| Alzheimer’s disease is degeneration of central nervous system, happened in the presenium and old age and feathered by progressive cognitive dysfunction and acts harm.The exact pathogenesis of AD was unclear, but the dominant position and mature mechanism was the unbalance about generation and clear of (3-amyloid protein. Previous studies have shown long-term use of statins decrease the risk of AD, non-cholesterol pathway is unclear. Studies have shown non steroidal anti inflammatory drug affected the location of RhoA in cell membrane and the activation of downstream protein kinase ROCK. Statins decreased the secretory of A(342through RhoA/ROCK pathway is unclear. Mevalonic acid is the promosa of the isoprenoid, literature suggested that extracellular with high cholesterol，low concentration of mevalonic acid could recover the road of isoprenoid.The extracellular with full cholesterol.we grouped the controls、statins、statins+mevalonic acid. then test the RhoA in membrane and the activity of ROCK.Extract and test the Aβ in extracellular fluid, to analyze the prevention of AD.Objective:Ensure the culture environment full of cholesterol, observe the effect of simvastatin on the production of beta amyloid peptide via RhoA/ROCK pathway.Methods: 1.BACE1-HEK293culture and experimental group:RIPM1640medium with100U/ml penicillin and100μg/ml streptomycin, showed10%fetal bovine serum full of cholesterol, cultured in37℃、5%co2incubator. BACE1-HEK293cells were divided into five groups:control group,1μmol/L Sim,1μmol/L Sim+250μmol/L mevalonic acid,5μmol/L Sim,5μmol/L Sim+250μmol/L Mev.2. MTT method:Identified the viability of the cells,5×103cells were seeded in96-well plates, when the cells grow to the density of70%-80%, as the above grouping and24h drug treatment. MTT added to every hole,37℃hatched4h,t hen discared the cell culture, added100μl DMSO to every hole, shaked10min, detected the absorptiomethy value at the550nm wave-length.3. COD-PAP method:As the cells grows to80%of the hole, use the isopropanol to pick up total cholesterol in cells. The total intracellular cholesterol/total protein in cells as the standard of cholesterol.4. ELISA method:Collect the supernatant1ml, added10μl PMSF, high-speed centrifugation at12000rpm4℃, then fetch50μl as the sample. ELISA method was employed to detect extracellular Aβ42.5. Western blot method:Western blot method was used to detect the expression of RhoA in membrane and Thr696phosphorylated myosin-binding subunit of myosin phosphatase (p-MYPT1) in cytoplasm.Results1.The viability of BACE1-HEK293cells didn’t show significant change in all treatment groups.2.Compared with the control group, simvastatin did not significantly reduced the intracellular cholesterol level at24h and48h with FBS-containing medium. 3.Compared with control group, the secreted Aβ42was decreased in1μmol/L and5μmol/L Sim treatment groups, added the250μmol/L Mev could perform antagonistic effect on the function of Sim.4.The expression of RhoA in membrane and Thr696phosphorylated myosin-binding subunit of myosin phosphatase in cytoplasm were both decreased in1μmol/L and5μmol/L Sim treatment groups. Added250μmol/L Mev could perform antagonistic effect on the function of Sim.Conclusion1. Statins decerased the location of RhoA anchoring into cellular membrance and p-MYPT1in cytoplasm in cholesterol-independent mechanism.2. Statins decreased the secreted Aβ42may have the relation with RhoA/ROCK.SignificanceThis topic experiment proved the statins influenced the metabolic pathway of A(3, may concerned with the RhoA/ROCK signal transduction pathway, further provide theoretical basis for therapy of AD. |
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