Hepatocellular Damage in Normal and LBP-Deficient Rats after LPS-Administration
Inferior vena cava blood was collected from 3 normal and 3 LBP-deficient rats after LPS-induced at 0 h, 6 h, and 24 h respectively, then we analyzed the serum levels of ALT. In the normal groups, the serum ALT level reached a peak at 6 h after LPS administration (Fig. 1), suggesting the most severe liver injury. In contrast, less hepatocellular damage was observed in consistent with the obvious decreases in serum levels of ALT in LBP-deficient samples (Fig. 1).
Mapping and Annotation of RNA Sequencing Reads
The RNA sequencing technique integrated with bioinformatics analysis was used to characterize alteration in liver gene expression between WT and LBP-deficient samples triggered by LPS-induced systemic inflammation, and the analyze steps were sown in Fig. 2A. We obtained about 69.7 million (M) of 150 bp paired-end reads for each sample (ranging from 57.2 to 107.2 million reads) (Additional file 1). After ambiguous mapping (allowing for multi-hits) via STAR-2.5.3a , a total of ~ 64.4 M reads for each sample were mapped against the rat reference genome Rattus_norvegicus.Rnor_6.0 (Ensembl, ftp://ftp.ensembl.org/pub/release-96/fasta/rattus_norvegicus) (Fig. 2B, Additional file 1). Among the mapped reads, 92.1% of these reads were mapped to exonic regions, 4.5% mapped intergenic regions, and 3.4% mapped intronic regions (Fig. 2B).
To evaluate the segregation between WT and LBP-deficient samples during the different time after LPS administration, we conducted the neighbor-joining tree of samples based on the expression of all genes. As shown in Fig. 2C, a clear divergence between the time of LPS-treated (0 h, 6 h, and 24 h) was observed in this tree, and WT and LBP-deficient rats were also defined their respective separate clades, suggesting high fidelity of our RNA-Seq data.
Systemic Administration of Bacterial LPS Induces Global Changes in the Liver Transcriptome
To further characterize the DEGs from our RNA-Seq data, an analysis was performed to screen DEGs with P-value less than 0.05 and log2(fold change) higher than 1.5 using DESeq2 R package  (Fig. 3). In total, we identified 168, 284 and 307 significantly alternative genes respectively during the time of 0 h, 6 h, and 24 h between the normal and LBP-deficient samples. Then we clustered these DEGs via hierarchical heatmap (Fig. 3A-C) to depict the differential expression gene profile between the normal and LBP−/− rats. The most significantly DEGs with P-value < 0.001 and log2(fold change) > 1.5 were labeled in the volcano plots (Fig. 3D-F, Additional file 2). Among that, Lrp5 , Cyp7a1 , Nfkbiz , Sigmar1 , Fabp7 , and Hao1  (Additional file 3) have been reported in inflammatory response and lipid metabolic process, suggesting these genes may play an important role in modulating sepsis-induced system inflammation in WT and LBP-deficient rats after LPS injection.
Gene Annotation and Gene Ontology Analyses of DEGs
To further study of significantly overrepresented gene ontology terms involving these DEGs during 0 h, 6 h, and 24 h after LPS administration, functional annotations were performed with the DAVID Bioinformatics Resources 6.7 (https://david-d.ncifcrf.gov/) respectively . Selecting from the full enrichment data sets (Fig. 4A), we found ten representative terms with the exhibition of strong differential enrichment patterns were mainly related to inflammatory response, immune response and lipid metabolic processes (Fig. 4B). In further, to detect the most associated DEGs during those biological processes between healthy and LBP-deficient groups, we exhibited associated genes evolved in the representative terms and pathways (Fig. 5). Interestingly, we found that in the normal rats, LPS strongly upregulated genes involved in the processes of the inflammatory response and immunomodulation including Ifng , Cxcl10 , Serpine1 , and Lbp  (Additional file 4A-D) at 6 h after LPS injection, then proceed in lipid metabolic response including C5 , Cyp4a1 , and Eci1  (Additional file 4E-G) at 24 h (Fig. 5A). And the enriched pathways were in accordance with the results of gene ontology (Fig. 5B), which revealed that inflammatory pathways containing the toll-like receptor signaling pathways and natural killer cell-mediated cytotoxicity were enhanced in the normal groups at 6 h and lipid-related metabolism of peroxisome proliferator activated receptor (PPAR) signaling pathway was enriched at 24 h after LPS administration. Conversely, the functional enrichment of DEGs in LBP-deficient groups predominantly activated the lipid metabolic response instead of inflammatory or immunological response during the first two time points, enriching some up-regulated genes such as Dhrs7b  and Tysnd1  (Additional file 4H-I). And the PPAR signaling pathway was significantly over-represented at the first two time points, which suggesting modulating inflammation and bacterial killing after LPS challenge with the deficient of the LBP gene . Interestingly, the DEGs both in healthy and LBP−/− groups were over-represented in the processes of lipid metabolic and repeatedly enriched genes of Eci1, Pnpla3 , Apoa5 , and Fabp1  (Additional file 4J-L).
A Proposed Model of the Roles of NF-κB and PPAR Signaling Pathways in the WT and LBP-Deficient Rats after LPS Challenge
Based on the biological functions of above-mentioned genes and previous studies of nuclear factor kappa B (NF-κB) and PPAR signaling pathways, we presented a proposed model for the development of sepsis in rats (Fig. 6). At 6 h, the upregulation of Ifng, Cxcl10, Serpine1, and Lbp in WT rats trigger NF-κB signaling pathway induced inflammation response after LPS injection. And the activation of NF-κB signaling pathway is responsible for modulating the immune reaction via enhanced biosynthesis of large quantities of pro-inflammatory molecules, including cytokines, adhesion molecules, etc., which are frequently induce sepsis and cause tissue damage when their production is dysregulated and excessive [35, 36].
At 24 h, PPAR signaling pathway was found and may function as bacterial clearance via the formation of NET by highlighted genes of C5, Cyp4a1, and Eci1 in SD rats, and enhanced Dhrs7b and Tysnd1 in the LBP−/− rats after LPS administration. Just as reports revealed, PPARs are a large superfamily of nuclear receptors and incorporate three isoforms (PPAR-α, PPAR-β, and PPAR-γ), which are broadly involved in the regulation of metabolism, especially associated with lipid and glucose homeostasis [37, 38]. In the process of activating pathway, PPAR-γ negatively regulates the activity of the transcription factor to inhibit the expression of pro-inflammatory mediators such as tumor necrosis factor alpha (TNF-α), interleukin 12 (IL-12), and adhesion molecules which results in anti-inflammatory outcomes in the setting of sepsis induced by LPS .
Together, the proposed model reflects that invading LPS may interplay with LBP to activate NF-κB signaling pathway and trigger uncontrolled inflammatory response. However, when inhibiting the activity of NF-κB, lipid-related metabolism would make bacteria removal via the effect on PPAR signaling pathway in the absence of LBP gene.