| Traumatic brain injury(TBI) is one common severe disease in neurosurgery with the high incidence in trauma. The mortality and morbidity of TBI are at the head of various types of trauma. In addition to the primary brain injury, the secondary brain injury including neural inflammation, free radicals accumulation, excitatory amino acid poisoning, mitochondrial dysfunction and calcium overload can also damage the brain structure. Studies from animals indicated that the neural inflammation in the acute phase of TBI develops rapidly and lasts for a long time, which play a vital double-edged sword role in the secondary brain injury. The majority of researches suggested that the neural infla mmation in acute phase of brain injury is not conducive to survival and repair of neural cells especially neurons and can lead to aggravated injury in neural cells and tissues. The effective regulation on the neural inflammation in acute phase of brain inj ury of TBI is a significant way on the intervention of neural injury of TBI. Traditional Chinese Medicine is a great treasure for our medical workers. Curcumin, a major component extracted from the rhiome Curcuma longa, has been known as a Chinese herbal medicine for centuries. Curcumin is an effective active ingredient purified from its roots out and has been extensively studied because of its wide biological activities such as anti-infection, anti-oxidant, anti-inflammatory, and anti-tumor properties. Modern medical researches confirmed that curcumin has a strong anti-inflammatory effect, can promote the repair damage of tissue inflammation. Researches related to nerve system damage has been involved in recent years. Few studies observed that curcumin has significantly effect on the protection of neural injury in TBI. However, the mechanism that curcumin has the regulation of neural inflammation in the acute phase of TBI remains unclear.Monocyte-macrophage, the immune cells in peripheral circulation system, caused neural inflammations response due to enter injured brain through the blood-brain barrier. Moreover, the microglia, belong to monocyte-macrophage cell lines, clear away necrotic neural cellular debris in amoeba-like structure when stimulated by the endogenous stimuli releasing from injured neural cells. Inflammatory cytokines and cytokine released from microglia widely involve in the induction and regulation of neural immune and inflammatory response. Neuroinflammation regulation effect of curcumin on neurons is may mediated through the structure and function of microglia. Previous studies showed that toll-like receptors(TLRs) were widely expressed in the microglia membrane. TLRs recognize different pathogen-associated molecular patterns(PAMPs) like bacteria, fungus and virus through highly conserved microbial motifs, play an important role in the diseases infected by pathogen through the effect of innate immunity. In recent years, researche rs founded that many endogenous ligands released during tissue damage including the degradation products(heat shock proteins, polypeptide, nucleic acids, et al.) and intracellular components of ruptured cells, known as damage-associated molecular patterns(DAMPs), can also activate TLRs. And it is found that TLRS involved in recognition of DAMP and immune signal transduction by started noninfectious innate immune response. TLR4, the toll-like receptor subtypes, has been shown to play an important role in initiating the neural inflammatory response. There are two signal transduction pathways including myeloid differentiation factor 88(MyD88)-dependent and non-dependent pathways. In the MyD88-dependent signaling pathway, extracellular signal accepted from TLR4 leads to the activation of down-stream subsequent production of proinflammatory cytokines implicated in neurotoxicity such as interleukin-1 receptor associated kinases(IRAK), transforming growth factor-β activated kinase 1(TAK1). Following the phosphorylation degradation of inhibitor of nuclear factor-κB(IκB) causes NF-κB(particularly p65 subunit) is transferred from the cytoplasm to the nucleus, then starts classes of gene transcription and protein synthesis of inflammatory cytokines and participates in neural immune inflammatory response actively.In the present study, we used immunohistochemistry, western blot, transwell co-culture, enzyme-linked immunosorbent assay, FJB histochemistry and TUNEL staining for the examination. First, the neurological function and neuronal impairment as well as cytokine release were detected 24 hours following TBI in WT and TLR4-/- mice, which provided favorable evidence for the role of TLR4 in the acute inflammatory response stage of TBI and mediated neuronal death and function. Then curcumin injection intraperitoneally 15 minutes post-TBI was used to observe the neurological deficit and the changes of inflammatory mediators in the injured brain tissue 24 hours after TBI. The expression of TLR4 and related signal pathway in microglia also were detected post-TBI, which provided the role of TLR4 in the regulation of curcumin on the alleviation of excessive neural inflammation in the acute phase of TBI. At last, primary microglia and neurons co-culture was performed in vitro and administrated with bacterial lipopolysaccharide(LPS) and curcumin to observe the neuronal injury, morphology of microglia, expression of TLR4 in the membrane and changes of inflammatory mediators. It validates that curcumin alleviated excessive activation of microglia and reduced neuronal inflammatory injury via regulating microglial TLR4 signaling pathway, and investigates the possible mechanism.Main results:1. All mice were awaked fully in about 2-3 hours after TBI, and general activities and eating behavior were weakened. Head incision healed well in 24 hours post-TBI, no infection and no animal death were happened. Survival rate reached one hundred percent after operation.2. The expression of TLR4 was significantly increased in the injured tissue at six hours post-trauma. The expression further increased continuously and reached a maximum at 24 hours. Afterward, it decreased but also remained high at 72 hours post-trauma.3. Neurological function impairment and cerebral edema were observed obviously in both WT and TLR4-/- mice at 24 hours post-TBI. In TLR4-/- mice, the neurological deficit score and the brain water content of were significantly lower than that in WT mice at 24 hours post-trauma. In addition, both FJB-positive cells with neuronal morphology and TUNEL-positive cells were evident at 24 hours post-trauma in the injured tissue in both WT and TLR4-/- mice. However, significantly fewer TUNEL-positive cells were found in TLR4-/- mice than that in WT mice, and TLR4-/- mice also had significantly fewer FJB-positive neurons in the injured tissue when compared with WT mice.4. The level of inflammatory mediators including IL- 6, IL-1 β, MCP-1, TNF- α and RANTES in the damaged brain tissue were significantly increased in the TBI groups when compared with the sham groups. These mediators were all dramatically decreased in TLR4-/- mice when compared with that in WT mice.5. Fifteen minutes post-TBI, different concentrations of curcumin was administrated to mice intraperitoneally. 