Effects of hypoxia inducible factor-2α on promoting angiogenesis of residual hepatocellular carcinoma after high-intensity focused ultrasound ablation / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the dynamic features of angiogenesis in residual tumors after high intensity focused ultrasound (HIFU),and to determine the temporal effect and mechanism of hypoxia inducible factor-2 alpha (HIF-2a) in the angiogenic process of residual tumors.</p><p><b>METHODS</b>Xenograft tumors of HepG2 cells were generated by subcutaneously inoculating athymic BALB/c nu/nu mice with the hepatoma cells.About 30 days after inoculation,all mice (except in the control group) were treated by HIFU and assigned randomly to the following 7 groups according to various time intervals post-treatment:1st,3rd,5th day and 1st,2nd,3rd,4th week when the residual tumor tissues were obtained from the experimental groups.Protein levels of HIF-2a and vascular growth factor A (VEGF-A) were quantified by immunohistochemistry and western blotting,and mRNA levels were measured by (real-time quantitative) qPCR. Microvascular density (MVD) was calculated by counting the CD31-positive vascular endothelial cells identified by means of an immunohistochemical staining method.</p><p><b>RESULTS</b>Compared with results from the control group,the protein and mRNA levels of HIF-2a expression reached the highest level in the experimental mice at the 2nd week (P=0.000 and P < 0.01 respectively),and were decreased thereafter(3rd week and 4th week, P=0.000 and P < 0.05).VEGF-A expression in the residual tumor tissues group that received HIFU was significantly decreased,compared with the control group,at all time points uPto 1 week (all P=0.000 and P < 0.01),but the levels increased compared to controls in the 2nd through 4th week (all P=0.000, P < 0.05). Similar results were obtained for MVD.</p><p><b>CONCLUSION</b>HIFU treatment can inhibit angiogenesis in residual hepatoma tissues in the short-term (1 to 2 weeks post-treatment) in mice with hepatocellular carcinoma,but can promote angiogenesis overtime (2 to 4 weeks post-treatment); the angiogenic process may involve the HIF-2α/VEGFA pathway.</p>

Tetramethylpyrazine protect rats against the inflammation and Aβ25-35 induced ROS by targeting the RAGE-ERK1/2-p38-NFκB pathway / 中国药学杂志

METHODS: Rats received a hibateral intracerebroventricular fusion of Aβ25-35. Tetramethylpyrazine was administrated through i.v. injection at 20 and 60 mg��kg-1��d-1 for 21 d. The expression of RAGE and inflammatory factors(including IL-1β, IL-6, TNF-α)were assessed immunohistochemical and ELISA, respectively. Further, an AD cell model (primary cultured hippocampal neurons of SD neonatal rats) and a RAGE-over expressing cell model(primary cultured hippocampal neurons transfected with pCMV/hygro-RAGE) were used for investigating the mechanisms of tetramethylpyrazine's effects. Cell viability was determined by MTT assay and intracellular reactive oxygen species (ROS) was identified by DCFH-DA staining. Real time fluorescent quantitative PCR and Western blot method were employed to detect the transcriptional level(RAGE)and phosphorylation of targeted proteins (including ERK1/2, p38, IκB) in all groups.

Experimental study on vascular bundle implantation combined with cellular transplantation in treating rabbit femoral head necrosis / 中国骨伤

