Essence Of Large Intestine Dampness-heat Syndrome And Treatment Mechanisms Of Yujin Powder On It

Large Intestine Dampness-heat Syndrome(LIDHS)refers to the symptoms mainly showing diarrhea caused by dampness-heat accumulating knot in large intestine and leading to disorder of its conductive function.It is a common syndrome and happens generally in livestock,poultry and human clinics,which is seen in dampness-heat type diarrheal diseases.In modern livestock and poultry industry,diarrheal diseases become a type of seriously stubborn illness which block the development of modern livestock and poultry industry.And dampness-heat type diarrhea is one of the major type,the incidence of which is rising with the increase of intensive farming and mobility in livestock and poultry such as pigs,cattle and chickens.For human,with the development of social economy,improper diet due to the accelerated pace of work and life,changes of diet structure due to the improved living standards(indulgence in a richly fatty,sweet and full-flavored diet and drinking)and emotional stress and depression due to the increase of life pressure are easy to damage spleen-stomach,and further induce LIDHS.Yu Jin Powder(YJP),an old prescription,is one of the most classical prescription for treating the LIDHS in Traditional Chinese Veterinary Medecine(TCVM).TCVM clinical practices indicated that YJP had good treatment effects on dampness-heat type diarrheal diseases.In order to explore the essence of the LIDHS and the treatment mechanisms of YJP,the rat model of LIDHS was established by such complex factors as high-sugar and high-fat diet,improper diet,high temperature and humidity environment(HTHE),drinking and intraperitoneal injection of Escherichia coli.,which imitated the inducing conditions of LIDHS.It was treated with high,middle and low dose of YJP.Firstly,clinicopathological studies were performed;and then intestinal muscoal barriers of ileum and colon which were the leading lesions sites were studied;the proteomics of colon of which the lesions were more serious were studied;finally,the system pathological changes which showed in LIDHS were explored with the strategy of serum metabolomics.Methods and restlus were as follows:1.Clinicopathological studies Sixty Wistar rats(half males and half females)were randomly divided into six groups with 10 rats in each group: normal control group(NC group),LIDHS model group(Model group),self-healing group(Self-healing group),high-dose YJP group(HD-YJP group),middle-dose YJP group(MD-YJP group),low-dose YJP group(LD-YJP group).The rat model of LIDHS was established by such complex factors as high-sugar and high-fat diet,improper diet,high temperature and humidity environment(HTHE),drinking and intraperitoneal injection of Escherichia coli.,which imitated the inducing conditions of LIDHS.After the model was successfully established,which was treated with high,middle and low dose of YJP.Then the clinical symptoms and signs,blood routine,blood biochemistry,whole blood viscosity(WBV),serum inflammatory cytokines(TNF-?,IL-1?,IL-6,IL-10,IL-17 and IL-23)levels and the histopathological changes of main organs(ileum,colon,heart,liver,spleen,lung and kidney)were detected and observed,respectively.The results showed that the clinical symptoms and signs of the model rats were consistent with the diagnostic criteria of LIDHS,moreover,the blood lipid and WBV increased and there were obvious systemic inflammatory response and extensive congestion in multiple organs.And after treatment with YJP in different dosages,the clinical symptoms and signs of the rats with LIDHS were improved;the indexes of blood routine and blood biochemistry and inflammatory cytokines levels tended to be normal;the WBV decreased and histopathological changes of major organs were alleviated or returned to normal.There was an obvious dose-effect relationship,and the HD-YJP had the best treatment effects.2.Studies on intestinal mucosal barriers For NC,Model,Self-healing and HDYJP groups,the intestinal mucosal integrity of the ileum and colon was observed by the method of histopathology;the expression level of TFF3,Occludin,ZO-1 and MUC2 m RNA in the ileum and colon tissue were detected by the method of real-time quantitative PCR(RT-q PCR);diamine oxidase(DAO),D-Lactic(D-LA)and endotoxin(ET)in serum,Secretory Immunoglobulin A(SIg A)in the ileum and colon mucus and inflammatory cytokines(TNF-?,IL-1?,IL-6,IL-10,IL-17 and IL-23)in the ileum and colon tissue were detected by ELISA test.And the V3-V4 districts of 16 S r DNA of microbial flora in the feces of rats of NC,LIDHS and YJP groups were detected and analyzed through the Illumina Mi Seq high-through sequencing technologies.The Alpha diversities,species composition and microbial community structure,etc.were analyzed.The results showed that compared with NC group,the integrity of ileum and colon epithelium of the model rats was destroyed;the expression of Occludin and ZO-1 were significantly or extremely significantly decreased(P<0.01 or P<0.05),TFF3 was significantly increased(P<0.01)in ileum and colon and MUC2 had different changes in ileum and colon;DAO,D-LA and ET were all significantly increased(P<0.01);SIg A was significantly increased;pro-inflammatory cytokines TNF-?,IL-1?,IL-6,IL-17 and IL-23 were significantly or extremely significantly increased(P<0.05 or P<0.01)and anti-inflammatory cytokine IL-10 significantly or extremely significantly decreased(P<0.05 or P<0.01).After the treatment of high dose of YJP,all these indexes tended to or were closed to normal level.The bacteria in NC,Model and HD-YJP groups were identified 13 phylum,20 classes,28 orders,46 families and 75 genera.In the classification