Preliminary Study Of Mechanism Of Increased HO-1 Expression In Spinal Cord Of Formalin Inflammatory Pain Rat

Objective Previous studies in our laboratory have confirmed that the input of nociceptive information induced by formalin inflammatory pain could induce an increase in the expression of Heme oxygenase-1 (HO-1) in rat spinal cord. The increases of the HO-1expression and endogenous carbon monoxide (CO) production in spinal cord play an important role in the nociceptive perception and hyperalgesia. However, there have been few reports about the mechanism of up-regulation of HO-1 expression in the spinal cord during formalin inflammatory pain.Many research results have shown that the input of nociceptive information induced by formalin inflammatory pain could induce the release of the neurotransitter which are mainly composed of excitatory amino acids (EAAS) in spinal cord. Actived NMDA receptors in the neurons of spinal cord can induce the increases of production of signal transducer nitric oxide (NO) and activation of protein kinase C (PKC). These changes play an important role in the process of transfer of nociceptive formation and the production of hyperalgesia. We have observed the effects of N-methyl-D-aspartic acid (NMDA )receptor and PKC to NOS expresstion and production of NO in the spinal cord of rats with formalin Inflammatory pain by Intrathecal injection of NMDA receptor antagonist MK-801 or chelerythrine chloride (CH) which is the inhibitor of PKC. The results have proved that both of NMDA receptor activtion and PKC activtion could induce the increase of NOS expression and the production of NO in spinal cord of rats with formalin inflammatory pain. The results showed that the production of NO in dorsal horn induced by formalin inflammatory pain was regulated not only by the activation of NMDA receptor but also by the activation of signal transducer PKC .A lot of results have confirmed that there are close similarty between systems of NOS/NO and HO/CO. It needs to be further studied that whether or not the increase of HO-1 expression in spinal cord can been regulated by the NMDA receptor of EAAS and the signal transducer PKC during formalin inflammatory pain.In this study , We observed the effects of NMDA receptor antagonist MK-801 and PKC inhibitor (CH) to the HO-1 expression in the spinal cord of rats with formalin Inflammatory pain and the effects of NMDA receptor selective agonist NMDA and Phorbol 12-Myrstate-Acetate which is an agonist of PKC to HO-1 expression in the spinal cord of normal rats to investigate preliminary mechanism of increased HO-1 expression in spinal cord of Formalin Inflammatory Pain rats.1 Effect of activated NMDA receptor to HO-1 protein expression in the spinal cord of formalin inflammatory pain ratThirty five Male Sprague-Dawley rats, (280±20g in weight), which were provided by Hebei Medical University Animal Laboratory Center. Rats were randomly divided into 5 groups as follows:①control group (Control).②formalin group (FM).③formalin and normal saline group (FM + NS).④formalin and MK-801 group (FM + MK-801).⑤NMDA group. There were seven animals in each group. The rats in Control group were directly sacrificed without any treatment. Rats of formalin groups were subcutaneously injected with 0.2ml 5% formalin into the plantar surface of the right hind paw to induce periphery inflammatory pain and the L5 segment of spinal cord was dissected out 24h later. FM+NS and FM+MK-801 groups, respectively, NS and NMDA receptor inhibitors MK-801 solution intrathecally injected at 15 min prior to the formalin injection, then the dorsal horn of lumbar 5segment (L5) of spinal cord of the rats were dissected out after sacrificing the animals with decapitation at 24h. Rats of NMDA group were normal rats, they were intrathecally injected NMDA receptor agonist NMDA solution and 24h later the dorsal horn of lumbar 5segment (L5) of spinal cord of the rats were dissected out after sacrificing the animals with decapitation. The immunohistochemical method was used to observe the expression of HO-1 protein in the spinal cord.The results showed that the rats in Control group that HO-1 immunoreactive cells in laminaeⅠ-Ⅱof the L5 segment of spinal cord were rhombus, ellipse or round and that most of the cells were small in size. There are many clear protuberances and vimineous nerve fibers in part of cells.Compared with the Control group, FM Group the number of HO-1 immunoreactive cells significantly increased in theⅠ-Ⅱlayers of bilateral spinal dorsal horn (P <0.01), and the staining degree of these positive cells also was significantly increased (P <0.01), show that after injection of formalin, the expression of HO-1 could be increased in