| Based on our previous studies,we find that the traditional Chinese medicine formula Kai-Xin-San(KXS)significantly ameliorate depression-like behaviors of chronic unpredictable mild stress(CUMS)induced depression animal models.In studying literatures,we find that the cascade of Hypothalamus-Pituitary-Adrenal(HPA)axis stimulation significantly altering gut microenvironment and inducing central neuronal inflammation played an important role in pathogenesis of depression,which is summarized as regulation of"micro ecology-HPA-neuronal inflammation" axis.This hypothesis is also identified with TCM reorganization of depression,which is described as "deficiency of heart and spleen".Therefore,we developed CUMS depression mice model to evaluate the antidepressant effect of KXS based on regulating gut microbiota-stress-central neuronal inflammation system.Moreover,the anti-depression substance of KXS were screened on series of cell models.Afterwards,the screened substances were re-combined to be verified on CUMS animal model.The paper is divided into four chapters,and the main research content is summarized as follows:?.Review of previous studies on depression and KXS.1.In this part,etiology of depression was summarized.2.In this part,research of anti-depression mechanism of Chinese medicine formulae were summarized.3.In this part,history and anti-depression mechanism studies of KXS were introduced.?.Elucidating anti-depression mechanism of KXS on regulation of gut micro ecology-stress-central nervous inflammation system.1.Anti-depression efficacy evaluation of KXS.In this section,CUMS depression mice were used as animal model.A series of behavioral tests,such as sucrose preference test(SPT),forced swimming test(FST),tail suspension test(TST),and opening field test(OFT),were applied for evaluation of anti-depression effect of KXS with different compatibility ratios.2.The anti-depression mechanism of KXS in regulating gut micro ecology.In this section,the gut microbiota distribution was evaluated by 16sRNA technology by analyzing feces of animals.The inflammatory factors and gut barrier protein in the small intestine were determined by enzyme-linked immunosorbent assay(ELISA)and western blotting method.We found that KXS treatment could modify the disturbed microbiota distribution,downregulate the enhanced inflammatory factor expressions and upregulate the decreased gut barrier proteins.3.The anti-depression mechanism of KXS in regulating central neuronal inflammatory systemIn this section,expressions of inflammation cytokines were determined in brain and blood brain barrier proteins tissue and serum in model animals.KXS treatment could significantly decrease expressions of inflammation cytokines in brain tissues and serum.Meanwhile,the expressions of blood brain barrier proteins were also up-regulated by KXS treatment.Therefore,it seemed that the anti-depression effect of KXS was related to regulation of central neuronal inflammation system.4.The anti-depression mechanism of KXS in regulating the stress systemIn this section,the expression of stress factors in related organs of HPA axis of CUMS model animals were determined,including hypothalamus,adrenal gland,hippocampus and serum.The results showed that the enhanced expression of stress factors in organs and serum could all be down regulated by KXS treatment,which indicated that the anti-depression efficacy of KXS was related with HPA axis regulation.?.Validation of anti-depression efficacy of KXS in regulating gut micro ecology-stress-central inflammation system.In the animal experiment in this section,a mixed solution of antibiotics was added to animals'drinking water while the CUMS model was established.The antibiotic mix decreased the levels of intestinal bacteria in intestine of the animals.Then anti-depression efficacy of KXS treatment was impaired by antibiotics treatment,which implied that KXS exerted anti-depression effect via regulation of gut micro ecology-stress-central inflammation system.?.Screening the substance basis of anti-depression effects of KXS based on cell models.1.Screening of functional substances based on intestinal inflammation cell models.Mice RAW264.7 and IEC-6 cell lines were applied as cell models.The effect of KXS on expressions of inflammatory cy tokine and barrier proteins were evaluated under conditions of normal and LPS stimulation.Ginsenoside and poria polysaccharides were found to decrease over-expressions of inflammation cytokines led by LPS stimulation.2.Screening of functional substances based on the stress model.Mice BV2 cell lines and primary mouse cortical astrocytes(MCAST)were used for test expressions of cytokines and blood brain barrier proteins under cytokine stimulation.Besides,mice hippocampal neuronal cell lines were employed as cell model to test cell viability under cytokine damages.It was found that asarones and ginsenosides might be the active substances in regulating neuronal inflammation system.3.Screening of functional substances based on stress cell models.Rat PC 12 cells and human IEC-6 cell lines were applied as cell models.The effects of KXS on cell viabilities were evaluated under corticosterone stimulation.Polygalaxanthone ?,asarones and triterpene acids might be the active substance protecting cells from corticosterone damages.V.Verifying the material basis of the efficacy of KXS on animal model.Based on the experimental results of the above cytological studies,we selected four compounds derived from the four herbs respectively:ginsenoside Rg1,polyxanone ?,alpha-asarone and pachymic acid.We optimized the compatible ratio of four compounds by orthogonal test and applied FST and TST models to test the anti-depressive efficacy.Afterwards,the anti-depressive efficacy was further evaluated on CUMS animal model.It was found that the compound combination exerted obvious anti-depression effect,which was similar to KXS formula. |
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