Study On Antidepressant Mechanism Of Huazhuo Jiedu Shugan Decoction Based On PI3K/AKT/FOXO3a Mediated Autophagy Regulating Microglia Phenotype

Depression is a common mental disease,the pathogenesis is not clear,treatment is also a difficult problem.Microglia activation is considered to be an important step in leading to various neurological diseases,including depression.The classical activated M1 microglia cells have the function of promoting inflammation,which can damage neurons.The activated M2 microglia cells have the functions of phagocytosis,anti-inflammatory and neurotrophic.Autophagy is closely related to the activation of M2 microglia.After the body is stimulated by stress,the peripheral immune system induces the cytokines to transport to the central nervous system,which stimulates the activation of microglia;on the other hand,activated microglia secretes a variety of inflammatory cytokines and other inflammatory media to damage the adjacent neurons.Autophagy of microglia regulates the production and secretion of cytokines,and can eliminate the pathogens accumulated and attacked,which is the key to achieve the best immune function.Fox O3 a signal transduction plays a key role in the regulation of autophagy of microglia,and is the key molecule of regulation of autophagy downstream of pi3k/akt.Lc3 b is a autophagy specific protein,which participates in and regulates the formation of autophagy,and is a marker protein of the reactive autophagy activity.ATG5 has been proved to be an important part of the regulation autophagy,and its silence completely suppresses autophagy.Huaturbid Jiedu Shugan prescription is a self-made prescription based on turbid poison theory and established by many years of clinical practice and scientific research experiments.The preliminary clinical and animal experiments of this research group fully prove that Huazhuo Jiedu Shugan decoction has clear antidepressant effect.The purpose of this study is to explore the role of microglia autophagy and its phenotype transformation in the pathogenesis of depression,to explore the possible pathogenesis of depression;on this basis,to explore the antidepressant mechanism of Huazhuo Jiedu Shugan decoction,and to provide a new basis for its scientific connotation of antidepressant.Part one The regulation of microglia phenotype by PI3 K / AKT/FOXO3 a mediated autophagy by Huazhuo Jiedu Shugan decoction can improve the depression behavior induced by CUMSObjective: The previous research of the group fully proved that huaturbid Jiedu Shugan recipe can improve the depression like behavior of CUMS mice.This part of the study studied the regulation of autophagy by PI3 K / AKT/FOXO3 a pathway,observed the phenotype transformation and inflammatory factor level of microglia,and explored the antidepressant behavior mechanism of Huazhuo Jiedu Shugan decoction on CUMS mice.Methods: Methods: 60 ICR male mice were divided into normal group,model group,fluoxetine group,Huazhuo Jiedu Shugan decoction high-dose group,Huazhuo Jiedu Shugan decoction low-dose group.Enzyme linked immunosorbent assay(ELISA)was used to detect the inflammatory factors IL-1 β and TNF α in the hippocampus of mice,and Western blot was used to detect the inflammatory reaction The expression of PI3 K / AKT/ FOXO3 a pathway related proteins and autophagy related proteins lc3 b and ATG5 were detected by Western blot(WB);FOXO3a was detected by immunofluorescence to observe nuclear translocation;Iba-1 was detected to observe microglia activation;real-time quantitative PCR(real-time PCR)was used to detect the expression of PI3 K / FOXO3 a pathway related proteins lc3 b and ATG5 The M1 phenotype marker(i NOS,IL-1 β)and M2 phenotype marker(Ym1 / 2,Arg-1)were detected by PCR and RT-PCR,and the damage of hippocampal neurons was observed by Nissl staining.Results: 1.The results of general conditions,sugar water preference test,tail suspension test and open field test of cums model mice were consistent with depression like behavior,and the model was successfully established.The water maze showed the decline of learning and memory ability.After drug treatment,depression like behavior and learning and memory ability were improved.2.ELISA showed that inflammatory factors increased in the hippocampus of cums mice,WB combined with immunofluorescence showed that PI3 K / Akt was inhibited,the downstream FOXO3 a was dephosphorylated,and nuclear translocation occurred.Autophagy related proteins lc3 b and ATG5 were enhanced.RT-PCR combined with immunofluorescence showed that small glial cells in the hippocampus mainly expressed M1 phenotype.After drug treatment,inflammatory factors decreased,PI3 K / Akt activated,FOXO3 a phosphorylated expression decreased,autophagy related protein expression decreased,microglia in hippocampus mainly expressed M2 phenotype.3.Nissl staining showed that the number of neurons in CA3 and DG area of hippocampus in cums group decreased,Nissl bodies decreased and neurons were damaged.After drug treatment,neurons were repaired.Summary: 1.CUMS can successfully induce depression like behavior in mice,accompanied by the decline of learning and memory ability.The mechanism may be that inhibition of PI3 K / Akt can promote the dephosphorylation