Cellular Signal Transduction Pathway And Molecular Mechanism Of Neurolathyrism

Grass pea(Lathyrus sativas L.) is an excellent arid conditions adapted plant resources. Long-term over consumption of Lathyrus seeds will cause human and animal spastic paraparesis which called neurolathyrism. Neurolathyrism is considerd as a neurodegeneration caused by β-N-oxalyl-L-α, β-diaminoprop ionic (P-ODAP), but its cell and molecular mechanism remains unclear. In this study, human glial cell line M059K was used as in vitro experimental system, set glutamate as control. Cells were treated by concentration gradient of β-ODAP which was extracted from Lathyrus seeds. The concentration of drug was determined by analysing the cell survival rate and growth dynamics. The flow cytometry, confocal microscopy, immunocyto-chemistry and transcriptome sequencing and other related technologies were used to track and detect the β-ODAP dynamics and its express access and network impact of changes on cells in cell proliferation ability, changes in cell morphology and cell cycle, cell death manner and DNA damage dynamic, mitochondrial membrane potential and changes of cytoskeleton, calcium homeostasis status and gene differential expression. Finally, the data were analyzed by classical mathematical statistics methods, microscopy demonstration and molecular expression path analysis.The results were listed as following:1. Cells were treated using 10 concentration gradients of β-ODAP (2-22 mM) and Glu to obtain the inhibition ratio from OD values of different groups according to MTT assay. By drawing the cell inhibition rate curve, we calculated IC50 to be 17.70 mM. In order to better simulate the process of toxicity, avoid the interference of Glu toxic, 10 mM concentration as applied dose were determined using for further experiments. At that moment the cell inhibition rate caused by P-ODAP was 24.6%, while Glu was 0.2%.2. After treated cell by P-ODAP, using FITC marked Annexin V as a probe detected by flow cytometry, there was no significant apoptosis occured. The proportions of apoptotic of cells under different treatment time were all less than 2%. There was no cell cycle arrest phenomenon under flow cytometry test; moreover, no obviously DNA damage was observed by DNA fragmentation detection immunohistochemistry. The results of cell colony formation assay showed β-ODAP could affect cell proliferation and significantly reduced colony formation.3. After treated cell by β-ODAP, Fluo-3-AM and Mito-Tracker M7512 were used to mark intercellular Ca2+ and mitochondria respectively. Changes were observed via immunohistochemistry assay. Results showed the concentration of Ca2+ continuously increased and absorbed by mitochondria from 6 hours to 24 hours after P-ODAP treatment, however, great amount of mitochondria were inactived in 24 h post treatment. Using Rho-123 as indicator, the measurement of the mitochondrial membrane potential by FCM showed continuous degradation of membrane potential caused by β-ODAP.4. After treated cell by P-ODAP, cytoskeleton was marked using Phalloidin, the results showed P-ODAP could cause cytoskeleton deform and collapsed, the fluorescence intensity was significantly reduced, F-actin polymers flip and fold, cytoskeleton separate from nucleus, and breaks in intracellular actin connection.5. Gene detection and expression result indicated that after treated by β-ODAP for 24 hours, changes of cell transcriptome were measured by transcriptome sequencing and real time PCR technology. The results showed 16 genes differential expression in the pathway of extracellular matrix and adhesion spots which include Integrin β1, Laminin, Fibronectin, Paxillin genes. At the same time, it caused some key disease genes upregulated in variety of other neurodegeneration pathway.6. We pictured the cell signal pathway and molecular mechanism of neurolathyrism. Above-mentioned that neither apoptosis nor necrosis caused by p-ODAP was the major way of cell death, β-ODAP could somehow bind or activated AMPA receptor, increased the expression of β1 integration in cell surface, further increased the phosphorylation of focal adhesion kinase (FAK). Large numbers of FA unit formation on the cell micro filaments, induce the over expression of Paxillin, affect cytoskeleton polymerization, ultimately lead to neuronal dystrophy. Otherwise, a great number of Ca2+ caused the destruction of intercellular calcium homeostasis, decrease of cell viability and proliferation rate, mitochondria excessive intake of Ca2+ also cause d changes in mitochondrial membrane potential, block ATP synthesis, increase ROS formation, further damage the biomembrane and lead to cell injury and death.In conclusion, the signal pathway of neurolathyrism was complicated, it was involved in multigene regulation of different levels and in different ways. In this study the glioma cells were used as in vitro system to explore the pathway and molecular mechanisms of neurolathyrism. However, limited to the research techniques, the P-ODAP molecule cannot be marked and located; parts of the results were in great need of perfection in further experiments.