Inhibitory Effect And Mechanism Of Trilobatin On Beta-amyloid 25-35 Fragment-induced HT22 Cell Apoptosis

Objective:The present study was designed to explore the effect of trilobatin on beta-amyloid 25-35(Aβ_25-35)-induced HT22 cell apoptosis,and analyze its underlying mechanism.Methods:Cultured HT22 cells were randomly divided into blank control group（control）,control+TLB（50μM）group,model group(20μM Aβ_25-35)and Aβ_25-35+TLB（12.5μM,25μM,50μM）groups.The HT22 cells were treated with or without 12.5,25,and 50μmol/L TLB 20μmol/L Aβ_25-355-35 for 48 h.Thereafter,the effect of TLB on Aβ_25-35-induced the HT22 cells activity and cytotoxicity were determined by MTT assay and Lactate dehydrogenase（LDH）assay,respectively.Apoptosis of HT22 cells was detected by TUENL staining.ELISA assay and Mito-SOX staining were used to detect intracellular ROS and mitochondrial ROS content,respectively.Moreover,antioxidant enzyme activities were determined using ELISA assay,Furthermore,Bax,Bcl-2 and Sirt3protein expressions,cleaved caspase-3 level and p38 phosphorylation level were determined using Western blot analysis.Sirt3,caspase 3 and caspase 3/7 activities were detected according kits.Caspase 3 inhibitor Z-VAD-FMK was used to further validate the role of caspase 3 in TLB’s anti-Aβ-ls-induced apoptosis.Molecular docking was used to detect the binding energy between TLB and p38.Results:The results showed that the HT22 cell activity significantly decreased in the model group than that of control group（P<0.01）,and the LDH release significantly increase in in the model group than that of control group（P<0.01）.While,TLB significantly increased the HT22 cell activity and decreased the LDH release than those of model group.Moreover,the intracellular ROS and mitochondrial ROS content increased significantly,and the antioxidant enzyme activities decreased in model group than those of control group（P<0.01）.While,TLB not only decreased the intracellular ROS and mitochondrial ROS,but also and increased the antioxidant enzyme activities.In addition,p38 phosphorylation level,Bax protein expression,cleaved caspase-3 level and caspase 3/7activity significantly increased,and Bcl-2 and Sirt3 protein expression as well as Sirt3activity were significantly decreased in the model group than those of control group（P<0.01）.While,TLB reversed these change than those of model group.Furthermore,the cell viability and LDH release of Z-VAD-FMK and TLB treated group were increased and decreased,respectively,than those of Aβ_25-355-35 group or Aβ_25–35+TLB（50μM）group.Meanwhile,the binding energy of TLB with p38 was-6.05 kcal/mol,it may directly bound to p38 as evidenced by molecular docking.Conclusion:Under the current conditions,TLB effectively inhibits Aβ_25-35-HT22 cell apoptosis,and its mechanism is related to the regulation of ROS/p38/caspase 3 signaling pathway.