Treatment with β-Adrenoceptor Agonist Isoproterenol Reduces Non-parenchymal Cell Responses in LPS/D-GalN-Induced Liver Injury

Abstract

There is an increasing evidence indicating the involvement of the sympathetic nervous system (SNS) in liver disease development. To achieve an extensive comprehension of the obscure process by which the SNS alleviates inflammatory damage in non-parenchymal liver cells (NPCs) during acute liver failure (ALF), we employ isoproterenol (ISO), a beta-adrenoceptor agonist, to mimic SNS signaling. ISO was administered to C57BL/6J mice to establish an acute liver failure (ALF) model using LPS/D-GalN, which was defined as ISO + ALF. Non-parenchymal cells (NPCs) were isolated from liver tissues and digested for tandem mass tag (TMT) labeled proteomics to identify differentially expressed proteins (DEPs). The administration of ISO resulted in a decreased serum levels of pro-inflammatory cytokines, e.g., TNF-α, IL-1β, and IL-6 in ALF mice, which alleviated liver damage. By using TMT analysis, it was possible to identify 1587 differentially expressed proteins (DEPs) in isolated NPCs. Notably, over 60% of the DEPs in the ISO + ALF vs. ALF comparison were shared in the Con vs. ALF comparison. According to enrichment analysis, the DEPs influenced by ISO in ALF mice were linked to biological functions of heme and fatty acid metabolism, interferon gamma response, TNFA signaling pathway, and mitochondrial oxidation function. Protein-protein interaction network analysis indicated Mapk14 and Caspase3 may serve as potentially valuable indicators of ISO intervention. In addition, the markers on activated macrophages, such as Mapk14, Casp1, Casp8, and Mrc1, were identified downregulated after ISO initiation. ISO treatment increased the abundance of anti-inflammatory markers in mouse macrophages, as evidenced by the immunohistochemistry (IHC) slides showing an increase in Arg + staining and a reduction in iNOS + staining. Furthermore, pretreatment with ISO also resulted in a reduction of LPS-stimulated inflammation signaling markers, Mapk14 and NF-κB, in human THP-1 cells. Prior treatment with ISO may have the potential to modify the biological functions of NPCs and could serve as an innovative pharmacotherapy for delaying the pathogenesis and progression of ALF.

Keywords: acute liver failure.; hepatic non-parenchymal cells; isoproterenol; proteomic.

Fig. 1

Effect of ISO treatment on liver hepatic pathology caused by lipopolysaccharide (LPS)/D-galactosamine (D-GalN) in female mice. a Morphological observation of liver tissue. b Representative sections of hematoxylin–eosin (H&E) staining (100-fold) in liver tissues. c Serum levels of alanine transaminase (ALT) and aspartate transaminase (AST), in mice with LPS/D-GalN administration. d Survival curves in different group of mice and e biological analysis of TNF-α, IL-6, and IL-1β mRNA levels in liver tissues. Statistical difference was performed by Student’s t-test in each comparison. **P < 0.01, ***P < 0.001.

Fig. 2

Analysis of differentially expressed proteins (DEPs) in each comparison. a Volcano plots showing the fold changes of protein expressions in different comparisons: left: ALF vs. Con, middle: ISO + ALF vs. ALF, right: ISO + ALF vs. Con. b Statistical number of upregulated and downregulated DEP profiles in each comparison. c Venn diagram showing the interactions of all DEPs analyzed in three compared groups. d Hierarchical clustering heatmap analysis of difference in protein expression. Rank abundance plots presented the proteins in each comparison: e ALF vs. Con, f ISO + ALF vs. ALF, and g ISO + ALF vs. Con. The top 10 upregulated and downregulated proteins screened from the comparison of ISO + ALF vs. ALF were especially labeled out.

Fig. 3

Enrichment pathways with the negative/positive enrichment score after gene set enrichment analysis (GSEA) in different comparisons: a ALF vs. Con group, d ISO + ALF vs. ALF group, g ISO + ALF vs. Con group. Individual GSEA enrichment plots for top enriched categories: b Heme metabolism and c interferon gamma response in ALF vs. Con group, e fatty acid metabolism and f adipogenesis pathway in ISO + ALF vs. ALF group, and h coagulation pathway in ISO + ALF vs. Con group.

Fig. 4

Hierarchical clustering heatmap revealed five distinct temporal patterns of proteins expression identified by K-means clustering. Protein expression changes in a Cluster 1, b Cluster 2, c Cluster 3, d Cluster 4, and e Cluster 5 across different groups. Bubble diagrams displaying the top 10 items of Gene Ontology (GO) for f Cluster 1, g Cluster 4, and h Cluster 5.

Fig. 5

Bubble diagrams displaying the top 10 items Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of the three clustering differentially expressed proteins (DEPs): a Cluster 1, b Cluster 4, and c Cluster 5. The interaction network of the hub proteins presented in different clusters for Cluster 1 (d), Cluster 4 (e), and Cluster 5 (f).

Fig. 6

Exploration of macrophage activation in mice liver. a Hierarchical clustering heatmap analysis for the absolute quantification levels of DEPs correlated to macrophage activation and b bubble diagram displaying the enrichment analysis for cell compartments, tissues, and Wikipathways. Immunohistochemistry stainings of c iNOS and d Arg-1 for the markers of macrophages in mouse liver tissues. e Western blot for the protein expressions of inflammation-induced apoptosis in isolated non-parenchymal tissues.

Fig. 7

ISO repressed inflammation progress probably via p38 and NF-kB signaling in macrophage THP-1 cell line. RT-qPCR for the expressions of IL-10 (a), TNF-α (b), and IL-6 (c) mRNAs in THP-1 cells with LPS (1 µg/mL, 24 h) and ISO (100 nM, pretreated 2 h). Flow cytometry analysis of activation of THP-1 cells among control (d), LPS (1 µg/mL, 24 h; e), and ISO + LPS (100 nM, pretreated 2 h; then 1 µg/mL, 24 h; f) groups with surface antibodies CD14 (Pacific blue) and CD40 (FITC) was calculated with the bar chart (g). Western blot for the protein expressions of NF-κB (h, upper) and p38 MAPK (i, upper) in THP-1 cells was calculated with bar charts in bottom plots. **P < 0.01.