spinal dorsal horn. There was no significant difference between FM + NS Group and FM group, indicating that the expression of HO-1 had no significant change after intrathecal injection of solvent NS. MK-801 group showed the number and staining degree of HO-1 immunoreactive cells significantly declined (P<0.01).Prior intrathecal injection of MK-801 significantly inhibited the up-regulation of HO-1 expression induced by formalin injection .The result showed that block NMDA receptors, can inhibit expression of HO-1 protein during inflammatory pain after formalin injection. With the Control group, normal rats have intrathecal administered NMDA solution, animals have emerged a spontaneous nociceptive reaction, including flinches, scratching and biting the wounded paw. At the same time, the number and the staining degree of HO-1immunoreactive cells after NMDA injection was increased in laminaeⅠ-Ⅱof spinal dorsal horn.(P<0.01). This result further proved that the activation of NMDA receptor could increased the expression of HO-1 protein in spinal cord nerve cell.2 Effect of PKC activation on expression of HO-1 protein in spinal cord of the rats with formalin inflammatory painThirty five Male Sprague-Dawley rats, (280±20g in weight), which were provided by Hebei Medical University Animal Laboratory Center. Rats were randomly divided into 5 groups as follows:①control group (Control).②formalin group (FM).③formalin and DMSO group (FM+DMSO).④formalin and CH group (FM+CH ).⑤PMA group. There were seven animals in each group. The rats in Control group were directly sacrificed without any treatment. Rats of formalin groups were subcutaneously injected with 0.2ml 5% formalin into the plantar surface of the right hind paw to induce periphery inflammatory pain and the L5 segment of spinal cord was dissected out 24h later. FM+DMSO and FM+CH groups are respectively intrathecally injected with DMSO and 10nmol CH solution at 15 min prior to the formalin injection, then the dorsal horn of lumbar 5segment (L5) of spinal cord of the rats were dissected out after injected with formalin at 24h. Rats of PMA group were normal rats. They were intrathecally injected with 0.8nmo1 PKC agonist-PMA solution 24h later the dorsal horn of lumbar 5segment (L5) of spinal cord of the rats were dissected out after injected with PMA at 24h. The immunohistochemical method was used to observe the protein expression of HO-1 protein in laminaeⅠ-Ⅱof the L5 segment of spinal cord.The results showed that rat in FM and FM+DMSO group contrast with rats in Control group, the number and Staining degree of HO-1 immunoreactive cell significantly increased in theⅠ-Ⅱlayers of bilateral spinal dorsal horn of ratsi increased significantly (P<0.01). But there was no significant difference between FM and FM+DMSO group (P>0.05), indicating that the expression of HO-1 had no significant change after intrathecal injection of DMSO solvent. Rats in FM+CH group Contrast with rats in FM group, the number of HO-1 positive cells in theⅠ-Ⅱlayers of bilateral spinal dorsal horn was decreased significantly,and staining degree of positive cells and fibers were weakered. significantly,but higher than the level of Control group (P<0.01). This showed that inhibit the activation of PKC could inhibit the increase the expression of HO-1 protein during formalin-induced inflammatory pain. When intrathecal injection of PMA, animals have emerged a spontaneous nociceptive reaction, including, such as grasp, licking, biting and other spontaneous reaction of falling back. Expression of HO-1 protein in spinal cord changed significantly. Contrast to Control group, the number of HO-1 HO-1 immunoreactive cells increased in theⅠ-Ⅱlayers of bilateral spinal dorsal horn in PMA group(P<0.01). Positive cells'AOD also increased significantly (P<0.05). Further improved, the activation of PKC could improve HO-1 protein expression of neurons in spinal cord.Conclusion:Intrathecal injection of MK-801 could inhibit the expression of HO-1 protein in spinal cord induced by formalin inflammatory pain. Intrathecal injection of NMDA in normal rats could improve the expression of HO-1 protein in the spinal cord . These results showed that the expression of HO-1 protein in the spinal cord of rats with formalin inflammatory pain rats were regulated by the activation of NMDA receptor .Intrathecal injection of CH could inhibit the expression of HO-1 protein in the spinal cord of rats with formalin inflammatory pain. Intrathecal injection of PMA in normal rats could increase the expression of HO-1 protein in the spinal cord . These results showed that the expression of HO-1 protein were regulated by PKC activation in the spinal cord of rats with formalin inflammatory pain.