of FOXO3 a,thus enhancing autophagy,regulating the transformation of microglia to M1 type,promoting inflammatory response,damaging neurons and causing depression like behavior.2.Huazhuo Jiedu Shugan decoction can inhibit excessive autophagy by activating PI3 K / Akt and promoting FOXO3 a phosphorylation,thus regulating the transformation of microglia to M2 type,alleviating inflammatory response,protecting neurons and improving depression like behavior induced by CUMS.Part two The regulation of microglia phenotype by PI3 K / AKT/FOXO3 a mediated autophagy by Huazhuo Jiedu Shugan decoction can improve the depression behavior induced by LPSObjective: Injection of lipopolysaccharide(LPS)can induce inflammatory response and make animals show depression like behavior.LPS can also activate microglia.LY294002 is a common inhibitor of PI3 K and autophagy.In this part,we studied the effect of Huazhuo Jiedu Shugan Recipe on the behavior of LPS stimulated mice,the regulation of PI3 K / AKT/FOXO3 a pathway on the autophagy of microglia,and the effect of autophagy on the phenotypic change of microglia and the change of inflammatory factors,so as to explore the mechanism of Huazhuo Jiedu Shugan decoction.Methods: 1.In vivo experiment: 60 ICR male mice were divided into normal group,model group,fluoxetine group,Huazhuo Jiedu Shugan decoction group,LY294002 inhibitor group.LPS model was established by intraperitoneal injection.The depression state of mice was tested by behavioral study.The expression of pi3k/foxo3 a pathway and autophagy related proteins lc3 b and ATG5 were detected by WB;Iba-1 was detected by immunofluorescence;M1 and M2 phenotype marker were detected by RT-PCR.2.In vitro experiment: mouse microglia(BV2)were divided into blank control group,model group,medicated serum experimental group,LY294002 inhibitor group and medicated serum control group.LPS model was established and intervened with medicated serum and LY294002.The inflammatory factors were detected by ELISA,the expression of PI3 K / AKT/FOXO3 a pathway related proteins and autophagy related proteins were detected by WB,and M1 and M 1 of microglia were detected by RT-PCR 2phenotype marker.Results: 1.The general situation of LPS model mice,the results of sugar preference test and tail suspension test were consistent with depression like behavior.The model was successfully established,and the depression like behavior was improved after drug treatment.2.In vivo ELISA showed that the levels of inflammatory factors in the hippocampus of LPS model mice were increased,WB showed that PI3 K / Akt was activated,FOXO3 a was phosphorylated,and lc3 b and ATG5 were decreased.RT-PCR combined with immunofluorescence showed that the hippocampal microglia mainly expressed M1 phenotype.After drug treatment,the levels of inflammatory factors decreased,PI3 K / Akt was inhibited,FOXO3 a was dephosphorylated,and the expressions of lc3 b and ATG5 were increased.The M2 phenotype was mainly expressed in the hippocampal microglia of the treatment group.In the inhibitor group,the levels of inflammatory factors in hippocampus were higher,PI3 K / Akt pathway was inhibited,FOXO3 a phosphorylation,lc3 b and ATG5 expression were decreased,and M1 phenotype was dominant in hippocampus microglia3.In vitro ELISA showed that the inflammatory factors of BV2 cells treated with LPS were increased,WB showed that PI3 K / Akt pathway was activated,FOXO3 a phosphorylation,lc3 b and ATG5 expression were decreased,RT-PCR showed that BV2 cells in LPS group mainly expressed M1 phenotype.In the drug containing serum group,the inflammatory factors of BV2 cells were decreased,PI3 K / Akt pathway was inhibited,FOXO3 a was dephosphorylated,and the expression of lc3 b and ATG5 was enhanced,mainly M2 phenotype.In the inhibitor group,inflammatory factors were increased,PI3 K / Akt pathway was inhibited,FOXO3 a phosphorylation,lc3 b and ATG5 expression were decreased,mainly M1 phenotype.Summary: 1.LPS can successfully induce depression like behavior in mice.The mechanism may be to activate PI3 K / Akt,promote FOXO3 a phosphorylation,inhibit autophagy,regulate the transformation of microglia to M1 type,promote inflammatory response and cause depression like behavior.2.Huazhuo Jiedu Shugan decoction can enhance autophagy by inhibiting PI3 K / Akt and promoting FOXO3 a dephosphorylation,thus regulating the transformation of microglia to M2 type,alleviating inflammatory response and improving LPS induced depression like behavior.3.Huazhuo Jiedu Shugan decoction can enhance the autophagy of microglia by inhibiting the dephosphorylation of FOXO3 a induced by PI3 K /Akt,regulate the transformation of microglia to M2 type,and alleviate the inflammatory response induced by LPS stimulation.Conclusions:1.Huazhuo Jiedu Shugan decoction can improve the depression like behavior induced by CUMS and LPS,promote the neuron recovery and improve the learning and memory ability of cums model mice.2.Huazhuo Jiedu Shugan decoction can regulate the autophagy level of microglia through PI3 K / AKT/ FOXO3 a pathway,thus regulating the conversion of microglia to M2 phenotype,alleviating central inflammatory response and improving depression like behavior induced by CUMS and LPS.