| Objective: The present study was conducted to (1) observe the changes and the distribution of toll-like receptor 2 and 4 (TLR2, TLR4) mRNA in different tissues in a cecal ligation puncture (CLP) model; (2) investigate the relationship between the expression of TLRs mRNA and lipopolysaccharide-binding protein (LBP)/CD14 pathway as well as pro-inflammatory/anti-inflammatory cytokine formation; (3) evaluate the influence of treatment with recombinant bactericidal/permeability- increasing protein (rBPl2i) and sodium butyrate (NaB) on TLR2/TLR4 mRNA expression; and (4) clarify the potential role of TLR2/TLR4 in the pathogenesis of CLP-induced sepsis and subsequent multiple organ dysfunction syndrome (MODS).Material and methods: 120 male Wistar rats were randomly divided into four groups as follows: (1) normal control group (n=10); (2) sham operation group (n=10); (3) CLP group (n=60): being further divided respectively into 2h, 6h, 12h, 24h, 48h and 72h subgroups post-CLP; (4) rBPI treatment group (n=20): intravenous injection of rBP^i at a dose of Img/kg at 0.5h and 4h after CLP, being further divided respectively into 12h, 24h post-CLP subgroups; (5) NaB treatment group (n=20): intravenous injection of sodium butytate at a dose of 500mg/kg at 0.5h and 4h after CLP, being further divided respectively into 12h, 24h subgroups post-CLP. At serial time points in each group, animals were sacrificed, and blood and tissue samples from liver, lungs, kidneys and small intestine were harvested. Blood and tissue endotoxin concentrations were measured by the chromogenic Limulus Amebocyte Lysate (LAL), which was modified by perchloric acid (PCA) pretreatment for samples. Both blood andtissue TNF-cc, IL-10 protein levels were determined by enzyme-linked immunosorbent assay (ELISA). TLR2, TLR4, LBP, CD 14, TNF-a, HMG-1, and IL-10 mRNAs were semi-quantitated by the reverse transcription polymerase chain reaction (RT-PCR) taking GAPDH as an internal standard. The major organ functional indices, pulmonary myeloperoxidase (MPO) and small intestinal diamine oxidase (DAO) activities were also measured.Results: 1. Endotoxin levels in plasma, liver and lungs increased significantly following CLP with double peaks at 2-12h and 48-72h, respectively. Treatment with BPI could significantly reduce endotoxin levels in liver, lungs and kidneys except plasma at 12h after sepsis. Similarly, treatment with NaB could decrease endotoxin levels in liver, and kidneys without marked influence on pulmonary levels at 12h. 2. TLR2 and TLR4 mRNA could be detected in various tissues with low values in normal controls, and they were significantly increased at 6h after CLP. Tissue TLR4 mRNA gradually decreased 24h later, while TLR2 mRNA levels kept high values up to 72h. In comparison with CLP animals, treatment with BPI significantly decreased TLR2 mRNA in various tissues at 12h and 24h, so did tissue TLR4 mRNA at 12h, without marked influence on TLR4 mRNA expression at 24h in liver, lungs and small intestine. Both TLR2 and TLR4 mRNA were significantly decreased at 12h in all tissues by NaB treatment. 3. LBP mRNA were significantly expressed in liver, lungs and kidney at 2~48h post-CLP, but they were elevated in small intestine only at 2h and 72h after CLP. LBP mRNA expressions were inhibited at 12h and 24h in liver and lung by BPI or NaB treatment. Concomitantly, CD 14 mRNA levels in various tissues increased to different extent, but it was inhibit in all tissues at 12h and in intestine at 24h by the treatment with rBPIn- Also, treatment with NaB could significantly down-regulate CD 14 mRNA expression in various tissues at712h but not at 24h. 4. TNF-a gene and protein expressions in all tissues as well as plasma samples were enhanced to certain extent with double peaks at 2-12h and 48-72h following CLP. Treatment with rBPI2i could markedly inhibit tissues TNF-a mRNA and tissues/blood TNF-a protein levels at 12h post-CLP, whereas no significant differences in TNF-a gene/protein expression were noted at 12h and 24h post-CLP. 5. HMG-1... |
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