| Lead is a common environmental pollutant and even continual low dose lead exposure can give rise to damage of human bodies, especially influence on the nervous system. Children are especially sensitive to lead, and the accumulation of lead in nervous system can cause dysfunctions in learning and memory ability, cognitive impairment, stunted growth and so on. Many studies on lead induced damage in hippocampus were conducted by using rats in developmental phase.The theory of learning and memory is based on synaptic plasticity, and the change of the synaptic transmission plays a vital important role in synaptic plasticity. Neurotransmission is conducted mainly by exocytosis of neurotransmitters. Soluble NSF attachment protein receptor(SNARE) composites play an important role in the exocytosis of neurotransmitters, and they are mainly involved in regulating the presynaptic membrane fusion function. Synaptic vesicle membrane protein 2(vesicle- associated be protein 2, VAMP2) is an important part of SNARE complex, distributed in vesicle membrane, synaptic corpuscle related proteins with(synaptosomal associated protein of molecular mass 25 kd, SNAP25) and synaptic fusion protein syntaxin constitute SNARE protein, play an important role in the neurotransmitter release.Micro RNAs(mi RNA) are small noncoding RNAs, about 22 nucleotides in length, which binds to their target genes in transcription level to promote their degradation or inhibit their translation. In recent years, studies have shown that mi RNAs are involved in regulating functions of the nervous system and related diseases. Reports show that mi RNAs may be involved in maintaining normal brain functions, such as learning and memory, the differentiation of neurons, synaptic plasticity, and neuron generation, and so on. Many neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s disease, ataxia, Huntington’s disease and schizophrenia are related to mi RNAs. In our lab, we used mi RNA chips technology to investigate the expression of mi RNA after lead exposure, and found that mi R-34 b may be involved in the role of lead neurotoxicity, but the specific mechanism is not clear.In our study, we investigated the expression of mir-34 b and VAMP2 expression after lead exposure in vivo and in vitro, and how mir-34 b regulated the expression of VAMP2 in lead induced dysfunction in formation of neurotransmitters, Our study will help elucidate the molecular mechanisms of lead neurotoxicity. Objective:This research focused on the expression of mir-34 b and VAMP2 expression after lead exposure in vivo and in vitro, and the role of mir-34 b in regulating the expression of its target molecule VAMP2 in lead induced dysfunction in formation of neurotransmitters. This study will help elucidate the molecular mechanisms of lead neurotoxicity. Methods:1. Establishment of lead exposure in vivo models and in vitro models.(1) In vivo model: 21 SD male rats were randomly divided into 3 groups: control group, Pb exposure group were given 100 ppm or 300 ppm lead acetate in drinking water for 8 weeks.(2) In vitro model: PC12 cells and HT22 cells were treated with Pb(AC)2 at different concentrations 0, 5, 10 mmol/L.2. Using Morris water maze methods to test spatial learning and memory abilities in rats.3. The expression of mi R-34 b and vamp2 m RNA were determined by q RT-PCR. Western blot was used to detect the expression level of VAMP2 protein4. Using luciferase reporter gene system to test the effect of mi R-34 b and vamp2 m RNA 3 ’UTR on the stability of the region.5. Observing the effect on the VAMP2 m RNA and protein expression after inhibiting the expression of mi R-34 b in PC12 cells by RNAi technology,6. Using immune electron microscopy technology to detect the expression of synaptic vesicles VAMP2 membrane protein in rat hippocampal.7. Flow cytometry methods were used to detected VAMP2 protein expression level in PC12 cells.8. Using transmission electron microscopy technology to detect synaptic vesicles formation in rat hippocampal after lead exposure. Results:1. Lead exposure affected spatial learning and memory abilities in the developmental rats.Results of blood lead concentrations showed that blood lead levels increased significantly in a dose-dependent way in lead exposure group. Morris water mazes data showed that escape latency increased significantly after lead exposure(p< 0.05); After removal of platform, the time of going through the platform in lead exposure group was much less than the control group, and the difference has statistical significance(p < 0.05).2. Lead exposure influenced the expression of mi R-34bReal-time fluorescent quantitative q RT- PCR detection showed that lead exposure up-regulated mi R- 34 b expression in rat hippocampus, and 300 ppm lead group was much higher than 100 ppm dose exposure, and the difference is statistically significant(p < 0.01). In vitro, lead exposure also increased the expression of mi R-34 b in PC12 cells and HT22 cells, and the expression level increased with the increase of lead dose(p< 0.05).3. mi R-34 b regulated the expression of target molecules VAMP2By using bioinformatics method to analyze the target genes of mi R-34b(three target genes predicted website: Targetscan and pic Tar and mi RNA.org), we found that VAMP2 might be the target genes of mi R-34 b. Luciferase reporter gene system test showed that mi R-34 b could bind to the 3’UTR of vamp2. Real time-PCR and western blot were used to detect the protein expression of VAMP2 after treatment with mir-34 b inhibitor in vitro, and the results showed that down-regulated mir-34 b could increase the expression of VAMP2.4. Lead exposure reduced the formation of synaptic vesicle by mediating membrane protein VAMP2 and mi R-34 b.q RT-PCR and Western blot results showed that lead reduced the expression of VAMP2 in vitro and in vivo. Flow cytometry assay results displayed that lead reduced the protein level of VAMP2.Transmission Electron Microscopy detection results show that lead exposure in developmental phase could significantly reduce the number of synaptic vesicles in rat hippocampus neurons; Immune electron microscopy methods showed that synaptic structures in hippocampus areas reduced significantly after down-regulating VAMP2 protein expression. Conclusion:1. Lead exposure could affect the expression of mi R-34 b in hippocampus tissues, PC12 cells and HT22 cells.2. Mi R-34 b regulated the stability of synaptic vesicle membrane protein vamp2 m RNA.3. Lead exposure reduced the expression of VAMP2 and the formation of synaptic vesicles in hippocampus neurons by regulating the expression of mir-34 b. |
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