<p><b>OBJECTIVE</b>To discuss the feasibility of vascular bundle implantation combined with allogeneic bone marrow stromal cells (BMSCs) transplantation in treating rabbit femoral head osteonecrosis and bone defect, in order to explore a new method for the treatment of femoral head necrosis.</p><p><b>METHODS</b>Thirty-six New Zealand rabbits were randomly divided into three groups,with 12 rabbits in each group. Bilateral femoral heads of the rabbits were studied in the experiment. The models were made by liquid nitrogen frozen, and the femoral heads were drilled to cause bone defect. Group A was the control group,group B was stem cells transplantaion group of allograft marrow stromal,and group C was stem cells transplantation group of allograft marrow stromal combined with vascular bundle implantation. Three rabbits of each group were sacrificed respectively at 2, 4, 8, 12 weeks after operation. All specimens of the femoral heads were sliced for HE staining. Furthermore ,vascular density and the percentage of new bone trabecula of femoral head coronary section in defect area were measured and analyzed statistically.</p><p><b>RESULTS</b>In group C,new bone trabecula and original micrangium formed at the 2nd week after operation; new bone trabecula was lamellar and interlaced with abundant micrangium at the 8th week;at the 12th week,the broadened,coarsened bone trabecula lined up regularly,and the mature bone trabecula and new marrow were visible. At the 2nd week after operation,there was no statistical significance in the percentage of new bone trabecula of femoral head coronary section in defect area between group B and C. While at 4, 8, 12 week after operation, vascular density and the percentage of new bone trabecula of femoral head coronary section in defect area of group C was higher than that of group B.</p><p><b>CONCLUSION</b>Allogeneic bone marrow stromal cells cultured in vivo can form new bone trabecula, and can be applied to allotransplant. Vascular bundle implanted into the bone defect area of femoral head necrosis could improve blood supply, and promote the formation of bone trabecula.</p>

Effects of soybean isoflavones on RAGE mediated signal transduction in the hippocampus of rats with Alzheimer's disease / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To investigate the effects of soybean isoflavones on receptor for advanced glycation end products (RAGEs) mediated signal transduction in the hippocampus of rats with Alzheimer's disease (AD).</p><p><b>METHODS</b>AD rat model was established by bilateral hippocampus injection of amyloid beta25-35 (Abeta25-35). Sixty rats were equally randomized into 5 groups, i.e., the model group, the high dose soybean isoflavones group (at the daily dose of 30 mg/kg), the low dose soybean isoflavones group (at the daily dose of 10 mg/kg), the estrogen group (at the daily dose of 0.4 mg/kg), and the sham-operation control group. Three days after modeling 2 mL soybean isoflavones or estrogen was respectively administrated to rats in the high dose soybean isoflavones group, the low dose soybean isoflavones group, and the estrogen group by gastrogavage. Equal volume of 0.5% CMC-Na was administered to rats in the model group and the sham-operation group. The treatment lasted for 21 days. The contents of RAGE and interleukin-6 (IL-6) in the hippocampus were measured by ELISA. The activity of cysteine-containing aspartate-specific protease-3 (Caspase-3) was measured by spectrophotometry. The expressions of phosphorylation extracellular signal-regulated kinase1/2 (P-ERK1/2) in the hippocampus were measured by immunohistochemical assay.</p><p><b>RESULTS</b>Compared with the model group, soybean isoflavones could significantly decrease the contents of RAGE and IL-6, the activity of Caspase-3, and the phosphorylation level of ERK1/2 in the hippocampus of AD model rats (P < 0.01).</p><p><b>CONCLUSION</b>Soybean isoflavones could down-regulate RAGE mediated inflammatory signal transduction in the hippocampus of AD rats, attenuate the inflammatory reactions, reduce the neurotoxicity of Abeta, and resist the apoptosis of hippocampal neurons.</p>

