Abstract
Purpose: To investigate the protective effect of floroindole against cecal ligation and puncture (CLP)- induced sepsis, as well as the underlying mechanism of action.
 Methods: Thirty-five 10–week-old male Wistar rats weighing 190 - 210 g (mean: 200.00 ± 10.10 g) were used for this study. The rats were randomly assigned to seven groups of five rats each, viz, normal control group, and six CLP groups. The CLP groups were those subjected to cecal ligation and puncture (CLP). The first 5 CLP groups received 2, 4, 6, 8 or 10 mg/kg floroindole, respectively, 1 h after CLP, via intraperitoneal route (i.p.) while the 6th CLP group served as untreated control. Western blotting, enzyme-linked immunosorbent assay (ELISA) and real-time quantitative polymerase chain reaction (qRT-PCR) were used for the assessment of the expression levels of tumor necrosis factor-α (TNF- α), interleukn-6 (IL-6), nucleotide-binding oligomerization domain 2 (NOD2) and p-NF-κB p65.
 Results: Cecal ligation and puncture (CLP) significantly and time-dependently upregulated the expressions of TNF-α, IL-6 and NOD2 in intestinal tissues of rats (p < 0.05). However, treatment with floroindole significantly, and dose-dependently down-regulated CLP-induced expressions of these proteins (p < 0.05). Treatment of rats with floroindole also significantly and dose-dependently inhibited CLP-induced phosphorylation of NF-κB p65 in rat ileum (p < 0.05).
 Conclusion: The results obtained in this study demonstrate that floroindole confers some degree of protection against CLP-induced sepsis via inhibition of NF-κB p65 phosphorylation.
Highlights
Sepsis is a complicated clinical syndrome caused by inflammatory reactions to microbial invasion [1]
The present study investigated the protective effect of floroindole against CLPinduced sepsis, and the underlying mechanism
The results of Western blotting and Quantitative real-time polymerase chain reaction (qRT-PCR) showed that cecal ligation and puncture (CLP) significantly and timedependently promoted NF-κB p65 phosphorylation in rat ileum relative to normal control group (p < 0.05; Figure 5)
Summary
Sepsis is a complicated clinical syndrome caused by inflammatory reactions to microbial invasion [1]. Two rats from each group were sacrificed at different time points: 2, 4, 6, 8 and 10 h of CLP after isoflurane anesthesia, and the ileal tissues excised and kept at -78 oC prior to use. The ileum of each rat treated with floroindole was excised under isoflurane anesthesia and washed twice with phosphate-buffered saline (PBS). The ileal tissues were homogenized on ice in HEPES buffer (10 mM, pH 8.0) which consisted of potassium chloride (10 mM), magnesium chloride (2 mM), EDTA (0.1 mM), dithiothreitol (1.0 mM) and PhCH2SO2F (0.5 mM). Total RNA from homogenized ileal tissues of rats was isolated using Trizol reagent. Electrophoresis was performed on 2 % agarose gel for separation of PCR products and normalization of the mRNA expression level was done with GAPDH. Values of p < 0.05 were considered statistically significant
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