A Preliminary Study On The Role Of Tnf-α In Rats During Early Inflammaion

Background and ObjectiveLipopolysaccharide (LPS) is a major integral structural component of the outer membrane of Gram-negative bacteria and one of the most potent initiators of inflammation. During inflammation, LPS entered into blood circulation, many inflammatory cells release inflamed mediators which can cause inflammation. When the body is inflamed, LPS could effected on membrane receptor through signal transmission change gene expression. LPS activates endothelial cells, smooth muscle cells, mechanocytes, epithelial cells and monocytes/macrophages to produce cytokines, chemotatic factors, growth factors and other factors, such as interleukins (IL) -1, tumor necrosis factor-alpha (TNF-α), that in turn, serve as endogenous inflammatory mediators.Tumor necrosis factor factor-alpha (TNF-α) is a multifunctional cytokine, which mediates immunity and inflamed action. After the damage of sciatic nerve, the Schwann cells were activated to produce TNF-α. However, the mechanism of TNF-αis still unknown. The effects of TNF-αare mediated through two receptors, TNF receptor type 1 (TNFR1, p55) and TNF receptor type 2 (TNFR2, p75).In order to invest whether LPS could induce rats glia cell to produce TNF-α, we used two animal models, in which SD rats were intraperitoneal injected or intrathecal injected with LPS respectively, and investigate if there were any cells produce TNF-αin sciatic nerves and spinal cords of rats, then find out the cell types.Extracellular regulated protein kinases (ERK) contain ERK1 and ERK2, which transduce extracellular stimuli into intracellular post-translational and transcriptional responses. Activated ERK transport from cytoplasm to nuclear, in order to mediate the transcription of ATF, NF-κB, Ap-1, c-fos and c-Jun. ERK participate in cell proliferation and differentiation, sustain cell appearance, build cystoskeleton, and even affect cell apoptosis and canceration. While there is growing evidence showing that LPS can cause hyperalgesia, so we used these two animal models to gain the data of the activation of ERK and TNF-αproduction following the onset of LPS injection, in purpose to provide a theoretical evidence for pathogenesis of neuro-inflammation disorders. Methods1. Animal model was preparated in rat by LPS injection intraperitoneal or intrathecal.2. The sciatic nerve and spinal cord tissues content of TNF-αprotein was assayed by ELISA, and the location of TNF-αprotein in the tissues were detected by double immumofluorescence.3. The spinal cord tissue content of ERK and pERK protein was assayed by Western blot, and the location of pERK protein in the tissues was detected by double immumofluorescence.Results1. 1) Administration of LPS resulted in remarkable upregulation of TNF-αin rat sciatic nerve, which mainly expressed in Schwann cell and macrophages.2) In the control group (normal rats), TNF-αstaining dispersed in the rat spinal cord. LPS injection resulted in obvious increase of TNF-α-immunoreactivity as compared to the control, especially in neurons and microglia cells.3) During the early time of LPS intrathecal injection, the level of TNF-αincreased in spinal cord, which mainly expressed in microglia cells of the injected side.2. 1) During the early time of LPS intraperitoneal injection, the pERK level reached peak at 1 h, and then decreased at 12 h, It appeared that blotting density of ERK was almost unchanged. LPS injection resulted in obvious increase of pERK-immunoreactivity as compared to the control, especially in microglia cells of rat spinal cord.2) During the early time of LPS intrathecal injection, the pERK level reached peak at 4 h, which was increased in microglia cells of the injected side of rat spinal cord, and few in the opposite side.Conclusions1. TNF-αdistributes a few in normal rat sciatic nerve and spinal cord tissues. Intraperitoneal administration of LPS alters TNF-αexpression, indicating that TNF-αis an inflammatory response molecule participating in the inflammatory process of CNS and peripheral nerve system (PNS).2. Observations of TNF-α-IR in Schwann cell and microglia in the early stage after LPS injection suggests that Schwann cell is most likely the main source of TNF-αin rat sciatic nerve after LPS stimulation. As glia are intimately involved in the process of systematical inflammation, our data demonstrated that microglia cell function as immunological cell in CNS.3. During the systemic inflammation, ERK participated in the cell signal transduction in spinal cord, which almost expressed in microglia cell. And its increased physiological function in microglia indicated that ERK signaling pathway-induced microglia activation plays a key role in inflammatory processes.4. After intrathecal injection of LPS, TNF-αand pERK both expressed in microglia in the injected side of rat spinal cord, which suggested that ERK signaling pathway was involved in the secretion of TNF-α, and microglia cells play a key role in this process.