expression, which attenuated neuropathic pain. microRNA-122 expression was decreased in the DRGs of CCI rats; in contrast, GCSF treatment upregulated microRNA-122 expression in the DRGs in the early stage after nerve injury. GCSF itself directly, and the upregulation of microRNA-122 expression by GCSF treatment indirectly suppressed MCP-1 expression in the DRGs in the late stage after nerve injury, which further attenuated neuropathic pain. (CCI = chronic constriction injury, GCSF = granulocyte colony stimulating factor, MCP-1 = monocyte chemoattractant protein-1, ↑: increase, ↓: decrease.).