| Intestinal health is an important guarantee for livestock and poultry production.The intestine is directly connected to the outside,and is one of the main battlefields that resist external pathogens.It is also the main way for the various nutrients needed by livestock and poultry,while the largest immune organ.A healthy gastrointestinal tract can provide optimal digestion and absorption function,effective barrier function and"stable"microflora for livestock and poultry.If intestinal problems occur,it will lead to indigestion,absorption disorders and even multi-system diseases,resulting in a series of problems such as low feed conversion rate,high mortality rate and reduced production performance.Most studies have reported that traditional Chinese medicine has antibacterial,anti-inflammatory,antiviral and immunomodulatory effects,and its advantages such as green,natural,non-toxic,no residue and no drug resistance have been increasingly concerned.Pudi powder,which is composed of Dandelion,Chrysanthemum and Violaceum,can effectively control intestinal inflammation and regulate the intestinal immune barrier.In this paper,the anti-inflammatory effect of Pudi powder was explored by LPS-induced RAW264.7 cells and the mouse inflammatory mode,and the regulatory effect of Pudi powder on intestinal immune function was studied through immunosuppression in mice induced by cyclophosphamide.Finally,a clinical trial will be conducted to study the effect of Pudi powder on the performance and gut health of laying and broiler chickens,so as to provide technical and theoretical basis for the later clinical application.The research content is divided into the following 6 parts:Experiment Ⅰ:Effect of Pudi powder on the production of inflammatory factors in LPS-induced RAW264.7 cell In order to study the anti-inflammatory effect of Pudi powder in vitro,MTT method was used to study the toxicity of Pudi powder on mouse peritoneal macrophages at first,while Griess reagent colorimetric method and ELl SA method were used to explore the effect of Pudi powder on inflammatory factors of mouse peritoneal macrophages induced by LPS.Experimental results showed that the maximum safe concentration of Pudi powder was 2000μg/m L through cytotoxicity test,At the safe concentration of 2000μg/m L,1000μg/m L and 500μg/m L,Pudi powder could significantly reduced the content of NO,TNF-α,IL-1βand IL-6 and increased IL-10 content in LPS-induced RAW264.7 cell.The results showed that Pudi powder could inhibit the inflammatory response of RAW264.7 cells stimulated by LPS.Experiment Ⅱ:Effect evaluation of Pudi powder on LPS-induced enteritis in mice In order to further study the effect of Pudi powder on intestinal inflammation in mice,acute enteritis in mice was induced through intraperitoneal injection of LPS,then the contents of cytokine in serum and DAO,SOD and MDA in jejunum were determined by ELISA.The morphological changes of jejunum and intestinal villi change were observed by HE.The expression of ZO-1 and MUC-2 proteins were observed by immunofluorescence.The results showed,that the Pudi powder could significantly reduce the content of TNF-α,IL-1βand IL-6,and increased the content of IL-10 in the serum of LPS-induced mice(P<0.05).The levels of DAO and MDA in the jejunum tissue of each group were significantly lower than LPS group(P<0.05),and the SOD content was significantly higher than LPS group(P<0.05).At the same time,histological observation showed that Pudi powder improved LPS-induced intestinal loss and villi destruction to a certain extent.LPS reduced the expression of ZO-1 and MUC-2 proteins in jejunum through immunofluorescence observation,while the expressions of ZO-1 and muc-2proteins in jejunum were increased to a certain extent in Pude power group.The results showed that Pudi powder could inhibit the inflammatory response and ameliorate the intestinal damage induced by LPS in mice.Experiment Ⅲ:Effects of Pudi powder on intestinal immune function of normal mice In order to study the effect of Pudi powder on the immune function of intestinal mucosa,the contents of sIgA and TNF-α,IL-1β,IL-6,IL-10 in duodenal intestinal juice were determined by ELISA.We calculate the thymus and spleen organ index and count the number of jejunal goblet cells and Peyer’s patches,and the expression of IgA~+cells in jejunum and CD4~+,CD8~+T cells in mesenteric lymph nodes were observed through immunofluorescence.The results showed that Pudi powder could significantly increase the spleen and thymus index of mice than those of the control group,and the content of sIgA,TNF-α,IL-6,IL-10 and IFN-γin the intestinal fluid of mice,as well as the number of jejunal goblet cells and the Peyer’s patche in small intestinal(P<0.05).Immunofluorescence results showed that Pudi power could increase the percentage of IgA+cells to a certain extent in the intestine,and the expressions of CD4+and CD8+T cells in mesenteric lymph nodes.The results indicated that Pudi powder could improve the immune function of intestinal mucosa in mice by regulating the development of immune organs,balancing cytokine secretion and promoting lymphocyte differentiation.Experiment Ⅳ:Effects of Pudi powder