Tenuifolin On D-Galactose Synergistically With Aβ_1-42 Induced Ameliorative Effect Of Alzheimer’s Disease-like Phenotype And Mechanism Study

Background:The risk of developing Alzheimer’s disease（AD）increases with age.The traditional Chinese herb polygala tenuifolia Willd has the function of tranquilizing and educating the mind.Tenuifolin（Ten）,as the main bioactive component of polygala tenuifolia,has the unique advantage of multi-pathway and multi-target,and the specific mechanism of the neuroprotective effect of tenuifolin to improve cognitive function is still unclear.Objective:In this study,we used D-galactose intraperitoneal injection synergistically with Aβ_1-42 hippocampal localization injection to induce AD mouse model,and D-galactose synergistically with Aβ_1-42 to induce HT-22 cell injury to establish AD cell model,aiming to investigate the effects of tenuifolin on AD in vivo and ex vivo models from multiple perspectives of synaptic injury,mitochondrial oxidative stress,calpain system,and ferroptosis,and by addition of calpain inhibitor calpeptin to determine the potential link between calpain and neuronal cell death patterns.Methods:In this study,we used D-galactose intraperitoneal injection in conjunction with Aβ_1-42 hippocampal localization injection to induce AD mouse model,and evaluated the effect of tenuifolin on learning memory of AD mice by behavioral experiments and Western Blot method and used D-galactose synergistically with Aβ_1-42to construct AD cell model to explore the action mechanism of tenuifolin.The MTT method was used to determine the optimal modeling concentration and the concentration of tenuifolin administration,and the mortality rate of HT-22 cells was detected by trypan blue dye exclusion assay;the senescence of HT-22 cells was detected byβ-galactosidase staining;the extent of mitochondrial damage in HT-22 cells was observed by Mito-Tracker fluorescence labeling;the mitochondria of HT-22 cells were detected by rhodamine 123 staining flow cytometry membrane potential changes;colorimetric assay to detect the alteration of ultra-trace total ATPase content in HT-22cells;microplate assay to detect LDH leakage level in HT-22 cells.The effects of tenuifolin on D-galactose synergistically with Aβ_1-42 induced proliferation,mitochondrial oxidative stress,calpain system and ferroptosis in hippocampal neuronal cells HT-22 by fluorescent staining,flow cytometry and Western Blot,and added calpeptin to inhibit the expression of calpain,and detected the effects of HT-22 cells ferroptosis related protein and synaptic related protein expression changes,as well as the effect of tenuifolin on HT-22 cell apoptosis using Annexin V-FITC/PI double-stained flow cytometry.Results:The results of animal experiments revealed that tenuifolin ameliorated D-galactose synergistically with Aβ_1-42 induced learning memory decline and synaptic impairment in mice.Further,the results of cellular experiments revealed that compared with the model group,tenuifolin（10,20,40μmol/L）also significantly increased D-galactose synergistically with Aβ_1-42 induced HT-22 cell survival and decreasedβ-galactosidase positive expression.Protective effect of tenuifolin on D-galactose synergistically with Aβ_1-42 induced HT-22 cell death and synaptic damage.In addition,D-galactose synergistically with Aβ_1-42 induced a decrease in the expression of calpastatin,ferroptosis-associated protein GPX4 and SLC7A11 in the hippocampus of mice and increased the expression of calpain and ferroptosis-associated protein ACSL4,however,all these changes were reversed after treatment with tenuifolin administration.By inhibiting D-galactose synergistically with Aβ_1-42 induced calcium overload in neuronal HT-22 and regulating the expression of m-calpain and calpastatin（which is the only intracellular protein that inhibits calpain activity）in the calpain system,tenuifolin increased D-galactose synergistically with Aβ_1-42 induced intracellular ATPase activity in HT-22,prevented the decrease of mitochondrial membrane potential,oxidative stress damage and ferroptosis.Interestingly,when the calpain inhibitor calpeptin was added it did not block the effect of tenuifolin on ferroptosis,but blocked D-galactose synergistically with Aβ_1-42 induced apoptosis and synaptic damage in HT-22 cells.Conclusion:These results suggest that tenuifolin has a protective effect on HT-22 cells damaged by D-galactose synergistically with Aβ_1-42 in a certain dose range,and its protective mechanism may be through inhibiting synaptic damage in hippocampal neurons,enhancing mitochondrial energy metabolism,preventing mitochondrial oxidative stress damage,preventing the occurrence of ferroptosis,maintaining the stability of the calpain system and inhibiting neuronal apoptosis.Thus,it improved the learning and memory function of mice.This finding points to the potential of a new therapeutic strategy for neurodegenerative disorders,including AD,with the use of tenuifolin.