Expressions and significance of COX-2 and P-gp in human hepatocellular carcinoma tissues / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the effect of the COX-2 gene on the oncogenesis and development of the hepatocellular carcinoma and the influence of COX-2 gene on the expression of P-gp protein.</p><p><b>METHOD</b>Fifty-two pieces of the hepatocellular carcinoma samples and 20 cases of normal liver samples were collected from the patients operated from October 2003 to June 2005. RT-PCR and immunohistochemistry staining were employed to detect the COX-2 mRNA as well as P-gp protein in the normal liver tissues and the carcinoma tissues. Meanwhile, the expression of the mdr 1 mRNA in the carcinoma tissues was also determined and the correlation between the expressions of the COX-2 and P-gp was investigated.</p><p><b>RESULTS</b>No expression of the COX-2 in the normal liver tissue was detected. The positive expression of COX-2 in the low and middle differentiated carcinoma was elevated significantly as compared with that in the high differentiated carcinoma tissue (x2 = 6.80, P less than 0.01). The positive expression of the COX-2 in the HBSAg (+) carcinoma tissue was significantly higher as compared with that in the HBSAg (-) carcinoma (x2 = 4.70, P less than 0.05), and the carcinoma in combination with cirrhosis also showed significantly higher in expression of COX-2 than the carcinoma without cirrhosis (x2 = 7.51, P less than 0.01). The mdr1 mRNA was found both expressed in the normal and carcinoma tissues. The expression of COX-2 mRNA was found in the carcinoma, but not found in the normal tissues. The COX-2 mRNA and mdr1 mRNA was found both expressed in the normal and carcinoma tissues. The correlation coefficient between COX-2 and mdr1 mRNA was 0.563 ( P less than 0.01).</p><p><b>CONCLUSION</b>The results indicated that Cox-2 gene might involved in the multidrug resistance of the hepatocellular carcinoma mediated by P-gp.</p>

Reversal of multidrug resistance in hepatocellular cell line HepG2R by mdr1-antisense RNA / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate whether multidrug resistance gene 1(mdr1) could reverse multidrug resistance (MDR) in HepG2R cells.</p><p><b>METHODS</b>An adenovirus vector, Adeno-asmdr, containing the antisense RNA driven by AFP promoter, was construct. Adeno-EGFP was transfected into HepG2, an AFP producing cell line, L02, a normal human liver cell line, and HeLa, a human cervical cancer cell line, the EGFP transcription level was detected by RT-PCR. Adeno-asmdr was transfected into HepG2R cells, the expression of P-gp170 was detected by western blotting, apoptosis was detected using TUNEL and flow cytometry, cell cycle was analyzed by flow cytometry.</p><p><b>RESULTS</b>EGFP was highly expressed in HepG2 cells, however, its expression in L02 or HeLa cells was very weak. Western blot showed that the P-gp170 was marked down-regulated 48h after transfection with Adeno-asmdr, and the expression of P-gp170 was detectable at least 7d post-transfection. Compared with control cells, Adeno-asmdr transfected HepG2R cells were more sensitive to different chemicals, as indicated by TUNEL staining and flow cytometry. Chemical treatment arrested the cells in S and G0/M phase.</p><p><b>CONCLUSION</b>The recombinant adenoviral vector, Adeno-asmdr, can block the expression of mdr1, and reverse MDR in HepG2R cells.</p>

Reversal of adriamycin resistance of hepatocellular carcinoma by targeting it with recombined adenovirus carrying antisense multidrug resistance gene 1 RNA / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate if an adenovirus vector carrying antisense multidrug resistance gene 1 (MDR1) could reverse multidrug resistance (MDR) of HepG2/ adriamycin (ADM) cells in tumors transplanted in athymic mice.</p><p><b>METHODS</b>An adenovirus vector carrying AFP promoter and antisense MDR1 was constructed. HepG2 MDR cells (HepG2/ADM) were induced by graded resistance to ADM and were subcutaneously inoculated into athymic mice to construct the transplanted tumor. After adeno-asmdr1 was injected, the volume of the transplanted tumor and the apoptotic body in the xenograft tumor cells were observed and reverse transcriptase polymerase chain reaction was employed to investigate the expression of the mdr1-mRNA from the mouse transplanted tumor cells.</p><p><b>RESULTS</b>Following injection with adeno-asmdr1, the tumor volumes in this mice group did not increase. However the tumor volume in the PBS plus ADM group did increase significantly (P less than 0.05). In the tumor xenograft cells, mdr1 mRNA in the xenografts was assessed by RT-PCR and found to be reduced at week 1, and at week 4 in the ADM+asmdr1 group, but it was stable in the ADM group. It was only 20% in the ADM+asmdr1 group compared to the ADM group at the 4th week. Evidence of apoptosis was observed in the tumor xenograft cells treated with adeno-asmdr1, but there was rarely any apoptosis in the group treated with ADM and PBS.</p><p><b>CONCLUSION</b>Adenovirus carrying antisense mdr1 RNA can partially reverse the MDR of HepG2/ADM cells and inhibit tumor growth by down-regulating mdr1 mRNA resulting in tumor cell apoptosis.</p>

