Inflammation And Oxidative Stress-Based Approach To Investigate Ginsenoside Re Regulating Depression-Like Behaviors And Related Mechanism

Background:Depression is a common,highly fatal,and highly recurrent disease that imposes a significant burden on both medical care and finance to society.Synthetic medicines that have been reported to be used to treat depression have many side effects,such as headaches,nausea,and weight gain,but limited treatment efficacy.Therefore,it is crucial to develop a new type of antidepressant that is more natural,effective,and less toxic.In addition to depression-like behavior and BDNF dysfunction,depression is associated with elevated pro-inflammatory cytokines and increased environmental ROS.Ginsenoside Re is one of the main bioactive substances extracted from ginseng and has a protective effect to maintain cell redox balance on the nervous system.However,research on the antidepressant effect and molecular mechanism of ginsenoside Re has not been conducted.In order to investigate the effect and molecular mechanism of ginsenoside Re on depression,this study started with in vivo and in vitro experiments,combined with behavioral pharmacology,molecular biology and network pharmacology experiments to verify the anti-depression effect and mechanisms of ginsenoside Re comprehensively.Purpose:Based on the cytokine hypothesis of depression,this study utilized H₂O₂-induced oxidative stress neural cells as an in vitro model and reserpine-induced inflammatory depression mice as an in vivo model to comprehensively investigate the effects and molecular mechanisms of ginsenoside Re on depression,especially the protective role of ginsenoside Re during the oxidative stress process on the neurons via experimental methods such as behavioral pharmacology,molecular biology,and immunohistochemistry.The findings provide a new theoretical basis for elucidating the protective and antidepressant effects of ginsenoside Re,and promote the exploitation of new antidepressant drugs;and reveal the underlying mechanisms of depression.Methods:First,the antioxidant capacity of ginsenoside Re was tested in vitro to preliminarily identify its protecting ability for nerve cells.Subsequently,mouse neuronal cells HT22were stimulated with H₂O₂ to simulate the inflammatory environment in the brains of depression patients.The two groups,with and without ginsenoside Re pretreatment,were compared to comprehensively analyze the protective effect of ginsenoside Re on nerve cells in vitro.The BDNF/Trk B pathway was verified by Western Blot and RT-q PCR.The protective effects and the specific mechanism were thoroughly analyzed by cell proliferative ability,intracellular ROS level,cell apoptosis degree,cell lipid peroxidation level,mitochondrial membrane potential,and inflammatory cytokines expression.Next,the anti-depressent effect and neuronal protect of ginsenoside Re were evaluated in vivo.The a reserpine-induced inflammatory depression mice model was established,and those depressed mice were compared through the behavioral pharmacology experiments to determine the function of ginsenoside Re on the depression-like behavior induced by reserpine.Then,immunoblotting and immunofluorescence experiments were used to analyze the effect of ginsenoside Re on the expression of depression-and inflammation-related proteins in lipopolysaccharide-induced inflammatory depressed mice.Following,the mechanism of the antidepressant effect of ginsenoside Re was demonstrated by detecting the level of lipid peroxidation in the hippocampus,suggesting that the antidepressant effect of ginsenoside Re may be achieved through antioxidative and neuroprotective effects.Finally,based on the network pharmacology results and previous experiments,the mice were processed with the BDNF receptor inhibitor-K252a,and behavioral pharmacology experiments were performed to further investigate the mechanism of the antidepressant effect of ginsenoside Re,and the results were validated through Western Blot.Results:In the intracellular,it was verified that ginsenoside Re can significantly improve cell survival induced by H₂O₂-induced oxidative stress model,restore cell proliferation ability,reverse the mass production of oxidative stress markers（including ROS production and lipid peroxidation levels）and the occurrence of cell apoptosis（Caspase3 protein）.Moreover,it can reduce the expression of pro-inflammatory cytokines IL-1β,IL-6 and TNF-α,and restore the abnormal mitochondrial membrane potential,indicating that ginsenoside Re can mediate the normal operation of intracellular antioxidant enzyme metabolism system by restoring mitochondrial dysfunction,and finally produce the protective effect on nerve cells.The BDNF/Trk B/ERK/CREB pathway was successfully activated to play an antidepressant role.Subsequently,the anti-depressive effect of ginsenoside Re was verified at the animal level.We found that ginsenoside Re not only has a regulatory effect on neuroinflammation,but also affects the expression of BDNF in the brain.Ginsenoside Re has been preliminarily verified to play an antidepressant effect with fewer side effects by improving impaired neurogenesis in depression,inhibiting microglia overactivation and increasing BDNF signaling pathway.Ginsenoside Re therapy can play a neuroprotective and multitarget antidepressant role in reserpine-induced acute inflammatory depression in mice.Finally,Trk B was selected as an effective target of ginsenoside Re by network pharmacology combined with molecular docking,and Trk B inhibitor K252a was given to verify the important role of BDNF/Trk B in the anti-depression and anti-oxidation process of ginsenoside Re by behaviours experiment and Western Blot.Conclusion:1.The results of cellular and in vivo experiments have proved that Ginsenoside Re plays a neuroprotective and potential antidepressant role by reducing inflammatory factors,anti-apoptosis and promoting proliferation.2.Network pharmacology results revealed that ginsenoside Re and Trk B have strong binding force,which may activate the downstream pathway by directly acting on Trk B molecules.Combined with behavioral pharmacology and molecular experimental evidence prove that ginsenoside Re exerts neuroprotective and antidepressant role through Trk B/CREB/BDNF pathway.