Red Nucleus Interleukin-6 Evokes Tactile Allodynia in Male Rats Through Modulating Spinal Pro-inflammatory and Anti-inflammatory Cytokines

Abstract

Our previous studies have clarified that red nucleus (RN) interleukin (IL)-6 is involved in the maintenance of neuropathic pain and produces a facilitatory effect by activating JAK2/STAT3 and ERK pathways. In this study, we further explored the immune molecular mechanisms of rubral IL-6-mediated descending facilitation at the spinal cord level. IL-6-evoked tactile allodynia was established by injecting recombinant IL-6 into the unilateral RN of naive male rats. Following intrarubral administration of IL-6, obvious tactile allodynia was evoked in the contralateral hindpaw of rats. Meanwhile, the expressions of pro-inflammatory cytokines tumor necrosis factor-α (TNF-α), IL-1β, and IL-6 were elevated in the contralateral spinal dorsal horn (L4-L6), blocking spinal TNF-α, IL-1β, or IL-6 with neutralizing antibodies relieved IL-6-evoked tactile allodynia. Conversely, the levels of anti-inflammatory cytokines transforming growth factor-β (TGF-β) and IL-10 were reduced in the contralateral spinal dorsal horn (L4-L6), an intrathecal supplement of exogenous TGF-β, or IL-10 attenuated IL-6-evoked tactile allodynia. Further studies demonstrated that intrarubral pretreatment with JAK2/STAT3 inhibitor AG490 suppressed the elevations of spinal TNF-α, IL-1β, and IL-6 and promoted the expressions of TGF-β and IL-10 in IL-6-evoked tactile allodynia rats. However, intrarubral pretreatment with ERK inhibitor PD98059 only restrained the increase in spinal TNF-α and enhanced the expression of spinal IL-10. These findings imply that rubral IL-6 plays descending facilitation and produces algesic effect through upregulating the expressions of spinal pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 and downregulating the expressions of spinal anti-inflammatory cytokines TGF-β and IL-10 by activating JAK2/STAT3 and/or ERK pathways, which provides potential therapeutic targets for the treatment of pathological pain.

Keywords: allodynia; interleukin-10; interleukin-1β; interleukin-6; red nucleus; spinal cord; transforming growth factor-β; tumor necrosis factor-α.

FIGURE 1

Red nucleus IL-6-evoked tactile allodynia. (A) Experimental schemes for studying the expressions of spinal pro-inflammatory and anti-inflammatory cytokines in rubral IL-6-evoked tactile allodynia rats. (B) Intrarubral injection of 20 ng IL-6 evoked significant tactile allodynia in the contralateral hindpaw of naive rats compared with normal saline (n = 7 rats/group). (C) Footprint analysis indicated that intrarubral injection of 20 ng IL-6 did not affect the locomotion of naive rats (n = 6 rats/group). (D) The injection site was verified to be within the RN by 2% direct blue 1. **P < 0.01 and ***P < 0.001. RN, red nucleus; Aq, aqueduct of midbrain.

FIGURE 2

Actions of spinal pro-inflammatory and anti-inflammatory cytokines in rubral IL-6-evoked tactile allodynia. (A) Experimental schemes for studying the actions of spinal pro-inflammatory and anti-inflammatory cytokines in rubral IL-6-evoked tactile allodynia. (B–D) Intrathecal pre-injection of 2.0 μg anti-TNF-α-Ab, 1.0 μg anti-IL-1β-Ab, or 1.0 μg anti-IL-6-Ab alleviated rubral IL-6-evoked tactile allodynia compared to the normal saline (n = 7 rats/group). (E,F) Intrathecal pre-injection of 10 ng TGF-β or 300 ng IL-10 lessened rubral IL-6-evoked tactile allodynia compared with normal saline control (n = 7 rats/group). (G) Intrathecal injection of anti-TNF-α-Ab, anti-IL-1β-Ab, anti-IL-6-Ab, recombinant TGF-β, or IL-10 did not alter the locomotion of naive rats (n = 6 rats/group). *P < 0.05, **P < 0.01 and ***P < 0.001.

