Study on Alleviating Effects of Branched-Chain Fatty Acids on Lipopolysaccharide-Induced Inflammatory Response in IPEC-J2 Cells
This experiment aimed to study the effects of branched-chain fatty acids(BCFA)on the antioxi-dant,anti-inflammatory,anti-apoptotic,and barrier-protective capabilities of porcine intestinal epithelial cells(IPEC-J2 cells)induced by lipopolysaccharide(LPS).A single-factor experimental design was used,it was divided into 8 groups:control group,LPS induction group,iso-C14∶0+LPS group,iso-C15∶0+LPS group,iso-C16∶0+LPS group,iso-C17∶0+LPS group,anteiso-C 15∶0+LPS group,anteiso-C17∶0+LPS group,with three repetitions for each group.Before officially commencing the experiment,the optimal conditions for trea-ting IPEC-J2 cells with six BCFA monomers(iso-C14∶0,iso-C15∶0,iso-C16∶0,iso-C17∶0,anteiso-C15∶0,anteiso-C17∶0)and LPS were obtained.The activity of IPEC-J2 cells was determined using the CCK-8 meth-od,the reactive oxygen species(ROS)content of IPEC-J2 cells was measured with a ROS detection kit,and the impact of each BCFA monomer on IPEC-J2 cells was examined through real-time quantitative PCR(RT-qPCR).The results were showed as follows:1)through gradient testing,it was ultimately determined that cells were treated with six BCFA monomers at a concentration of 1.5 μmol/L for 24 h,and cells in each group were treated with LPS at a concentration of 10 µg/mL for 24 h to establish a cellular inflammatory model.2)Examination of the effects of six BCFA monomers on the activity of IPEC-J2 cells induced by LPS revealed that,compared with the LPS-only stimulation group,BCFA pretreatment significantly alleviated the reduction in IPEC-J2 cell viability caused by LPS stimulation(P＜0.05).Among the BCFA monomers,iso-C17∶0 pre-treatment resulted in the highest cell viability,reaching 91.55％.3)Testing of ROS content in cells using a kit found that,compared with the LPS group,ROS content in IPEC-J2 cells was significantly reduced in the BC-FA group(P＜0.05).4)After BCFA pretreatment,the relative gene expression levels of interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α),and interferon-γ(IFN-γ)in cells were lower than those in the LPS group(P＜0.05).For the relative gene expression level of interleukin-6(IL-6),the treatment with iso-C14∶0,iso-C17∶0 and anteiso-C15∶0 was significantly lower than that in the LPS group(P＜0.05).As for the relative gene expression level of interleukin-8(IL-8),only the treatment with iso-C17∶0 was significantly lower than that in the LPS group(P＜0.05).Except for the iso-C14∶0+LPS group,the relative gene expression level of interleukin-10(IL-10)in cells of other BCFA+LPS groups was significantly higher than those in the LPS group(P＜0.05).5)Compared with the LPS group,the relative gene expression levels of Toll-like receptor 4(TLR4)and myeloid differentiation factor 88(MyD88)in IPEC-J2 cells were significantly reduced due to the addition of BCFA(P＜0.05).Except for anteiso-C15∶0,other BCFA also significantly reduced the relative gene expression level of nuclear factor-KB(NF-κB)in cells(P＜0.05).6)Compared with the LPS group,BCFA monomers were able to increase the relative expression levels of zonula occludens-1(ZO-1),Occludin,Claudin-1,and Claudin-4 genes in IPEC-J2 cells stimulated by LPS(P＜0.05).7)The addition of BCFA could reduce the relative expression level of Caspase-8 gene in IPEC-J2 cells after LPS stimulation(P＜0.05).Compared with the LPS group,the relative expression level of Caspase-3 gene in cells was significantly re-duced after treatment with iso-C14∶0,iso-C16∶0,iso-C17∶0 and anteiso-C17∶0(P＜0.05).After treatment with iso-C14∶0 and iso-C17∶0,the relative expression levels of Caspase-9 gene in cells were also significantly reduced(P＜0.05).8)Compared with the LPS group,the relative expression levels of BCL-2-associated X protein(BAX)gene in cells was significantly reduced after adding other BCFA monomers except anteiso-C15∶0(P＜0.05).After the addition of BCFA,the relative expression level of B-cell lymphoma-2(BCL-2)gene was significantly higher than those in the LPS group(P＜0.05).In conclusion,BCFA can reduce the ac-cumulation of ROS in IPEC-J2 cells,regulate the secretion of inflammatory cytokines,suppress the overactiva-tion of the TLR4/NF-κB signaling pathway,enhance the expression of genes related to tight junctions,regu-late the expression of apoptosis genes,and alleviate the inflammatory response in IPEC-J2 cells caused by LPS.[Chinese Journal of Animal Nutrition,2024,36(9):5935-5948]
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