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Effects Of Pine Pollen Polysaccharides On Intestinal Mucosal Immunity Of Chickens And Immunomodulatory Of Macrophages

Posted on:2022-07-08Degree:DoctorType:Dissertation
Country:ChinaCandidate:Z ShaFull Text:PDF
GTID:1483306320994549Subject:Prevention of Veterinary Medicine
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Pine pollen has been used as a dietary supplement in traditional medicine for hundreds of years,polysaccharides(PS)in pine pollen with a wide range of benefits are one of the key active ingredients,and it has been used to improve and regulate immune functions and many other fields.The mucosal immune system is an important part of the entire immune network of animals(especially poultry)and the first line of defense against foreign antigen infections.The intestinal mucosa is a key part of food digestion and nutrient absorption,a large number of immune cells and intestinal microorganisms together form a strict defense barrier.Macrophages(m(?)),like immune cells,which have the functions of chemotaxis,phagocytosis,secretion and antigen presentation,playing an important role in the process of anti-infection,anti-tumor and regulating the body’s immunity.In this thesis,pine pollen polysaccharides(PPPS)as the research object isolated and purified from pine pollen in Tai’an City,Shandong Province,using molecular biology and other techniques to study the effects of PPPS on intestinal mucosal immunity in vivo;using 16S high-throughput sequencing analyzes the effect of PPPS on the structural abundance of the intestinal flora;macrophages(HD11)are used as target cells to reveal the immunomodulatory effect of PPPS on it and preliminary explore its mechanism of action.The purpose of this research is to provide an experimental and theoretical basis for the development of new immune-enhancing preparations with immune-enhancing effects on mucosal immunity.The main research contents and results are as follows:1.The effects of PPPS on mucosal immunityIn this study,a total of 120 1-day-old SPF chickens were selected and randomly divided into PBS group,LPPPS group,MPPPS group,and HPPPS group.They were orally inoculated with PBS and 3 different doses of PPPS(10 mg/m L,20 mg/m L and 40 mg/m L)respectively,0.2 m L per chicken a day for 21 days.The intestinal tissue,feces and blood samples of each group were collected on the 7,14 and 21 days of the experiment.The immunological indicators of each group were detected by ELISA method,flow cytometry,CCK-8 reagent,real-time quantitative PCR(q PCR)and other technologies,including SIg A and Ig G antibody levels,CD4+,CD8+T lymphocyte levels,T lymphocyte transformation rate,cytokines(interleukin 2,interleukin 4,interferonγ),mucosal immune-related molecular genes(CD86,CD80,MHC-I,MHC-II).The results showed that PPPS significantly increased the levels of mucosal antibody SIg A and serum antibody Ig G,and effectively promoted the secretion of cytokines and the level of T lymphocytes,and up-regulated the m RNA levels of mucosal immune-related genes.The effects of PPPS on the structure of small intestine villi were observed by HE staining,the duodenum,jejunum,and ileum tissues of each group were collected to prepare paraffin sections,and the length,width,crypt depth,and V/C(length/crypt depth)value of intestinal villi were measured and statistically analyzed.The results show that PPPS effectively improves the development of intestinal villi to enhance the physical barrier of the intestinal mucosa against pathogens.To order study the protective effect of PPPS on the intestinal mucosa,on the 21st day of the experiment,each group of chickens was nasally inoculated with 100μL of 106TCID50NDV virus solution.The weight changes and mortality rate after the challenge were recorded,and each segment of the small intestine and lung was taken to observe the pathological changes by HE staining,and the intestinal viral load was detected by q PCR.The results showed that the MPPPS group and HPPPS group showed a significant reduction in weight loss and mortality caused by the NDV virus.The protective effect of PPPS on respiratory mucosa was explored by infection with H9N2 virus.After 7 days of oral administration of PPPS at a dose of 40 mg/m L,5 chickens in the PBS group and PPPS group were nasally inoculated with 100μL 104TCID50H9N2 virus solution.Lung tissues were collected for HE staining on the 3rd,5th,and 7th day,the amount of lung virus was detected by q PCR,the virus was located by immunofluorescence,and the fluorescence intensity was analyzed.The results showed that PPPS significantly reduced the amount of virus in the lungs and reduced pathological changes in the lungs.It is proved that PPPS not only can effectively protect the intestinal mucosa from damage but also has a protective effect on the respiratory mucosa.2.The influence of PPPS on the intestinal floraTo study the effects of PPPS on the intestinal flora,the chickens were orally administered with PPPS for 21 days,samples of feces from each group were collected,and16S high-throughput sequencing was used to analyze and study the composition and diversity of chicken flora.The results showed that there was a certain difference