Involvement Of Spinal Orphanin FQ/Nociceptin In Inflammatory Pain And Electroacupuncture Analgesia In Rats

Chronic inflammatory pain is one of the most common pathologic pains in clinic. Inflammatory pain arises as a debilitating consequence of injury to the peripheral tissue, which is characterized by combination of spontaneous burning pain, hyperalgesia and allodynia. The researches about its peripheral and central mechanism have been the important focus of the international pain research organization. Substantial evidences have indicated that a number of bioactive substances involved in the information and modulation of inflammatory pain. Unfortunately, the etiological mechanism of neuropathic pain has been poorly understood.Orphanin FQ/ nociceptin (N/OFQ), a member of opioids family found in 1995, is an endogenous ligand of opioid receptor like receptor (NOP), which has been identified as a member of opioids receptor family. N/OFQ is a 17 amino-acid peptide (FGGFTGARKSARKLANQ). In spite of its structural homology with the endogenous opioid peptides (especially dynorphin A), N/OFQ has high affinity to NOP receptor but does not show appreciable binding either μ, δ or κ receptors. N/OFQ and NOP receptor are distributed widely in central nervous system, and have been shown to play a role in lots of physiological functions, such as pain modulation, cardiovascular controls, pituitary functions, immune, feeding, learning and memory. In pain modulation, the role of supraspinal N/OFQ and spinal N/OFQ is different. Substantial evidences have indicated that spinal N/OFQ produced analgesia and potentiated morphine analgesia but supraspinal N/OFQ induced hyperalgesia and antagonized morphine analgesia. The previous studies in our lab showed that spinal N/OFQ produced analgesic effect in the neuropathic pain rats through NOP receptor. Spinal N/OFQ-NOP receptor system was involved in neuropathic pain. However, much less is known about the effects of N/OFQ on CFA-induced hyperalgesia, the changes of expression of N/OFQ and NOP receptor in the spinal cord during different time course of chronic inflammatory pain, and whether N/OFQ and NOP receptor in the spinal cord were involved in inflammatory pain.In clinic, management of chronic inflammatory pain is one of the most difficultchallenges. Electroacupuncture (EA) has long been used to relieve pain. Numerous reports have indicated that EA had potent analgesic effect on pathological pain with few side effects in patients and rat models. Studies have showed that EA plays its analgesic role via activating the endogenous pain modulating systems, i.e., opioid system. However, the role of N/OFQ, a member of endogenous opioid peptides, playing in EA analgesia in neuropathic pain is still unclear. Therefore, to investigate the role of N/OFQ in chronic inflammatory pain and EA analgesia is the main target of the present study.New research implicates spinal cord astrocytes as one of the key players in the chronic pathological pain. Potential activation of glia was observed at the lumbar spinal cord, which involved in hypersensitivity and persistent pain induced by inflammation and damage to peripheral tissues, peripheral nerves, spinal nerves and spinal cord. Upon activation, a variety of algesic substances are released by glia that may potentiate pain transmission by neurons. Of these glial products, proinfiammatory cytokines, such as interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-a (TNF-a), have been demonstrated to be common mediators of allodynia and hyperalgesia. In addition, NOP receptor is densely distributed in some brain nuclei involved in pain modulation and spinal dorsal horn. Furthermore, recently it has demonstrated that NOP receptor was expressed on astrocytes of rat brain, indicating there might be a relationship between N/OFQ-NOP receptor system and astrocytes. However, it was also unclear whether NOP receptor was expressed on astrocytes of rat spinal cord, whether astrocytes played a role in spinal N/OFQ analgesia. Then, another part of our studies was to explore the relationship of N/OFQ and astrocytes in order to investigate the mechanism of spinal N/OFQ analgesia.By using the molecular biological techniques, including RT-PCR, immunohistochemistry, Western-Blot and astrocytes cultures, the present study was designed to explore: (1) the role of spinal N/OFQ-NOP receptor system in