| In the treatment of sepsis, early fluid therapy appears to be essential to optimize hemodynamics and obtain suitable tissue perfusion. However, much controversy still exists about the role of crystalloids and colloids in fluid therapy. Clinicians are often confused about which fluid fraction is most appropriate for septic patients.Hydroxyethyl starch (HES) is one of the most frequently used plasma substitutes and derived from maize, sorghum or potato. According to its in vitro molecular weight (MW) and molar substitution (MS), HES is classified into different starch preparations. HES130/0.4, the third generation of HES, was developed with lower MW (130) and MS (0.4) to enhance degradation and to minimize side effects.In addition to maintaining the stability of hemodynamics, previous studies have shown that HES may have anti-inflammatory effects, such as down-regulating systemic inflammatory mediators and reducing capillary permeability through inhibiting leukocyte adhesion and infiltration. However, the accurate mechanism of the anti-inflammatory effect of HES is still unclear.The Toll-like receptors (TLRs)/nuclear factor-kappa B (NF-кB) signaling pathway plays an important role in initiating the inflammatory response in sepsis. TLR family members are transmembrane proteins, which can specifically recognize pathogen-associated molecular patterns. Among 10 mammalian TRLs, TLR4 functions as a receptor for lipopolysaccharide (LPS) from Gram-negative bacteria, and TLR2 is involved in the recognition of multiple products of Gram-positive bacteria. TLR signals finally activate the transcription factor NF-kB and permit the transactivation of proinflammatory cytokine genes. Our experiments were designed to investigate the anti-inflammatory effect of HES 130/0.4 in septic rats induced by cecal ligation and puncture (CLP) and the possible mechanism related with TLRs/NF-kB signaling pathway.Section one:Effect of HES130/0.4 on plasma cytokines in septic rats and possible mechanism related with TLR4/NF-kB signaling pathwayIn addition to expanding blood volume, HES130/0.4, the third generation of HES colloidal solutions, has been reported to exert an anti-inflammatory effect. However, the accurate mechanism is still obscure. The aim of the experiment was to observe the effect of HES130/0.4 on plasma cytokines in septic rats and investigate the possible mechanism related with LPS-TLR4-NF-kB signaling pathway. Rats with sepsis induced by CLP were treated with HES 130/0.4 (7.5,15, or 30 ml/kg) intravenously, then, rat plasma and peripheral blood monocytes were isolated from blood at 5h after CLP. The plasma levels of LPS and cytokines (tumor necrosis factor [TNF]-a and interleukin [IL]-6), NF-kB activities and mRNA and protein levels of TLR4 in peripheral blood monocytes were determined by limulus amebocyte lysate test, enzyme-linked immunosorbent assay, electrophoretic mobility shift assay, reverse transcription-polymerase chain reaction and western blotting test, respectively. The results showed that HES 130/0.4 dose-dependently reduced the plasma levels of TNF-a and IL-6 in rats with sepsis. As compared with 7.5 and 30 ml/kg HES130/0.4,15 ml/kg caused greater reduction of plasma TNF-a and IL-6 concentrations. HES 130/0.4 also significantly inhibited NF-kB activation and TLR4 expression in monocytes. The results suggest that during sepsis HES 130/0.4 could down-regulate the inflammatory response, possibly through inhibiting the TLR4/NF-kB signaling pathway, and could be one more appropriate plasma substitute in sepsis.Section two:Effect of HES130/0.4 on pulmonary capillary permeability in septic rats and possible mechanism related with TLRs/NF-kB signaling pathwaySepsis frequently causes multiorgan dysfunction and the lung is the most vulnerable organ that is often affected. According to different severities, lung injury is classed into acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Previous studies indicated that HES could inhibit leukocyte adhesion and infiltration due to its anti-inflammatory effect, and then reduce capillary permeability. However, the accurate mechanism is obscure. The aim of the experiment was to observe the effect of HES 130/0.4 on pulmonary inflammatory mediators, leukocyte infiltration and pulmonary capillary permeability and further investigate the mechanism related with TLRs (TLR2, TLR4)/NF-kB signaling pathway. Rats with sepsis induced by CLP were treated with HES130/0.4 or succinylated gelatin (15 ml/kg, intravenously), then, rat lungs were isolated from blood at 5 h after later. The pulmonary levels of inflammatory mediators (TNF-α, IL-1βand intercellular adhesion molecule [ICAM]-1), NF-kB activities and mRNA and protein levels of TLRs (TLR2 and TLR4) were determined by enzyme-linked immunosorbent assay, electrophoretic mobility shift assay, reverse transcription-polymerase chain reaction and western blotting test, respectively. At 12 h after CLP, the rat lungs were collected for determination of capillary permeability and myeloperoxidase (MPO) activities. The results showed that HES130/0.4 significantly reduced pulmonary edema and capillary permeability, down-regulated pulmonary inflammatory mediators including TNF-a, IL-1βand ICAM-1, and inhibited pulmonary NF-kB activation and TLRs (TLR2 and TLR4) expressions. Compared with HES130/0.4, gelatin could also decrease pulmonary levels of inflammatory mediators and reduce capillary permeability, but not impact NF-kB activation and TLRs expression. The results of the experiment indicated that HES 130/0.4 could reduce pulmonary capillary permeability possibly through down-regulating inflammatory mediators and inhibiting TLRs (TLR2, TLR4)/NF-kB signaling pathway in septic rats.ConclusionThe aim of the experiments was to observe the effect of HES 130/0.4 on systemic inflammatory response and pulmonary capillary permeability in septic rats and further investigate the possible mechanism related with TLRs/NF-kB signaling pathway. The results definitely demonstrated that HES 130/0.4 could down-regulate the septic rat systemic inflammatory mediators, reduce pulmonary capillary permeability and inhibit TLRs expression and NF-kB activation in peripheral monocytes and lung tissue, which suggests that the mechanism on anti-inflammatory effect of HES 130/0.4 in sepsis might be related with inhibiting TLRs/NF-kB signaling pathway.As one colloidal solution, the main pharmaceutical effect of HES is still to expand blood volume and improve hemodynamic parameters. However, compared with other many solutions in pharmaceutical market, the effects of anti-inflammatory and reducing capillary permeability would strengthen the competitive capacity of HES. As the results of the second experiment showed, HES 130/0.4, compared with the same dose of succinylated gelatin, could more significantly reduce pulmonary capillary permeability in septic rats. If the conclusion of our animal experiments was further conformed using clinical trials, the clinicians would consider the above pharmaceutical advantages of HES 130/0.4, which is the utmost clinical significance of our experiments.
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