Role Of Spinal Lipoxin In Neuropathic Pain And Electroacupuncture Analgesia In Rats

Neuropathic pain has been redefined as "pain arising as a direct consequence of a lesion or disease affecting the somatosensory system". Neuropathic pain induced by peripheral nerves injury is a prevalent, persistent, and debilitating problem. Chronic neuropathic pain can be caused by many diseases such as trigeminal neuralgia, postherpetic neuralgia, diabetic nephropathy., stroke, Parkinson’s disease, multiple sclerosis etc. In china, about 0.9 hundred million people have been suffered from neuropathic pain, according to the incidence with 8% of the globle people.Neuropathic pain causes suffering and disability for many patients, and is an important public health problem. It is significant to explore the pathogenesis and therapy of neuropathic pain in clinical and basic aspects.As we know, neuroinflammation in the spinal cord plays a critical role in the processing of various chronic pain. Neuropathic pain could be induced by the peripheral nerve injury. Inflammatory mediators released from damaged nerves and tissue are responsible for triggering ectopic activity in primary afferents and that, in turn, provokes increased spinal cord activity and the development of ’central sensitization’. The excitatory substances such as PGE2、ATP、NO and so on were significantly increased in the spinal cord of neuropathic pain.It had been shown that suppression of inflammation in spinal cord could inhibit the development of neuropathic pain and attenuate the pain response.It has been reported that Lipoxins,as mediators of key events in endogenous anti-inflammation and resolution, are eicosanoids derived from sequential lipoxygenase (LO) metabolism of arachidonic acid. LXA4 and LXB4are the main components of this series. Lipoxin has been served as the first family of endogenous "braking signals" in inflammation in vivo. ATL, an analogue of LXA4, shares many anti-inflammatory activities and the same receptor affinity as the native LXA4. They exert their anti-inflammation through binding to the specific G protein-coupled receptor known as the LXA4 receptor (FPR2/ALX) in inflammatory pain. So, the question of the spinal role of ATL in neuropathic pain has been our first part of our research.The JAK2-STAT3 pathway is one of the important signaling pathways downstream of cytokine receptors,which has been reported to play a variety of roles in cellular physiological function such as cell proliferation, differentiation and survival. The suppressor of cytokine signaling 3 (SOCS3) protein acts as feedback inhibitor of the JAK2/STAT3 pathway. SOCS1 and SOCS3 are the best-studied family members and have been shown to act as negative feedback inhibitors of the JAK2/STAT3 signaling pathway, involved in the signaling regulation of cytokines, growth factors and endocrines. STAT3, a member of the JAK2/STAT3 signaling family, is expressed in the CNS. It has been shown that rapid activation of JAK2/STAT3 signaling may play an important role in the induction and development of neuropathic pain in SNL rats.In addition, accumulating evidence showed that the activation of astrocytes depends on the phosphorylation state of JAk2-STAT3.So the other part of our research would be concerned in the role of JAk2-STAT3-SOCS pathway in regulating the spinal neuroinflammation of CCI rats and the effect of ATL.Electroacupuncture (EA), as a traditional clinical method, is widely accepted in neuropathic pain clinics, which shows the better analgesic and anti-inflammation effect in previous research. Previous reports indicated that arachidonic acid (AA) derivatives and relative synthetases such as PGs, COX-2, Leukotrienes play vital roles in pain status and analgesic effect of EA.5-lipoxygenase (5-LO) as the key enzyme in synthesis of LXA4 was involved in the development of neuropathic pain.Annexins are a well-known multigene family of Ca(2+)-regulated phospholipid-binding and membrane-binding proteins. Annexin-Al (ANXA1, lipocortin-1) is an anti-inflammatory protein whose expression is modulated by glucocorticoids, involved in inflammatory pain and displayed its anti-nociceptive effect through FPR2/ALX. Therefore, the third research would be focused on the role of spinal 5-LO, AnnexinAl and ALX in the effect of EA in the modle of CCI rats.According to the reported animal model of neuropathic pain, the presented study was aimed to:(1) the effect of intrathecal injection of ATL on mechanical allodynia and spinal inflammatory cytokine in neuropathic pain model on day 7 afterCCI surgery.(2) the effect of JAK2-pSTAT3 in the neuropathic pain with intrathecal injection of ATL.