| 1 BackgroundThe syndrome of stagnation of liver qi and spleen deficiency is a common syndrome of traditional Chinese medicine(TCM),which can be widely found in the process of some refractory diseases,such as depression,diarrhea irritable bowel syndrome,chronic liver disease,sub-health,et al.The Western medicine and TCM played important roles in their own ways in the treatment of these refractory diseases.Owing to well understanding the diagnosis and pathophysiology of diseases,the Western medicine is good at dealing with the symptoms and focusing on the disease itself,but TCM is good at syndrome differentiation so that it payed more attention to illness related syndromes.The basic study aiming at the syndrome of stagnation of liver qi and spleen deficiency can help us to explore its essence and better use in clinical treatment.At present,the development of TCM "micro syndrome" provides a new idea for the study of syndromes.The micro-indicators in the micro syndrome are more easily quantified and standardized when compared with the symptoms and signs and other information,so,some research interesting in the biomarkers has been more attention with the development of science and technology.This is of great significance to the combination of TCM syndrome differentiation and TCM diagnosis and treatment.Brain-gut peptide(BGP)is a kind of endocrine signaling molecule co-expressed in the central nervous system(CNS)or gastrointestinal tract,and participates in feeding regulation and energy balance through brain-intestinal axis(intestinal-brain axis).Recently,it has been reported that Nesfatin-1,as a BGP,could across the blood-brain barrier freely and have an effect of inhibition of appetite.In addition,nesfatin-1 was also shown to be involved in stress response and was positively correlated with depressive status.It is believed that there is a nesfatin-1-POMC/OT network system in the hypothalamus.The proopiomelanocortin(POMC)neurons in the arcuate nucleus(ARC)can be activated by exogenous(from the peripheral or paraventricular nucleus)or endogenous(ARC autologous)nesfatin-1,and acts as a suppress food through the melanocortin pathway or the activated oxytocin(OT)pathway in paraventricular nucleus(PVN).The network system ensures nesfatin-1 regualte the feeding function in the physiological state,but once the balance is broken,there will be a series of neuropsychiatric-related symptoms and gastrointestinal symptoms.Our previous study showed that there were extensive changes in BGP level in rats with liver depression and spleen deficiency,which may be one of the causes of brain-intestinal interaction dysfunction.However,the relationships between nesfatin-1 and nesfatin-l-POMC/OT network and liver depression and spleen deficiency have not yet been studied.Xiaoyaosan is commonly used in the treatment of syndorme of liver qi and spleen deficiency,which has been widely researched in the syndrome of liver and spleen deficiency.Our previous study has been proven that Xiaoyaosan could effectively regulate the syndorme of liver qi and spleen deficiency induced by the 21-day chronic constriction stress.Also,the mechanism of the action may be related to the regulation of multiple BGP.But,it is not yet clear that whether Xiaoyaosan could improve the syndrome of liver and spleen deficiency related to the regulat the nesfatin-1 and nesfatin-l-POMC/OT networks.2 ObjectiveIn this study,we analyzed the changes of brain-intestinal peptide nesfatin-1 and its related signals factors,to explore the biological basis of liver depression and spleen deficiency syndrome as well as the interaction between "liver depression" and "spleen deficiency".First of all,to determine whether the presence of nesfatin-1 signal in rats with liver-depression and spleen-deficiency syndrome,and then to further analyze the source of its changes and the role of pathways.Finally,we analyzed how nesfatin-1 signal to regulate the nesfatin-l-POMC/OT network in the hypothalamus,and the regulation of Xiaoyao Powder was observed.3 MethodsThis study is mainly composed of four parts.Part oneFirst of all,combined with the previous experimental study,21-days chronic restraint stress method was used to replicate the rat model of liver and spleen deficiency syndrome.The model was evaluated by observing the general condition,monitoring the body weight and food intake,conducting the open field test and sugar preference test,detecting the D-xylose absorption rate and corticosterone levels in peripheral blood,and observing the effects of Xiaoyao Powder on the syndrome and fluoxetine on the symptoms.Part twoThe expressions of nesfatin-1 and GHRL in serum were measured by enzyme immunoassay(EIA)and enzyme linked immunosorbent assay(ELISA)respectively,and the correlation between them was analyzed.Then,the effects of Xiaoyao Powder on the regulation of peripheral nesfatin-1 were determined.Part threeFirstly,we screened the internal reference genes which were suitable for the real-time quantitative polymerase chain reaction(RT-qPCR)technique used in this experiment.Then,the gene expressions of NUCB2/nesfatin-1 in the hypothalamus and fundus of stomach and the gene expressions of related signal molecules in nesfatin-l-POMC/OT in the hypothalamus were determined by the RT-qPCR and normalized the suitable reference genes to observe the effects of Xiaoyaosan.Part fourThe protein expressions of NUCB2/nesfatin-1,POMC,OT,and melanocortin receptor subtype 4(MC4R)in the hypothalamus were determined by Western blot(WB)and or immunochemistry method.Meanwhile,the immunofluorescence double staining method was used to-co-target these related proteins in the hypothalamic nesfatin-l-POMC/OT network.The purpose of these experiments was to investigate the changes of nesfatin-l-POMC/OT network in the rat hypothalamus of liver depression and spleen deficiency syndrome and the regulation effects of Xiaoyao Powder.4 ResultsPart oneRats exposed to chronic restraint stress showed gradually decreases in food intake and body weight,the yellow hair,lazy less moving,dry stool dry and wet appearance.The results of behavioral studies:the rats exposed to chronic restraint stress showed that the total distance of movement and the time spending in the central area were decreased in the open field experiment,and the sugar preference was reduced.The results of microscopic index:the contents