| Manganese is an essential trace element for human beings. It is an important component of a variety of enzymes and plays an important role in maintaining the body's physiological function and growth. However, excess manganese exposure can cause functional abnormalities which are like Parkinson's disease(PD). These changes are collectively known as manganese poisoning. Patients of manganese poisoning appear like those of Parkinson's disease changes, which include bradykinesia, tremor, rigidity and postural abnormalities. In rodent and primate animal models, excess manganese exposure can cause gathered manganese, ?- aminobutyric acid(GABA) and dopaminergic(DA) neurotransmission damage, glial cell activation in brains, ultimately resulting in reduced number of neurons. Application of manganese in the life of a very wide range, such as steel welding, fungal agents, agricultural fertilizers, batteries and so requires the presence of manganese, precisely because of the widespread use of manganese, manganese so that the possibility of human exposure greatly increased, excess manganese exposure on the body impact becomes an issue of concern. The application of manganese covers a very wide range, such as steel welding, fungal agents, agricultural fertilizers, manufacture of batteries and so on. It is because of the widespread use of it, the possibility of human manganese exposure greatly increases and the impacts of excess manganese exposure on bodies become an issue of concern.Regarding to the mechanism of manganese toxicity studies, some researchers focused on a direct toxic effect of manganese on neurons. They found that the manganese-induced neuronal oxidative stress and mitochondrial dysfunction could lead to neuronal apoptosis. But there are some other researchers' findings suggest that manganese-induced inflammatory responses could also affect neurons. After manganese exposure, nitric oxide(NO) increased in striatal and pallidal regions as well as chemokines(Ccl2, Cxcl2) and inflammatory cytokines(IL-1?)increased in striatal region, which all suggest that the neurotoxicity of manganese may be related to the various cytokines produced by glial cells.Microglia are the resident immune cells within the brain, accounting for 10%-15% of the total volume of the brain, mainly in the dense gray area, such as the hippocampus and the basal ganglia. Under normal physiological conditions, microglia are in a resting state, but can continuously stretch out to monitor the changes of the surroundings. In the face of external stimuli or pathological changes, microglia can be quickly activated microglia and release inflammatory factors, such as tumor necrosis factor(TNF)-?, interleukin(IL)-1?, IL-6, etc. In addition, in conjunction with the astrocytes, microglia are able to release reactive oxygen species(ROS), nitric oxide(NO) and prostaglandin(PG) E2. The previous studies of our lab have proved that manganese can induce microglial activation and thereby cause inflammatory respense.Tumor necrosis factor receptor factor(TRAF) 3 is a member of the TRAF family, mainly involved in TNF-? receptor family signaling pathway. Its structure is characterized by a twist in the C-terminal helix structure and a conserved TRAF domain. TRAF3 has multiple functions. The study for TRAF3 knockout mice found that such mice can't survive long after birth, which confirmed the important role of TRAF3 in maintaining normal growth and development and physiological functions. Later studies further proved that TRAF3 could regulate NF-?B signaling pathway and MAPK signaling pathway negatively as well as regulate the production of interferon positively. In recent years, a new study has found that TRAF3 might be able to affect the activity of calmodulin phosphatase. In summary, TRAF3 plays an important role in immune signaling pathway.Objective: Through the establishment of Mn exposure model of mice microglia cells, we discussed the effects of Mn on microglia cells' morphology and inflammatory response. Using microarray technology, we studied the influences of manganese exposure on gene expression profile in central nervous system and further investigated the molecular mechanisms of Mn induced inflammatory response. Our work has successfully provided an experimental basis for the study of manganese neurotoxicity mechanisms.Methods: 1. Establish Mn exposure microglia model; 2. Using MTT(MTT) method to draw cell growth curve and to determine the optimal seeding density; 3. Using immunofluorescence staining method to identify whether BV2 cell can represent microglial cell and to determine the effects of Mn exposure on its shape; 4. Using real-time quantitative PCR method to detect the changes of m RNAs of chemokines mcp-1 and cxcl1, inflammatory cytokine tnf-? and immune signaling pathway molecules traf3, lrrk2 and akr1b3 in BV2 cells after Mn exposure. 5. The enzyme-linked immunosorbent assay(ELISA) was used to determine the protein level of chemokine MCP1 and CXCL1 in microglia and to determine the secretion level of inflammatory cytokines TNF-? after Mn exposure; 6. Microarray was used to study the effects of Mn exposure on gene expression profiles in central nervous system. 