(3) the effect of Electroacupuncture (EA) on mechanical allodynia and thermal hyperalgesia and the analgesic mechanism in spinal cord. The results are as follows:Prat One The effect of ATL on the neuropathic pain of CCI ratsl.Chronic constriction injury rats induced mechanical allodynia and thermal hyperalgesiaSD male rats were divided into Normal group, Sham operation group and a model group subjected to chronic constriction injury of right sciatic nerve, n=8, respectively. Neuropathic pain was induced according to the procedure described by Bennett and Xie (1988). Briefly, rats were anaesthetized with 0.4ml/kg i.p.10%chloral hydrate. Under aseptic conditions, the right common sciatic nerve was exposed at the level of the middle thigh by blunt dissection. Proximal to the trifurcation, the nerve was carefully freed from the surrounding connective tissue and four chromic cat gut ligatures (4-0) were tied loosely around proximal to the sciatica’s trifurcation at 1 mm intervals. After hemostasis was confirmed, the incision was closed in layers and disinfected with penicillin.The animals were allowed to recover from surgery and then housed one per cage with free access to water and standard laboratory chow. Sham surgery was carried out by exposing the right sciatic nerve without ligation. The paw withdrawal thresholds and latency were detected from day2 to day 21 after CCI surgery.The results suggested that compared to the normal and sham group rats, there was significant decrease of paw withdrawal thresholds and latency in ipsilateral limb in model rats.2. The cellular localization and expression of ALX in the CCI rats.To identify what types of cells that expressed ALX in spinal cord, we used double immunofluorescence of ALX with frequently used cell-specific markers: NeuN(neurons), GFAP (astrocytes) and CD11b (microglia). In the spinal cord of Normal,Sham and CCI group of rats, ALX was co-expressed with astrocytes and neurons, almost not co-expressed with microglia. Preliminary immunohistochemistry experiments showed that in CCI rats, nerve injury resulted in more accumulation of GFAP-IR mainly in the superficial and medial laminae (I-V) compared with normal and sham rats. Western Blot and immunofluorescence of ALX showed that no change of ALX expression was found in the CCI rats.3. The effect of intrathecal and tail vain injection ATL on the mechanical pain in CCI ratsSingle intrathecal ATL 200ng could obviously enhance the mechanical response threshold in CCI-POD7 rats from 1h to 4h after injection, however there was no effect on sham rats with equal doses of ATL. We also observed the effect of different signle intrathecal doses of ATL with 50,100,200ng on CCI-POD7 rats, dose dependent change of mechanical response threshold was found and dose of 200ng ATL showed the best effect compared with other two doses. Given with LXA4 receptor antagonist BOC-2, the effect of ATL could be reversed from 1h to 2.5h after injection. In addition, we found that system administration with different doses of ATL could also alleviate the mechanical allodynia and the effect could last to 4h after tail vein injection of ATL.4.The effect of intrathecal injection ATL on the spinal inflammatory cytokine in CCI ratsTo observe the effect of ATL on the attenuation of neuroinflammation of neuropathic pain, we detected the mRNA levels of IL-1β, IL-6, TNF-a in the L4-L5 spinal cord of CCI rats. The mRNA levels of IL-1β, IL-6, TNF-a were significantly increased on day 7 after CCI surgery compared with the control group, and single intrathecal injection with ATL inhibited the rising tendency of inflammatory factors (IL-1β, IL-6, TNF-a) induced by the neuropathic pain after 1h and 4h administration.Summary:The model rats displayed significant mechanical allodynia and thermal hyperalgesia on ipsilateral side of hind limbs after CCI surgery induced neuropathic pain. Signle intrathecal injection ATL could inhibit the spinal neuroinflammation and reduce the mechanical allodynia in CCI rats throuth the LXA4 receptor ALX in spinal astrocyte.In addition, ATL could attenuate the mechanical pain via systematic administration.Part Two The role of JAK2-STAT3 on the anti-inflammation and analgesic effect of ATL in CCI rats1. The effect of ATL on the expression of p-STAT3 and SOCS1-3 in spinal cord in CCI ratsWestern Blot and immunofluorescence showed that p-STAT3 expression was significantly increased in the bilateral spinal dorsal horn.RT-PCR analysis showed that the mRNA expression of SOCS2,3 was significantly increased while the SOCS1 mRNA had no change in CCI rats. After single intrathecal doses of 200ng, ATL could significantly attenuate the increased levels of p-STAT3 induced by the neuropathic pain of CCI rat. (*p< 0.05 compared with CCI+NS group).Doses of 200ng ATL could significantly increase the levels of SOCS3. (**p< 0.01 compared with CCI+NS group).2.The role of JAK2-STAT3 on the neuropathic pain of CCI rats2.1 The effect of JAK2 inhibitor AG490 on the mechanical pain in CCI ratsAfter three intrathecal doses of the JAK2 inhibitor AG490(1,5ug), Significant mechanical threshold was increased in POD2, POD 4, POD 7 of CCI+AG490-5ug group.(**p< 0.01 compared with CCI +DMDO group,.