The Role and Mechanism of Anisodamine in Alleviating Neuronal Damage by Regulating Oxidative Stress

CHEN Xuesong; SHI Xiaojing

doi:10.3969/j.issn.2096-305X.2025.04.005

Abstract：Objective To explore the protective mechanism of macelignan(MLN)against oxidative stress-induced damage in PC12 neuronal cells induced by hydrogen peroxide(H2O2).Methods The oxidative stress model of PC12 cells was induced by H2O2.The cells were divided into the Control group,the H2O2 group(150 μmol/L),and the H2O2+MLN group(10 μmol/L).Cell Counting Kit-8(CCK8)was used to determine the H2O2 induction concentration and the optimal administration concentration,and to detect the toxicity of MLN.The expression levels of malondialdehyde(MDA),superoxide dismutase(SOD),glutathione peroxidase(GSH-Px),and catalase(CAT)were detected by the kit.The DCFH-DA kit was used to detect the reactive oxygen species(ROS)levels in each group.The mitochondrial membrane potential kit JC-1 was used to detect the mitochondrial membrane potential in each group.Immunofluorescence was used to detect the apoptotic factor Cleaved-capase3.Western Blot was used to detect the expression levels of Cleaved-capase3,B lymphoma-2(Bcl-2),and Bcl-2 related x protein(Bax)in each group.Results The CCK8 results showed that the optimal administration concentration of MLN was 10 μmol/L at an H2O2 induction concentration of 150μmol/L.This concentration had no toxic effect on the cells.Compared with the Control group,the level of oxidative stress factor MDA in the H2O2 group was higher than that in the Control group,while the levels of SOD,GSH-Px,and CAT decreased.Compared with the H2O2 group,the MDA level in the MLN treatment group decreased,and the levels of SOD,GSH-Px,and CAT increased.The results of the ROS kit assay indicated that there was an excessive accumulation of ROS within the cells in the H2O2 group.After MLN treatment,the level of ROS in the cells significantly decreased.The JC-1 results showed that the mitochondrial membrane potential in the H2O2 group was lower than that in the Control group.After MLN intervention treatment,there was a tendency for the mitochondrial membrane potential to increase.The immunofluorescence results showed that the fluorescence intensity of Cleaved-capase3 in the H2O2 group was significantly higher than that in the MLN treatment group.The WB results indicated that the protein levels of Cleaved-capase3 and Bax increased in the H2O2 treatment group,while the protein level of Bcl-2 decreased.After MLN treatment,the protein levels of Cleaved-capase3 and Bax decreased,while the protein level of Bcl-2 increased,and all of these showed statistical significance(P＜0.05).Conclusion MLN regulates cellular oxidative stress to inhibit apoptosis and alleviate H2O2-induced neuronal damage in PC12 cells.