in the composition of the intestinal flora between the PBS group and the PPPS group.The Venn diagram showed that the OTU value of the PPPS group was significantly increased.Analysis at the phylum level,the Firmicutes,Bacteroidetes,and Proteobacteria of the PPPS group showed greater changes than that of the PBS group.At the family level,Lactobacillaceae,Bacteroidaceae,Lachnospiraceae,and Rikenellaceae have more obvious changes.At the genus level,compared with the PBS group,the average abundance of Enterococcus,Bacteroides,Alipipes,Helicobacter,Intestinimonas in PPPS group has increased.At the species level,the abundance of Lactobacillus_reuteri,Enterococcus_cecorum,Bacteroides_uniformis,Bacteroides_thetaiotaomicro,Lachnospiraceae_bacterium_615,Alistipes_finegoldii,Lachnospiraceae_bacterium_M18-1,in PPPS group has increased.The analysis ofαdiversity and UPGMA clustering tree found that the species diversity of the flora in the PPPS group was significantly higher than that in the PBS group.Therefore,PPPS can improve the structure and abundance of intestinal flora.3.The immunomodulatory effect of PPPS on HD11 cellsTo study the immunomodulatory effects of PPPS on HD11,firstly,a CCK-8 reagent was used to detect the proliferation of different concentrations of PPPS on HD11 cells,and neutral red and FITC-dextran to detect the effect of the phagocytic activity of HD11 cells.Griess assays the levels of NO secreted by HD11 cells,the reactive oxygen species ROS were determined by fluorescence probe method,and the content of cytokines(IL-1β,TNF-α)in the supernatant of HD11 cell culture medium was detected by ELISA method.The m RNA levels of i NOS,IL-1βand TNF-αwere detected by the q PCR method.The results show that the concentration is higher than 800μg/m L,PPPS has a certain inhibitory effect on the proliferation of HD11 cells,and the concentration is lower than 400μg/m L,which can significantly promote the proliferation effect.Therefore,PPPS with a concentration range of50-400μg/m L was selected for subsequent experiments.Neutral red and FITC-dextran tests showed that PPPS can significantly increase the phagocytic activity of HD11 cells,but there is no significant difference among the different concentrations.PPPS promoted the secretion of NO,IL-1β,and TNF-αin HD11 cells in a dose-dependent manner,and up-regulated i NOS,IL-1β,and TNF-αm RNA levels.HD11 cells treated with 200μg/m L PPPS for 24 h and the cells in the PBS group were used for transcriptome sequencing to analyze and compare the differently expressed m RNA and mi RNA and signal pathways.The results showed there were 2358 up-regulated genes and2028 down-regulated genes in PPPS group,the main immune-related pathways in the enrichment analysis were Endocytosis,Adherens junction,Protein processing in the endoplasmic reticulum,Cytokine-cytokine receptor interaction,Transporter,Toll-like receptor,MAPK,Notch,C-type lectin receptor,NOD-like receptor,RIG-I-like receptor and other signaling pathways.The results of mi RNA showed that 10 mi RNAs were highly expressed in the PPPS group,and 21 mi RNAs were down-regulated.The main pathways of KEGG pathway enrichment analysis are Endocytosis,Tight junctions,Lysosomes,Adhesion junctions,Cell adhesion molecules,Protein processing in the endoplasmic reticulum,Notch and other signaling pathways.In order to explore the receptors that PPPS acts on HD11 cells and the signal pathways of immune regulation,after the treatment of HD11 cells with PPPS,the m RNA levels of surface receptors Toll-like receptors 2 and 4,mannose receptor(MR)and NF-κB molecules were detected by the q PCR method.After pretreatment of the cells with three subunits of TLR4,MAPK(p38,JNK,ERK1/2)and NF-κB inhibitors,the levels of NO were detected by the Griess method,and the secretion of IL-1βand TNF-αwas detected by the ELISA method.The results showed that PPPS up-regulated the m RNA levels of cell surface receptors TLR4and nuclear factor NF-κB at different time points,proving that TLR4 is an important receptor for PPPS to activate HD11 cells,and NF-κB is a key nuclear factor for PPPS to promote the activation of HD11 cells.TLR4,MAPK,and NF-κB inhibitors can significantly reduce the secretion of NO,IL-1βand TNF-α,indicating that TLR4,MAPK,and NF-κB are all involved in the immune enhancement effect of PPPS on HD11 cells.The results indicated that PPPS activates HD11 cells to produce immune responses through the TLR4/MAPK/NF-κB signaling pathway.In summary,PPPS significantly promotes intestinal mucosal immune function and small intestinal villi development,effectively reduces the pathological damage of intestinal and lung tissues caused by NDV and H9N2,improving the abundance of intestinal flora,activating HD11 cells with TLR4/MAPK/NF-κB signaling pathway.This study not only explores the effects of PPPS on mucosal immunity and intestinal flora,but also reveals the immunomodulatory effect on macrophages.Therefore,PPPS as a natural immune enhancer has good development potential and application prospects.
Keywords/Search Tags:pine pollen polysaccharides, mucosal immunity, immune regulation, intestinal flora, macrophages, transcriptome, signaling pathway
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