High Glucose Induced Myosin Light Chain Kinase And Its Relationship To Microfilament Cytoskeleton In Hippocampal Neurons And Its Underlying Mechanism

Objective: Different factors and ultrastructures were observed under the hippocampal neuron morphology;Western blotting was used to detect the MLCK and p-MLC expression level. Then F-actins which were stained by immunofluorescence were observed under confocal laser scanning microscope after the primary culture of SD neonatal rats’ hippocampal neurons;All above was to investigate the correlation between MLCK and the actin cytoskeleton in cultured hippocampal neurons cultured under high glucose in vitro, and to explore the mechanism of the changes in the cognitive dysfunction of diabetic encephalopathy.Method: 1. Primary cultured neonatal rat hippocampal neurons which were observed by inverted phase contrast microscope every day. The activity of hippocampal neurons was detected by CCK-8 every 2 days, and the purity was detected by NSE immunohistochemistry. 2. The cells were divided into control group,high glucose group and ML-7 group. After 5 days, the high glucose group was cultured with 45mmol/L sugar concentration for 48 hours, the ML-7 group added 20mol/L MLCK inhibitor(ML-7)effect for 8 hours when the cells were cultured with45mmol/L sugar concentration for 48 hours. 3. The changes of the composition of F-actin in each group were observed under laser scanning confocal microscope, by using immunofluorescence staining technique. 5. The expression of MLCK and p-MLC levels were detected by western blotting in each group. 4. The ultrastructural changes of hippocampal neurons were observed by transmissionelectron microscope. Result: 1. Successfully cultured primary hippocampal neurons by Trypsin digestion. Culture until the 7th day, cells in the inverted microscope appear as: mature neuronal morphology, larger cell volume, aggregation of survival, obvious halo, developed neurite extended outwardly, mutually connected to form a dense network. After 7 days of culture, the activity of hippocampal neurons reached the highest, when was followed by a short period of plateau, and the activity of 11 d decreased gradually. The identification of the hippocampal neurons purity was more than 90%. 2. Compared with the control group, the synapsis forthe hippocampal neurons of high glucose group became shorten, and the ultrastructure of hippocampal neurons showed high glucose group cell membrane shrinkage, irregular nucleus,nuclear chromatin, uneven distribution of particles, agglutinate and margination. At the same time,the endoplasmic reticulums and mitochondrias appearred vesicle expansion, with autophagy phenomenon, and changes of microfilament cytoskeleton structure obviously. Compared with the high glucose group, ML-7 group had no obvious change in cellular and mitochondrias, autophagy rarely appearred in the cell,and there’s little change in endoplasmic reticulums and more abundant microfilaments.3. The expression of p-MLC and MLCK were up-regulated in high glucose group(P< 0.05) and the expression of p-MLC and MLCK in ML-7 group was lower than that in high glucose group(P < 0.05). 4. Immunofluorescence showed that the cytoskeletons of F-actins in high glucose group rearranged and cell morphologieswere abnormal, butthe cytoskeletons of ML-7 group had recoverred. Conclusion: 1. High glucose culture can made the expression of p-MLC and MLCK. 2. High glucose culture can change the morphology and ultrastructure of hippocampal neurons.3.The elevated expression of p-MLC and MLCK depolymerizes the F-actins, which also reconfigures neuronal cytoskeletons and lead to neuronal cell dysfunctions eventually.