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. 2019 Oct;18(20):2614-2628.
doi: 10.1080/15384101.2019.1653106. Epub 2019 Sep 1.

Ethyl pyruvate inhibits LPS induced IPEC-J2 inflammation and apoptosis through p38 and ERK1/2 pathways

Na Dong  1 , Xinyao Xu  1 , Chenyu Xue  1 , Chensi Wang  1 , Xinran Li  1 , Chongpeng Bi  1 , Anshan Shan  1
Affiliations

Ethyl pyruvate inhibits LPS induced IPEC-J2 inflammation and apoptosis through p38 and ERK1/2 pathways

Na Dong et al. Cell Cycle. 2019 Oct.

Abstract

The endotoxin of Gram-negative bacteria threatens the intestinal health of livestock. Ethyl pyruvate (EP) has been shown to regulate intestinal immunity and protect against cell and tissue damage. In this study, it was first verified that EP could reduce the secretion of IL-8, TNF-α, IL-6 and IL-1β in LPS-induced IPEC-J2 cells. Then, we used RNA sequencing (RNA-seq) to analyze the differentially expressed genes (DEGs) of inflammatory factors induced by LPS in IPEC-J2 cells. It was found that LPS induced the upregulation of 377 genes and the downregulation of 477 genes compared to Vehicle; LPS+EP induced the upregulation of 258 genes and the downregulation of 240 genes compared to Vehicle; and LPS+EP induced the upregulation of 373 genes and the downregulation of 188 genes compared to LPS (fold change > 1.5 and FDR < 0.01). Their enrichment pathways included the MAPK signaling pathway, PI3K-Akt signaling pathway, Toll-like receptor signaling pathway, and other pathways. Furthermore, the mRNA level of cytokines associated with inflammation and apoptosis enriched in the MAPK pathway was verified by qRT-PCR. Western blots and immunofluorescence revealed that EP significantly inhibited phosphorylated p38 and phosphorylated-ERK1/2 protein expression levels (P < 0.05). The apoptosis due to LPS reduced by EP was significantly inhibited, as shown by Annexin V-FITC/PI staining. According to the results, EP inhibited the expression of IL-8, TNF-α, IL-6 and IL-1β as well as apoptosis by inhibiting the phosphorylation of p38 and ERK1/2 in LPS-induced IPEC-J2 cells.

Keywords: Apoptosis; Ethyl pyruvate; MAPK; Porcine jejunal epithelial cells; RNA-seq.

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Figures

Figure 1.
Figure 1.
Effects of EP on secretion of inflammatory factors in LPS induced IPEC-J2 cells. (a) Concentration of TNF-α in IPEC-J2 cells; (b) Concentration of IL-8 in IPEC-J2 cells; (c) Concentration of IL-6 in IPEC-J2 cells; (d) Concentration of IL-1β in IPEC-J2 cells. IPEC-J2 cells were treated by 10 μg/mL LPS, 10 μg/mL LPS+2.5 mM EP or 2.5 mM EP respectively for 12 h. #p means significant compared with control, *p means significant between LPS group. Values are mean ± SD.
Figure 2.
Figure 2.
Go Terms of biological function analysis among Vehicle, LPS and LPS+EP groups.
Figure 2.
Figure 2.
(Continued).
Figure 3.
Figure 3.
Up-regulation genes and down-regulation genes. (a)Numbers of DEGs between groups compared each other. (b-d) Vocano plot of DEGs. The x-axis indicates the difference in expression level on a log2 (fold change). The y-axis represents corresponding false discovery rate on a negative Lg (FDR). Red represents up-regulation gene and green means down-regulation genes.
Figure 4.
Figure 4.
(a) Heatmaps of all the differential genes in KEGG inflammatory pathways. (b-d) Bubbles of KEGG pathways for differential gene enrichment. The circle presented gene number. The color of circles indicates the Q value.
Figure 5.
Figure 5.
Validation of the RNA-Seq expression profiles of genes randomly selected from DEGG by RT-qPCR. Black bars represent FPKM, white bars represent fold change. FPKM (Fragments Per Kilobase Million) normalized values as gene expression in RNA-seq. #p means significant compared with control, *p means significant between LPS group. Values are mean ± SD.
Figure 6.
Figure 6.
(a-b) Transient expression of the p-p38 and p-ERK1/2 protein in IPEC-J2 cells by fluorescence microscopy. IPEC-J2 cells were incubated overnight and treated by 10 μg/mL LPS, 10 μg/mL LPS+2.5 mM EP or 2.5 mM EP respectively for 12 h. P-p38 and p-ERK1/2 protein fluorescence was determined by fluorescence microscope.
Figure 7.
Figure 7.
Phosphoprotein levels of p38 and ERK1/2. (a-b) Phosphoprotein levels of p38 in IPEC-J2 cells. (c-d) Phosphoprotein levels of ERK1/2 in IPEC-J2 cells. Cells were treated by 10 μg/mL LPS, 10 μg/mL LPS+2.5 mM EP or 2.5 mM EP respectively for 12 h. #p means significant compared with control, *p means significant between LPS group. Values are mean ± SD.
Figure 8.
Figure 8.
Effects of p38 and ERK1/2 inhibitors on the secretion of inflammatory factors by EP in LPS induced IPEC-J2 cells. ip38 represents p38 inhibition and iERK1/2 represents ERK1/2 inhibition. (a-b) Effects of p38 and ERK1/2 inhibition on secretion of TNF-α. (c-d) Effects of p38 and ERK1/2 inhibition on secretion of IL-8. (e-f) Effects of p38 and ERK1/2 inhibition on secretion of IL-6. (g-h) Effects of p38 and ERK1/2 inhibition on secretion of IL-1β. Cells were pre-incubated with 10 μM p38(SB203580) and 20 μM ERK1/2(U0126) inhibitors for 1 h and then treated by 10 μg/mL LPS or 10 μg/mL LPS+2.5 mM EP respectively for 12 h. Different values between groups are shown with capital letters (a, b, c; p < 0.05). Values are mean ± SD.
Figure 8.
Figure 8.
(Continued).
Figure 9.
Figure 9.
Effect of EP on LPS-induced apoptosis of IPEC-J2 cells. IPEC-J2 cells were incubated and treated by 10 μg/mL LPS, 10 μg/mL LPS+2.5 mM EP or 2.5 mM EP respectively for 12 h, then cells were incubated with Annexin V-FITC/PI kit for 10 min. Finally, observed apoptosis fluorescence by fluorescence microscope.
Figure 10.
Figure 10.
Signaling pathways of inflammation and apoptosis in LPS induced cells by EP regulation.
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Grants and funding

This work was supported by the Heilongjiang Provincial Science and Technology Department [QC2015018];National Natural Science Foundation of China [31472104];National Natural Science Foundation of China [31501914];National Natural Science Foundation of China [31672434];Postdoctoral Research Foundation of China [2015M571385].