Protection Of Chloral Hydrate On LPS/D-GalN-induced Lethal Liver Injury And Zymosan-induced Peritonitis In Mice And Its Mechanism

BackgroundAcute or serious inflammation induced by infection or uninfection has been considered as a very important issue in clinical and the research on pathogenesis and development of new medicines. Scientists are doing great efforts to find new leads of medicine including chemical compounds, plant extracts, even combinatory protein or peptide. In addition, people found that some of the medicines which were approved being used in other fields have good anti-inflammatory activities. Recently, several anesthetics have been found to possess anti-inflammatory and anti-infective effects apart from their functions of pain relief and muscle relaxation. For example, lidocaine, ketamine, isoflurane and pentobarbital can significantly improve the survival of mice and rats with endotoxic shock and protect them against liver and renal injury resulting from cecal ligation and puncture (CLP)-induced septic peritonitis .In the previous work, we occasionally found that chloral hydrate used as an amimanl anesthetic can improve the hemodynamic parameters of mice with endotoxin shock and anaphylactic shock. Therefore, we want to know whether chloral hydrate can function as an inhibitor of inflammation, because there are no any reports about the effect of chloral hydrate on acute inflammation. What made us interested at this issue is that chloral hydrate as a sedative and anesthetic has been used in clinical for over one hundred years, and it is currently used in pediatric manipulations like magnetic resonance imaging (MRI) and echocardiogram on children and in animal experiments at many countries. Furthermore, the evaluation of pharmacologic mechanism and safety of chloral hydrate have been still emphasized in human which suggests that chloral hydrate would not be obsolete even today.In the present study, we sought to investigate the effects of chloral hydrate on the acute lethal liver injury induced by lipopolysaccharide/D-galactosamine (LPS/D-GalN) or on the acute peritonitis, a mild inflammatory response induced by zymosan A, which have been considered as well defined animal models . Especially, LPS/D-GalN-induced acute lethal liver injury in mice is a widely used as one of experimental animal models for more than 20 years, by which the mechanisms of lethal hepatic failure and development of the effective therapeutic strategies against endotoxin challenge have been investigated, because endotoxemia or sepsis is also associated with fulminant hepatic failure in human Moreover, the manifestation of systemic inflammatory response syndrome (SIRS) in the absence of infection may occour the similar syndrome to sepsis Therefore, our work could provide useful evidence used in research of LPS/D-GalN-induced acute lethal liver injury and sepsis. Another classic model used in our work is acute peritonitis induced by zymosan A, a kind of mild non-lethal acute inflammation. The mice with lethal acute liver injury induced by LPS/D-GalN can live a short period of time and caused an extreme serious state in order to observe the protection of chloral hydrate. The protection of chloral hydrate on peritonitis, a mild non-lethal inflammation is more close to clinical.We also intended to explore the mechanisms of the protective effects against LPS/D-GalN-induced acute lethal liver injury and peritonitis by chloral hydrate, including the profiles of proinflammatory cytokines and the change of activities of nuclear factorκappaB (NF-κB). Assessment and selection of the parameters for present work including levels of IL-6, TNF-a and MCP-1 were according to the work on prognosis-related cytokines in patients with septic shock as reported by Bozza. Among them, TNF-a has been proved to be closed associated with survival of mice challenged with LPS only or LPS/GalN and MCP-1 is an important cytokine, which can balance the proinflammatory and anti-inflammatory reactions in acute lethal inflammation like sepsis.Based on the protection of chloral hydrate in vivo, we also selected RAW264.7 cell and mouse peritoneal macrophages as models to study how chloral hydrate affects the activity of NF-κB and the production of inflammatory factors.PartⅠ. Protection of chloral hydrate on LPS/D-GalN-induced lethal liver injury and zymosan-induced peritonitis in miceSince chloral hydrate (CH) treatment in this work is once injection and maintaining for short period, we chose a more serious acute inflammation model induced