7. Western Blot(Western Blot) was used to detect changes of TNF-? and TRAF3 in microglia after Mn exposure;8. Using cell transfection method, we detect the effects of TRAF3 high expression on the inflammatory response in microglia.Results: 1. Drawing BV2 cell growth curve and determining the optimal seeding density. BV2 cells were seeded in 96 well plates in different cell densities during the logarithmic growth phase. After cultured at different times, MTT solution and dimethyl sulfoxide(DMSO) were added. Then absorbance value(A) was measured by enzyme-linked immunosorbent assay to draw the cell growth curve and further to determines the optimum cell seeding density. 2. BV2 microglial cell morphological changes induced by exposure to Mn. Using immunofluorescence staining methods to detect the morphological changes of BV2 cells after Mn exposure and BV2 cells with such changes were counted. The results show that after Mn exposure the number of BV2 cells with morphological changes is much higher, and the difference can represent the statistical alteration(P<0.05). And the 50?M exposed group is higher than 100?M exposed group, and the difference can represent the statistical alteration(P <0.05). 3. Mn exposure changed the m RNA expression of chemokines mcp-1 and cxcl1 in BV2 cells. By q RT-PCR test, we find that compared with the control group, Mn exposure can cause chemokines tnf-? m RNA increases in BV2 cells, and the difference can represent the statistical alteration(P<0.05). And 100?M Mn exposure group is higher than 50?M Mn exposure group, and the difference can represent the statistical alteration(P<0.05). 4. Mn exposure increased the expression and secretion of inflammatory factor TNF-? in BV2 cells. By q RT-PCR test, we find that compared with the control group, Mn exposure can cause inflammatory cytokines tnf-? m RNA increases in BV2 cells, and the difference can represent the statistical alteration(P<0.05). And 100?M Mn exposure group is higher than 50?M Mn exposure group, and the difference can represent the statistical alteration(P<0.05). By Western Blot, we find that compared with the control group, Mn exposure can cause inflammatory cytokines TNF-? protein increases in BV2 cells, and the difference can represent the statistical alteration(P<0.05). And 100?M Mn exposure group is higher than 50?M Mn exposure group, and the difference can represent the statistical alteration(P <0.05). 5. Microarray showed that Mn exposure could affect the gene expression profile in nigra. After establishing the manganese poisoning rats model by stereotaxic injection method, we used Affymetrix Rat Gene 2.0 ST microarray to determine the changed genes, among which, 1107 genes were upregulated and 633 genes were downregulated compared with control group. The functions of these genes showed that many signaling pathways were involved in Mn poisoning, including neurotransmitters, neurodevelopment, signal transduction, neuro immunomodulation, material transport and metabolism. The fold change(FC) of TRAF3, which is an upstream regulator of TNF-?, is-4.56, which means TRAF3 may play an important role in the expression and secretion of TNF-? in BV2 cells. 6. The expression of TRAF3 reduced in BV2 cells after Mn exposure. The q RT-PCR test results showed that exposure to Mn can cause reduced m RNA expression of traf3 in BV2 cells and the difference can represent the statistical alteration(P <0.05). And 100?M Mn exposure group is lower than 50?M Mn exposure group, and the difference can represent the statistical alteration(P <0.05). The Western Blot test results showed that exposure to Mn can cause reduced protein expression of TRAF3 in BV2 cells and the difference can represent the statistical alteration(P <0.05). And 100?M Mn exposure group is lower than 50?M Mn exposure group, and the difference can represent the statistical alteration(P <0.05). 7. TRAF3 overexpression can affect the Mn-induced expression of chemokines in BV2 cells. TRAF3 was transfected into BV2 cells, so that it is highly expressed. Then q RT-PCR was used to determine the effects of TRAF3 overexpression in mcp-1 and cxcl1 expression in BV2 cells. The results showed that after TRAF3 overexpression, manganese-inducedmcp-1 and cxcl1 increases were rescued compared with the control group, and the difference can represent the statistical alteration(P <0.05). 8. TRAF3 overexpression can affect the Mn-induced expression and secretion of inflammatory factor TNF-? in BV2 cells. After transfecting TRAF3 overexpression plasmid, ELISA was used to determine the effects of TRAF3 overexpression in TNF-? secretion in BV2 cells. The results showed that after TRAF3 overexpression, manganese-induced TNF-? increases were rescued compared with the control group, and the difference can represent the statistical alteration(P <0.05).Conclusions: 1. Mn exposure can lead to morphological changes in BV2 cells, which are characterized by the changes from resting state to activating state. 2. Mn exposure increases the expression of chemokines MCP-1 and CXCL1, and increases the expression and secretion of cytokine TNF-?, indicating that Mn neurotoxicity may be related to the inflammatory response in microglia. 3. After Mn exposure, the expression of TRAF3 decreased and overexpression of TRAF3 can affect the expression of chemokines and expression and secretion of inflammatory factors, indicating that TRAF3 signal pathwasy may play a regular role in the inflammatory response in microglia. |
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