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.

Lipopolysaccharide induced synthesis of CD14 protein and its gene expression in hepatocytes during endotoxemia

AIM:To observe synthesis of CD14 protein and expressionof CD14 mRNA in hepatic tissue and hepatocytes of ratsduring endotoxemia.METHODS:The endotoxemia model of Wistar rat wasestablished by injection of a dose of lipopolysaccharide(LPS)(5mg·kg^(-1),Escherichia coil O111:B4)via the tailvein,then the rats were sacrificed after 3,6,12 and 24 h inbtaches.Hepatocytes were isolated from normal and LPS-injected rats by in situ collagenase perfusion technique andwere collected to measure the expression of CD14 mRNAand synthesis of CD14 protein by reverse transcription-polymerase chain reaction(RT-PCR)or Western blotanalysis.The binding of fluorescein isothiecyanate(FITC)-CD14 polyclonal antibody to isolated hepatocytes was alsoassessed by flow cytometric analysis(FCM).RESULTS:In the rats with endotoxemia,the expressions ofCD14 mRNA in hepatic tissue and isolated hepatocytes werestronger at 3,6,and 12 h than that in control rats(3.48±0.15,5.89±0.62,4.33±0.18,vs 1.35±0.14 in hepatictissue,P<0.01;4.12±0.17,6.24±0.64,4.35±0.18,vs1.87±0.15 in hepatecytss,P<0.01).The synthesis of CD14protein in hepatic tissue and isolated hepatoeytes increasesalso obviously in 6 and 12 h when compared to that incontrol rats(13.27±1.27,17.32±1.35,11.42±1.20,vs 7.34±0.72 in hepatic tissue,P<0.01;14.68±_+1.30,17.95±1.34,11.65±1.19,vs 7.91±0.70 in hepatocytoes,P<0.01).FCM showed that mean fluorescence intensity(MFI)andnumbers of FITC-CD14 positive cells in the rats withendotoxemia increased obviously at 3,6,12 and 24h whencompared with normal control group(43.4%,70.2%,91.4%,32.6% vs4.5%,P<0.01).CONCLUSION:LPS can markedly promote the synthesis ofCD]4 protein and up-regulate the expression of CD14 mRNAin isolated hepatocytes and hepatic tissue.Liver might be amain source for soluble CD14 production duringendotoxemia.

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.