The Protective Mechanism Of Hippophae Rhamnoides Polysaccharides On The Inflammatory Damage Of Intestinal Porcine Epithelial Cells Induced-by Lipopolysaccharide In Piglets

Early weaning stress can decrease immunity,cause intestinal structural abnormalities and dysfunction,and seriously threaten the healthy growth of piglets.Early weaning stress usually induced intestinal diseases and increased the risk of disease in piglets.Hippophae rhamnoides polysaccharide(HRP)as immunostimulants can enhance the anti-inflammatory of immune system and host defense response.However,little is known about the protective effect of HRP on the intestinal immune stress in early weaned piglets.The mechanism of HRP inhibits the intestinal inflammatory in early weaned piglets was also unknown.Intestinal porcine epithelial cell(IPEC-J2)is an important cell line,which study intestinal immunity and inflammation,and is also a model system for studying immune response in vitro.Therefore,this study selected IPEC-J2 as the research object,established inflammatory damage model of IPEC-J2 induced by lipopolysaccharide(LPS)to simulate early weaning stress of piglets in vivo.We explored the protective effect and molecular mechanism of HRP on LPS-induced inflammatory damage in IPEC-J2 cells,and clarified whether HRP has anti-inflammatory effect on weaned piglets' intestinal tract.Our study laid a theoretical foundation for the application of HRP as an anti-inflammatory immunostimulants in piglet production.This study consists of four chapters.The main conclusions are as follows:(1)The effect of HRP on the proliferation,apoptosis and immune function of IPEC-J2 cellsFirstly,in order to obtain the safe concentrations and optimal time for HRP treatment of IPEC-J2 cells.IPEC-J2 cells were pre-treated with HRP of 0,200,400,600,and 800 ?g/m L.MTS method was used to detect the proliferation rate of IPEC-J2 cells pre-treated with HRP for0,6,12,24 and 48 h.The results showed that after HRP at a concentration of 200?600 ?g/m L pre-treated the cells,the cell proliferation rate increased with the increase of the concentration and the proliferation rate of IPEC-J2 cells reached the maximum at 24 h.Therefore,we used200?600 ?g/m L and 24 h as the safe concentration and the best time for HRP to treat cells.Subsequently,we explored the effect of HRP on the immune regulation of IPEC-J2 cells.The results showed that 200?600 ?g/m L HRP increased IL-1?,IL-6,IL-8,IL-10,TNF-?,Ig M,Ig A and Ig G contents in IPEC-J2 cells to varying degrees,which promoted the proliferation of immune cells and improved the ability of cellular immune response.200?600 ?g/m L HRP reduced the ROS content and apoptosis rate in IPEC-J2 cells to varying degrees,and 600 ?g/m L HRP exerted an effective protective effect.(2)Construction of IPEC-J2 cell inflammatory damage model in vitroFirstly determine the optimal concentration and optimal time for LPS treatment of IPEC-J2 cells.IPEC-J2 cells were treated with LPS of 0,0.1,1,10 and 100 ?g/m L.MTS method was used to detect the proliferation rate of IPEC-J2 cells treated with LPS for 0,4,8,12,16 and 24 h.The results showed that the optimal conditions for LPS-induced IPEC-J2 cells to construct an inflammatory damage model are the concentration of 10 ?g/m L and the duration of action for 16 h.In order to further determine the optimal concentration of LPS,IPEC-J2 cells were treated with LPS at concentrations of 0,0.1,1,10 and 100 ?g/m L for 16 hours to explore the effects of different concentrations of LPS on IPEC-J2 cells.The results show that 0.1?100 ?g/m L LPS increased the contents of IL-1?,IL-6,IL-8 and TNF-?,and decreased the contents of IL-10,Ig M,Ig A and Ig G in IPEC-J2 cells to varying degrees.On the basis of not affecting subsequent experiments,the result of 10 ?g/m L LPS played a better effect.0.1?100 ?g/m L LPS increased the ROS content and apoptosis rate in IPEC-J2 cells to varying degrees,and reduced the number of IPEC-J2 cells to survive.On the basis of not affecting subsequent experiments,the result of10 ?g/m L played a better effect.