| BackgroundThe syndrome of stagnation of liver qi and spleen deficiency is a common traditional Chinese medicine syndrome,which is mainly related to the dysfunction of liver controlling conveyance and dispersion with spleen in transportation.As it says in Suwen:'when people became angry,qi reversed flow,and impairing liver','spleen storing idea,and belong to thinking','worry impairing spleen','all diseases resulting from qi disorder,......,pensiveness leading to qi knotting';in Jinkui Yaolue:'the liver disease transferred to spleen,it should enhance the function of spleen to resist exopathogens'.All of these records indicate that the syndrome of stagnation of liver qi and spleen deficiency is closely related to the change of emotion and the function of liver and spleen,emotional stress can lead to physiological changes,neuroendocrine disorders,immune dysfunction and so on.Depression is one of the most serious mental diseases in the world and endangered people's life and health.Clinically,the pathological mechanism of depression is not clear and the therapeutic effect is not consistent,so it is considered as a heterogeneous disease,while the syndrome of stagnation of liver qi and spleen deficiency is one of its main syndrome types.Inflammation is a kind of defensive immune response that the body resists the external damage or the occurrence and development of diseases,involving many diseases and the pathological changes of various systems of the human body.Chronic inflammation leads to cell dysfunction,and then leads to organ dysfunction and disease,which is the key link of disease development.The mechanism of inflammation leading to a variety of chronic diseases has become one of the hot topics in modern research.In modern research,it is found that the pathogenesis of depression includes the activation of the immune system and the release of inflammatory cytokines,moreover,the syndrome of stagnation of liver qi and spleen deficiency is related to many kinds of diseases in TCM,such as internal,external,women,children and so on.Its pathogenesis is very complex,and it is related to the occurrence of chronic inflammation.Therefore,to study the material basis of chronic inflammation is an important way to analyze the mechanism of syndrome of stagnation of liver qi and spleen deficiency.In recent years,with the development of research on the mechanism of cell death,it has been found that cell necrosis is not disordered,but a process of programmed necrosis with precise molecular mechanism.In addition to causing significant and extensive inflammatory response,necroptosis is also an important way of neuronal death,involving a variety of central nervous system injury mechanisms,but also included in the mechanism of other related diseases such as liver cell death and liver injury.As a common clinical syndrome,the pathogenesis of syndrome of stagnation of liver qi and spleen deficiency involves the changes of many body system and function such as neuroendocrine,immunity,digestion,metabolism and so on.Therefore,we can speculate that the occurrence and development of syndrome of stagnation of liver qi and spleen deficiency may be related to the chronic inflammation mediated by necroptosis.To study the mechanism of chronic inflammation and necroptosis can provide a bridge to explore the relationship between necroptosis and the syndrome of stagnation of liver qi and spleen deficiency,so as to further enrich its scientific connotation.ObjectiveAccording to the current research,the action sites of Lipocalin-2 are highly overlapped in the mechanisms of depression,syndrome of stagnation of liver qi and spleen deficiency,as well as the medicinal mechanism of xiaoyaosan,which involve the central nervous system,immune inflammatory response,liver function,glucose and lipid metabolism and energy metabolism,and may be related to the necroptosis pathway.Therefore,the purpose of this study is to explore whether the modern biological basis of the syndrome of stagnation of liver qi and spleen deficiency includes the mechanism of chronic inflammation mediated by necroptosis pathway,observe whether xiaoyaosan can regulate the necroptosis pathway,and whether.the inflammatory related functional protein Lipocalin-2 can be used as a potential target of xiaoyaosan in the treatment of depression with syndrome of stagnation of liver qi and spleen deficiency.In order to provide new theoretical basis and experimental support for the application of xiaoyaosan,at the same time,provide a new way of thinking with formula-syndrome relation for the modern research of the syndrome of stagnation of liver qi and spleen deficiency.Methods(1)The model of depression with syndrome of stagnation of liver qi and spleen deficiency in mice was established by chronic unpredictable mild stress(CUMS).The manifestation of'stagnation of liver qi' was objectively evaluated by means of general state,sucrose preference test,force swimming test,novelty-suppressed feeding test and open field test,then,the manifestation of 'spleen deficiency' was evaluated by serum D-xylose,body weight and food intake.At the same time,observe the therapeutic effect of xiaoyaosan on depression with syndrome of stagnation of liver qi and spleen deficiency model mice based on syndrome detecting from formula used.