The Modern Mechanism Of Functional Diarrhea And Spleen Deficiency Syndrome Based On Brain-gut Peptide Regulation And The Intervention Effect Of Traditional Chinese Medicine

Functional diarrhea(FD)is a kind of functional bowel disease(FBDs),and FBDs is one of functional gastrointestinal disorder(FGIDs)that is intestinal disorders in the middle and lower digestive tract.FD is a syndrome which appears loose stool(mushy dung)or water feces consistently and repeatedly and not accompanied by abdominal pain or abdominal discomfort symptoms.FD should be diagnosed at least 6 months prior to the onset of symptoms,and the symptoms are consistent with the above criteria in the last 3 months.The incidence of the disease is high.There is no organic disease,but the long-term discomfort symptoms seriously affect the quality of life of patients.Its pathogenesis is still not clear,and targeted therapeutic drugs is also lack.Therefore,it is a hot spot to study the pathogenesis of FD and find effective therapy.Traditional Chinese Medicine(TCM)has a good curative effect on the treatment of FD,and there is established animal model of spleen deficiency syndrome about FD successfully in the basic research.Modern research shows the trend of development from the whole level to the cell level and the molecular level.Therefore,the present study explores the modern mechanism and possible mechanism of TCM intervention on the level of isolated cells in spleen deficiency syndrome about FD.The theoretical studyStudy 1 The recognition of FD in TCMThis study is conducted on FD from TCM disease name,etiology,pathogenesis and other aspects for further understanding.FD is due to spleen and stomach disease-Xiexie in TCM.The pathogeny of FD is feeling external evil,diet injuries,emotional disorders,insufficient natural endowment,weak organs and so on.The basic pathogenesis of FD is spleen deficiency and wetness hyperactivity.FD occurs in bowel and the key of disease lies in spleen and wet.The course of FD is longer,and the main method of relieving diarrhea is spleen deficiency.The research focus is spleen deficiency about FD and Shenling Baizhu decoction--Pixu Sihao Fang(PXSHF,No.4 Formula for Spleen Deficiency).Study 2 Study on the correlation between FD and spleen deficiency syndromeThis study attempts to explore the correlation between FD and spleen deficiency syndrome from the aspects of diagnostic criteria,clinical symptoms,etiology and pathogenesis.The clinical symptoms and the longer course of FD are similar to the spleen deficiency syndrome manifesting as diarrhea mainly.Spleen governing movement and transport is the one of the main physiological functions of the spleen,and the main cause of spleen deficiency syndrome is spleen deficiency with abnormal movement and transport.Spleen deficiency with abnormal transport can reduce the ability of decomposition and absorption of fine materials,as well as the ability to transport water and wetness,which results in the symptoms of diarrhea.The key pathogenesis of FD is spleen deficiency with wetness hyperactivity,so the treatment of FD should be promoting spleen for removing wetness.The experimental studyStudy 1 Rebuilt and evaluation of animal model combined disease and syndrome--spleen deficiency syndrome about FDObjectives Rebuilt and evaluate to the animal model combined disease and syndrome--spleen deficiency syndrome about FD.Methods Using the method combined high lactose diet and standing on small platform with water environment,we observe the general condition of rats,body weight,food intake,water intake,and measure diarrhea index,serum amylase activity,D-xylose,serum lactate content as the reference index to evaluate objectively for combined animal model.Result Compared with the control group,rats in the model group appears pale yellow dilute stool or liquid stools,lusterless fur,weary,tired lying lazy,lethargy,squinting arch back,ears pale and other symptoms of spleen deficiency syndrome,and there is a significant difference in higher diarrhea index(P<0.01),lower weight(P<0.01),weight gaining slowly,less water intake,more food intake,lower serum amylase activity(P<0.01),lower D-xylose(P<0.05),higher serum lactate content(P<0.01).Conclusions Succeed to rebuilt and evaluate the combined animal model of spleen deficiency syndrome about FD.Study 2 Preparation and generation in primary culture of isolated cells from modeled rat of spleen deficiency syndrome about FDObjectives To prepare and culture colonic smooth muscle cells and hypothalamic nerve cells from modeled rat of spleen deficiency syndrome about FD in vitro.Methods Use the enzyme decomposition method to isolate colonic smooth muscle cells and hypothalamic nerve cells from combined animal model.Results The growth state of colonic smooth muscle cells and hypothalamic nerve cells in the control group and model group is relatively stable,which can be used for later experimental detection.Conclusions Succeed to prepare and culture isolated cells about colonic smooth muscle cells and hypothalamic nerve cells from modeled rat of spleen deficiency syndrome about FD in vitro.Study 3 The mechanism of PXSHF containing serum on isolated cells from modeled rat of spleen deficiency syndrome about FDObjectives To investigate the therapeutic effect and possible mechanism of PXSHF containing serum on colonic smooth muscle cells and hypothalamic nerve cells