L-lactate Produces Antidepressant Effect By Reducing The Expression Of High Mobility Group Box-1

[Background]With the development and progress of society,the incidence of depression is increasing year by year,which has already caused a serious burden on society.The main means of treatment of depression are medication and psychological intervention.Recent studies have pointed out that exercise,as a healthy and effective antidepressant treatment,has attracted more and more attention.A study by Carrard et al.in 2018 found that peripheral injection of L-lactate(LA)has an antidepressant effect,and Lactate is a massively produced metabolic end product during exercise.Therefore,we imagine that lactate may be a modulator of exercise antidepressant.So what is the mechanism of lactate’s antidepressant effect? The pathogenesis of depression is still divergent,but many studies have reported that inflammation of the central nervous system may be one of the key causes of depression.Recently,reports on inflammation-induced depressive-like behaviors pointed out that high mobility group box-1(HMGB-1)is considered to be an important inflammatory molecule that can cause depression.HMGB-1 is a richly expressed and migratory nuclear protein.It is expressed in most cell types and is associated with many inflammatory diseases.Many studies have pointed out that after cells are stressed,HMGB-1 will be released from the nucleus to the extracellular environment in large quantities,and by binding to pattern recognition receptors(PRRs)of other cells,such as toll-like receptors,activates the occurrence of inflammatory response and participates in the pathogenesis of depression.Hippocampus is the most frequently studied brain area in depression research.The neurogenic hypothesis believes that major depressive disorder(MDD)is related to neuronal disorders in the adult hippocampus.Neurons perform the most basic functions in the central nervous system,such as signal transmission and network integration,and are the main targets of emotional memory disorders.Neurons are one of the cells that mainly express HMGB-1 in the central nervous system.Then,if neurons receive stress signals,will they induce inflammation in the central nervous system through HMGB-1? Microglia is one of the main cells involved in inflammation in the central nervous system.Various PRRs,such as TLR-4,are expressed on the cell membrane of microglia.When the body is stressed,these receptors can receive stress signals and activate a series of inflammatory responses,such as activating nuclear factor kappa-B(NF-κB)phosphorylation and inducing inflammasome NOD-like receptor protein 3(NLRP3),Apoptosis-associated speck-like protein containing CARD(ASC),Cysteinyl aspartate specific proteinase-1(Caspase-1).Or induce inflammatory factors interleukin-1β(Interleukin-1β,IL-1β)and tumor necrosis factor-α(Tumor necrosis factor,TNF-α)to increase.Ultimately these factors lead to the occurrence of depression.[Research purposes]1.Is HMGB-1 a regulatory factor in the occurrence of depression-like behaviors?2.Does neuron HMGB-1 play a regulatory role in the increase of inflammatory molecules in microglia?3.Does LA produce antidepressant effects by inhibiting the expression of HMGB-1?[Method]1.C57BL/6 mice were given different concentrations of lipopolysaccharide(LPS)(0.4,0.5,0.7,0.8 mg/kg)intraperitoneally to establish an LPS depression model.Depressive-like behavioral tests were performed on mice: tail suspension test(TST),forced swimming test(FST),Sucrose preference test(SPT)to verify whether the model was successfully constructed.And detect the expression of inflammatory indicators such as HMGB-1,TLR-4,P-NF-κB,NLRP3,ASC,Caspase-1 and IL-1β,TNF-α in the hippocampus of mice.2.To explore the effect of intraperitoneal administration of different concentrations(20,30,40 mg/kg)of HMGB-1 inhibitor Glycyrrhizic acid(GZA)on the behavior of LPS mouse depression model and the effect of hippocampal HMGB-1.3.To explore the effects of different concentrations of GZA on the expression of TLR-4,P-NF-κB,NLRP3,ASC,and Caspase-1 in the hippocampus of the LPS mouse depression model.4.Explore the effect of LPS on the expression and release of HMGB-1 after processing neurons.Subsequently,the neuron supernatant was collected to observe its effects on the expression of TLR-4,P-NF-κB,NLRP3,ASC,Caspase-1 protein and IL-1β and TNF-α in microglia.5.Culture primary neurons and microglia in vitro to explore the effect of GZA on the expression and release of HMGB-1 after LPS-treated neurons? Collect the neuron supernatant