Screening of naturally occurring chemicals by enhancing Neprilysin activity and potential applications in treatment of Alzheimer′s disease

Objective To obtain potential chemicals useful for chemotherapy of Alzheimer′s disease(AD) by screening naturally occurring compounds which could increase the Neprilysin(NEP) activity.Methods Neuroblastoma cells were cultured(SK-N-SH cells) and pcDNA3.1-sweAPP was transfected into the SK-N-SH cells with LipofectamineTM2000,and G418 was used for screening.The stably transfected cells were confirmed with Western blot and enzyme linked immunosobent assay(ELISA). MTT was used to identify the optimal drug concentration and time.The effects of drugs on NEP activity were determined by the NEP peptidase assay,and the Aβ levels were determined by ELISA.Results Western blot results showed that SK-N-SH cells transfected with pcDNA3.1-sweAPP highly expressed APP proteins,and ELISA results showed that Aβ40 and Aβ42 in culture media were significantly increased respectively by 7.26 and 3.27 fold after transfection.NEP peptidase assay showed that tea polyphenols,ginsenoside Rb1 and Rg3 could significantly enhance the NEP activity by 117±2.8%,131±4.0% and 121±1.6% respectively.ELISA results showed that they could significantly decrease Aβ40 and Aβ42 in culture media of SK-N-SH cells by 82±0.78% and 85±2.21%,74±1.61% and 99±3.31% and 83±5.31% and 76±6.74% respectively.Conclusion The AD model cells were successfully constructed,and tea polyphenols,Ginsenoside Rb1 and Rg3 can effectively decrease Aβ40 and Aβ42 in culture media.Our findings offer potent theoretical supports for application of these drugs in treating AD.