(2)To detect the occurrence of necroptosis pathway in the hippocampus of model mice.Necrotatin-1 was used to block necroptosis,Western Blot and Immunohistochemistry were used to observe the expression of necroptosis key proteins including RIPK1,RIPK3 and MLKL,while the phosphorylation level of MLKL was observed by Western Blot,Immunohistochemistry and Immunofluorescence to confirm that the necroptosis was activated by RIPK1/3-mediated MLKL.(3)The expressions of IL-1 ?,Lipocalin-2 and Iba-1 in hippocampus,and the expressions of IL-1 P,Lipocalin-2 and MC4R in hypothalamus were detected,in order to explore the relationship between the activation of microglia in the hippocampus as well as the food inhibition pathway of MC4R by Lipocalin-2 and the central inflammation in the syndrome of stagnation of liver qi and spleen deficiency.(4)The levels of serum ALT,AST,DBIL,TBIL and TBA were detected,and then the expressions of IL-1? and Lipocalin-2 were observed to explore the relationship between the expression of Lipocalin-2 and the inflammatory liver injury related to syndrome of stagnation of liver qi and spleen deficiency.(5)To observe the regulatory effect of xiaoyaosan on chronic inflammation and Lipocalin-2 function mediated by necroptosis pathway,analyze the effects of xiaoyaosan in the treatment of depression with syndrome of stagnation of liver qi and spleen deficiency from the perspective of inflammation,in order to further explain the scientific connotation of syndrome of stagnation of liver qi and spleen deficiency.Results(1)In the first study,a 6-week CUMS was used to establish the depression with syndrome of stagnation of liver qi and spleen deficiency mice model.The behavioral evaluation methods,such as the general state of mice,the change of body weight and food intake,the content of D-xylose in serum,the sucrose preference test,force swimming test,novelty-suppressed feeding test and open field test,then combined with the syndrome detecting from formula used of xiaoyaosan,were used to evaluate the model.(2)In the second study,we confirmed that the activation of necroptosis pathway is closely related to the dysfunction of hippocampus in CUMS induced model mice,and discussed the possible mechanism of necroptosis in the pathogenesis of depression with syndrome of stagnation of liver qi and spleen deficiency.At the same time,we found that xiaoyaosan may improving the expressions of RIPK1,RIPK3,MLKL and the phosphorylation level of MLKL,then regulate the necroptosis pathway,so as to alleviate the neuroinflammation and microglia activation in hippocampus.(3)In the third study,we found that the expression of MC4R in the hypothalamus of model mice may be related to the activation of necroptosis pathway.Meanwhile,it was found that the effect of xiaoyaosan on feeding regulation of model mice might be related to improving the level of necroptosis,regulating the expression of Lipocalin-2 and intervening the appetite regulation pathway related to MC4R in paraventricular nucleus of hypothalamus.(4)In the fourth study,we observed the changes of liver function in model mice,and further confirmed that necroptosis was related to liver dysfunction,then it was found that xiaoyaosan has a significant therapeutic effect on liver damage in model mice,which may be related to the improvement of the level of necroptosis and the regulation of the expression of Lipocalin-2.ConclusionIn this study,the depression with syndrome of stagnation of liver qi and spleen deficiency mice model was successfully established,and the effect of xiaoyaosan on the general state and behavioral changes of the model mice was used to verify this model based on syndrome detecting from formula used.Concurrently,it was found that the necroptosis inhibitor also had a certain regulatory effect on the behavior changes of model mice,suggesting that there may be activation of necroptosis pathway in the pathogenesis of depression and syndrome of stagnation of liver qi and spleen deficiency.It is also found that the occurrence of hippocampal neuroinflammation and the excessive activation of microglia were related to the occurrence of necrotic apoptosis.Moreover,necroptosis may be involved in the mechanism of appetite dependent regulation of MC4Rand the mechanism of liver function damage related to chronic inflammation through the regulation of Lipocalin-2 in depression and syndrome of stagnation of liver qi and spleen deficiency.At the same time,the effect of xiaoyaosan on depression and syndrome of stagnation of liver qi and spleen deficiency may be related to the above pathway of necroptosis. |
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