on intestinal immune function of immunosuppressed mice In order to explore intestinal mucosa Immunomodulatory function of Pudi powder,we established an immunosuppressed mouse model by injected intraperitoneally with cyclophosphamide.In this study,the contents of sIgA and TNF-α,IL-1β,IL-6,IL-10 in duodenal intestinal juice were determined by ELISA.Calculate the thymus and spleen organ index and count the number of jejunal goblet cells and Peyer’s patches.The expression of IgA~+cells,ZO-1 and MUC-2 proteins in jejunum and CD4~+,CD8~+T cells in mesenteric lymph nodes were observed by immunofluorescence.The results showed as follows,the Pudi powder could significantly increase spleen and thymus index,sIgA and contents of cytokinein,and significantly increased the number of goblet cells in jejunal and Peyer’s patches in immunosuppressed mice(P<0.05).Pudi powder could significantly increase the percentage of IgA~+cells through immunofluorescence observation(P<0.05).Simultaneously,the expressions of ZO-1 and MUC-2 proteins in jejunum and CD4~+,CD8~+T cells in mesenteric lymph nodes were increased to a certain extent in Pudi powder group.The results showed that Pudi powder could regulate the immune function of the mouse body and intestinal mucosa by improving the development of immune organs,balancing cytokine secretion,promoting the recovery of intestinal mucosal barrier structure and promoting lymphocyte differentiation in immunosuppressed mice.Experiment Ⅴ:Study of Pudi powder on production performance and intestinal healthy of laying hens In order to study the effect of Pudi powder on the production performance and intestinal health of laying hen,we observed the effects of the dead panning rate,egg production rate,feed-to-egg ratio of laying hen through adding Pudi powder to the feed.The contents of TNF-α,IL-1β,SOD,MDA and CAT in serum and sIgA in duodenum was measured by ELISA.HE staining was used to observe the shape of intestinal villi.The test results showed that compared with the control group,Pudi powder significantly reduced the dead rate,average daily feed intake,feed-to-egg ratio,broken egg rate and sand-shell egg rate of laying hens(P<0.05),and increased the egg production(P<0.05).At the same time,Pudi powder can significantly reduced the levels of TNF-α,IL-1βand MDA in serum,and increase the contents of SOD,CAT in serum and sIgA in duodenal(P<0.05).HE staining analysis showed that the jejunum in the Pudi powder group was relatively intact compared with the control group,and Pudi powder could significantly increase the VH value and VH/CD ratio,and decrease the CD value(P<0.05).In conclusion,the results showed that dietary supplementation of Pudi powder could reduce the death rate and improve egg production performance of laying hens,and it could improve the overall health of laying hens by reducing the level of inflammatory cytokines and lipid peroxidation,and increasing the content of antioxidant enzymes and intestinal immunoglobulins.Experiment Ⅵ:Study of Pudi powder on production performance and intestinal healthy of of broilers In order to study the effect of Pudi powder on the growth performance and gut health of broilers,we observed the effects of the survival rate,feed-to-meat ratio and other growth performance of broilers by adding Pudi powder to the feed.The organ indexes of liver,spleen,thymus and bursa of fabricius were calculated.The contents of sIgA,IFN-γ,TNF-αand IL-6 was measured by ELISA method.HE staining was used to observe the shape of intestinal villi,and 16S r DNA sequencing was used to investigate the changes of intestinal flora of broilers.The test results showed that compared with the control group,Pudi powder could significantly improve the survival rate of broilers(P<0.05),and increase the contents of sIgA,IFN-γ,TNF-αand IL-6 in the duodenum(P<0.01).HE staining analysis of intestinal tissue showed that the duodenal morphology of broilers in Pudi powder group was relatively complete and without obvious inflammatory infiltration compared with the control group.Finally,identified by 16S r DNA sequencing analysis,it was found that the number of Firmicutes in the Pudi powder group was higher than that in the control group,the number of lactic acid bacteria was slightly higher than that in the control group,and the number of Bacilli was slightly lower than that in the control group.The slaughter performance and meat quality results showed that Pudi powder could improve chicken slaughter performance,but there was no significant difference compared with the control group,while Pudi powder had no significant effect on chicken quality.In conclusion,the results showed that dietary supplementation of Pudi powder could reduce the mortality rate of broilers,improve growth performance and intestinal function of broilers,and reduce intestinal inflammation.In conclusion,Pudi powder had anti-inflammatory and immunomodulatory functions,which could improve the intestinal health of chickens,reduce the occurrence of intestinal diseases,and improve production efficiency.Therefore,Pudi powder could be developed as a new feed additive for chickens to replace the use of antibiotics in the breeding process. |