100mg/kg curcumin significantly reduced TLR4 expression compared with TBI alone and 50 mg/kg curcumin administrated mice. 200 mg/kg curcumin also reduced TLR4 expression compared with TBI alone but had no significant difference compared with 100mg/kg curcumin.6. At 24 hours post-trauma, the neurological deficit scores and brain water content in 100mg/kg curcumin administrated mice were significantly lower than that in vehicle-treated mice. Moreover, the percentage of TUNEL-positive cells with DAPI positive cells and the FJB-positive neurons in curcumin administrated mice were significant decreased than that in invehicle-treated mice.7. At 24 hours post-trauma, the concentrations of inflammatory mediators including IL-1β, IL-6, TNF-α, MCP-1 and RANTES in the injured brain tissue were significantly increased in the curcumin-treated mice and vehicle-treated mice. However, these mediators were all dramatically decreased in curcumin-treated mice when compared with vehicle-treated mice.8. At 24 hours post-trauma, a large number of co-expression of TLR4-positive and CD11b-positive cells were observed in the injured brain tissue of vehicle-treated and curcumin-treated mice. These microglia exhibited robust TLR4 positive immunoreactivity. Many TLR4-activated microglias, CD11b-positive cells, had large cell bodies and thickened, short neurites post-truma. The number of TLR4-positive microglia in curcumin-treated mice was significant lower than vehicle-treated mice.9. TLR4, MyD88, p-IκB-α and NF-κB protein expression in the injured brain tissue were all increased dramatically post-TBI in both vehicle-treated mice and curcumin-treated mice when compared with the sham control groups. However, the expression of TLR4, MyD88, p-IκB-α and NF-κB protein in curcumin-treated group were significantly lower than vehicle-treated animals. The expression of IκB-α protein was significantly increased in curcumin-treated mice when compared with vehicle treated mice and decreased in the two TBI groups when compared with the two sham groups.10. The concentrations of 0.5, 1, 2, 5μM curcumin were administrated separately to primary cultured microglia and the cell viability in the different concentration treatment groups did not significantly differ from that in the control group. However, the administration of 10μM curcumin significantly reduced microglial viability when compared with the control group. Moreover, neuronal viability reduced significantly by both 5μM and 10μM curcumin treated when compared with the control group. Therefore, 2μM was the optimal concentration for the cells transwell co-culture system.11. In the single-culture neurons, many neuronal bodies and neurites were destroyed or can’t form network structure after LPS administration. When the cells were treated with curcumin after LPS stimulation, a slight improvement of damaged structure was observed when compared with vehicle treated group, and the change was not significant. In the co-culture group more serious neuronal damage was observed than that in the single-culture group. When the cells were treated with curcumin after LPS stimulation, serious neuronal damage was improved obviously in the co-culture groups.12. In single-culture neurons, the caspase-3 protein level was significantly increased after LPS stimulation, and curcumin treatment after LPS stimulation did not significantly change the elevated caspase-3. The protein level was significantly higher in co-cultured neurons than that in the single-culture cells, and curcumin treatment after LPS administration significantly decreased the elevated of caspase-3.13. LPS stimulation induced numerous of co-expression of TLR4-positive and CD11b-positive microglia and these microglia also showed robust TLR4 immunofluorescence intensity, which was demonstrated a larger cell body and thickened, shorter neurites,. When cells were treated with curcumin after LPS stimulation, majority of co-expression of microglia were also observed but lower TLR4 immunofluorescence intensity were detected. The larger cell body and thickened, shorter neurites were ameliorated significantly.14. In the transwell co-culture cells, 24 hours after LPS stimulation induced the level of inflammatory mediators such as IL-1β, IL-6, TNF-α, MCP-1 and RANTES were all increased significantly, but only IL-6, IL-1β and RANTES were significantly decreased in the curcumin treated group than vehicle treated group.15. The expressions of TLR4, MyD88, p-IκB-α and NF-κB proteins, the molecules in the TLR4 downstream signaling pathways, were significantly increased after LPS stimulation and IκB-a protein expression was significantly decreased in contrast when compared with control groups. However, the upregulation of TLR4, MyD88, p-IκB-α and NF-κB proteins were significantly decreased significantly and the downregulation of IκB-a protein was also significantly decreased when curcumin administration than the vehicle-treated group after LPS stimulation.Main conclusions1. TLR4 has a key role in the acute neuroinflammation after traumatic brain injury.2. Effective modulation of microglial TLR4/MyD88/NF-κB signaling pathway is one of the mechniasm by which curcumin attenuates acute inflammatory injury.The present study illustrates preliminarily the relationship between neural inflammation response and neural injure in the acute phase of TBI. Further indicates that the regulated role of TLR4 on the neural inflammation and neural injure in the acute phase of TBI, which provides a new target for the exploration of TBI treatment. Moreover, this study demonstrates that the improvement of curcumin, major component extracted from Chinese herbal medicine Carcuma longa, on the neural injure in the acute phase of TBI at least is mediated partly by microglial TLR4 signaling pathway affecting neu ral inflammation response. The study expands the therapeutic range of curcumin, but also will provide new experimental evidences for the further investigation of possible pharmacological mechanisms of TBI treatment by curcumin. |
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