Expression changes of interleukin-1 receptor associated kinase-4 during endotoxin tolerance development in kupffer cells / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To explore the mechanism of endotoxin tolerance (ET) through observing the expression of interleukin 1 receptor associated kinase-4 (IRAK-4) during endotoxin tolerance development in Kupffer cells (KCs).</p><p><b>METHODS</b>Isolated KCs of Balb/c mouse were divided into two groups: the non-endotoxin tolerance (NET) group and the endotoxin tolerance (ET) group, which were pretreated with 10 ng/ml lipopolysaccharide (LPS) for 24 h. Then, the two groups were treated with 100 ng/ml LPS. The expressions of IRAK-4 gene and protein level were determined by RT-PCR and Western blot. The activities of NF-kappaB of KCs and the TNFalpha level were estimated by ELISA at 0 h, 1 h, 3 h, 6 h and 12 h after LPS stimulation.</p><p><b>RESULTS</b>The ultimate level of IRAK-4, the activities of NF-kappaB and the TNFalpha level were evidently lower in the ET group than those in the NET group (t = 12.4, 17.4 and 138.9 respectively, P<0.01).</p><p><b>CONCLUSIONS</b>Pretreatment with LPS on KCs could induce endotoxin tolerance of KCs and inhibition of IRAK-4 expression may be one of the reasons for its development.</p>

The mechanism and treatment phases chosen of glycine for inhibition lipopolysaccharide induced Kupffer cells activation / 中华外科杂志

<p><b>OBJECTIVE</b>To explore the possible mechanism and optimal treatment phase of glycine for inhibition lipopolysaccharide (LPS) induced Kupffer cells (KCs) activation.</p><p><b>METHODS</b>The KCs were isolated from 40 BALB/c mice and divided into four groups: the endotoxin group, the prevention group, the early treatment group and the later treatment group (n = 10). The endotoxin group was treated with 10 mg/L LPS, and in the other three groups, glycine (1 mmol/L) was given 24 h before, or at 0 h or 4 h respectively after LPS stimulation. At 0 h, 1 h, 2 h, 6 h and 12 h after LPS stimulation, the mRNA levels and protein expression of interleukin-1 receptor associated kinase-4 (IRAK-4) were determined by reverse transcription polymerase chain reaction (RT-PCR) and Western blot respectively, and nuclear factor-kappaB (NF-kappaB) activities as well as tumor necrosis factor alpha (TNF-alpha) levels were also detected by enzyme-linked immunosorbent assay (ELISA).</p><p><b>RESULTS</b>The climax values of IRAK-4, NF-kappaB and TNF-alpha were significantly higher in the endotoxin group and the later treatment group than that in the other two groups (t = 3.17, 4.33, 2.47, 126.73, P < 0.01).</p><p><b>CONCLUSION</b>The results indicated that prophylactic or simultaneous treatment with glycine could effectively inhibit LPS-induced KCs activation by inhibiting IRAK-4 expression.</p>

