| In this experiment we used the intestinal porcine epithelial cells(IPEC-J2)vitro culture model to elaborate the regulation mechanism of seaweed polysaccharide(SWP)on the barrier function of IPEC-J2 cells.The result provided a theoretical basis and guidance for the application of plant extract seaweed polysaccharide in livestock production.Experiment ?: Effect of seaweed polysaccharide on cytotoxicity,proliferation and apoptosis of IPEC-J2 cellsIn this study,IPEC-J2 cells were studied to investigate the effects of seaweed polysaccharides on cytotoxicity,apoptosis and proliferation of IPEC-J2 cells.Different concentrations of seaweed polysaccharides were selected in the experiment to determine their effects on the vitality of IPEC-J2 cells and LDH release rate,and to screen the appropriate dosage and action time of seaweed polysaccharides.On this basis,the effects of SWP on the apoptosis and proliferation of IPEC-J2 cells were determined by flow cytometry.The results showed that:(1)When treating IPEC-J2 cells with SWP for 12 h,the activity of IPEC-J2 cells could be significantly promoted by SWP at the concentration of 8-480 ?g/mL(P<0.05).The activity of IPEC-J2 cells was significantly increased at the concentrations of 60 ?g/mL,120 ?g/mL,and 240 ?g/mL for 24 h after treatment with SWP,and reached the maximum at 240 ?g/mL.At concentrations greater than 240 ?g/mL,cell viability began to decline.At the same time,the release rate of LDH could be significantly reduced in the range of 30-3640 ?g/mL(P<0.05).Therefore,the concentration ranges of 60 ?g/mL,120 ?g/mL and 240 ?g/mL were selected for subsequent experiments.(2)Compared with the control group,the addition of SWP 60 ?g/mL,120 ?g/mL and 240 ?g/mL significantly reduced cell apoptosis(P<0.05),and the 120 ?g/mL and 240 ?g/mL group significantly promoted cell proliferation and division(P<0.05).In summary,SWP had the effect of reducing IPEC-J2 cytotoxicity,promoting proliferation and reducing apoptosis in IPEC-J2 cells.Experiment ?: Effect of adding SWP on the barrier function of IPEC-J2 cellsThe aim of this study was to investigate the regulatory effect of polysaccharides from sea algae on the mechanical barrier and immune barrier function of IPEC-J2 cells in 60 ?g/mL,120 ?g/mL and 240 ?g/mL cells.The regulatory mechanism of SWP on the barrier function of IPEC-J2 cells was investigated by measuring the expression of related tight junction proteins and cytokines.The results showed that:(1)SWP of 60 ?g/mL and 120 ?g/mL significantly up-regulated the expressions of Occludin and ZO-1 mRNA of IPEC-J2 cells compared with the control group(P<0.05),and SWP of 120 ?g/mL and 240 ?g/mL significantly up-regulated the expressions of Claudin-1 mRNA of IPEC-J2 cells(P<0.05).At the same time,the addition of SWP had no significant effect on the expression of Occludin protein(P>0.05).The addition of 120 ?g/mL and 240 ?g/mL of SWP could significantly up-regulate the tight junction proteins expression of Claudin-1 and ZO-1 of IPEC-J2 cells(P<0.05).(2)Compared with the control group,the addition of 240 ?g/mL seaweed polysaccharide significantly down-regulated the mRNA expressions of TLR-4 and I?B?(P<0.05),60 ?g/mL,120 ?g/mL,and 240 ?g/mL seaweed polysaccharide significantly down-regulated the mRNA expressions of MyD88 and p65(P<0.05),showed no significant difference in the expressions of p65,p-p65,and p-p65/p65,There was no significant effect on the expression of inflammatory factors IL-6 and TNF-? in the cell supernatant(P>0.05).In conclusion,the addition of SWP for 24 h could promote the expression of IPEC-J2 cell tight junction protein and down-regulate the relative gene expression of NF-?B signaling pathway,but there was no significant difference in cytokine secretion.Experiment ?: Effect of SWP on barrier function of E.coli infected with IPEC-J2 cellsThe purpose of this study was to study after 24 h treatment of small intestinal epithelial cells IPEC-J2 with SWP,the effect of E.coli infection IPEC-J2 cells,the number of adhesion invasiveness,expression of tight junction protein,and the effect of NF-?B signaling pathway related genes expression,and to further explore the protective mechanism of polysaccharides from SWP on IPEC-J2 cells.The results showed that:(1)At 90 min of E.coli infection,240 ?g/mL of SWP could significantly alleviate the decreased permeability of IPEC-J2 cells caused by bacterial infection,at 120 min of E.coli infection,the addition of 60 ?g/mL,120 ?g/mL and 240 ?g/mL could significantly alleviate the decreased permeability of IPEC-J2 cells caused by E.coli infection,with a dose trend(P<0.05).(2)E.coli infection significantly reduced the expression of IPEC-J2 cells tight junction protein Occludin,Claudin-1 and ZO-1,but the pretreatment with seaweed polysaccharide significantly inhibited the expression of IPEC-J2 cell tight junction protein caused by E.coli infection(P<0.05).The expression of Occludin-1,Claudin-1,and ZO-1 decreased after 2 h of E.coli infection,but the difference was not significant.E.coli infection after 4 h,significant decrease is closely connected to the expression of the protein Occludin and Claudin-1,but there was no significant difference to TJs proteins expression of the protein ZO-1.Compare to E.coli infection group,60 ?g/mL,120 ?g/mL and 240 ?g/mL SWP significantly inhibited the expression of Occludin in IPEC-J2 cells caused by E.coli infection(P<0.05),and 240 ?g/mL SWP significantly inhibited the expression of Claudin-1 and ZO-1 in IPEC-J2 cells caused by E.coli infection(P<0.05).(3)When infected with E.coli for 2 h,the SWP of 60 ?g/mL,120 ?g/mL and 240 ?g/mL significantly promoted the expression of inflammatory cytokines in cell supernatant IL-6 and TNF-? compared with the group infected with E.coli(P<0.05),the SWP with 60 ?g/mL,120 ?g/mL and 240 ?g/mL significantly inhibited the expression of inflammatory factors compared with the E.coli infection group(P<0.05).(4)SWP had certain inhibitory effect on the activity of E.coli.120 ?g/mL and 240 ?g/mL SWP could reduce the number of viable E.coli,the number of adhesion of escherichia coli to IPEC-J2 cells and the number of invasive viable E.coli(P<0.05).To sum up,the addition of SWP could significantly inhibit the destruction of tight junction proteins by E.coli infection.When E.coli was infected for 2 h,SWP played a pro-inflammatory role in IPEC-J2 cells.When the E.coli was infected for 4 h,the polysaccharides from sea algae IPEC-J2 cells played an anti-inflammatory effect,and the SWP could reduce the activity of E.coli and the number of viable bacteria that adhered to and invaded IPEC-J2 cells. |
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