level of phylum,the bacteroidetes,proteobacteria and firmicutes in the three groups were dominant.However,the abundance of them had significant differences in the three groups.Compared with NC group,the abundance of bacteroidetes and proteobacteria significantly increased(P<0.05),but firmicutes significantly decreased in the model group(P<0.05).After treatment with YJP,the abundance of them were significantly back-regulated except bacteroidetes.In the classification level of families,compared with NC group,the abundance of porphyromonadaceae,desulfovibrionaceae,sutterellaceae,bacteroidetes,erysipelothrichaceae,acidaminococcaceae,enterobacteriacea,odoribacteraceae and helicobacteraceae all increased,but prevotellaceae,lachnospiraceae,rumen bacteria,eubacteriaceae and lactobacteriaceae all decreased in model group.After treatment with YJP,the abundance of all families were significantly back-regualted(P<0.05)except prevotellaceae continued to rise.3.Proteomics Analysis on Colonic Tissue The most severe lesion site colon tissues were selected to perform quantitative proteomics analysis based on i TRAQ.The differential proteins between Model and NC groups and between HD-YJP and Model groups were detected and analyzed and the pathways that these proteins participated in were analyzed.A total of 152 proteins were differentially expressed between LIDHS model and normal rats(84 were up-regulated and 68 down-regulated).And the further pathways analysis indicated that some pathways were significantly affected,which were mainly responsible for Fat digestion and absorption,AMPK signaling pathway,Oxidative phosphorylation,Retinol metabolism,Intestinal immune network for Ig A production,Fc epsilon RI signaling pathway,Complement and coagulation cascades,and Arachidonic acid metabolism etc.After treatment with YJP,a total of 221 proteins significantly changed(139 up-regulated and 68 down-regulated)and 55 proteins were directly back-regulated.Meanwhile,the above abnormal pathways were significantly improved.4.Serum metabolomics analysis The serum metabolites of rats in NC,Model and HD-YJP groups were detected using UPLC-QTOF-MS/MS technologies.Data preparation were performed with Progenesis QI software.The data after preparation were performed univariate and multivariate analysis by Metabo Analyst.According to the principle of fold changes ?1.5 or 0.677,q-value <0.05 and variable importance in the projection(VIP)?1 in the partial least squares-discriminant analysis(PLS-DA)pattern,the serum differential metabolites between Model and NC groups and between HD-YJP and Model groups were screened.And then the correlation and heatmap clustering analysis were performed for the differential metabolites.Finally,the differential metabolites were entered into the KEGG database and the related pathways were seeked.The results showed that a total of 41 differential metabolites between Model and NC groups were screened,including Aminoethylsulfonic acid,Taurochenodesoxycholic acid,L-Carnitine,L-Acetylcarnitine,Oleamide,Glycerol 3-phosphate,Hippuric acid,PC,Lyosc PC,etc.,which were mainly related to lipid metabolism,hexosamine synthesis pathway,phenylalanine metabolism and tryptophan metabolism,etc.A total of 20 differential metabolites between YJP and model groups were screened,including D-Glucose,D-Lactic acid,3-Hydroxybutyric acid,Betaine,Stearoylcarnitine,Lyosc PC,etc.,which were mainly related to catabolism of fat and fatty acid ?-oxidation tryptophan metabolism and phenylalanine metabolism,etc.The present study demonstrated that the rat model of LIDHS was successfully established through imitating the inducing conditions.The essence of the LIDHS mainly showed in four aspects:(1)the major clinical manifestation of LIDHS was diarrhea,the main lesion area was in ileum and colon,mucosa injury,inflammation and congestion of the large intestine with systemic inflammatory response and congestion were the most typical pathological characteristics;(2)the intestinal mechanical,chemical,immune and biological barriers in main ileum and colon were destroyed to different degrees;(3)the key of occerence of LIDHS was that the disorder of lipid metabolismin,the microcirlation,engery metabolism and retinol metabolism and biological factors etc.induced the disorder of intestinal immune network and destroy of homeostasis and made the body allergic and finally,intestine injury and inflammatory response occurred;(4)the disorder of lipid metabolism such as biosynthesis of cholic acid and bile secretion,biosynthesis and catabolism of fat,phospholipid metabolism and arachidonic acid metabolism,etc.and hexosamine synthesis pathway,phenylalanine metabolism and tryptophan metabolism,etc.was closely related to the occerence of LIDHS.The treatment of YJP on LIDHS mainly showed in four aspects:(1)YJP exerted the comprehensive effects of anti-diarrhea,antiinflammation,lowering blood lipid,relieving blood stasis,and regulation and protection of multiple organs;(2)repaired intestinal mechanical,chemical,immune and biological barriers;(3)regulated lipid metabolismin,microcirculation and engery metabolism,retinoicacid metabolism and intestinal immune network and homeostasis;(4)enhanced the catabolism of fat,fatty acid ?-oxidation and glucose aerobic oxidation;regulated tryptophan metabolism and phenylalanine metabolism etc.These findings provided not only important information for understanding the essence of LIDHS but also the guidance of clinical drug use for LIDHS;laid the foundation for seeking targets of treating dampness-heat type of diarrheal diseases,expanding the clinical applation of YJP and developing new-drugs for treating dampness-heat type of diarrheal diseases.