CFA-induced inflammatory pain and EA analgesia; (2) the changes of expression of N/OFQ and its receptor in spinal dorsal horn during EA treatment in peripheral inflammation; (3) the expression of GFAP and cytokines was characterized with respect to the development of thermal sensitivity during various phases of CFA-induced peripheral inflammation; (4) the mechanism of N/OFQ.The results are following:1. The role of spinal N/OFQ-NOP receptor system in inflammatory pain and EA analgesia1.1 Effects of i.t. N/OFQ on hyperalgesia of the inflammatory pain ratsOn day 4 after a subcutaneous injection of 100 μl CFA, rats received intrathecal injections with 1.5, 5 and 15 nmol of N/OFQ. The percentage changes of PWL to thermal stimulation were measured prior to and then 10, 20, 30, 60 and 90 min after the administration. The results indicated that N/OFQ at 15 nmol could significantly alleviate inflammation-induced hyperalgesia.1.2 Effects of i.t. NOP receptor antagonist, [Nphe¹]nociceptin(1-13)NH₂, on N/OFQ-induced anti-nociceptionOn day 4 after a subcutaneous injection of CFA, rats received the intrathecal injection of 15 nmol of N/OFQ, following 5 min later by intrathecal administration of 20 nmol of [Nphe¹]nociceptin(1 13)NH₂. The percentage changes of PWL to thermal stimulation were tested at the above same time course after intrathecal administration. The results showed that NOP receptor antagonist had no significantly effect on hyperalgesia of inflammatory pain rats but reversed N/OFQ analgesia.1.3 Effect of EA on CFA-induced hyperalgesiaOn day 4 after a subcutaneous injection of CFA, rats received EA treatment. Compared to control (CFA) and sham EA groups, EA significantly lengthened PWL of the CFA-injected hind paw at 10, 20, 30 and 60 min post-EA treatment, suggesting the effect of EA anti-hyperalgesia.1.4 Effect of i.t. [Nphe¹]nociceptin(1 13)NH₂ on EA anti-hyperalgesiaOn day 4 after a subcutaneous injection of CFA, rats received intrathecal administration of 20 nmol of [Nphe¹]nociceptin(1 13)NH2, followed 10 min later by EA treatment. The percentage changes of PWL to thermal stimulation were tested before and 10, 20, 30, 60 and 90 min after EA treatment. Compared with the saline plus EA control group, the effect of EA anti-hyperalgesia was partly reversed by [Nphe¹]nociceptin(1 13)NH₂.1.5 Effect of the combination of N/OFQ with EA on hyperalgesiaOn day 4 after a subcutaneous injection of CFA, rats received intrathecaladministration of 15 nmol of N/OFQ, at the same time EA treatment was given. The percentage changes of PWL to thermal stimulation were measured at the above same time points after the co-administration. Compared to the saline plus EA group and 15 nmol N/OFQ group, the anti-hyperalgesic effect of the combination of N/OFQ with EA was significantly greater than the effect of EA or N/OFQ alone, suggesting that N/OFQ could enhance the effect of EA-produced anti-hyperalgesia.Summary: i.t. N/OFQ at 15 nmol could significantly alleviate inflammation-induced hyperalgesia. NOP antagonist could block the anti-nociceptive effect of N/OFQ, suggesting that i.t. N/OFQ played its analgesic effect through NOP receptor. EA produced the significant anti-hyperalgesia induced by CFA. I.t. NOP receptor antagonist partly reversed EA analgesia and spinal N/OFQ enhanced EA analgesia, indicating that spinal N/OFQ-NOP receptor system was involved in EA analgesia.2. Changes of expression of N/OFQ and its receptor in spinal dorsal horn in peripheral inflammatory pain and EA treatmentThe experiment animals were divided into four groups: Normal group, inflammatory pain group (CFA), EA group and sham-EA group. After a subcutaneous injection of CFA and EA treatment every other day, (1) accumulative analgesic effect of repetitive EA on CFA-induced thermal hyperalgesia was observed by behavioral test; (2) the changes of spinal N/OFQ and NOP receptor in rat lumber spinal dorsal horn were observed by RT-PCR, immunohistochemistry and western blot.2.1 Accumulative analgesic effect of repetitive EA on CFA-induced thermal hyperalgesiaFollowing injection of 100ul CFA into the left hind paw, animals showed evident signs of inflammation, which were assessed by localized erythema and an increase in the swelling of the paw. EA treatment every other day could significantly increase hyperalgesia score, an accumulative anti-hyperalgesic effect, from the fifth time post-EA