An experimental study of the inhibitory effects on the activation of endotoxin-induced Kupffer cells through short hairpin RNA targeting interleukin-1 receptor associated kinase-4 gene / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To explore the inhibitory effects on the activation of endotoxin-induced Kupffer cells (KCs) through short hairpin RNA (shRNA) targeting interleukin-1 receptor associated kinase-4 (IRAK-4) gene.</p><p><b>METHODS</b>Two effective transfection shRNA plasmid (pSIIRAK-4-A, pSIIRAK-4-B) and one invalidated plasmids (pSIIRAK-4-C) targeting IRAK-4 gene were constructed. The isolated mouse KCs were divided into three groups: the normal control group, the RNAi control group (pSIIRAK-4-C) and the RNAi effective group (pSIIRAK-4-A, pSIIRAK-4-B). Then KCs were stimulated with 0.1 microg/ml lipopolysaccharide (LPS) after 24 h transfection with the constructed plasmid. The expression of IRAK-4 gene and protein level were determined by RT-PCR and Western blot at 6 h after LPS stimulation, and the activities of NF-kappaB in KCs and the TNFalpha level were estimated by ELISA at 0 h, 1 h, 3 h, 6 h and 12 h.</p><p><b>RESULTS</b>The level of IRAK-4, the activities of NF-kappaB and the TNF-alpha level in the RNAi effective group were evidently lower than those in normal and RNAi control groups (P < 0.01) at 1 h, 3 h, and 6 h. Especially, the pSIIRAK-4-A group in which the changes of the above indices were of no difference (P > 0.05), had better inhibited effects than that of the pSIIRAK-4-B group (P < 0.01).</p><p><b>CONCLUSION</b>The shRNA targeting IRAK-4 gene could effectively inhibit the activation of endotoxin-induced KCs.</p>

The effect of glycine on CD14 and NF-kappa B in Kupffer cells from rat liver grafts after ischemia-reperfusion injury / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the effect of glycine on CD14 and NF-kappa B in Kupffer cells from rat liver grafts after ischemia-reperfusion injury (IRI).</p><p><b>METHODS</b>The rats were randomly divided into an IRI group, saline solution preconditioning group, and glycine preconditioning group. Their survival rates, graft functions, and hepatic histopathologic examinations were observed after IRI. Kupffer cells (KCs) following IRI were isolated and cultured to detect CD14 mRNA, NF-kappa B binding activity, and the TNF alpha and IL-1 level in the supernatant of the media.</p><p><b>RESULTS</b>(1) Glycine preconditioning greatly enhanced the one-week survival rate (chi2 = 6.67 and 8.57 respectively), improved graft function, and ameliorated the histopathologic signs of injury. (2) The CD14 mRNA expression level (F = 7.64), NF-kappa B binding activity (F = 11.47), TNF alpha and IL-1 production (F = 14.08 and 9.56 respectively) in the glycine group were significantly lower than those in the other two groups.</p><p><b>CONCLUSION</b>Glycine could efficiently protect rat liver grafts from ischemia-reperfusion injury by repressing the expression of CD14 and NF-kappa B binding activity in Kupffer cells and inhibiting the productions of TNF alpha and IL-1.</p>

Study on ligustrazine in reversing multidrug resistance of HepG2/ADM cell in vitro / 中国中药杂志

<p><b>OBJECTIVE</b>To study the reverse effect of ligustrazine (TMP) on HepG2/ADM, a herd of hepatocellular carcinoma cell, multidrug resistance (MDR) and the influence of P-gp170 expression.</p><p><b>METHOD</b>The reverse effect of ligustrazine on HepG2/ADM cell was observed, with the methods of cell culture, MTT's analyze, RT-PCR and Flow cytometric, etc.</p><p><b>RESULT</b>Ligustrazine could make MDR of cell line of HepG2/ADM reduce the expression of P-gp170, enhance the density of adriamycin in cell and increase the adriamycin's cytotoxicity. With the Flow cytometric, the results of RT-PCR showed the transcriptional activity of the MDR1 decreased.</p><p><b>CONCLUSION</b>Ligustrazine can reverse MDR of HCC cell line of HepG2/ADM and has prospect in clinical use.</p>

