Enzyme-digested Colla Corii Asini (E’jiao) prevents hydrogen peroxide-induced cell death and accelerates amyloid beta clearance in neuronal-like PC12 cells

As an aging-associated degenerative disease, Alzheimer's disease is characterized by the deposition of amyloid beta (Aβ), oxidative stress, inflammation, dysfunction and loss of cholinergic neurons. Colla Corii Asini (CCA) is a traditional Chinese medicine which has been used for feebleness-related diseases and anti-aging. CCA might delay aging-induced degenerative changes in neurons. In the present study, we evaluated antioxidant activity, cytoprotective effects, and Aβ removability of enzyme-digested Colla Corii Asini (CCAD). Oxygen radical absorbance capacity (ORAC) activity assay showed that, as compared to gelatins from the skin of porcine, bovine and cold water fish, CCA exhibited the highest ORAC activity. The ORAC activity of CCA and CCAD was increased gradually by the length of time in storage. Ultrastructure analysis by scanning electron microscopy showed that among CCA manufactured in 2008, 2013, 2017 and gelatin from cold water fish skin, CCA manufactured in 2008 presented the smoothest surface structure. We further tested the protective effects of CCAD (manufactured in 2008) and enzyme-digested gelatin from cold water fish skin (FGD) on hydrogen peroxide (H₂O₂)-induced cell death in nerve growth factor-differentiated neuronal-like PC12 cells. Presto blue assay showed that both FGD and CCAD at 0.5 mg/mL increased cell viability in H₂O₂-treated neuronal-like PC12 cells. The protection of CCAD was significantly superior to that of FGD. Acetylcholinesterase (AchE) assay showed that both FGD and CCAD inhibited AchE activity in nerve growth factor-differentiated neuronal-like PC12 cells to 89.1% and 74.5% of that in non-treated cells, respectively. The data suggest that CCAD might be able to increase the neurotransmitter acetylcholine. Although CCAD inhibited AchE activity in neuronal-like PC12 cells, CCAD prevented H₂O₂-induced abnormal deterioration of AchE. ELISA and neprilysin activity assay results indicated that CCAD reduced amyloid beta accumulation and increased neprilysin activity in Aβ_1-42-treated neuronal-like PC12 cells, suggesting that CCAD can enhance Aβ clearance. Our results suggest that CCA might be useful for preventing and treating Alzheimer's disease.