treatment, while sham EA showed little anti-hyperalgesia.2.2 Time course of ppN/OFQ mRNA expression during CFA-induced inflammation and EA treatmentAt day 4, day 10 and day 14 following peripheral administration of CFA, corresponding to 2, 5, 7 times of EA treatment, respectively, lumbar spinal cords were removed. Time course of ppN/OFQ mRNA expression during CFA-induced inflammation and EA treatment was observed using RT-PCR. The results showed that increased expression of ppN/OFQ mRNA at the ipsilateral lumbar spinal cord was seen at day 4 following peripheral administration of CFA, and remained elevated during the whole phases (day 10 and day 14) of inflammation, which was further enhanced by EA treatment.2.3 Time course of the number of N/OFQ-like immunoreactive (N/OFQ-LI) neurons during CFA-induced inflammation and EA treatmentAt day 4, day 10 and day 14 following peripheral administration of CFA, corresponding to 2, 5, 7 times of EA treatment, respectively, lumbar spinal cords were removed and frozen sections were cut. Time course of the number of N/OFQ-like immunoreactive (N/OFQ-LI) neurons during CFA-induced inflammation and EA treatment was observed using immunohistochemistry. The results showed that N/OFQ-LI signals were mainly limited to the superficial layers of the spinal dorsal horn. CFA-induced chronic inflammation elicited a dramatic decrease of N/OFQ immunoreactivity in the ipsilateral spinal dorsal horn from 4 days after CFA injection. After EA treatment, the number of N/OFQ-LI neurons was significantly increased compared with that of inflammatory pain rats.2.4 Time course of NOP receptor mRNA expression during CFA-induced inflammation and EA treatmentAt day 4, day 10 and day 14 following peripheral administration of CFA, corresponding to 2, 5, 7 times of EA treatment, respectively, lumbar spinal cords were removed. Time course of NOP receptor mRNA expression during CFA-induced inflammation and EA treatment was observed using RT-PCR. The results showed that increased expression of NOP mRNA in CFA-injected rats was significant during the whole phases (day 4, 10 and day 14) of inflammation, which was further enhanced by EA treatment.2.5 Time course of NOP receptor protein expression during CFA-induced inflammation and EA treatmentAt day 4, day 10 and day 14 following peripheral administration of CFA,corresponding to 2, 5, 7 times of EA treatment, respectively, lumbar spinal cords were removed. Time course of NOP receptor protein expression during CFA-induced inflammation and EA treatment was observed using Western blot. The results showed that increased expression of NOP receptor protein in CFA-injected rats significantly remained elevated during the different phases (day 4, 10 and day 14) of inflammation compared with that of normal rats, which was further enhanced by EA treatment.Summary: The ppN/OFQ mRNA level, both mRNA and protein levels of NOP receptor were significantly increased following CFA-induced inflammatory pain, suggesting that spinal N/OFQ and NOP receptor might be involved in the different time course of inflammation. Furthermore, The N/OFQ immunoreactive cells in lumber spinal dorsal horn were reduced, and the reason for the result might be that the N/OFQ protein synthesis couldn't be compensated for increasing release of N/OFQ in neuropathic pain. The ppN/OFQ mRNA level, both mRNA and protein levels of NOP receptor, and N/OFQ immunoreactive cells in lumbar spinal dorsal horn were increased after EA treatment, suggesting the synthesis of N/OFQ was further enhanced to compensate the low level of N/OFQ in lumbar spinal dorsal horn of inflammatory pain rats. The results indicated that spinal N/OFQ and NOP receptor were involved in the whole time course of inflammatory pain and EA analgesia.3. Changes of spinal glial activation in the whole time course of CFA-induced inflammatory painThe experiment animals were divided into two groups: Normal group, inflammatory pain group (CFA). The expression for the astroglial marker, GFAP, and proinflammatory cytokines (IL-1β , IL-6, TNF-α) during various phases of inflammation was observed using immunohistochemistry, Western blot, RT-PCR.3.1 Time course of glial activation during CFA-induced inflammatory pain At day 4, day 10 and day 14 following peripheral administration of CFA, lumbar spinal cords were removed and frozen sections were cut. Time course of the number of