Synthesis of endotoxin receptor CD14 protein in Kupffer cells and its role in alcohol-induced liver disease

AIM: To observe the synthesis of endotoxin receptor CD14 protein and its mRNA expression in Kupffer cells (KCs), and evaluate the role of CD14 in the pathogenesis of liver injury in rats with alcohol-induced liver disease (ALD).METHODS: Twenty-eight Wistar rats were divided into two groups: ethanol-fed group and control group. Ethanol-fed group dextrose instead of ethanol. Two groups were sacrificed at 4 wk and 8 wk, respectively. KCs were isolated and the synthesis of CD14 protein and its mRNA expression in KCs were determined by flow cytometric analysis (FCM)or the reverse transcription polymerase chain reaction (RTPCR) analysis. The levels of plasma endotoxin and alanine transaminase (ALT) were measured by Limulus Amebocyte Lysate assay and standard enzymatic procedures respectively, and the levels of plasma tumor necosis factor (TNF)-α and interleukin (IL)-6 were both determined by ELISA. The liver pathology change was observed under light and electric microscopy.RESULTS: In ethanol-fed group, the percentages of FITCCD14 positive cells were 76.23 % and 89.42 % at 4 wk and 8 wk, respectively. Compared with control group (4.45 %and 5.38 %), the difference was significant (P＜0.05). The expressions of CD14 mRNA were 7.56±1.02 and 8.74±1.37 at 4 wk and 8 wk, respectively, which were significantly higher compared with the control group (1.77±0.21 and 1.98±0.23)(P＜0.05). Plasma endotoxin levels at 4 wk and 8 wk increased dramatically in ethanol-fed rats (112±15 IU/L and 147±22 IU/L) than those in the control animals (31±12 IU/L and 33±9 IU/L) (P＜0.05). In ethanol-fed rats, the levels of wk, respectively which were significantly higher than those fed rats, there were marked pathological changes including steatosis, cell infiltration and necrosis. No marked pathological changes were seen in control group.CONCLUSION: Ethanol administration led to a significantsynthesis of endotoxin receptor CD14 protein and its gene expression in KCs, which maybe result in the pathological changes of liver tissue and hepatic functional damages.

Protective effect of nitric oxide induced by ischemic preconditioning on reperfusion injury of rat liver graft

AIM: Ischemic preconditioning (IP) is a brief ischemic episode,which confers a state of protection against the subsequent long-term ischemia-reperfusion injuries. However, little is known regarding the use of IP before the sustained cold storage and liver transplantation. The present study was designed to evaluate the protective effect of IP on the long-term preservation of liver graft and the prolonged anhepatic-phase injury.METHODS: Male Sprague-Dawley rats were used as donors and recipients of orthotopic liver transplantation. All livers underwent 10 min of ischemia followed by 10 min of reperfusion before harvest. Rat liver transplantation was performed with the portal vein clamped for 25 min. Tolerance of transplanted liver to the reperfusion injury and liver damage were investigated. The changes in adenosine concentration in hepatic tissue and those of nitric oxide (NO) and tumor necrosis factor (TNF) in serum were also assessed.RESULTS: Recipients with IP significantly improved their one-week survival rate and liver function, they had increased levels of circulating NO and hepatic adenosine, and a reduced level of serum TNF, as compared to controls. Histological changes indicating hepatic injuries appeared improved in the IP group compared with those in control group. The protective effect of IP was also obtained by administration of adenosine,while blockage of the NO pathway using Nco-nitro-L-arginine methyl ester abolished the protective effect of IP.CONCLUSION: IP appears to have a protective effect on the long-term preservation of liver graft and the prolonged anhepatic-phase injuries. NO may be involved in this process.

Role of NF-kB in multiple organ dysfunction during acute obstructive cholangitis

AIM:To elucidate the role of NF-kB activation in the development of multiple organ dysfunction(MOD)during acute obstructive cholangitis(AOC)in rats. METHODS:Forty-two Wistar rats were divided into three groups:the AOC group,the group of bile duct ligation(BDL group),and the sham operation group(SO group).All the animals in the three groups were killed in the 6th and 48th hour after operation.Morphological changes of vital organs were observed under light and electron microscopy.NF-κB activation was determined with Electrophoretic Mobility Shift Assay(EMSA).Arterial blood gas analyses and the serum levels of lactate dehydrogenase(LDH),alanine aminotransferase(ALT),blood urea nitrogen(BUN)and creatinine were performed.The concentrations of TNF-α and IL-6 in plasma were also measured. RESULTS:The significant changes of histology and ultrastructure of vital organs were observed in AOC group. By contrast,in BDL group,all the features of organs damage were greatly reduced.Expression of NF-κB activation in various tissues increased in AOC group when compared to other two groups.At 6 h,the arterial pH in three groups was 7.52±0.01,7.46±0.02,and 7.45±0.02,and the blood pCO_2 was 33.9±0.95 mmHg,38.1±0.89 mmHg,38.9±0.94 mmHg,there was difference in three groups(P<0.05).At 48 h,the blood pH values in three groups was 7.33±0.07, 7.67±0.04,and 7.46±0.03,and blood HCO_3^- was 20.1±1.29 mmol·L^(-1),26.7±1.45 mmol·L^(-1)and 27.4±0.35 mmol·L^(-1),there was also difference in three groups(P<0.05).In AOC group, Levels of LDH,ALT,BUN and creatinine were 16359.9±2278.8 nkat·L^(-1),5796.2±941.9 nkat·L^(-1),55.7±15.3 mg/dl,and 0.72± 0.06 mg/dl,which were higher than in SO group(3739.1± 570.1 nkat·L^(-1),288.4±71.7 nkat·L^(-1),12.5±2.14 mg/dl,and 0.47±0.03 mg/dl)(P<0.05).Levels of plasma TNF-α and IL-6 in AOC at 48 h were 429±56.62 ng·L^(-1)and 562±57 ng·L^(-1), which increased greatly when compared to BDL group (139±16 ng·L^(-1),227±43 ng·L^(-1))and SO group(74±10 ng·L^(-1), 113±19 ng·L^(-1))(P<0.05). CONCLUSION:The pathological damages and the NF-κB activation of many vital organs exised during AOC.These findings have an important implication for the role of NF-κB activation in MOD during AOC.