A total of 240 wild-type AB strain zebrafishes were randomly selected at 1 dpf, and 30 zebrafishes per well were plated in 6-well plates. Control and model groups were established as described above. Five different concentrations of 3 mL water-soluble Triphala (0.123, 0.370, 1.11, 3.33, and 10.0 μg/mL) were applied to treat the 10 mM acrylamide-induced fishes at the same time. Glutathione (GSH) powder (SLCF2362; Sigma-Aldrich, Shanghai, China) was diluted into 615 μg/mL, and 3 mL GSH was used to stimulate the acrylamide-induced fishes together, regarded as a positive control group to compare the effects of Triphala. Then, the eight groups of fishes were incubated at 28°C for 24 h. Then, the fishes were stained with acridine orange (AO) (494-38-2, Sigma, China). After that, ten zebrafishes from each group were randomly selected, which were observed and imaged using a fluorescence microscope (VertA1; Shanghai Tucson Vision Technology Co., China). The mean fluorescence intensity was analyzed using NIS-Elements D 3.20 image software. The statistical analysis of the fluorescence intensity was used to evaluate the neuroprotective efficacy of Triphala.

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