GFAP-immunoreactive astroglial cells during CFA-induced inflammation was observed using immunohistochemistry. The results showed that at day 4 after intraplantar CFA administration, a large number of GFAP-positive astroglial cells exhibited intense immunoreactivity and appeared hypertrophied with thick processes.Time course of GFAP protein level in CFA-injected rats was observed using Western blot. The results showed that increased expression of GFAP level at the lumbar spinal cord in CFA-injected rats was significant during various phases of CFA-induced inflammation.3.2 Time course of proinflammatory cytokines expression during CFA-induced inflammatory painAt day 4, day 10 and day 14 following peripheral administration of CFA, lumbar spinal cords were removed. Time course of proinflammatory cytokines mRNA expression during CFA-induced inflammation was observed using RT-PCR. The results showed that the increased expression of IL-1β, IL-6 and TNF-α level at the lumber spinal cord was significant throughout the phases of inflammationSummary: The expression of GFAP and IL-1β, IL-6, TNF-α was increased in various phases of inflammation, suggesting that astrocytes and proinflammatory cytokines in the spinal cord played an important role during the different time course of CFA-induced inflammatory pain.4. The mechanism of spinal N/OFQ analgesia4.1 Effect of i.t. N/OFQ on the expression of proinflammatory cytokines in CFA-injected ratsThe experiment animals were divided into four groups: Normal group, inflammatory pain group (CFA), CFA + N/OFQ group, CFA + N/OFQ + [Nphe¹]nociceptin(1-13)NH₂ group. Using RT-PCR to observe the effect of N/OFQ on the expression of proinflammatory cytokines. The results showed that cytokines mRNA transcripts were expressed at low levels in control group. However, these levels were significantly increased following CFA, and antagonized by treatment with N/OFQ. This effect was antagonized by co-treatment with a NOP receptor antagonist.4.2 Spinal astrocytes expressed NOP receptorRT-PCR, immunocytochemistry and Western blot were performed to determine whether NOP receptor was expressed on cultured astrocytes of rat spinal cord. These results demonstrated that cultured astrocytes expressed NOP receptor.4.3 Effect of N/OFQ on proinflammatory cytokines gene expression in cultured astrocytesUsing RT-PCR to observe: (1) the characterization of LPS-induced proinflammatory cytokines release; (2) the effect of N/OFQ on IL-1β, IL-6, TNF-α gene expression in cultured astrocytes. These results indicated that (1) the IL-1β, IL-6, TNF-α gene expression significantly increased after exposure to LPS, suggesting proinflammatory cytokines were released by glia upon activation; (2) The increased IL-1β, IL-6 and TNF-α gene expression induced by LPS could be inhibited by N/OFQ. The reduced proinflammatory cytokines gene expression was significantly abolished by (Nphe¹)nociceptin (1 13)-NH2, indicating the effect was attributable to the NOP receptor pathway.Summary: The expression of proinflammatory cytokines was significantly increased following CFA, and antagonized by i.t. N/OFQ; Spinal astrocytes expressed NOP receptor; The increased IL-1β, IL-6 and TNF-α gene expression induced by LPS could be inhibited by N/OFQ, which was abolished by (Nphe¹)nociceptin (1 13)-NH2. The effect indicated that the nociception modulation of N/OFQ might be dependent on inhibiting the expression of proinflammatory cytokines through NOP receptor.Conclusion:1. Spinal N/OFQ produced analgesic effect in the inflammatory pain rats through NOP receptor. Spinal N/OFQ-NOP receptor system was involved in EA analgesia.2. Cumulative EA has potent analgesic effect on inflammatory pain of rats.3. Changes of spinal N/OFQ and NOP receptor took place during the various inflammatory pain induced by CFA and EA treatment, suggesting spinal N/OFQ-NOP receptor system was involved in inflammatory pain and EA analgesia.4. The expression of GFAP and proinflammatory cytokines was increased in various phases of inflammation, suggesting that astrocytes and proinflammatory cytokinesin the spinal cord played an important role during the different time course of CFA-induced inflammatory pain.5. I.t. N/OFQ inhibited the proinflammatory cytokines gene expression. The nociception modulation of N/OFQ might be dependent on inhibiting the expression of proinflammatory cytokines through NOP receptor.