Regulation Effects Of Tea Tree Oil On The Immune Stress Of Ileal Epithelial Cells Of Piglets Induced By Iipopolysaccharide

In actual production,piglets are prone to stress syndrome after weaning,which causes intestinal immune stress of piglets.How to improve the immune function of piglets and relieve the immune stress of weaned piglets through nutrition regulation is a research hotspot.The study of our research group found that Tea Tree Oil(TTO)added into the diet can improve the intestinal mucosal immune function of piglets and reduce the diarrhea rate of piglets,but the mechanism of Tea Tree Oil on intestinal mucosal immune function of piglets is still unclear.Therefore,in this study,Lipopolysaccharides(LPS)was used to induce IPI-2I immune stress in pig ileum epithelial cells,and an immune stress model was established to further explore the regulatory effect of Tea Tree Oil on IPI-2I immune stress in pig ileum epithelial cells induced by Lipopolysaccharides,so as to provide theoretical basis for Tea Tree Oil as a nutrient to relieve weaning stress in piglets.The overall study consists of the following two parts:Experiment 1:Effect of Tea Tree Oil on growth performance and intestinal immune function of weaned pigletsIn actual production,antibiotics are often used to alleviate the immune stress of weaned piglets,but antibiotics have a variety of negative effects.With the ban of antibiotics,new health green additives are getting more and more attention.As a feed additive,tea tree oil has the effects of improving growth performance and immune function.The purpose of this experiment was to explore the effect of adding tea tree oil in the diet on the growth performance,diarrhea and immune function of the ileal mucosa in weaned piglets.The test selected 48 healthy Duro×Landrace × Yorkshire ternary cross-weaned piglets of 21-day-old body weight(6.83±0.13 kg),randomly divided into 2 groups,namely the control group(CON group,basic diet),tea tree oil In the group(TTO group,basic diet+100 mg/kg tea tree oil),4 replicates per group,6 piglets per replicate,half male and female,21 days in the test period.The effects of tea tree oil on growth performance,diarrhea rate,ileal tissue morphology and immune function of weaned piglets were determined.The results showed that:1)Compared with the CON group,the average daily weight gain(ADG)of the TTO group increased,and the diarrhea rate and diarrhea index decreased significantly(P<0.05).2)The intestinal villi in the TTO group are longer,the structure is more regular and well developed,and the microvilli in the ileal epithelium of the TTO group are significantly longer,neat and denser than the CON group;the number of ileal goblet cells is significantly higher than that of the CON group.3)Compared with the CON group,the content of cytokines IL-1β and TNF-α in the ileal mucosa of the TTO group decreased,but the difference was not significant(P>0.05),and the content of IFN-y increased significantly(P<0.05).In summary,tea tree oil has the effect of improving the growth performance of weaned piglets,reducing the rate of diarrhea,and promoting intestinal development and immune functionExperiment 2:Establish the immune stress model of IPI-2I cellsHow to relieve the immune stress of piglets during weaning is a difficult problem in pig production.In this study,lipopolysaccharide(LPS)was used to induce immune stress on IPI-2I cells,to explore the optimal stimulation time and concentration,and to establish an immune stress model.Concentration and time to a stimulus through cell vitality index after preliminary screening,this test set up four groups:CON(DMEM),LLPS group(DMEM+10 ug/mL LPS),MLPS group(DMEM+50 ug/mL LPS),HLPS group(DMEM+100 ug/mLLPS),each group time is 3 h,6 h,9 h and 12 h,determination of cytokines in the supernatant content and immune related genes expression in cell volume.The results showed that:1)with the increase of LPS concentration,the activity of IPI-21 cells decreased.Compared with CON group,both MLPS group and HLPS group significantly reduced the activity of IPI-2I cells(P<0.01).The HLPS group showed the greatest decrease in IPI-2I cell activity,but the HLPS group was able to cause a large number of IPI-2I adherently cells to fall off and the suspension cells to increase.With the increase of LPS