The present study demonstrated that i.t. treatment with LXA4, LXB4 or ATL could significantly alleviate the mechanical allodynia in CIBP. ATL showed the longest and most potent analgesic effect compared to LXA4 and LXB4. Further analysis showed that the increased expression of IL-1β and TNF-α in CIBP were significantly inhibited after i.t. injection with ATL. Immunohistochemistry revealed that ALX was mainly co-localized with the astrocytes and partly co-localized with the neurons but not with microglia. The present study suggests that ATL might exert its anti-neuroinflammation effects in CIBP through the ALX receptor expressed on astrocytes and/or some neurons at the spinal cord.
Many reports suggest that the ALX was identified and cloned in various cell types, including polymorphonuclear cells, monocytes, activated T cells, intestinal enterocytes and synovial fibroblasts [14, 22]. In our study, the analgesic effects observed after i.t. injection with LXA4, LXB4 and ATL indicated that this class of lipid mediators acts on specific targets in the spinal cord. Recently, evidence from two independent groups revealed the existence of ALX expression in rat primary astrocytes [24, 32] and microglia  at both the mRNA and protein levels. However, the present results showed that the ALX was mainly co-localized with astrocytes, sometimes co-localized with neurons, and did not co-localize with microglia, which is inconsistent with the previous reports. These discrepancies may be due to the different models and contexts (in vivo or in vitro). It has been demonstrated that non-neuronal cells may play an important role in the spinal facilitation of pain processing [34, 35], and LXs may act through ALX distributed on astrocytes and neurons to participate in the development and maintenance of chronic pain. This point needs to be elucidated in further investigations.
The significantly superior analgesic effect of ATL compared to the other drugs may have stemmed from the trihydroxytetraene structure of native lipoxins, which is sensitive to metabolic inactivation by dehydrogenation, but ATL is more resistant to metabolic inactivation than is the native LXs [21, 22]. It has been reported that i.v. treatment with LXA4, LXB4 or ATL significantly alleviated the heat hyperalgesia in a carrageenan-induced inflammatory pain model . The present study revealed similar effects of LXs and analogues on CIBP.
Furthermore, repeated i.t. injection of ATL had a therapeutic analgesic effect on neuropathic pain in a chronic compression of dorsal root ganglia (CCD) model . Since the multiple effects of LXs and analogues include anti-inflammatory and anti-cancer effects [36–38], the possible therapeutic effect of chronic systemic administration of LXs and analogues needs to be assessed soon.
Early reports demonstrated that LXs play an important role in pain processing by regulating communication between the immune and sensory nervous systems , which has been supported by research regarding the analgesic effects of LXs on inflammation pain and neuropathic pain [23, 24, 40]. It has been reported that LXA4 and ATL could interfere with the mitogen-activated protein kinase (MAPK) signaling pathway, inhibit the activation of NF-kappa B and AP-1, and consequently control the expression of pro-inflammatory cytokines [21, 22, 41]. Therefore, ATL may alleviate mechanical allodynia in CIBP by inhibiting the MAPK signaling pathway and NF-kappa B activation to inhibit the production of pro-inflammatory mediators. However, our current in vivo study revealed little effect of ATL on the spinal MPAK signaling pathways (data not shown). These warrant further study and invitro studies are underway by culturing spinal neurons and glial cells, respectively.
Interestingly, it has been reported that an LX analogue can elevate the mRNA of both suppressors of cytokine signaling–1 (SOCS-1) and SOCS-2, two of the endogenous inhibitors of cytokine-elicited signaling pathways, in the kidney in ischemic acute renal failure in mice . Our preliminary experiments showed that ATL-treated rats also displayed increased spinal mRNA levels for SOCS-1 (data not shown). Since the putative role for SOCSs as endogenous inhibitors of cytokine bioactivities transduced through JAK-STAT signal transduction pathways [43, 44], the finding of decreased mRNA levels for IL-1β, IL-6 and TNF-α in association with increased expression of SOCS-1 suggests a possible mechanism through which lipoxins could modulate cytokine bioactivity and, hence, attenuate spinal neuroinflammation conditions in rats with CIBP. The LXs may exert their analgesic effect through the ALX on astrocytes and neurons via its multipronged effects on the neuroinflammation milieu as well as neural activity in the spinal cord. However, our in vivo study revealed little effect of ATL on these signal pathways (data not shown). These warrant further study and in vitro studies are being conducted by culturing spinal neurons and glial cells, respectively.
Taken together, the results of the present study demonstrated for the first time that i.t. injection with LXs could strongly attenuate the mechanical allodynia in CIBP. The increased expression of pro-inflammatory mediators in CIBP was significantly attenuated by i.t.ATL. This study indicates that LXs and analogues could alleviate CIBP with sustained efficacy and these findings point to novel therapeutic targets for analgesia in CIBP.