Effects Of Pinus Pollen Polysaccharides On Intestinal Microenvironment And Colitis In Mice

Plant polysaccharide is a kind of biological macromolecule with complex structure,which has a wide range of biological activities,including mediating signal transduction,antibody-antigen recognition,resistance to bacterial invasion,etc.Long-term studies on pine pollen polysaccharides(PPPS)in our laboratory have confirmed that PPPS can be used as an immune enhancer to reduce the immune suppression of chickens caused by Avian leukosis virus subgroup B(ALV-B).SPF chickens co-infected with Avian Leukemia Virus and Bordetella avium showed gradual improvement in immune function and developmental status after treatment with PPPS.PPPS act as immune adjuvant in the recombinant vaccine,which can stimulate the body to produce immune response.The relationship between PPPS and the effects of intestinal microenvironment and ulcerative colitis in mice is still unclear.By studying the PPPS in mice gut bacteria,mucosal immune and intestinal epithelial cells,reveal the interaction between PPPS and gut in mice,on this basis to explore the PPPS in mice ulcerative colitis adjustment ability,and for the development of PPPS provide theoretical foundation.In this study,after oral PPPS treatment for 14 days,16S rRNA high-throughput sequencing technology was used to analyze the changes of bacteria in mouse feces.The sequencing results showed that when the number of sequences reached 30000,the dilution curve gradually flattened out,indicating that the sequencing depth met the requirements and included the vast majority of microorganisms in the sample.Alpha diversity showed that the gut bacteria richness of mice treated with PPPS was higher than PBS group.The relative abundance result of intestinal flora showed that the top 10 phyla with higher richness were the most abundant in taxonomic level.Compared to the PBS group,Bacteroidetes,Proteobacteria,Actinobacteria,Deferribacteres,Cyanobacteria,Melainabacteria,Armatimonadetes and some Unidentified＿Bacteria increased in relative abundance in PPPS group,whereas the relative abundance ratio of Firmicutes and Tenericutes decreased in PPPS group.At the level of family classification,PPPS group compared with PBS group,the relative abundance ratio of Bacteroidaceae,Muribaculaceae,Xanthobacteraceae,Rikenellaceae,Prevotellaceae,Sphingomonadaceae,Bifidobacteriaceae and Paenibacillaceae increased,the relative abundance ratio of Lactobacillaceae and Lachnospiraceae decreased.q PCR verified the level of family classification,and the results showed that the relative abundance of Bacteroidaceae,Muribaculaceae,Rikenellaceae,Prevotellaceae,Bifidobacteriaceae,and Lachnospiraceae in the intestinal bacteria of mice treated with PPPS significantly increased,which was similar to the results of 16S rRNA sequencing.Most of these bacteria are some probiotics and important participants in intestinal environment stability,resistance to foreign pathogenic bacteria infection and intestinal damage repair.collectively,PPPS can affect the gut bacteria of mice,enhance the level of intestinal probiotics,and maintain intestinal bacteria dynamic balance.Peyer patches(PPs)is an important site of mucosal immune response,which belongs to lymphatic follicular tissue of small intestine mucosa.T,B and DC cells are enclosed by invaginated M cells.M cells take up antigens,DC recognizes and presents antigens and activates T and B lymphocytes.Activated T and B lymphocytes initiates intestinal mucosal immune response and can enter the blood circulation.Therefore,it not only participates in local immunity,but also is closely related to systemic immune response.When mice after injection of cyclophosphamide(CTX)in the immunosuppression state,the mouse oral polysaccharide reached the dose(125 mg/kg)can significantly reduce proportion of CD3~+T and CD3~+CD8~+T caused by CTX,can make the CD4~+/CD8~+T cell percentage returned to normal level,improve the proportion of B cells and intestinal sIgA levels.In vitro experiments,PPPS can stimulate lymphocytes in PPs to secrete GM-CSF and IL-6,promote the proliferation of bone marrow cells and the differentiation of myeloid stem cells,thus acting on systemic immunity and exerting immunomodulatory activity.The Caco2 cell model is a human cloned colorectal adenocarcinoma cell,which is similar in structure and function to differentiated small intestinal epithelial cells,with microvilli and other structures,and contains enzyme lines related to the small intestinal brush border epithelium,with the same cell polarity and tight connection.Lipopolysaccharide(LPS)damage model was established to investigate the role of PPPS in intestinal barrier damage.The results showed that LPS could reduce the expression and distribution of tight junction protein,and damage the barrier function of Caco2 cells.PPPS can significantly improve the abnormal expression of tight junction protein in LPS-induced Caco2 cells,increase the expression of Occludin and ZO-1 proteins,and improve the barrier function of Caco2 cells.When the concentration reaches 200ug/m L,the difference is extremely significant.This suggests that PPPs can play a good protective role in the intestinal epithelial barrier injury and maintain the dynamic balance of the intestinal tract.In the study of the effect of PPPS on mouse colitis,we used sodium dextran sulfate(DSS)to establish the mouse model of ulcerative colitis.The experimental results showed that DSS-induced colitis in mice could be significantly prevented by taking PPPS orally at medium dose(125mg/kg).First,the mice lost less weight and had a lower clinical score.At day 8,the colon length was significantly longer,and histology showed less tissue damage and inflammation in the colon.PPPS can reduce proinflammatory factor TNF-α,IL-1β,IL-6 m RNA transcription level,at the same time can improve the anti-inflammatory factor IL-10 and IL-4 m RNA,also can improve the level of IL-22 m RNA transcription,the result showed that PPPS can reduce the inflammatory response in the colitis,strengthen intestinal damage repair ability.Translocation bacteria showed a significant reduction in the number of beneficial and harmful bacteria entering the blood and liver through intestinal mucosal damage,which also reduced the risk of systemic infection in the mice.In addition mechanism of increased IL-22 in colitis has explored,our results show that PPPS can promote the expression level of IL-22,but not the extra to raise the level of the expression of IL-22RA1,it suggests that PPPS not affect IL-22RA1 expression changes,in the cells of the activity of producing IL-22(ILC3 and Th17),we found that PPPS can improve Th17 secretion of IL-22,and improve the intestinal repair,but PPPS is not able to impact the ILC3 change in IL-22.ELISA results also confirmed that the level of IL-23 in the colon of mice treated with PPPS was significantly reduced,suggesting that PPPs promoted the secretion of IL-22 by Th17 in a non-IL-23 pathway,and that low level of IL-23 effectively inhibited the inflammatory response in colitis.In summary,PPPS can increase the richness and diversity of gut bacteria in mice,can adjust the intestinal immunosuppression status and alleviate the damage to Caco2 cells induced by LPS,in the study of the role of colitis shows that PPPS can promote the Th17 secretion of IL-22 in a non-IL-23 pathway,which promote intestinal mucosal damage repair.In addition,the low level of IL-23 effectively reduce the inflammation in the colitis environment,indicating that PPPS can play a positive role in gut.