FIGURE 3

Increased expressions of spinal TNF-α, IL-1β, and IL-6 in rubral IL-6-evoked tactile allodynia rats. (A) qRT-PCR showed that intrarubral injection of IL-6 significantly enhanced the mRNA levels of contralateral (but not ipsilateral) spinal TNF-α (n = 6 rats/group), IL-1β (n = 6–8 rats/group), and IL-6 (n = 6 rats/group) compared with normal saline control or IL-6 pre-neutralized with anti-IL-6-Ab control. (B,C) Western blotting showed that intrarubral injection of IL-6 significantly increased the protein expressions of contralateral (but not ipsilateral) spinal TNF-α, IL-1β, and IL-6 compared with normal saline or IL-6 pre-neutralized with anti-IL-6-Ab control (n = 6 rats/group). (D,E) Immunohistochemistry demonstrated that intrarubral injection of IL-6 increased the expressions of spinal TNF-α (n = 4–5 rats/group), IL-1β (n = 4 rats/group), and IL-6 (n = 4 rats/group) in the contralateral spinal dorsal horn (n = 4 rats/group). (F) Schematic diagram of the Rexed’s laminae of the spinal dorsal horn. *P < 0.05, **P < 0.01, and ***P < 0.001. Contra, contralateral to intrarubral injection of IL-6; Ipsi, ipsilateral to intrarubral injection of IL-6. Scale bars = 100 μm.

FIGURE 4

Decreased expressions of spinal TGF-β and IL-10 in rubral IL-6-evoked tactile allodynia rats. (A) qRT-PCR showed that intrarubral injection of IL-6 decreased the mRNA levels of contralateral (but not ipsilateral) spinal TGF-β (n = 6–8 rats/group) and IL-10 (n = 6–8 rats/group) compared with normal saline, although they did not show statistical significance. Administration of IL-6 pre-neutralized with anti-IL-6-Ab even significantly promoted the mRNA expression of spinal TGF-β. (B,C) Western blotting showed that intrarubral injection of IL-6 significantly decreased the protein expressions of contralateral (but not ipsilateral) spinal TGF-β and IL-10 compared with normal saline or IL-6 pre-neutralized with anti-IL-6-Ab control (n = 6 rats/group). (D,E) Immunohistochemistry exhibited that intrarubral injection of IL-6 decreased the expressions of spinal TGF-β (n = 4 rats/group) and IL-10 (n = 4 rats/group) in the contralateral spinal dorsal horn. *P < 0.05, **P < 0.01, and ***P < 0.001. Contra, contralateral to intrarubral injection of IL-6; Ipsi, ipsilateral to intrarubral injection of IL-6. Scale bars = 100 μm.

FIGURE 5

Red nucleus IL-6 induced the contralateral spinal TNF-α, IL-1β, and IL-6 by activating JAK2/STAT3 and/or ERK pathways. (A) qRT-PCR displayed that intrarubral pre-injection of JAK2/STAT3 inhibitor AG490 significantly downregulated the mRNA expressions of contralateral spinal TNF-α (n = 6–8 rats/group) and IL-1β (n = 6–8 rats/group), but ERK inhibitor PD98059 only notably lowered the mRNA of spinal TNF-α compared with DMSO control. (B) Western blotting showed that intrarubral pre-injection of JAK2/STAT3 inhibitor AG490 significantly downregulated the protein levels of contralateral spinal TNF-α, IL-1β, and IL-6, ERK inhibitor PD98059 only significantly reduced the protein levels of spinal TNF-α compared with DMSO control (n = 6 rats/group). (C,D) Immunohistochemistry demonstrated that intrarubral pre-injection of JAK2/STAT3 inhibitor AG490 downregulated the protein levels of spinal TNF-α (n = 4–5 rats/group), IL-1β (n = 4 rats/group), and IL-6 (n = 4 rats/group), and ERK inhibitor PD98059 reduced the protein levels of spinal TNF-α in the contralateral spinal dorsal horn. *P < 0.05. Contra, contralateral to intrarubral injection of IL-6. Scale bars = 100 μm.

FIGURE 6

Red nucleus IL-6 inhibited the contralateral spinal TGF-β and IL-10 by activating JAK2/STAT3 and/or ERK pathways. (A) qRT-PCR showed that intrarubral pre-injection of JAK2/STAT3 inhibitor AG490 significantly promoted the mRNA expression of contralateral spinal TGF-β (n = 6–8 rats/group), while ERK inhibitor PD98059 significantly enhanced the mRNA expression of IL-10 (n = 6–8 rats/group) compared with DMSO control. (B) Western blotting showed that intrarubral pre-injection of JAK2/STAT3 inhibitor AG490 promoted the protein expressions of the contralateral spinal TGF-β and IL-10, but ERK inhibitor PD98059 only enhanced the protein expression of IL-10 compared with DMSO control (n = 6 rats/group). (C,D) Immunohistochemistry exhibited that intrarubral pre-injection of JAK2/STAT3 inhibitor AG490 promoted the protein expressions of spinal TGF-β (n = 4 rats/group) and IL-10 (n = 4 rats/group), and ERK inhibitor PD98059 enhanced the protein expression of IL-10 in the contralateral spinal dorsal horn. *P < 0.05 and **P < 0.01. Contra, contralateral to intrarubral injection of IL-6. Scale bars = 100 μm.