Experimental Research Of The Three Kinds Of Natural Products’ Intervention On Cytotoxicity Of PC12 Cells Caused By Aβ_1-40

Objective: Alzheimer’s disease（AD）is a neurodegenerative disease characterized by cognitive dysfunction.It is characterized by amyloid β-protein（Aβ）deposition and neurofibrillary tangles in the brain.Proanthocyanidins,flavonoid and saponin of Boussingaultia gracilis are natural plant extract products,which have antioxidant effect and a protective effect on the nervous system.Mitochondria are the cell energy producers,the main place for aerobic respiration.Mitochondrial damage is closely related with the pathogenesis of AD.Lysosome is the digestive organ which can swallow macromolecules,aging organelles,pathogens and other substances.It is closely associated with the lysosomal autophagy.Studies preformed on AD patients and AD mice suggested that the neurons haveoxidative stress damage and accumulation of autophagosomes which may cause AD.In neurons,nuclear factor-E2-related factor 2（Nrf2）plays a vital role in anti-oxidative stress and regulating cell autophagy by regulating mitochondrial function.In this study,we aim to explore whether the three natural products have protective effect on the PC12 neurotoxicity induced by Aβ_1-40 and the underlying mechanisms.Methods: PC12 cells were selected as test system.MTT was used to detect the toxic effects of Aβ and the three natural products on PC12 cells.Fluorescence microscopy was used to observe the intracellular ROS levels,lysosomal membrane stability and mitochondrial membrane potential.We used Transmission electron microscope（TEM）to observe the autophagosomes.Western blot was used to detect the intracellular protein level of microtubules associated protein light chain 3（LC3）,nuclear factor-E2-related factor 2（Nrf2）,substrate protein p62.SPSS 17.0 statistical software was used for statistical analysis.Results: After treated with Aβ_1-40 24 hours,the cell activity of Aβ treatment group was significantly decreased compared with the control group,and the cell activity of the proanthocyanidins group,flavonoid group and saponin group were significantly increased compared with Aβ treatment group.The mitochondrial membrane potential of Aβ treatment group was significantly decreased compared with the control group,and the mitochondrial membrane potential of the proanthocyanidins group,flavonoid group and saponin group were significantly increased compared with Aβ treatment group.The DCF fluorescence intensity of Aβ treatment group was significantly increased compared with the control group,and the DCF fluorescence intensity of the proanthocyanidins group,flavonoid group and saponin group were significantly decreased compared with Aβ treatment group.The lysosomal membrane stability of Aβ treatment group was significantly decreased compared with the control group,and the lysosomal membrane stability of the proanthocyanidins group,flavonoid group and saponin group were significantly increased compared with Aβ treatment group.The nucleus Nrf2 protein expression of Aβ treatment group was no significant difference compared with the control group,the nuclear Nrf2 protein expression of proanthocyanidins group,flavonoid group,saponin group were significantly increased compared with Aβtreatment group.The LC3-IIprotein expression of Aβ treatment group was increased compared with the control group,the LC3-IIprotein expression of proanthocyanidins group and saponin group were decreased compared with Aβ treatment group,the LC3-II protein expression of flavonoid group had no significant difference compared with Aβ treatment group.Electron microscopy showed that,the autophagosomes of Aβ treatment group was increased compared with the control group,the autophagosomes of proanthocyanidins group and saponin group were decreased compared with Aβ treatment group,the autophagosomes of flavonoid group had no significant difference compared with Aβ treated group.The p62 protein expression of Aβ treatment group was increased compared with the control group,the p62 protein expression of proanthocyanidins group and saponin group were decreased compared with Aβ treatment group,the p62 protein expression of flavonoid group had no significant difference compared with Aβ treatment group.Conclusion: Proanthocyanidins,flavonoid and saponin of Boussingaultia gracilis can inhibit PC12 cell damage induced by Aβ_1-40.Proanthocyanidins,saponin of Boussingaultia gracilis can relieve the dysfunction of autophagosomes eliminate.Flavonoid of Boussingaultia gracilis has no effect on the dysfunction of autophagosomes eliminate.