#p< 0.05 ompared with CCI+ AG490-1ug group).2.2 The effect of AG490 on the spinal inflammatory cytokine in CCI ratsAfter three doses of 5ug intrathecal injection,AG490 could significantly attenuate the increased mRNA levels of inflammatory factors(IL-1, IL-6 and TNF-a) induced by the neuropathic pain of CCI rat. (**p< 0.01 compared with normal group, ##p< 0.01 compared with CCI+DMSO group).2.3 The effect of AG490 on the SOCSmRNA in spinal cord in CCI ratsRT-PCR analysis of SOCS1, SOCS2, SOCS3 in the L4-L5 spinal cord of the control group and the model group on day 7 after CCI surgery. After three doses of 5ug intrathecal injection,AG490 could significantly increase the mRNA levels of SOCS1,3 of CCI rat. (**p< 0.01 compared with normal group,#p< 0.05 and ##p< 0.01 compared with CCI+DMSO group).Summary:JAK2-STAT3-SOCS3 signaling pathway in spinal cord was involved in the induction and development of neuropathic pain. Single intrathecal injection ATL could inhibit the phosphorylation of STAT3 and spinal neuroinflammation, which could reduce the mechanical allodynia in CCI rats.Part Three The spinal mechanism of the analgesic and anti-inflammatory effect of EA in CCI rats.1. The spinal molecular and mechanical threshold of single EA in different time-course of CCI surgery.1.1 The effect of single EA on the mechanical allodynia and thermal hyperalgesia in different time-course of CCI surgery.We used von Frey hair and Radiant heat pain measurement instrument to detect the change of mechanical and thermal response threshold of the ipsilateral paw (Right paw) in the received CCI surgery after single electropuncture therapy.In POD3 of CCI rats, mechanical withdrawal thresholds and paw withdrawal latency were significantly increased after EA therapy from 30mins to 90mins, compared with CCI and CCI+Sham-EA group. In POD7 of CCI rats, mechanical withdrawal thresholds and paw withdrawal latency were significantly increased after EA therapy from 30mins to 60mins, compared with CCI and CCI+Sham-EA group. E-F. In POD21 of CCI rats, mechanical withdrawal thresholds and paw withdrawal latency were significantly increased after EA therapy from 45mins to 60mins, compared with CCI and CCI+Sham-EA group. (**p< 0.01,*p<0.05compared with CCI group).There was no effect on the mechanical withdrawal thresholds and paw withdrawal latency after single sham EAin CCI rats of POD3,7,21.1.2 The effect of single EA on the expression of 5-LO and Annexin Al in spinal cord in different time-course of CCI surgery.RT-PCR analysis of 5-LO and AnnexinAlwere conducted in the L4-L5 spinal cord of the normal group,CCI group and CCI+EA group on day 3,7,21 after CCI surgery.Compared with normal group, the mRNA levels of 5-LO was significantly decreased in POD21 of CCI rats (**P<0.01).Given single EA therapy, t he mRNA levels of 5-LO was significantly increased compared with CCI group (##P<0.01) Compared with normal group, the mRNA levels of AnnexinAl was significantly decreased in POD3,7,21 of CCI rats (**P<0.01).Given single EA therapy, the mRNA levels of 5-LO was significantly increased compared with CCI group in POD7,21 (**P<0.01,*P<0.05)2.The effect of ALX on the EA theray in the CCI rats2.1 The effect of ALX on the mechanical pain in the CCI rats with EA therayAfter electropuncture therapy and. intrathecal injection ALX antagonist BOC-2. In POD7 of CCI rats, mechanical withdrawal thresholds were significantly increased after single EA therapy from 30mins to 60 mins, compared with CCI and CCI+Sham-EA group(*P<0.05). After single intrathecal injection of BOC-2, mechanical withdrawal thresholds were significantly decreased compared with CCI+ EA group(*P<0.05).Given multiple EA therapy from day3 to day7 after CCI surgery, mechanical withdrawal thresholds were significantly increased compared with CCI and CCI+Sham-EA group in POD7(*P<0.05).. After multiple intrathecal injection of BOC-2, mechanical withdrawal thresholds were significantly decreased ccompared with CCI+MEA group in POD7(*P<0.05).2.2 The effect of ALX on the spinal inflammatory cytokine in CCI rats with EA therayRT-PCR analysis of IL-1β, IL-6,TNF-a in the L4-L5 spinal cord of the normal group and the model group on day 7 after CCI surgery with multiple EA and intrathecal injection of BOC-2. Compared with CCI group, multiple EA could significantly enhance the mRNA levels of inflammatory factors(IL-1β,IL-6 TNF-a.). Compared with CCI+MEA group, multiple intrathecal injection of BOC-2 could increase the decreased mRNA levels of IL-1β and IL-6 induced by MEA therapy(**p < 0.01 compared with CCI group,##p< 0.01 compared with CCI+MEA group).Summary:EA therapy could enhance the mechanical and thermal response threshold in part through the ALX in spinal cord.5-LO and AnnexinAl were involved in the induction and development of neuropathic pain, which regulate the analgesic effect of EA.Conclusion:1.The LXA4 receptor ALX mainly in the spinal astrocytes. ATL could inhibit the spinal neuroinflammation, which could reduce the mechanical allodynia through the spinal ALX in CCI rats.In addition, ATL could exert the analgesic effect through the vein administration.2.The upregulation of p-STAT3, SOCS2, SOCS 3 in spinal cord occurred in the induction of neuropathic pain.Inhibition of spinal JAK2- p-STAT3 could reduce the neuroinflammation and the mechanical allodynia. ATL could down-regulate the expression of p-STAT3 and enhance the expression of SOCS3 mRNA.In a word, JAK2-STAT3-SOCS3 signaling pathway was involved in the induction of neuropathic pain.3. EA could enhance the mechanical and thermal response threshold in part through the ALX in spinal cord.5-LO and AnnexinAl were involved in the induction and development of neuropathic pain, which regulate the analgesic effect of EA.