Neuroprotective Effects Of Potassium2-(1-hydroxypentyl)-benzoate And It’s Effects On Transgenic Mice Of AD

Potassium2-(1-hydroxypentyl)-benzoate (dl-PHPB), derivated from3-n-butylphthalide (dl-NBP), was a novel compound synthesized by Institute of Materia Medica. Previous studies showed that dl-PHPB reduced infarct volume and improved regional cerebral blood in cerebral ischemic rat model. Furthermore, it has been shown that dl-PHPB significantly improved the learning and memory deficits in the cerebral hypoperfused rats, AP25-35intracerebroventricularly-infused rats and the senescence-accelerated mice (SAM). The aims of the present study were to evaluate the neuroprotective effects of dl-PHPB and to investigate its possible anti-AD mechanisms.Part I. The anti-apoptotic effect of potassium2-(1-hydroxypentyl)-benzoate on hydrogen peroxide damaged SK-N-SH cellsApoptosis, an autonomous, programmed cell death, can occur in physiological and pathological conditions. Recently, it has become more clearly that apoptosis may play a pivotal role in the pathogenesis of stroke and neurodegenerative diseases. Meanwhile oxidative stress is one of the main reasons of neuronal apoptosis. Thus, the therapeutic drugs of inhibiting oxidative stress-induced neuronal death appear to be promising in the treatment of neurodegeneratice disease and stroke. The aims of the present study were to evaluate the neuroprotective effects of dl-PHPB on H2O2-induced apoptosis and to investigate the anti-apoptosis mechanisms of dl-PHPB.Firstly, the effect of dl-PHPB on H2O2-induced cell death and apoptosis was evaluated. Cell viability was measured by MTT method. The results showed that150μM H2O2markedly decreased the cell viability. However, when the cells were preincubated with10μM dl-PHPB, H2O2-induced cell damage was significantly attenuated. In order to determine the cells undergoing the apoptosis following150μM H2O2treatment, cells were stained with Hoechst33342nuclear stain. In the control group, apoptotic neurons were only comprised3.6%of the total number of cells. After exposure to150μM H2O2for24h, the number of apoptotic cells was significantly increased, when the cells were preincubated with0.1μM,1μM and10μM dl-PHPB, H2O2-induced cell apoptosis was concentration-dependently attenuated.To gain insight into the anti-apoptotic mechanism of dl-PHPB, the expression of apoptosis-related gene was measured. Real-Time PCR results showed H2O2treatment significantly reduced the expression level of Bcl-2and increased the gene expression of Bax. The pretreatment of dl-PHPB at10μM significantly prevented H2O2-induced decrease of Bcl-2and increase of Bax level. At the meantime, western blot was used to measure the protein expressions levels of Bax, Bcl-2, Caspase3and PKCa. Compared with control group, after exposure to150μM H2O2for24h, the expression of Bax and cleaved caspase3were obviously increased, dl-PHPB significantly decreased the expression levels of Bax and active form caspase3at10uM. Treatment with150μM H2O2significantly reduced the protein expression of Bcl-2and PKCa, dl-PHPB partly reversed the effects of H2O2on Bcl-2and PKCa expression.In conclusion, the results indicated that dl-PHPB exhibited significant protective effects against H2N2-induced apoptosis in human neuroblastoma SK-N-SH cells, and provided evidence that the neuroprotective effects of dl-PHPB were mediated by gene and protein regulation of the Bcl-2-related family, which possibly partly through upstream PKCa signaling pathway. Part â…¡. The mechanism study of potassium2-(1-hydroxypentyl)-benzoate on anti-Alzheimer’s disease in APP/PS1transgenic miceExtracellular senile plaques composed of β-amyloid protein, as well as the intracellular neurofibrillary tangles composed of hyperphosphorylated tau protein, are the two major pathological features of Alzheimer’s disease. In this study, we investigated the effects of dl-PHPB on Tau protein, β-amyloid protein and the related regulation enzyme expression in cortex and hippocampus of APP/PS1trangenic mice.In this study, western blot was used to measure the protein expression. Compared with wild-type mice, the expression of phosphorylated Tau ptotein at the sites of Ser396and Thr205were significantly elevalted in cortex and hippocampus of transgenic mice with15months of age. Treatment with30mg/kg dl-PHPB