from modeled rat of spleen deficiency syndrome about FD.Methods To prepare PXSHF containing serum for interventing the isolated cells from modeled rat of spleen deficiency syndrome about FD.Detecte modeled cell proliferation activity by CCK-8,as well as check the content of brain gut petide(CCK/VIP/SS/Ghrelin/SP/CGRP)in cell supernatant by ELISA and the expression of brain gut petide(CCK/VIP/SS/Ghrelin/SP/CGRP)mRNA by Real time PCR after PXSHF containing serum intervention.Results CCK-8 Results:About colonic smooth muscle cells,the OD value in the model group is very significantly lower than that of the control group(P<0.01),which is very significantly higher after PXSHF and Montmorillonite powder containing serum intervening(P<0.01).About hypothalamic nerve cells,the OD value in the model group is very significantly lower than that of the control group(P<0.01),which is very significantly higher than that of the PXSHF high-dose group(P<0.01),and other groups have the increased trend.ELISA Results:In colonic smooth muscle cells supernatant,the CCK/VIP/SS/Ghrelin/SP content of the model group is very significantly lower(P<0.01)and the CGRP content is significantly lower(P<0.05)than that of the control group,which is very significantly higher or significantly higher(P<0.01,P<0.05)in the PXSHF groups on the contrary.In hypothalamic nerve cells supernatant,the VIP/Ghrelin content of the model group is very significantly lower(P<0.01)and the CCK content is significantly lower(P<0.05)than that of the control group,but the SS/SP/CGRP content of the model group is very significantly higher(P<0.01)than that of the control group.By contrast,the CCK/VIP/Ghrelin content is very significantly higher(P<0.01)and the SS/SP/CGRP content is very significantly lower or significantly lower(P<0.01,P<0.05)after PXSHF containing serum intervening.Real time PCR Results:About colonic smooth muscle cells,the CCK/VIP/Ghrelin/SP/CGRP mRNA expression of the model group is very significantly lower(P<0.01)and SS mRNA expression is significantly lower(P<0.05)than that of the control group,which is very significantly higher or significantly higher(P<0.01,P<0.05)than that of the PXSHF groups,and SS mRNA expression has the increased trend.About hypothalamic nerve cells,the CCK/VIP mRNA expression of the model group is very significantly lower(P<0.01)than that of the control group,as well as the Ghrelin mRNA expression has the decreasing trend,but the SS/CGRP content of the model group is very significantly higher or significantly higher(P<0.01,P<0.05)than that of the control group as well as the Ghelin mRNA expression has the increased trend.By contrast,the CCK mRNA expression is significantly higher(P<0.05)as well as the VIP/Ghrelin mRNA expression has the increased trend in the PXSHF groups,and the SS/CGRP mRNA expression is very significantly lower or significantly lower(P<0.01,P<0.05),as well as the SP mRNA expression has the decreasing trend after PXSHF containing serum intervening.Conclusions Though the proliferation activity of colonic smooth muscle cells and hypothalamic nerve cells in model group is decreased significantly compared with blank control group,but PXSHF containing serum can effectively promote the proliferation activity of modeled cells.The test shows that the content and the gene expression of brain gut petide appears abnormity in modeled colonic smooth muscle cells and hypothalamic nerve cells,which can be regulated effectively after PXSHF containing serum intervening effect.Brief summaryBased on the above research results,FD is due to spleen and stomach disease-Xiexie in TCM,and its main pathogenesis is spleen deficiency and wetness hyperactivity.FD is closely related to spleen.Spleen disease is mainly about deficiency syndrome,focusing on abnormal transport.FD is consistent with the spleen deficiency syndrome manifesting as diarrhea mainly.The key pathogenesis of FD is spleen deficiency and wetness hyperactivity with abnormal transport.Through rebuilt and evaluation of animal model combined disease and syndrome--spleen deficiency syndrome about FD successfully,we establish a stable method of preparation and generation in primary culture of isolated cells from modeled rat about colonic smooth muscle cells and hypothalamic nerve cells.PXSHF containing serum can effectively promote the proliferation activity of the modeled cells by testing results.It is suggested that PXSHF containing serum has some protective effects on isolated cells from modeled rat about colonic smooth muscle cells and hypothalamic nerve cells in primary culture.PXSHF containing serum can affect the content and gene expression of brain gut peptide in the modeled isolated cells,which may improve the symptoms of spleen deficiency syndrome about FD by the increase or decrease of the content and gene expression of brain gut peptide in colonic smooth muscle cells and hypothalamic nerve cells.And regulation of brain gut peptides may be the target of therapeutic mechanism about PXSHF.The disturbance of brain gut peptide is closely related to spleen deficiency syndrome,and the imbalance of brain gut peptide may be the modern mechanism of spleen deficiency syndrome due to abnormal transport.The research on cell level not only broadens the modern research of spleen deficiency syndrome about FD,but also enriches the development of intervention therapy of PXSHF in TCM.