The Role Of GPR30 In The Spinal Cord Injury

Background and objectiveSpinal cord injury ( SCI ) is a serious trauma of nervous system in our live or the war.It often leading to different levels of paralyzed limbs of patients or sphincter disturbances, not only cause pain to patients, but also increase the social and family pressure. The overall incidence rate of SCI in the world was 20-40 people / million / year,and the United States was 28 to 55 people / million / year.There was about ten thousand new cases each year, and the average age was 31.7 years. We spend more than seven billion U.S. dollars on caring and treatment of these patients each year. China did not have comprehensive Statistics, but the result of Investigation in Beijing the incidence rate of spinal cord injury was 60 / 1 million in 2002, much higher than the overall level of the United States and the world.With the economic development, the SCI increasingly caused by transportation and construction accidents affect the people's health and quality of life. SCI caused disability is currently no effective treatment, and the recovery of neurological function has been a big clinical problem. So, the research about repairing of spinal cord injury, is very necessary and urgent. Current research on spinal cord injury has made considerable progress, summarized in the following aspects: ( 1 ) Research of neuroprotection After the spinal cord injury except the primary trauma, through many mechanisms ( such as ischemia and hypoxia, excitatory amino acid toxicity, ion homeostasis disruption, calcium overload, apoptosis and inflammation, etc.) leading to the secondary injury is more important.It is the main reason for the disfunction of spinal cord. Primary injury is difficult to save, while secondary injury can be rescued. (2) Research of neuroregeneration In this area we got large progress in the last century 90s. The main consequents is that find the central nervous system has regenerative capacity, but the external micro-environment can cause inhibition of nerve regeneration, such as nerve regeneration inhibitory molecules, glial scars and cysts such as physical and chemical barrier. (3) Neurotransplantation It mainly through the peripheral nerve, tissue engineering, and promoting regeneration to repair the injured spinal cord. ( 4 ) Neuroplasticity Many examinations found that patients after SCI can restore some functions by theirselves. As long as remaining small number of nerve fibers, it can help the functional restoration of spinal cord. So we often use rehabilitation and physiotherapy to help the reconstruction patients spinal cord function. In clinical.\Although these strategies have achieved good results in animal experiments, but the clinical efficacy is not satisfactory.The evidence from evidence-based medicine shows drugs for secondary damage ,such as the NMDA receptor antagonist, ganglion glycoside lipid, free radical scavenging agents are ineffective against SCI. So looking for the new breakthrough and new therapeutic targets about spinal cord injury and repairing is very important in medical value and social significance.People have noted the fact that female with functional recovery and prognosis is often better than men after central nervous system injury.Further clinical studies (including epidemiology) and animal experiments found similar results, and extended it to neural trauma and neurodegenerative diseases (such as Alzheimer's disease, Parkinson's disease). Many experts believe that this is mainly related to estrogen. For a long time, soluble intracellular estrogen receptor ERα/β(ER) has been considered to the only receptor for its role. This kind of estrogen receptor with the combination of conformational change occurs, and transferred to the nucleus to specific DNA Series - estrogen response elements binding, regulation of transcription and expression of a gene, thus affecting the development, proliferation and apoptosis of cell. This way is classical pathway of estrogen and it will spengd much time on it . However, a large number of studies have shown that estrogen can be onset very quickly such as vascular tone regulation, rapid promotion of NO release, Ca2 + mobilization. All that show expect the classic slow gene effects, it are still not known by non-genetic mechanism of rapid onset. In 2005, Science published an article to the existence of sufficient evidence that a novel estrogen receptor-GPR30 (a kind of G protein-coupled receptor)-mediated rapid estrogen pathway, so as mediating rapid effects of estrogen membrane receptor in estrogen effect on spinal cord injury may be an important role, is to study spinal cord injury repair is an important new target.AimThis issue wanted to define the distribution of GPR30 in each section of rat spinal cord with Immunohistochemistry .Then using the immunofluorescence technique to determine localization of GPR30 in the spinal cord cells. Which may provide a good foundation for the further study about Intention and mechanism. Contradistinction the lower limb motor functions and the expression protein level of GPR30 after SCI in normal group, saline group, E2 group, E2-BSA group and G-1 group, which reveals the role of GPR30 in the spinal cord injury.MethodsAll experiment in this issue will use adult male SD rats to study. First of all , to define the spatial distribution of GPR30 in the cervical, thoracic, lumbar and sacral segments of rat by immunohistochemistry staining method. Then using immunofluorescence method to determine the location of GPR30 in the cells of rat spinal cord . Inference GPR30 may play an important role in spinal cord injury. And we establish the experimental model of spinal cord injury .Around the