markedly reduced the expression levels of phosphorylated Tau ptotein at the sites of Ser396and Thr205. In the cortex, no significant difference in the protein expression level of P-Tau (S199) was revealed between wild-type mice and transgenic mice. Compared with wild-type mice, the protein expression of P-Tau (S199) site was significantly increased in hippocampus of transgenic mice, dl-PHPB significantly inhibited the protein expression of P-Tau (S199) site. No difference of phophorylated Tau protein at the site of Ser404could be found in cortex and hippocampus among the groups.In order to understand the mechanism involved in the regulation of phosphorylation of Tau protein of dl-PHPB, protein expression of protein kinase GSK-3β, CDK5and protein phosphatase PP2A, which regulate the phosphorylation of Tau protein, was measured. Compared with wild-type mice, the protein expression level of P-GSK-3β (Y216) site was significantly increased in hippocampus of transgenic mice, dl-PHPB significantly reduced its expression. The protein expression of P-CDK5(S159) sites in cortex and hippocampus of transgenic mice were significantly higher than those in wild-type mice, dl-PHPB significantly reduced protein expression of P-CDK5(S159) in cortex and hippocampus of transgenic mice. No difference was seen in the levels of total-GSK-3β, total-CDK5, P-GSK-3β (S9), P-GSK-3α (Y279) and PP2A in cortex and hippocampus among different groups.Immunohistochemistry results showed that there were a large number of β-amyloid deposition in cortex and hippocampus of transgenic mice, dl-PHPB did not show significant change on β-amyloid levels. Compared to wild-type mice, TBS soluble, TBST soluble and Guanidine soluble A(31-42in cortex and hippocampus of transgenic mice significantly increased,dl-PHPB has no effect on the levels of three forms of Aβ1-42.Compared with wild-type mice, the expression of P-APP (T668) was significantly elevated in cortex of APP/PS1trangenic mice, treatment with dl-PHPB significantly reduced P-APP (T668) expression level. The protein expression of APP, a-APP, ADAM17and ADAM10did not show significant difference among groups.Our study suggested that dl-PHPB could regulate the expression of phosphorylated Tau protein and reduce the expression of phosphorylated APP, which may be associated with anti-Alzhimer’s disease effect of dl-PHPB. GSK-3β and CDK5possibly involved in the regulation effect of dl-PHPB on phosphorylation of Tau protein.Part â…¢. The regulation effect of Tau protein phosphorylation of potassium2-(1-hydroxypentyl)-benzoate in neuroblastoma SK-N-SH cells over-expressing wild type human Amyloid precursor protein695Neuronal toxicity induced by Tau protein phosphorylation is considered as an important pathological mechanism of Alzheimer’s disease. In this study, the protein expression levels of phosphorylated Tau protein at the site of Ser396, Thr205and Serl99were measured in neuroblastoma SK-N-SH cells over-expressing wild type human Amyloid precursor protein695, and the effects of dl-PHPB on these three kinds of phosphorylated Tau protein expression were evaluated. In order to understand the anti-Alzheimer’s disease mechanism of dl-PHPB, the expression of protein kinase GSK-3â'‰and CDK5was measured, which regulated the phosphorylation of Tau protein.Western blot showed that the preotein expression of phosphorylated Tau protein at the site of Ser396, Thr205and Serl99in SK-N-SH APP695cells were higher than that in wild-type cells, treatment with dl-PHPB significantly reduced the proteins expression levels of P-Ser(396), P-(Thr205) in SK-N-SH APP695cells, but no difference was seen in the level of P-Tau(S199).The results showed that1μM dl-PHPB significantly increased the protein expression of P-GSK-3β (S9) in SK-N-SH APP695cells. Furthermore, at the dose of0.1μM,1μM and10μM, dl-PHPB markedly reduced the protein expression of P-GSK-3β (Y216), P-GSK3α (Y279) and P-CDK5(S159) in SK-N-SH APP695cells. No significant difference in the levels of total GSK-3β and CDK5was revealed in SK-N-SH APP695cells treated with dl-PHPB compared with vehicle treated SK-N-SH APP695cells.Our studies demonstrated that dl-PHPB inhibited the phosphorylation of Tau protein by the regulation of GSK-3β and CDK5phosphorylation levels, which may be associated with anti-Alzhimer’s disease effect of dl-PHPB.