The Molecular Mechanisms Of β-asarone Resisted To Synaptic Impairment By Targeting ROCK Pathway In Alzheimer’s Disease

ObjectiveAlzheimer’s disease is a neurodegenerative disorder, characterized by progressive dementia and companied with synapse impairment and loss, which be critical for learning and memory process, and defined histologically with the presence of the two hallmarks:extracellular senile plaques constituted with amyloid- β (A β)proteins and intracellular inclusions of hyperphosphorylated microtubule-associated tau protein, refered as neurofibrillary tangles. In the early stage of Alzheimer’s diseases, the learning and memory loss was directly connected with synaptic impairment in hippocampus. Amyloid-β, as a factor associated closely with AD pathological process, had confirmed the relationship with the ROCK pathway, when A 0 were higher expression in AD brain, which can enhance the expression of ROCK pathways to inhibit the synaptic plasticity, meanwhile, the overexpressions of ROCK pathway reversely promote A β anomaly concentration. Therefore, we consider the ROCK pathway may as a drug target for the treatment study in AD.Rhizoma acori graminei is an herb with the effect of dissipating dampness and harmonizing stomach, calm the nerves and benefiting memory, which the taste was characterized with acrid opening, bitter dryness of dampesss and wentong, and rhizoma acori graminei always be combination with other drugs to strengthen the expectorant work in treatment. In the previous study, we found that rhizoma acori graminei, as one of effective components, has a protective effect on the synaptic damage in AD, but the inhibition of A β 42 abnormal accumulation was not obvious, at the same time, the results found that it may be associated with ROCK expression, but the mechanism remains unclear. To explore the molecular mechanism of β-asrone targeted ROCK pathway for effection on synapstic damages in AD, we performed in two spects of AD animal and cell models.Methods1. To explore the protection of β-asarone on AD treatment, we choose 5-month-old APP/PS1 mice as the AD animal model, meanwhile take the wild type mice as blank control group, by using Morris water maze test to detect the behavior change, with gorky silver dye and Congo red staining to observe the morphology changes in AD and the effects of beta asarone on the pathological morphology in APP/PS1 mice;By using Western blot method to detect the expressions of synaptic-associated protein like the Synapsin protein 1 (SYN1) and postsynaptic density 95 (PSD95) in the hippocampus of APP/PS1 mice and observe the synapses protection of β-asarone; To observe the effect of β-asarone on the ROCK signaling pathways by detecting the expressions of ROCK pathways related protein (ROCK2, LIMK1).2. By the miRNA microassay technology, to screen out the special expression of microRNA in the hippocampus of APP/PS1 mice, and identify with the qRT-PCR methods, we can find the influence of β-asarone on miRNA and which may be associated with ROCK pathways.3. By constructing ROCK2 promoter luciferase reporter gene and transfecting PC12 cells, to observe the effect of β-asarone on ROCK2 promoter transcriptional activity when the cells was treated with Aβ25-35,.4. Using immunofluorescence and Western blot method to detect the expressions of nerve Growth Associated Protein Growth Associated Protein 43 (GAP43), synaptic SYN1 and Rho related Protein kinase 2 antibodies (ROCK2) in the model in AD cells for observing the protective effection of β-asarone on AD cells model and influence on the expression of ROCK2.5. To observe whether the effect of β-asarone be dependent of ROCK2 pathway with transfecting the pc12 cell with ROCK2-siRNA and ROCK2-overexpression plasmid.6. By the construction of a specific expression of microRNA mimics and inhibitors (like miR-335, miR-574), and with the bioinformatics prediction about them to detect the miRNA target, and also through the construction of ROCK2-3’UTR region containing binding sites of miR-335 dual luciferase reporter gene to further determine the function of microRNAs.Results1.β-asarone can effectively improved the spatial learning and memory ability in 5-months-old APP/PS1 mice.2. β-asarone prevented the synaptic-associated protein SYN1 and PSD95 loss, at the same time, can effectively reduce the expression of ROCK2 and LIMK1 protein in hippocampus of APP/PS1 mice.3. with the miRNA microassay analysis, compared with the APP/PS1 mice, there were 60 upregulated in the β-asarone group, and 53 downregulated. With qRT-PCR detection, compared with wild type mice, miR-335 was down-regulated in the miRNA expression of APP/PS1 mice, but miR-574 and miR-711 up-regulated, While β-asarone treated, the miRNA level of miR-335, miR-574 and miR-711 were effectively changed. With the bioinformatics prediction, miR-335, miR-574 and miR-711 were respectively base complementary with ROCK2, neuritin (Nrn1) and p21 activated kinase 2 antibodies (PAK2) mRNA.4. β-asarone can effectively down-regulated the transcriptional activity of ROCK2 promoter in PC12 cells which Aβ25-35 induced.5. β-asarone can increased the expressions of SYN1 and GAP43 protein in PC12 cells and original hippocampal neurons induced by A β 25-35, and inhibited the expression of ROCK2 protein, at the same time, with the transfection ROCK2-siRNA and ROCK2-overexpression plasmid, β-asarone has no significant effect on SYN1 in PC12 cells which ROCK-siRNA transfection, but the expression of SYN1 protein was changed obvious in the ROCK2 overexpression group.6. β-asarone inhibited the expression of ROCK2 mRNA by preventing the decrease of miR-335.Conclusionβ-asarone resisted the synaptic damage by inhibiting the expression of ROCK pathway in Alzheimer’s diseases, and prevented the decrease of miR-335 to enhance the inhibition of ROCK in its translation process.