| Liver failure in chronic hepatitis B (CHB) patients is a severe life-threatening condition. Intestinal endotoxemia play a significant role in the progresses of the liver failure. High mobility group box-1 (HMGB1) protein is involved in the process of endotoxemia. Regulatory T (Treg) cells can maintain the immune tolerance and contribute to the immunological hyporesponsiveness against HBV infection. Ethyl pyruvate (EP) has been shown to ameliorate hepatic, renal and intestinal mucosal injury, and down-regulate expression of several proinflammatory mediators in a wide variety of preclinical models of critical illnesses. On the basis of above-mentioned research background, this study firstly aims to explore the roles of HMGB1 and Treg cells in the pathogenesis of liver failure in CHB patients, and the effects of HMGB1 on immune activity of Treg cells in vitro; and then, aims to investigate the protective effects of EP on acute-on-chronic liver failure (ACLF) in rat model, and to observe the level changes of multiple proinflammatory proteins in vivo.(1) In vitro:15 liver failure patients with chronic HBV infection,20 CHB patients and 10 healthy controls were selected in this study. Peripheral blood mononuclear cells (PBMCs) from above-mentioned patients and controls were obtained via density gradient centrifugation. The levels of HMGB1 expression in serum, PBMCs and liver tissue were detected by ELISA, real-time RT-PCR, and Western-blot respectively. The percentage of CD4+CD25+CD127low Treg cells within the CD4+cells were detected by Flow cytometry in liver failure patients with chronic HBV infection, CHB patients and healthy controls. Then, isolated CD4+CD25+CD127low Treg cells from the PBMCs of CHB patients were stimulated with HMGB1 in different concentrations or at various time periods. The effect of HMGB1 on immune activity of Treg cells was observed by a suppression assay of allogeneic mixed lymphocyte response (MLR). The levels of forkhead box P3 (Foxp3) expression in Treg cells treated with HMGB1 were detected by RT-PCR and Western-blot. The results showed that the serum HMGB1 levels were significantly greater in the liver failure patients compared with the CHB patients (82.6±20.1μg/L vs.34.2±13.7μg/L, t=8.48, P<0.01), and there was no significant differences in serum HMGB1 levels between the CHB patients and healthy controls (34.2±13.7μg/L vs.27.8±6.5μg/L, t=1.73, P>0.05). The results of RT-PCR and Western blot further confirmed that a significantly higher expression of HMGB1 mRNA in PBMCs and HMGB1 protein in the liver tissues of the liver failure patients compared with the CHB patients (5.21±0.42 vs.1.78±0.18,t= 29.43, P<0.01; 4.86±1.07 vs.1.43±0.35, t=13.46, P<0.01, respectively). CHB patients had a significantly higher percentage of Treg cells within their population of CD4+ T cells in PBMCs than healthy controls (9.52%±3.89% vs.3.21%±1.21%,t= 4.97, P<0.01). While for the liver failure patients with chronic HBV infection, a significantly decreased proportion of Treg cells relative to CD4+ T cells were shown compared with CHB patients (4.55%±1.34% vs.9.52%±3.89%,t=4.73, P<0.01). The immune activity of Treg cells was significantly weakened and the levels of Foxp3 expression were reduced as a dose- or time-dependent manner, when Treg cells were stimulated with HMGBl in vitro. These experimental results concluded that the high level of HMGB1 and the low percentage of Treg cells play an important role in the pathogenesis of the liver failure patients with chronic HBV infection. Moreover, HMGB1 can weaken the immune activity of Treg cells. It is suggested that effectively inhibiting the HMGB1 expression could be expected a feasible way to treat the liver failure by suppressing endotoxemia and enhancing Treg cells activity.(2) In vivo:An ACLF model was induced by human serum albumin, D-galactosamine and lipopolysaccharide in rat. Animals were randomly divided into three groups:control group, model group and EP treatment group. The rats in EP treatment group received EP (40 mg/kg) at 3 h,6 h,12 h and 24 h time point after induction of ACLF. Animals were sacrificed, and samples were obtained at 48 h after induction of ACLF. Serum high mobility group box-1 (HMGB1), alanine aminotransferase (ALT), tumor necrosis factor (TNF)-α, interferon (IFN)-γ, interleukin (IL)-10 and IL-18 levels were measured. The changes of liver histology were examined, and the expressions of HMGB1 mRNA and protein in liver tissue were detected. The effects of EP on survival times of ACLF rats were also observed. The results showed that the serum levels of HMGB1, ALT, TNF-α, IFN-γ, IL-10 and IL-18 were significantly increased in model group compared with control group, and liver tissue of rats in model group presented severe pathological injury at 48 h after induction of ACLF, which showed that the structure of hepatic lobules was in disorder with many periportal inflammatory cells infiltration, hepatic plate was dissociated, and hepatocytes were swelling or lytic necrosis. However, EP administration significantly improved the hepatic histopathology, which showed obvious decrease of inflammatory cell infiltration, well-arranged hepatic cord, alleviation of hepatocytes swelling, and less pyrenolysis, and reduced the serum levels of HMGB1, ALT, TNF-α, IFN-γ, IL-10 and IL-18, and tissue levels of HMGB1 in EP treatment group regardless of treating time point at 3 h,6 h,12 h or 24 h after induction of ACLF. ACLF rats had a median survival time of 60 h. EP treatment significantly prolonged the median survival time of ACLF rats to 162 h,120 h,102 h and 78 h at 3 h,6 h,12 h and 24 h treating time point respectively. These experimental results concluded that EP administration is able to inhibit HMGB1 expression, decrease the levels of multiple inflammatory factors including TNF-α, IFN-γ, IL-10 and IL-18, reduce inflammatory cells infiltration, ameliorate hepatocellular injury, improve survival, and resultantly protect acute-on-chronic liver failure in rat. It is suggested that EP is a potential novel therapeutic agent for severe liver injury.
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