normal group, saline group, SCI + E2 group, SCI + E2-BSA group, SCI+G-1 group , between groups for the following research: 1, functional studies: comparison the lower extremity motor function of each group, which can infer to the role of GPR30 after SCI. 2, using Western-blot technique to detect the protein expression levels of GPR30 in each groups, which can further reveal the effect of GPR30 after SCI.Results1.We find that rhe GPR30 can be seen in all segments of the rat spinal cord, and expressed mainly in the spinal cord gray matter. The distribution of white matter in each segment was basically similar to the uniform distribution, no expression of dense area.There are some differences in distribution of GPR30 in gray matter. In the cervical spinal cord gray matter, GPR30 express mainly in the ventral horn, and around the central canal is also seen some expressed, but in the dorsal horn of the spinal cord was no expression of GPR30.But we can find GPR30-positive signals in the gray matter near the dorsal horn. In thoracic spinal cord gray matter, GPR30 is still distributed mainly in the ventral horn, and around the central canal see a very small amount of distribution, in the dorsal horn and around gray matter ,the GPR30 were no significant positive signal. In the lumbar spinal cord gray matter GPR30 is still distributed mainly in the ventral horn, and the joint department of gray matter can be seen a very small amount of the distribution, but in dorsal horn and periaqueductal gray can see some positive signals. In the sacral, GPR30 mainly express in the ventral horn of gray matter and the distribution is relatively rare, no obvious aggregation, and no obvious distribution in the dorsal horn. Location view from the cell structure, GPR30 in various segments of spinal cord gray matter cells are expressed in the cytoplasm membrane, the nucleus was not detected the positive signal of GPR30.2.With immunofluorescence technique ,we find that GPR30 express specifically in neurons in spinal cord, and in oligodendrocytes and microglial cells also expressed, but is not expression in astrocytes cells.3.Functional studies show that: The motor function recovery in E2 group, E2-BSA group and G-1group are better than saline group.In E2-BSA and G-1 group the recovery of motor function are worse than the E2 group, and between E2-BSA group and G -1 group are no clear difference .4.Protein level: Western-blot showed that in saline group, E2 group, E2-BSA group and the G-1 group, the GPR30 protein levels are significantly higher than control group. Contradistinction the protein expression of the each intervention groups can find it was significantly lower in saline group than the E2 group, E2-BSA group and G-1 group, and the saline group protein growth trend is consistent with the E2 group. The protein level of E2 group increased slowly in the early stages after SCI, and at the 14th reached the highest value, then dilatory reducing. The protein level of E2 group, are significantly higher than the E2-BSA group at 1and 3 days, but we can see the protein level rapidly increased at 7th day in E2-BSA group, and significantly higher than the expression of E2 group. The protein level of E2-BSA group also increased slowly within 3 days after SCI, but around the 7th day the expression reached the highest value. The protein level of G-1 group increased rapidly in early stage after SCI, and at third day had significantly higher than other groups, at 7th day to reach the highest value, then protein expression began to rapidly reduce, and the expression was significantly lower than E2 group from 14th day.Conclusion1.The membrane estrogen receptor GPR30 can express in both gray and white matters in all segments of spinal cord, mainly in the ventral horn (movement angle) gray matter and in the central canal of the spinal cord is also a small amount of expression .Except the lumbar spinal cord dorsal horn has a small amount of GPR30-positive signals, the remaining segments were no significant GPR30-positive signals. In white matter distribution of GPR30 was uniform, and the GPR30 was expressed mainly in the cytoplasm of cells, no expression in the nucleus. So GPR30 maybe mediate the impact damage and recovery of motor neurons through non-genetic effects of estrogen, while to the impact of sensory neurons in dorsal horn may be relatively weak. In the white matter can find the expression of GPR30, indicating GPR30 may mediate the proliferation, secretion and other effects of glial cells.2.Estrogen membrane receptor GPR30 is explicit expression in neurons of the spinal cord, and indicate that estrogen can directly affect neurons by GPR30-mediated effects of it. In the white matter of spinal cord, the oligodendrocytes and microglia also have GPR30 expression, but in astrocytes is not expressed. So, estrogen may directly affect on microglia cells and oligodendrocyte , which may affect the accumulation of inflammatory factors and glial cell neuroendocrine function, and may indirectly affect other cells.3.The estrogen have clear effect on motor function recovery after SCI. The GPR30-mediated nongenomic estrogen effects on spinal cord injury was an important part, and the classical nuclear estrogen receptor-mediated gene effect was also to play a certain role.4.The protein expression levels of GPR30 are showing a rising trend after SCI. And using E2, E2-BSA, G-1 and other interferences factors can significant increase the protein expression level of GPR30, which indicate that GPR30 was mediated the protective effect in spinal cord injury. As the specific GPR30 agonist G-1 which can induced the GPR30 expression, and its ability is significant stronger than normal estrogen for GPR30...