(3)The protective effect of HRP on LPS-induced IPEC-J2 cell inflammatory damageThe IPEC-J2 cells were divided into control group,LPS group and 3 HRP treatment groups on the basis of the experiments in chapters 1 and 2.Cells without added reagents were used as control.In the LPS group,cells induced for 16 h with 10 ?g/m L LPS.In the HRP treatment group,cells pre-treated with 200?600 ?g/m L HRP for 24 h followed by treatment with 10?g/m L LPS for another 16 h.We explored whether HRP plays a protective effect on LPS-induced inflammatory damage in IPEC-J2 cells.The results showed that the HRP treatment group significantly reduced the levels of pro-inflammatory cytokines(IL-1?,IL-6,IL-8,TNF-?),ROS content,apoptosis gene levels(caspase-3,caspase-8,caspase-9 and BAX)and the rate of apoptosis in IPEC-J2 cells(P<0.05),and significantly increased IL-10 level,immunoglobulin levels(Ig M,Ig A and Ig G),tight junction protein levels(ZO-1,Occludin and claudin-1)in IPEC-J2 cells(P<0.05).HRP protected the damage caused by LPS to the cytoskeleton structure and improved the integrity of the cell structure.(4)Proteomics analysis revealed the protective mechanism of HRP on LPS-induced inflammatory damage in IPEC-J2 cellsAs described in Chapter 3,HRP has an effective immunomodulatory effect on the inflammatory damage of IPEC-J2 cells induced by LPS.No proteomics data showed that protein changes induced by LPS after HRP pre-treatment of IPEC-J2 cells.Therefore,in this study,we used DIA quantitative proteomic technique to study the key proteins and cell pathways related to pre-treatment with HRP followed by challenge with LPS in IPEC-J2 cells.This reveals the protective mechanism of HRP on LPS-induced inflammatory damage in IPEC-J2 cells.The results of Chapter 3 show that 600 ?g/m L HRP has the best protective effect on the inflammatory damage of IPEC-J2 cells induced by LPS.Therefore,in this experiment,IPEC-J2 cells were divided into control group,LPS group and HRP treatment group.The control group and the LPS group process the cells in the same way as the experiment in Chapter 3.In the HRP treatment group,cells pre-treated with 600 ?g/m L of HRP for 24 h followed by treatment with10 ?g/m L LPS for another 16 h.The results showed that the analysis of the DIA data results identified 18,768 proteins according to the DDA reference database.The GO,KEGG,and COG/KOG libraries totaled 17,962 proteins,and the number of jointly annotated proteins was6,084.Significant differentially expressed proteins between the groups were screened out with Q value < 0.05 and fold change absolute value > 1.5 double.Among them,there are 2052(1720up-regulated and 332 down-regulated),358(262 up-regulated and 96 down-regulated)and 1532(314 up-regulated and 1218 down-regulated)significant differentially expressed proteins were identified in C vs.L,C vs.H6-L,and L vs.H6-L,respectively.GO enrichment analysis revealed that binding,catalytic activity,cellular process,single-organism process,metabolic process,cell,cell part and organelle were the most enriched pathways for significant differentially expressed proteins.KEGG enrichment analysis indicated that the top 20 pathways in the L-vs-H6-L comparison group related to immunity were the tight junction,MAPK signaling pathway,PI3K-Akt signaling pathway,rap1 signaling pathway,HIF-1 signaling pathway,ras signaling pathway and Fc gamma R-mediated phagocytosis.On the basis of proteomic analysis,we screened 42 key proteins related to the immune signaling pathway of HRP alleviating LPS-induced IPEC-J2 cell damage in the L-vs-H6-L comparison group.Compared with the LPS group,after HRP pretreatment,most of the 42 proteins were down-regulated,and HRP completely reversed the expression trend of these proteins.It indicated that HRP alleviated LPS-induced inflammatory response in IPEC-J2 cells by inhibiting the activation of the above signal transduction pathways and the expression of related differential proteins.Western blotting analyses confirmed that pre-treatment with HRP significantly reduced the levels of the phosphorylated MAPK7,phosphorylated RELA,phosphorylated NF-?B1 and phosphorylated NF-?B2,and increased the levels of CLDN2,IGF2 in cells(P<0.05).In conclusion,HRP protected IPEC-J2 cells from LPS-induced inflammatory damage by mainly inhibiting the MAPK/NF-?B signaling pathway and its related differentially expressed proteins,and reduced the levels of inflammatory cytokines and apoptosis-related genes,increased the levels of immunoglobulins and tight junction proteins,enhanced the immune regulation function of cells,and protected the integrity of cell barrier function.