by the intraperitoneal (i.p.)injection of 10μg LPS-2630 (Escherichia coli serotype 2630) plus 800 mg of D-galactosamine hydrochloride (D-GalN, Sigma-Aldrich, St. Louis, MO, USA)×kg-1 of body weight, which median survival time is 9.2 h. The effect with CH treatment at different doses (80,160 and 320 mg/Kg) and different time points (0,1,3 and 5 h after the challenge by LPS/D-GalN) were observed, based on the safety proved by 320 mg/Kg CH on mice in preliminary test.The results showed that CH treatment improved the median survival of mice in a dose-dependent manner, especially that in group which received 160(11.68±2.62 h) and 320 mg/Kg (13.78±1.21 h) was significantly improved compared with controls (9.17±1.10h) (P=0.024 and P=0.001),and CH administered at 0,1 and 3 h after the challenge by LPS/D-GalN significantly improved the median survival of these mice compared with controls(13.70,13.30 and 11.60h vs.9.40h, P=0.002, P=0.004 and P=0.002, respectively). CH treatment attenuated the rises of serum MCP-1,IL-6 and TNF-a levels at 3h after LPS/D-GalN challenge compared with those control group (P=0.001,P=0.009 and P=0.015,respectively). In contrast to the control group, however, the levels of IL-6 and TNF-αunderwent a sharp rise in the treatment group at 6 h after LPS/D-GalN challenge, which was significantly higher than that of the control group. The levels of ALT in mice with LPS/D-GalN-induced acute lethal liver injury remained significantly lower in the CH treatment group than those of the controls at 3,6 and 9 h after challenged with LPS/D-GalN(P=0.023,P=0.003 and P=0.042. respectively),while AST levels were significantly lower than those in the control group at 9 h after challenged with LPS/D-GalN (P=0.007). Gross examination of the liver showed a marked swelling, congestion and hemorrhage in the control group at 9 h after the challenge with LPS/D-GalN, while the liver had only mild congestion in the CH treatment group. And the liver sections stained by H&E showed that LPS/D-GalN-induced necrosis of hepatocytes in both control and CH-treated groups, but more severe liver pathologic changes induced by LPS/D-GalN were occurred in the control group than in the CH-treated group; especially the markedly differences appeared at 9 h after challenge, in which the structure of the liver lobules was destroyed and serious necrosis of hepatocytes and hemorrhage in the control group occurred, while only mild pathologic changes were shown in the CH-treated group.The treatment with CH (320 mg/Kg) on zymosan-induced peritonitis in mice significantly decreased inflammatory response. The peak of leukocytes count in the peritoneal exudates in mice treated by CH was delayed at least 16h compared to control group. The leukocytes count in the CH treatment group was significantly lower than the control group at 5 and 8 h (P=0.03,P=0.006, respectively). Also the peak of the protein in the peritoneal exudates in the CH treatment group was lowered and delayed 4 h, which also differed between the two groups at 0.5,1 and 8 h after zymosan A challenge.The levels of TNF-α,MCP-1 and IL-6 in the serum of CH treatment group was significantly lowered at 1,2 and 8 h after zymosan A challenge compared to the control group.In this part, it was found that CH (320 mg/Kg) treatment on acute lethal liver injury of mice induced by LPS/D-GalN and acute peritonitis induced by zymosan A significantly decreased the levels of inflammatory cytokines.PartⅡ. Chlorate hydrate inhibited the activity of NF-κB in vitro and in vivoBased on the changes of the levels of TNF-a, IL-6 and MCP-1 by CH treatment in mice with LPS/D-GalN-induced lethal liver injury and zymosan-induced peritonitis, we intended to detect activity of NF-κB in NF-κB-RE-luc (Oslo) luciferase reporter transgenic mice using in vivo bioluminescence imaging technology and in RAW264.7 cells transfected with NF-κB luciferase plasmid in vitro.The effect of CH (320 mg/Kg) treatment on the activation of NF-κB was examined in six of the NF-κB-RE-luc (Oslo) luciferase reporter transgenic mice after challenged by LPS/D-GalN. The results suggested that LPS/D-GalN challenge increased the NF-κB activities in mice treated only by NS, which peaked around 4 h after challenged with LPS/D-GalN. CH, however, significantly attenuated the rise of NF-κB activities at 3,4,5 and 6 h time point compared to control group (All P=0.000). Examination of individual organs for NF-κB activities further revealed the highest NF-κB activities in the liver followed intestines and lungs, while the NF-κB