of D-xylose in serum were decreased,and the CORT levels were increased in the model group.But,the changes were reversed by the pre-treatment of Xiaoyao Powder and Fluoxetine.Part twoThe result of EIA and ELISA:Compared with the control group,the concentration of nesfatin-1 in the peripheral blood of the model group was significantly increased,and the content of GHRL was significantly decreased,which were reversed by the pre-treatment of Xiaoyao Powder.Additionally,the serum nesfatin-1 levels were negatively correlated with GHRL concentrations in each group.Part threeThe results of screening of internal reference genes showed that Peptidylprolyl isomerase A/cyclophilin A(PPIA)was the most suitable gene for the detection of hypothalamic gene changes in this experiment.Then,the results of RT-qPCR showed that there were no significant changes in the NUCB2/nesfatin-1 mRNA expression of hypothalamus between the control group and the model group.The genes expression of POMC,OT and MC4R in the hypothalamus and fundus of stomach in the model group were significantly higher than those in the control group.However,these changes in POMC,OT and MC4R genes were reversed by the pre-treatment of Xiaoyao Powder.Part fourThe results of WB were accordance with the genes expression in the results of part three.The results of immunohistochemistry showed that NUCB2/nesfatin-1 positive neurons were widely distributed in arcuate nucleus(ARC)and paraventricular nucleus(PVN),while POMC was only distributed in ARC,and OT in PVN.The results of immunohistochemical analysis:The positive cell rate and mean optical density of POMC in ARC in the model group were significantly increased when compared with the control group,which were reversed by the pre-treatment of Xiaoyao Powder.But,there were significant changes in the NUCB2/nesfatin-1 of hypothalamus between the control group and the model group.Similarily,the positive cell rate and mean optical density of OT and NUCB2/nesfatin-1 in PVN in the model group were significantly increased when compared with the control group,which were reversed by the pre-treatment of Xiaoyao Powder.The results of immunofluorescence double staining:NUCB2/nesfatin-1 neurons were widely and granularly distributed in ARC and PVN;POMC neurons were only distributed in ARC;OT neurons were only distributed in PVN.The results of image merge showed that POMC neurons and NUCB2/nesfatin-1 neurons were co-expressed in the ARC and the OT neurons and NUCB2/nesfatin-1 neurons were co-expressed in the PVN.5 ConclusionsIn part one,the results indicate that we successfully established a rat model of liver depression and spleen deficiency syndrome through conducting chronic restraint stress for 21 days.The model mimicked some symptoms of liver depression and spleen deficiency syndrome including depression(depressive-like behavior),less abdominal distension(less weight loss and lower appetite),poor digestion and absorption(D-xylose absorption rate decreased),and loose stools(stool shape changes).Meanwhile,the improvement effects of the pre-treatment of Xiaoyao Powder and fluoxetine hydrochloride could further confirm the successful establishment of the model.In part two,the rat model of liver depression and spleen deficiency syndrome presented the changes in the nesfatin-1 and GHRL that were a pair of mutual constraint dietary regulation factors in the body,resulting in the imbalance of suppression food and promotion food.In the chronic stress state,the peripheral imbalance of suppression food and promotion food is broken,resulting in the influence the feeding regulation area in the central nervous system that is worth our further study.In part three,the hypothalamic PPIA gene had the highest stability under the influence of chronic restraint stress stimulation,and the treatment of Xiaoyao Powder and fluoxetine,which will provide a reliable internal reference for RT-qPCR detection in the future similar experiments.In the subsequent RT-qPCR test,the NUCB2/nesfatin-1 mRNA in the fundus of stomach of the model group was significantly increased,indicating that the increase of serum nesfatin-1 level in the liver and spleen deficiency state was likely to be secreted from gastric tissue.Although the gene expression of NUCB2/nesfatin-1 in the hypothalamus of the model group did not change significantly compared with the control group,the nesfatin-1-POMC/OT network was excessively activated in the model group.These indicated that the peripheral nesfatin-1 signal could pass through the blood circulation to reach the hypothalamus to play an inhibitory effect on the appetite.But,the treatment of Xiaoyao Powder can improve the gastrointestinal symptoms while adjusting the emotional change,so improving the imbalance in varying degrees.In part four,the results of WB further confirmed the results of RT-qPCR.Under the sustained stress,the excessive nesfatin-1 in peripher can activate the nesfatin-1-POMC/OT network in the hypothalamus,and this process may be achieved through the brain-intestinal axis.The treatment of fluoxetine or Xiaoyaosan can reduce the peripheral nesfatin-1 level and inhibit the activation of hypothalamic POMC/OT,which exerted the effects on the dispersing stagnated liver qi for relieving qi stagnation and regulating the appetite.In summary,the syndrome of liver depression and spleen deficiency syndrome existed the increased concentration of nesfatin-1 in serum and overactive nesfatin-1-POMC/OT network in the hypothalamus.This signaling pathway activated by chronic stress brought about the loss of food intake and body weight,and accompanied by depression-like emotional state.The effects of Xiaoyao Powder on the relevant indicators suggests that the regulation effects of the peripheral nesfatin-1 signal and the hypothalamic nesfatin-1-POMC/OT network may be the potential targets of Xiaoyao Powder that exerts the effects on the the dispersing stagnated liver qi for relieving qi stagnation and regulating the appetite.Therefore,this current study helps to reveal the underlying mechanism of liver and spleen deficiency syndrome showing imbalance of appetite regulation in CNS,and meanwhile to understand the targets and mechanism of Xiaoyao Power exerting the effects of dispersing stagnated liver qi for relieving qi stagnation and invigorating spleen. |
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