In vivo expression of lipopolysaccharide binding protein and its gene induced by endotoxin / 中华创伤杂志(英文版)

<p><b>OBJECTIVE</b>To investigate the expression of lipopolysaccharide binding protein (LBP) and its gene in rats with endotoxemia and explore the role of LBP in the response of host to endotoxin.</p><p><b>METHODS</b>Thirty Wistar rats were divided randomly into five groups: the normal group and the endotoxemia groups (1, 3, 6, 12 hours after LPS injection, respectively). The level of plasma endotoxin was determined by the Limulus Amebocyte Lysate assay. The expression of LBP mRNA in hepatic tissue was examined by reverse transcription polymerase chain reaction (RT-PCR). Plasma levels of LBP, tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 were measured by enzyme-linked immunosorbent assay (ELISA). Morphologic changes of hepatic tissue were observed under transmission electron microscope.</p><p><b>RESULTS</b>The level of plasma endotoxin peaked at 1 h after LPS injection, then declined, but was still higher than that of the normal group at 12 h; intrahepatic expression of LBP mRNA and plasma LBP increased with time after LPS stimulation; TNF-alpha and IL-6 in plasma increased with upregulation of LBP expression; there were significant differences between the normal group and endotoxemia groups (P<0.05). Activation of Kupffer cells and injury of hepatocytes could be seen in rats with endotoxemia.</p><p><b>CONCLUSIONS</b>LPS can upregulate the intrahepatic expression of LBP mRNA and increase the plasma LBP level. Under certain conditions, LBP may enhance the sensitivity of host to the toxic effects of LPS.</p>

The role of cyclooxygenase 2 and prostaglandin I2 in the development of portal hypertensive gastropathy / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To study the role of cyclooxygenase 2 (COX 2) and prostaglandin I2 (PGI2) in the development of portal hypertensive gastropathy (PHG).</p><p><b>METHODS</b>Forty Wistar rats were divided into surgery group (32) and control group (8). Partial portal vein ligation method was used to narrow the sectional area of portal vein to establish the experimental model of PHG in surgery group rats. Then they were divided into four groups (8 rats in each). The free pressure of portal vein was determined at the 1st, 2nd, 3rd, 4th weeks after the operation, and 8 rats were killed to observe the pathological change of gastric mucosa. The levels of 6-keto-PGF1 alpha, a stable metabolite of PGI2, were determined by radioimmunoassay in gastric mucosa homogenate and the blood of portal vein. The expression of COX 2 in gastric mucosa was determined by immunohistochemistry.</p><p><b>RESULTS</b>The free pressure of portal vein increased rapidly after partial portal vein ligation and maintained a high stable level after 1 week. They were (2.40+/-0.15) kPa, (2.38+/-0.17) kPa, (2.52+/-0.21) kPa, and (2.46+/-0.17) kPa at the 1st, 2nd, 3rd, and 4th weeks after partial portal vein ligation, while it was (0.90+/-0.16) kPa in control group (t>or=17.356, P<0.05). The gastric mucosa appeared pale, edema, hyperaemia, surface erosion, punctate hemorrhage and these lesions were more apparent with the time after the operation. The pathological examination showed that the gastric mucosa and submucosa thickened. The vessels of gastric mucosa and submucosa expanded and increased. There were lymphocytes and neutrophils infiltration around the vessels in the gastric mucosa and submucosa. The 6-keto-PGF1 alpha levels in gastric mucosa and the blood of portal vein increased rapidly and maintained a high level after partial portal vein ligation,which were higher than those in control group (104.52pg/ml+/-25.11pg/ml vs 73.62pg/ml+/- 20.33pg/ml, t=2.710, P<0.05; 180.21pg /ml+/-37.56pg /ml vs 142.11pg /ml+/-31.51pg /ml, t=2.198, P<0.05). The results of immunohistochemistry showed that the intensity and degree of the COX 2 staining in gastric tissue increased at the 1st, 2nd, 3rd, 4th weeks after partial portal vein ligation, while the COX 2 in control group rats was negative.</p><p><b>CONCLUSIONS</b>The expression of COX 2 and PGI2 in gastric tissue increased in portal hypertension. PGI2 as an inflammatory medium, damages the gastric mucosa by expanding vessels and other mechanisms in portal hypertension. It may be one of the important factors contributing to the development of PHG.</p>

