Abstract: AIM: To investigate the effects and protective mechanism of Tianma Gouteng (TGY) against rotenone (Rot) damage in PC12 cells. METHODS: PC12 cells were treated with Rot to establish nerve injury model and cell survival rate was determined by MTT colorimetry to determine the optimal modeling concentration of Rot and effective intervention concentration of TGY. Lipid reactive oxygen species (ROS) were detected by flow cytometry and fluorescence intensity was observed by inverted fluorescence microscope. The biochemical methods were used to detect, superoxide dismutase (SOD), glutathione (GSH) levels and activity and the contents of malondialdehyde (MDA). Transmission electron microscopy observed morphological change of mitochondria and protein expression of glutathione peroxidase 4 (GPX4), long chain lipoyl-coa synthase 4 (ACSL4) and lysophospholipid cholinyltransferase 3 (LPCAT3) were detected by western blot. RESULTS: The survival rate of cells treated with 0.6 μmol/L Rot for 24 h was close to 50%(56.7%±9.9%). Pretreatment with TGY for 12 h could inhibit the damage of Rot. At the same time, the leakage rate of lactate dehydrogenase (LDH) was reduced in a dose-dependent manner. Lipid ROS content increased after the treatment of Rot, whereas pretreatment with TGY effectively reduced lipid ROS content, decreased MDA level and increased SOD activity and GSH level in damaged cells in cells damaged by Rot. Transmission electron microscopy showed that the mitochondria of PC12 cells were shrunk after the damage of 0.6 μmol/L Rot and the mitochondrial morphology of PC12 cells was improved to some extent after preprotection of TGY compared with normal group. Western blot results showed that TGY pretreatment could increase the expression of GPX4 and reduce the expression of ACSL4 and LPCAT3 after damage of Rot to a certain extent. CONCLUSION: TGY can improve nerve damage by up-regulating the expression of GPX4 protein, down-regulating the expression of ACSL4 and LPCAT3 protein to inhibit the oxidation of unsaturated fatty acids, reduce the level of lipid peroxidation and ROS content.

Key words: Tianma Gouteng Yin, rotenone, ferroptosis, lipid metabolism, oxidative stress, neurodegenerative disease