stimulation time,the effect of LPS on decreasing the activity of IPI-2I cells became worse,among which,when the stimulation time was 6 h,the MLPS group had the best effect on decreasing the activity of IPI-2I cells.2)LPS stimulation of IPI-2I cells significantly increased the contents of IFN-γ,TNF-α and IL-1β in the supernatant(P<0.05).After stimulation of IPI-2I cells for 6 h,the IFN-y and IL-1β content in the MLPS group was significantly higher than that in the CON group,the LLPS group and the HLPS group(P<0.05),and the IFN-y and IL-1β content in the CON group was increased by 10.61 times and 7.83 times.3)LPS could significantly increase the TNF-y and IL-1β mRNA expression levels in IPI-2I cells(P<0.01),and significantly reduce the IL-10 mRNA expression levels(P<0.01).When IPI-2I cells were stimulated for 6 h,compared with CON group,LLPS group and HLPS group,IPI-2I cells in the MLPS group had the highest IL-1β,TNF-α,and IFN-y mRNA expression levels,and the gene expression levels of IPI-2I cells were significantly higher than those of other experimental groups(P<0.05).In conclusion,LPS can effectively reduce the activity of IPI-2I cells,increase the amount of TNF-α and IL-1β in the superfluid,and increase the expression of TNF-α and IL-1β mRNA in the cells,thus causing the immune stress of IPI-2I cells.In this study,the MLPS group,namely 50 ug/mL LPS,it was selected to stimulate IPI-2I cells for 6 hours to establish the immune stress model.Experiment 3:The regulatory effect of tea tree oil on the immune stress of IPI-2I cellsTea Tree Oil is a natural plant extract,which can be used as feed additive in piglets’ diets to improve intestinal immune function.This experiment was conducted to investigate the regulatory effect of Tea Tree Oil on the immune stress state of pig ileum epithelial cells IPI-21.1%DMSO was used to dissolve Tea Tree Oil in the experiment,and 5 groups were set in the experiment:CON group(DMEM),LPS group(DMEM+50 ug/mL LPS),LTTO group(DMEM+50 ug/mL LPS+0.01%TTO),MTTO(DMEM+50 ug/mL LPS+0.03%TTO),HTTO group(DMEM+50 ug/mL LPS+0.05%TTO),and the test time was 6 h.Cytokines,apoptosis,immune-related gene expression and TLR4/NF-κB mRNA were measured.The results showed that:1)Compared with CON group,the number of IPI-2I cells in LPS group was significantly reduced(P<0.05),and the number of IPI-2I cells in early and late apoptosis was significantly increased(P<0.01).Compared with the LPS group,the number of apoptotic cells at the late stage of IPI-2I cells in the LTTO group was significantly reduced(P<0.05).Compared with LPS group,the number of live cells in TTO group was significantly increased(P<0.05),and the number of IPI-21 early apoptosis was significantly reduced(P<0.01).2)Compared with the LPS group,the contents of IL-1β,TNF-α,and IFN-y in the supernatant of the TTO group were significantly reduced(P<0.01),while the contents of IL-1β,TNF-α,and IFN-y in the MTTO group were significantly lower than those in the LTTO and HTTO groups(P<0.05).3)Compared with the LPS group,the mRNA expression levels of IL-1β,TNF-α,and IFN-y in the TTO group were significantly reduced(P<0.01),while those of the MTTO group were significantly lower than those of the LTTO and HTTO groups(P<0.05).Compared with LPS group,the expression of IFN-y mRNA in LTTO group was significantly increased(P<0.05),and the expression of IL-10 mRNA was significantly increased(P<0.01).The expression levels of TLR4 and NF-κB mRNA were significantly increased in the LPS group compared with the CON group(P<0.01);the expression levels of TLR4 and NF-κB mRNA were significantly decreased in the TTO group compared with the LPS group(P<0.01);the decreased levels of MTTO and HTTO were higher than that in the LTTO group,but there was no significant difference between the MTTO and HTTO groups(P>0.05).In conclusion,tea tree oil by lowering the IPI-2I early apoptosis and cellular immune stress condition late apoptotic cells,increase the number of living cells,reduce the cell supernatant on IL-1β,TNFα secretion,reduce the beta cells IL-1β,TNF-α mRNA expression,thereby alleviating LPS stimulated for IPI-2I cells immune stress,this may be related to tea tree oil of TLR4/NF-κB signaling pathways regulating,0.03%of doses of tea tree oil to relieve IPI-2I cellular immune stress effect is best.