Effects And Mechanism Of Ox-LDL On Neural Cells Apoptosis Mediated By PCSK9/LRP1

Alzheimer's disease(AD) is one of the most common types of dementia, caused by a variety of factors and highly correlated with age. Progressive cognitive and memory damage in central nervous system is the major characteristic of AD. Neuronal apoptosis is one of its main pathological features. The incidence of AD is approximately 13% in individuals? 65 years and 45% in individuals?85 years of age. Classical pathological changes in AD include the following:(1) deposition of amyloid beta(A?) consisting of 39 to 43 amino acids forming extracellular senile plaques(SP);(2) excessive phosphorylation of the tau protein that shapes intracellular neurofibrillary tangles(NFT); and(3) neuronal loss accompanied by proliferation of astrocytes. Hyperlipidemia is a risk factor of AD, but the mechanism of hyperlipidemia and neurodegenerative diseases have not been fully clarified. Our researches focus on the effect and mechanism of neuronal apoptosis in hyperlipidemia and to explore the mechanism between hyperlipidemia and AD.A? and the expression of SREBP2,PCSK9 and LRP1 in PC12 cells Part?: Effect of Ox-LDL on lipid accumulations, apoptosis, secretion ofObjective: To indentify the effects of Ox-LDL on apoptosis change and the expression of SREBP2, PCSK9 and LRP1 in PC12 cells.Methods: PC12 cells were cultured in High glucose medium containing 10% fetal bovine serum. When the cells were grown to 70% in cell culture flasks, the medium was replaced with fresh fetal bovine serum-free medium containing different concentrations of Ox-LDL( 0mg/l?25mg/l?50mg/l?75mg/l?100mg/l) and then incubate for 24 h. We applied oil red O staining to detect lipid accumulations of PC12; Hoechst 33258 staining and flow cytometry were used to detect apoptosis change of PC12; Western blot were used to detect protein level of SREBP2, PCSK9 and LRP1, respectively. PC12 cells were cultured in High glucose medium containing 10% fetal bovine serum. When the cells were grown to 70% in cell culture flasks, the medium was replaced with fresh fetal bovine serum-free medium containing 75mg/l Ox-LDL for different times(0?6h?12h?24h?48h). Western blot were used to detect the protein levels of SREBP2, PCSK9 and LRP1 expressions.Results: The oil red O staining and Hematoxylin staining PC12 cells detected that PC12 cell lipid accumulation increased with the increase of Ox-LDL concentration, especially 75mg/l and 100mg/l groups. The Hoechst 33258 detected that PC12 cell apoptosis increased with the increase of Ox-LDL concentration, especially 75mg/l groups and 100mg/l. Western blot detected that Ox-LDL increased SREBP2 and PCSK9 and decreased LRP1 expression concentration and time dependently, especially 75mg/l groups and 24 hours.Summary: Lipid can induce PC12 cell apoptosis via increasing SREBP2 and PCSK9 expression and decreasing LRP1 expression.Part?:The mechanism of PCSK9 regulate apoptosis in PC12 cellObjective: To explore the molecular mechanism of PCSK9 regulate apoptosis in PC12 cell.Methods: Screening effective PCSK9 siRNA transfected into PC12 cell. After 48 hours transfected by PCSK9 si RNAs, PC12 cell was incubated for 24 hours with Ox-LDL. Hoechst 33258 staining and flow cytometry were used to detect apoptosis change of PC12. Western-blot was used to examine expression of PCSK9, LRP1, PI3 K, Akt, t- PI3 K, t-Akt, NF-?B, Bcl-2, Bax, Caspase9 and Caspase 3.Results: After transfection of PCSK9 si RNA for 48 h, PC12 cells were treated with Ox-LDL for 24 h, Hoechst 33258 staining and flow cytometer showed PCSK9 si RNA reduced apoptosis markedly; Moreover Western blot detected that the expression of Bax?Caspase9 and Caspase3 decreased while the expression of LRP1?Bcl-2?P-PI3 K and P-Akt increased.Summary: PCSK9 si RNA inhibited the decrease of LRP1 which was induced by 75mg/l Ox-LDL then to decrease PC12 apoptosis via activating PI3K/Akt and inhibiting NF-?B-Bcl-2/Bax-Caspase(9, 3) signaling pathways.Conclusions:Ox-LDL affects the different of the downstream signaling pathways by increasing the expression of PCSK9 to degrade the expression of LRP1. Ox-LDL plays a bidirectional regulation in the process of inducing neuronal apoptosis:1. To inhibit the activation of PI3K/Akt pathway, and inhibit PC12 cells to survive 2. To activate the NF-?B-Bcl-2/Bax-Caspase(3,9)pathway, and promote PC12 cells 3. To reduce A? secretion, and it may have the potential to inhibit A? induced by A? PC12 cells apoptosis...