Monascus pigment rubropunctatin derivative FZU-H reduces Aβ(1-42)-induced neurotoxicity in Neuro-2A cells

Abstract

Alzheimer's disease (AD) is an extremely complex disease, characterized by several pathological features including oxidative stress and amyloid-β (Aβ) aggregation. Blockage of Aβ-induced injury has emerged as a potential therapeutic approach for AD. Our previous efforts resulted in the discovery of Monascus pigment rubropunctatin derivative FZU-H with potential neuroprotective effects. This novel lead compound significantly diminishes toxicity induced by Aβ(1-42) in Neuro-2A cells. Our further mechanism investigation revealed that FZU-H inhibited Aβ(1-42)-induced caspase-3 protein activation and the loss of mitochondrial membrane potential. In addition, treatment of FZU-H was proven to attenuate Aβ(1-42)-induced cell redox imbalance and Tau hyperphosphorylation which caused by okadaic acid in Neuro-2A cells. These results indicated that FZU-H shows promising neuroprotective effects for AD.

Fig. 1. Chemical structures of lovastatin, simvastatin, rubropunctatin and rubropunctatin derivative FZU-H.

Fig. 2. Effect of FZU-H on cell viability in Aβ(1-42)-induced cytotoxicity. Each bar represents the mean ± SD from six independent experiments (n = 6, *p < 0.05 and **p < 0.01 versus control group; ^#p < 0.05 versus the group treated Aβ(1-42) alone).

Fig. 3. Effect of FZU-H on morphological characteristics of Neuro-2A cells. Morphological studies by inverted microscope at actual magnification 400×; (A). Normal control group; (B). Aβ(1-42) treatment group; (C). Aβ(1-42) + 50 μM FZU-H treatment group. Morphological observation with AO/EB staining at actual magnification 100×: (D). Normal control group; (E). Aβ(1-42) treatment group; (F). Aβ(1-42) + 50 μM FZU-H treatment group.

Fig. 4. Effect of FZU-H on Aβ(1-42)-induced apoptosis by flow cytometry; (A). Normal control group; (B). Aβ(1-42) treatment group; (C). Aβ(1-42) + 10 μM FZU-H treatment group; (D). Aβ(1-42) + 50 μM FZU-H treatment group; (E). Aβ(1-42) + 100 μM FZU-H treatment group. This is a representative experiment of three independent experiments.

Fig. 5. Inhibitory effect of FZU-H on Aβ(1-42)-induced caspase-3 expression in cultured Neuro-2A cells. The cells were incubated with 10 μM Aβ(1-42) and different concentration of H (10, 50 and 100 μM) for 24 h. The relative activities of caspase-3 shown are calculated from the average of three experiments. Each value was expressed as the ratio of caspase-3 activation level to control level, and the value of control was set to 100 (n = 3, *p < 0.05 and **p < 0.01 compared with normal control group; ^#p < 0.05 and ^##p < 0.01 compared with Aβ(1-42) treatment group).

Fig. 6. Effect of FZU-H on Aβ(1-42)-induced ROS overproduction. After treatment of cells with different concentration of FZU-H and 10 μM Aβ(1-42) for 24 h, the cells were incubated with 10 μM DCFH-DA at 37 °C for 30 min and then washed twice with PBS. The fluorescence intensity of DCF was measured at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. Relative fluorescence intensity obtained from three independent experiments (n = 3, *p < 0.05 and **p < 0.01 compared with normal control group; ^#p < 0.05 and ^##p < 0.01 compared with Aβ(1-42) treatment group).

Fig. 7. Effects of FZU-H on the inhibition of Aβ(1-42)-induced oxidative stress (n = 3, *p < 0.05 and **p < 0.01 compared with normal control group; ^#p < 0.05 and ^##p < 0.01 compared with Aβ(1-42) treatment group).

Fig. 8. Effect of FZU-H on mitochondrial membrane potential. Mitochondrial transmembrane potential was measured by JC-1 staining. Epics XL flow cytometer at FL1 (529 nm) for green fluorescence and FL2 (590 nm) for red fluorescence (EX = 488 nm). The results were expressed as the mean ± standard deviation (SD) from three independent experiments (*p < 0.05 and **p < 0.01 compared with normal control group; #p < 0.05 and ^##p < 0.01 compared with Aβ(1-42) treatment group).

Fig. 9. Western blot analysis of the effects of OA and FZU-H on the expression of Tau in Neuro-2A cells. β-Actin was used as an internal control. The relative quantity obtained by Bio-Rad Image Lab software (n = 3, *p < 0.05 and **p < 0.01 compared with normal control group; ^#p < 0.05 and ^##p < 0.01 compared with Aβ(1-42) treatment group).