Protective Effects Of Eleutheroside E On Barrier Function Of Small Intestinal Epithelial Cells In Piglets

The intestinal health of piglets is a very important part of piglet growth.Diarrhea in weaned piglets is mainly caused by impaired intestinal barrier function.Acanthopanax,as a Chinese herbal medicine feed additive,is also widely used in piglet production.In this paper,the effect of Eleutheroside E(EE)on the intestinal barrier function was studied.In vitro experiments were conducted on the intestinal epithelial cells(IPEC-J2)of piglet and the intestinal barrier function was studied.It provides theoretical support for the application of Chinese herbal medicine feed additives in piglet production.This study was divided into two parts.First,the effect of Eleutheroside on the barrier function of small intestinal epithelial cells in piglets under in vitro conditions was studied,and the mechanical barrier and immune barrier function of piglets' intestinal epithelial cells were studied.Then the injury model of piglet intestinal epithelial cells induced by soybean agglutinin(SBA)was used to explore the protective effect of Eleutheroside senticosus on the injury of intestinal mechanical barrier and immune barrier function.The content of this test is as follows:Pre-test,concentration screening of Eleutheroside E on effective proliferation activity of piglet intestinal epithelial cells.Add 0 mg/mL,0.05 mg/mL,0.1 mg/mL,and 0.2 mg/mL Eleutheroside E to the normal DMEM/F12 medium.Observe different concentrations of Acanthopanax using a real-time label-free cell analyzer.E-stimulated proliferation of IPEC-J2 cells.According to the test results,a significant increase in cell proliferation at 35-50 h was selected as effective concentration(P<0.01).Test 1,Effect of Eleutheroside E on the Permeability of Small Intestinal Epithelial Cells in PigletsThe IPEC-J2 cells which were in close proximity to the growth state were cultured with 0.5 mg/mL and 0.1 mg/mL of Eleutheroside E,The effects of Eleutheroside E on cell membrane permeability were measured by measuring the transepithelial resistance(TEER)values of cells grown in the six-well cell plate and the activity of alkaline phosphatase(AKP)in the supernatant of cell culture.The results showed that 0.1 mg/mL of Eleutheroside E significantly increased the cell TEER value(P<0.05),and the activity of AKP in the cell culture fluid decreased significantly(P<0.05).Test 2,Effect of Eleutheroside E on the expression of tight junction protein related to mechanical barrier function of small intestinal epithelial cells in pigletsSelect the 0.1 mg/mL Eleutheroside E treatment dose for the determination of the tight junction protein expression of IPEC-J2,The expression of Claudin-3,Ocludin,and ZO-1 in the tight junction between the experimental group and the control group was measured by real-time fluorescence quantitative PCR and Western blotting,respectively.The results showed that the addition of 0.1 mg/mL of the experimental group significantly increased the expression of Claudin-3 and ZO-1 genes and significantly increased the protein expression of Claudin-3 and ZO-1(P<0.05).Test 3: Effect of Eleutheroside E on immune barrier function of small intestinal epithelial cells in pigletsA 0.1 mg/mL dose of Eleutheroside E was used to determine the gene expression levels of inflammatory cytokines in small intestinal epithelial cells of piglets.The inflammatory cytokine IL-6 in test and control groups was determined by real-time fluorescent quantitative PCR.Gene expression of TNF-?,IFN-? and anti-inflammatory factors IL-10,TGF-?.The results showed that the gene expression of inflammatory cytokines IL-6,TNF-?,and IFN-? was significantly down-regulated after treatment with 0.1 mg/mL Eleutheroside E,and the genes for anti-inflammatory cytokines IL-10 and TGF-? Expression was significantly upregulated(P < 0.05).Test 4: Study on the protective effects of Eleutheroside E on injury of intestinal mechanical barrier and immune barrier functionIntestinal epithelial cells of piglets cultured in vitro were modeled.0 mg/mL EE was added to the control group in normal DMEM/F12 medium,0.1 mg/mL EE was the test group 1,and 0.5 mg/mL soybean lectin was the test group 2.0.1 mg/mL EE pretreatment and 0.5 mg/mL SBA were added as experimental group 3,and the cell proliferation activity,cell membrane permeability,tight junction protein gene expression and protein expression,and expression of major inflammatory cytokine genes were studied.Effects of Eleutheroside sbrevicatus on alleviating the injury of small barrier epithelial cells mechanical barrier and immune barrier function in piglets.The results showed that 0.1 mg/mL Eleutheroside senticosus can effectively prevent the damage caused by 0.5 mg/mL soybean lectin on cell mechanical barrier and immune barrier function.Combined with the above several experiments,it was shown that adding Eleutheroside E to the intestinal epithelial cells of piglets in vitro can promote the mechanical barrier function and immune barrier function of piglets,which is beneficial to the health of the intestine.Provide some data reference in the regulation of piglet nutrition.