and observe its effect on the protein expression of TLR-4,P-NF-κB,NLRP3,ASC,and Caspase-1 in microglia.6.Explore whether HMGB-1 sh RNA interferes with the expression of neuron HMGB-1 and whether it affects the effect of LPS on neuron HMGB-1.Collect the neuronal supernatant to observe whether it can affect the expression of TLR-4,P-NF-κB,NLRP3,ASC,and Caspase-1 protein in microglia.7.Explore the effects of human recombinant HMGB-1(r-HMGB-1)on microglia TLR-4,P-NF-κB,NLRP3,ASC,Caspase-1 protein,IL-1β,and TNF-α.8.Explore the effect of LA on the behavior of LPS depression model,HMGB-1,TLR-4,P-NF-κB,NLRP3,ASC,Caspase-1 protein and IL-1β,TNF-α.9.Explore the effect of LA on the expression and release of HMGB-1 after LPS-treated neurons,collect neuronal supernatants,and observe whether it can affect microglia TLR-4,P-NF-κB,NLRP3,ASC,Caspase-1 Protein expression and IL-1β and TNF-α release.[Result]1.Intraperitoneal administration of different concentrations of LPS(0.5,0.7,0.8mg/kg)can induce depressive-like behavior in mice.And after 0.5 mg/kg LPS at different time points can induce hippocampal tissue HMGB-1,TLR-4,P-NF-κB,NLRP3,ASC,Caspase-1 and IL-1β,TNF-α expression increased(p<0.05).2.GZA pretreatment for three days significantly shortened the immobility time of mice in LPS depression model TST and FST,and increased the sucrose consumption rate of mice in SPT(p<0.05).And it reduced the increase of HMGB-1 protein expression in hippocampus induced by LPS(p<0.05).3.GZA pretreatment reduced the increase of TLR-4,P-NF-κB,NLRP3,ASC,Caspase-1 protein expression in hippocampus induced by LPS(p<0.05).4.LPS can induce an increase in the expression and release of HMGB-1 in neurons(p<0.05).The results of immunofluorescence showed that the distribution of HMGB-1 in the nucleus decreased and the distribution of HMGB-1 in the cytoplasm increased with the increase of the time of LPS processing neurons.And the supernatant after LPS treatment of neurons can induce the increase of TLR-4,NLRP3,P-NF-κB,ASC,and Caspase-1 protein expression in microglia(p<0.05).5.Both 25 and 50 ng/m L GZA can inhibit LPS-induced increase in neuronal HMGB-1 release(p<0.05).GZA at 50 ng/m L can inhibit the increase in LPS-induced neuronal HMGB-1 expression.As a result,the supernatant cannot induce the increase in microglia TLR-4,NLRP3,P-NF-κB,ASC,and Caspase-1and protein expression(p>0.05).6.HMGB-1 sh RNA can inhibit the increase in the expression and release of HMGB-1 in neurons induced by LPS.As a result,the supernatant could not induce the increase of TLR-4,P-NF-κB,NLRP3,ASC,and Caspase-1 protein expression in microglia(p>0.05).7.r-HMGB-1 treatment of microglia increased the protein expression of TLR-4,P-NF-κB,NLRP3,ASC,and Caspase-1(p<0.05).And the content of IL-1β and TNF-α in the supernatant increased(p<0.05).8.LA can significantly reduce the immobility time in the LPS depression model TST and FST(p<0.001),and significantly increase the sucrose preference rate(p<0.01).It also inhibited the expression of LPS depression model HMGB-1,TLR-4,P-NF-κB,NLRP3,ASC,Caspase-1(p<0.05)and inflammatory factors IL-1β and TNF-α(p<0.05).9.Different concentrations of LA can inhibit the increase of HMGB-1 concentration in neuron supernatant induced by LPS(p<0.05).We chose 30 m M LA for follow-up experiments.Western blot showed that 30 m M LA can inhibit the increase of HMGB-1 protein in neurons induced by LPS(p<0.05).10.LA can inhibit the supernatant of LPS-treated neurons to induce the increase of TLR-4,P-NF-κB,NLRP3,ASC,Caspase-1(p<0.05)and IL-1β and TNF-α in microglia(p<0.05).[Conclusion]1.At the animal level,LPS can induce depressive-like behavior in mice and the expression of inflammatory proteins such as HMGB-1.At the cellular level,LPS can induce neurons to release HMGB-1 and increase the expression of inflammatory proteins in microglia.2.At the animal level,reducing the expression of HMGB-1 can alleviate depressive-like behavior and the expression of inflammatory proteins in mice.At the cellular level,reducing the expression of HMGB-1 in neurons can inhibit the expression of inflammatory proteins in microglia.3.The use of r-HMGB-1 can induce an increase in the expression of inflammatory proteins in microglia,indicating that HMGB-1 can regulate the expression of these inflammatory proteins.4.At the animal level,LA can inhibit LPS-induced depressive-like behavior in mice and the expression of inflammatory proteins such as HMGB-1.At the cellular level,LA can inhibit LPS-induced neuron release of HMGB-1 and increase the expression of inflammatory proteins in microglia.