Abstract:

Objective: To discuss the changes of expressions of filamentous actin(F-actin),Calponin 3 and Rho-associated coiled-coil-containing protein kinase 2(ROCK2) in neurons of the epileptic mice induced by pilocarpine, and to study the relationship among RhoA/ROCK2 pathway activation and Calponin 3 and the depolymerization and rearrangement of F-actin.Methods: The cortical neurons from ICR neonatal mice were isolated by using papain digestion and were identified with immunohistochemistry staining using microtubule associated protein 2(MAP2) antibody on the 7th day.The neurons cultured for 7 d were divided into control and epilepsy groups. The neurons in control group were cultured in normol neurobasal-A medium, and the neurons in epilepsy groups were cultured in medium with final concentrations of 2,3,and 4 mmol·L^-1 of pilocarpine respectively which were replaced by normol neurobasal-A medium after 24 h. The neurons of mice in various groups were fixed at different time points after modeling, then immunohistochemistry and fluorescence staining were performed. The fluorescence intensity of F-actin in neurons was observed by phalloidine staining labeled by Alex-546, as well as the expressions and distributions of Calponin3, ROCK2 and p-ROCK2 in neurons were observed by immunohistochemical staining.Results: The results of light microscope observation and F-acitn fluorescence staining showed that the neurons in 2 mmol·L^-1 pilocarpine group had no obviously changes 24 h after modeling compared with control group;the F-actin deconstruction and neurite disappearance partly of neurons in 3 mmol·L^-1 pilocarpine group were found, which gradually restored in next 7 d after the culture medium was replaced by normal one;the severel and irreversibe damage of F-actin structure and neurite disappearance in 4 mmol·L^-1 pilocarpine group were found.The results of immunohistochemical staining showed that Calponin 3 and ROCK2 dispersively distributed in cytoplasm in control group;as for epilepsy model group(3 mmol·L^-1 pilocarpine),Calponin 3 and ROCK2 obviously aggregated beneath the cell membrane 1 d after modeling, and their expression levels were gradually increased with the prolongation of culture time. The expressions of p-ROCK2 in epileptic groups were significantly higher than that in control group, and they were increased with the prolongation of culture time.Conclusion: The neuron epilepsy models are successfully established by using 3 mmol·L^-1 pilocarpine. 3 mmol·L^-1 pilocarpine may activate ROCK2 to p-ROCK2,and up-regulate the expression levels of ROCK2 and p-ROCK2 in neurons, so it can promote the depolymerization of F-actin and result in neurite disappearance partly. p-ROCK2 may activate Calponin 3 phosphorylation and promote the F-actin reconstruction and the restoration of the morphology of epileptic neurons gradually.

Key words: epilepsy, calponin 3, Rho-associated coiled-coil-containing protein kinase 2, neurons, pilocarpine, filamentous actin