| Due to the strong toxic and side effects of many chemically synthesized drugs,people are paying more and more attention to the development of anti-inflammatory drugs with less toxic and side effects from natural extracts,and seeking safe and effective anti-inflammatory extracts is a new idea to prevent and treat such diseases.It is also a new hot spot for the development of new anti-inflammatory feed additives.In this experiment,LPS-induced inflammatory response regulation signaling pathway was the main line to study the effect of gardenia on inflammatory stress piglet inflammation pathway and cytokine levels.In vitro establishment of piglet intestinal epithelial cell stress model to study gardenia extract geniposide The effects of glycosides on the inflammatory pathways and tight junctions of pig intestinal epithelial cells provide new ideas for preventing piglet immune stress and various intestinal diseases.In the in vivo test,24 weaned piglets of similar weight were randomly divided into four groups,with 6 pigs in each group,pre-fed for 3 days,and 3 gardenia added groups,each with gardenia powder 1.25,2.5,5 g added to the basic diet every day.Drink water and eat freely,weigh on the 10 th day,inject LPS after blood collection from the anterior vena cava,and collect blood 3h and 24 h after injection.Separate serum and lymphocytes.The expression levels of NF-κB pathway and IAP protein in lymphocytes,relative expression of m RNA and the changes of 8 cytokines in serum were measured.In the in vitro test part,IPEC-J2 cells were plated with the appropriate concentration of cell liquid,which were blank group and model group,0.1,1,10 mmol/L concentration of geniposide added group,37℃,5% CO2 incubator for 24 h In addition to the control group,add 20 μL of 0.1 mg/m L LPS to each well for 6 h,collect the cells and detect the NF-κB pathway and tight junction protein levels and relative m RNA expression levels.The test results are as follows: Piglet experiment part:(1)Growth performance changes: the daily gain and daily feed intake of weaned piglets in the gardenia added group were significantly reduced(P<0.05),and the feed weight ratio of the low and medium dose gardenia added group was not significantly increased(P>0.05).(2)Protein change level: The p65 and IκB phosphorylation levels in the gardenia added group were significantly decreased before and after inflammation(P<0.05),and the expression level of IAP protein in the low-medium-dose gardenia added group was significantly lower than that in the inflammation control group(P< 0.05);before inflammation,the expression of p65 and IκB protein in the low-dose group was lower than that in the inflammation control group(P<0.05),and at 3 h and 24 h after inflammation,the expression of IκB protein in the low-dose group was lower than that in the inflammation control group(P <0.05).(3)Changes in relative expression of m RNA: NF-κB p65 in the low and medium dose group was significantly lower than that in the inflammation control group(P<0.05);IκB and IKK in the middle and high dose group were significantly lower than that in the inflammation control group(P<0.05);low and high dose COX-2 in the group was significantly lower than that in the inflammation control group(P<0.05);IAP in the low-medium dose group was significantly lower than that in the inflammation control group(P<0.05).(4)The level of cytokine changes in serum: Compared with the non-inflammatory group,the levels of IL-1β,IL-10,and IFN-γ increased significantly after inflammation caused by LPS(P<0.05).In the case of inflammation,IL-1β and TNF-α were significantly lower in the low-dose gardenia addition group at 3 h after inflammation(P<0.05),and IL-10 was significantly higher at 24 h than the control group(P<0.05),IFN-γ and TNF-α were significantly reduced(P<0.05);IL-1β and TNF-α were significantly reduced(P<0.05)at the middle-dose addition group at 3 h and 24 h after inflammation(P<0.05),IL-4.IL-10 increased significantly(P<0.05);the content of IL-1b,IL-6,IL-8,IL-12 and IFN-γ in the high-dose addition group at 3 h and 24 h was significantly lower than that in the control group(P<0.05),IL-4 and IL-10 increased significantly(P<0.05),and the content of TNF-α at 24 h was significantly lower than that of the control group(P<0.05).In vitro cell experiment part:(1)The level of protein changes: IKK in the model group and the low-medium concentration addition group was significantly higher than that in the control group(P<0.05),and the p65 and IKK phosphorylation levels in the inflammation model group were significantly increased(P<0.05).Glycoside can significantly reduce the expression level of phosphorylated p65 protein in cells(P<0.05),and the phosphorylation of p65 and IKK in the remaining test groups returned to normal levels;the three tight junction proteins were significantly lower than the control group after LPS stimulation(P<0.05),the OCCLUDIN protein expression in the drug addition group was significantly higher than that in the control group(P<0.05),the medium and high concentration drug addition group ZO-1 protein expression was significantly higher than the control group(P<0.05),the medium concentration drug addition group The three tight junction proteins were significantly improved(P<0.05).(2)Changes in relative m RNA expression: the relative expression of p65,IKK,and IκB m RNA in the LPS-inflamed group was significantly higher than that in the control group(P<0.05),and the low-concentration group p65 and IKK were significantly higher than the control group(P<0.05),IκB in the high-concentration group was significantly lower than that in the control group(P<0.05);COX-2 in the inflammatory group was significantly higher than that in the control group(P<0.01),the three drug-added groups had a significant recovery effect,and COX-2 in the middle and high-concentration groups It was significantly higher than the control group(P<0.05);the ZO-1 and CLAUDIN-1 in the inflammation group were significantly lower than the control group(P<0.05),and the tight connection of the low concentration group returned to normal levels.Conclusion: 1.The three gardenia added groups can all reduce the increase of inflammatory pathway factors in the inflammatory response,alleviate the changes of proinflammatory and inflammatory cytokines caused by the inflammatory response,thereby improving the immune stress of LPS on weaned piglets.2.The intervention effect of geniposide reduces the expression of inflammatory pathway proteins and relative m RNA expression in IPEC-J2 cells,restores the relative expression of COX-2 m RNA to normal levels,and improves the expression of tight junction protein and m RNA Relative expression.It shows that the addition of geniposide can relieve inflammation and improve the level of tight junction of cells. |
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