Part 2 Neuroprotective Effects Of Phenylethanoid Glycosides in An in Vitro Model Of Alzheimer's Disease
Mar 10, 2022
Is the neuroprotective effect of Cistanche real?
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Discussion
Oxidative stress is the major mechanism underlying Aβ-mediated neurotoxicity in AD (11-13). Therefore, targeting oxidative stress may represent an approach for the treatment of AD. In the present study, an in vitro model of AD comprising Aβ1-42- and H2O2-induced PC12 cell injury was successfully established. Results of the MTT, LDH, and MDA assays showed that PhGs increased the cell viability, and decreased LDH and MDA release by PC12 cells subjected to injury. It can be concluded that PhGs have significant neuroprotective effects on PC12 cells.
Cistanche has neuroprotective effects
In order to reduce the effects of PhGs themselves on PC12 cell growth and prevent abnormal proliferation, the safe dose of PhGs was determined in a screening assay. The results showed that PhGs at 75, 100, 125, 150, 175 and 200 µg/ml had a significant inhibitory effect on PC12 cells (P<0.05, P<0.01), while cell viability remained >80% at concentrations of 5, 25 and 50 µg/ml. Thus, PhGs at the concentration of 5, 25, and 50 µg/ml were safe for PC12 cells.
The injury by Aβ1-42 was affected by certain factors, including the solvent, incubation time, and product quality. In the present study, Aβ1-42 peptide was dissolved in water (100 µg/ml) and incubated at 37˚C for 4 days in a CO2 incubator prior to use. PC12 cells were treated with Aβ1-42 at concentrations of 0.5, 1, 1.5, and 2 µM. The results showed that the cell viability was decreased with the increase of Aβ1-42, and the viability was <50% in the 1, 1.5, and 2 µM Aβ1-42 injury groups. Thus, treatment of PC12 cells with 0.5 µM Aβ1-42 for 48 h was determined to be the optimal condition for establishing the AD model. Aβ25-35 has been commonly used to establish AD models due to its low cost and simple operation (27-29). The neurotoxicity of Aβ1-42 is significantly higher than that of Aβ25-35, and Aβ1-42 is, therefore, the optimal Aβ fragment for establishing an AD model (1,8-10).
H2O2 is an oxidizer and excessive H2O2 may cause oxidative damage and induce cell apoptosis (30). In the present study, PC12 cells were treated with 25-500 µM H2O2 dissolved in DMEM with or without PBS. The results showed that H2O2 dissolved in DMEM without PBS caused an abnormal proliferation of PC12 cells. Thus, treatment of PC12 cells with 25 µM H2O2 dissolved in DMEM with PBS was the optimal condition for establishing the AD model. The aβ1-42-induced injury was greater than H2O2-induced injury due to poor stability of H2O2 and solvent effects. When the cell is damaged, LDH leakage into the culture medium is significantly increased. ROS is known to cause the production of MDA. The content of MDA and LDH, therefore, reflect the amount of oxidative damage. In the present study, damage-induced LDH and MDA activity was decreased with increasing doses of PhGs. These results indicated that PhGs have a significant neuroprotective effect on PC12 cells. The MTT assay showed that PhGs exhibited a dose-dependent neuroprotective effect on PC12 cells.
In conclusion, an in vitro model of AD comprising Aβ1-42- and H2O2-induced PC12 cell injury was successfully established. Treatment with PhGs increased the cell viability and decreased LDH and MDA release by PC12 cells treated with Aβ1-42 or H2O2. PhGs had a significant neuroprotective effect on Aβ1-42- or H2O2-induced cell injury.
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