activities in these three organs in the control group were significantly higher than those in the CH treatment group(P=0.002, P=0.026,P=0.015, respectively).RAW264.7 cells transfected with NF-κB luciferase plasmid and peritoneal macrophages were chosen as models in vitro. CH (0.5 and 1 mg/ml) treatment significantly inhibited the activity of NF-κB and decreased the production of IL-6 and TNF-a by peritoneal macrophages after stimulated by LPS/LTA.In this part, it was found that CH treatment significantly inhibited the activity of NF-κB in mice stimulated by LPS/D-GalN and the production of inflammatory cytokines stimulated by LPS/LTA in vitro and in vivo.Part III. Chloral hydrate induced the apoptosis of mononuclear cells and its mechanismThe different concentrations of CH (0.25,1 and 2 mg/ml) were used to treat RAW264.7 cells and peritoneal macrophages and the change of cell shape and function (eg, phagocytosis) were observed using detection of early apoptosis, Hochest 33258 staining, DNA ladder and other methods.The results showed that CH treatment induced the apoptosis of RAW264.7 cells, in which. From the typical spindle shape of cells gradually became to round and finally shedding suspended under the treatment of CH; the treatment with CH (0.5 mg/ml) treated for 2,3,5 h and 7 h significantly induced early apoptosis (P=0.000) and typical DNA ladder of RAW264.7 cells. The photos usting Hochest 33258 staining showed that CH treatment induced the typical nuclear features of apoptosis: nuclear pyknosis and density markedly significantly increased, cohesion and fracture, while NS treatment in control showed nucleus homogeneous blue fluorescence. The phagocytic activity of RAW 264.7 were reduced treated by CH(P=0.000).CH treatment also induced the apoptosis of peritoneal macrophages. Early apoptosis of spleen mononuclear gradually increased in LPS/D-GalN induced liver injury mice and the apoptotic rate of in group with CH (320 mg/Kg) treatment was higher than control group at 2 and 3 h time point(P=0.001 and P=0.000, respectively).We chose RAW264.7 cells as in vitro model and spleen tissues of normal mice, CH (320 mg/Kg) treated mice challenged by LPS/D-GalN as in vivo model to illustrate the mechanism of CH induced the apoptosis of target cells. The results showed that CH treatment induced the expression of Fas not FasL on RAW264.7 cells, and increased the expression of Fas on spleen tissues of mice receiving CH (320 mg/Kg) for 9 h, compared with normal spleen tissues expressing a less Fas. No significant difference in Fas expression between control group and CH (320 mg/Kg) treatment group of mice under challenge with LPS/D-GalN, but the expression in both group were higher than that in normal mice.In this part, several methods were used to detect the apoptosis of RAW264.7 cells and peritoneal macrophages in vitro and spleen mononuclear cells and spleen biopsy in vivo, the results showed that the expression of Fas by apoptotic RAW264.7 cells and spleen tissues of normal mice under treatment with CH (320 mg/Kg) significantly higher than normal without CH.StatisticsAll results were expressed as (Mean±SD).The survival rates were estimated using SPSS 13.0 Kaplan-Meier method and tested for statistical significance using Breslow-Gehan-Wilcoxon test. Comparisons between control and CH treated group were performed using Independent-student T test. Comparisons among control and CH treated groups on RLU (relative light unit) were performed using one-way ANOVA. P<0.05 was considered to be significant. ConclusionIn conclusion, it has been demonstrated that CH can attenuate and delay the inflammatory response of mice with LPS/D-GalN-induced acute lethal liver injuryor peritonitis and improve the survival of mice with LPS/D-GalN-induced acute lethal liver injury. The changed inflammatory response is associated with an inhibiting effect of CH against the increase of NF-κB activities and in the serum levels of the proinflammatory cytokines. It is also worth to emphasize that the anti-inflammatory effect of CH can be exerted even with administration at 1 to 3 h after the challenge of LPS/D-GalN. Moreover, CH treatment also can decrease the activity of NF-κB in RAW264.7 cells transfected with NF-κB luciferase plasmid stimulated by LTA/LPS in vitro, and the production of IL-6 and TNF-αby peritoneal macrophages. In addition, we have found that the expression of Fas on apoptotic RAW264.7 cells and spleen tissues of normal mouse under the treatment with CH (320 mg/Kg) are significantly higher than normal controls.