Synthesis of Toll-like receptor 4 in Kupffer cells and its role in alcohol-induced liver disease / 中华医学杂志(英文版)

<p><b>OBJECTIVES</b>To observe the synthesis of Toll-like receptor (TLR) 4 protein and its mRNA expression in Kupffer cells (KCs) and evaluate the role of TLR 4 in liver injury to rats through alcohol-induced liver disease.</p><p><b>METHODS</b>Twenty-eight Wistar rats were divided into two groups: ethanol-fed (group E) and control (group C). Group E rats were given ethanol at a dose of 5 - 12 g x kg(-1) x d(-1), while group C received dextrose. Animals from both groups were killed at 4 and 8 weeks. The KCs were isolated and synthesis of TLR 4 protein was determined by laser scanning confocal microscopy. TLR 4 mRNA expression in KCs was determined by reverse transcription polymerase chain reaction (RT-PCR) analysis. The levels of endotoxin, tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) in plasma were determined. Changes in liver pathology were observed.</p><p><b>RESULTS</b>Laser scanning confocal microscopy showed that the intensity of fluorescence of TLR 4 protein in group E was stronger than group C. Ethanol administration led to a significant increase in TLR 4 mRNA expression in group E compared with group C (P < 0.05). The concentrations of plasma endotoxin, TNF-alpha and IL-6 were higher in group E than in group C (P < 0.05). Liver sections from rats in group E demonstrated marked pathological changes.</p><p><b>CONCLUSION</b>Ethanol administration can lead to the synthesis of TLR 4 protein and its gene expression in KCs, indicating that TLR 4 may play a major role in the development of alcohol-induced liver injury.</p>

Expression of CD(14) protein in liver sinusoidal endothelial cells during endotoxemia / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To observe the expression of CD(14) protein and CD(14) gene in liver sinusoidal endothelial cells (LSECs) of rats during endotoxemia and the role of CD(14) protein in the activation of lipopolysaccharide (LPS)-induced LSECs.</p><p><b>METHODS</b>Wistar rat endotoxemia model was established by injection of a dose of LPS (5 mg/kg, Escherichia coli O111:B4) via the tail vein of the rats, then sacrificed immediately, at 3, 6, 12, and 24 h, respectively. LSECs were isolated from normal and LPS-injected rats by the in situ collagenase perfusion technique. The isolated LSECs were incubated with anti CD(14) polyclonal antibody, then followed by staining with goat anti-rabbit IgG conjugated fluorescein isothiocyanate (FITC). The percentage and mean fluorescence intensity (MFI) of CD(14)-positive cells were detected by the flow cytometric analysis (FCM). LSECs were collected to measure the expression of CD(14) mRNA by the in situ hybridization analysis. The isolated LSECs from normal rats were divided into two groups. Group of LPS: LSECs were induced with different concentration of LPS (0, 0.01 microg/ml, 1 microg/ml, 10 microg/ml, and 100 microg/ml). Group of anti-CD(14) blockade: LSECs were pre-incubated for 30 min with CD(14) antibody before different concentrations of LPS were added. The supernatants of these cells were then collected for measuring the levels of tumor necrosis factor (TNF)- alpha and interleukin (IL)-6.</p><p><b>RESULTS</b>In rats with endotoxemia, LSECs displayed a strong MFI distinct from that of control rats. The number of FITC-CD(14) positive LSECs was 54.32%, 65.83%, 85.61%, and 45.65% at 3, 6, 12, and 24 h, respectively, which increased markedly when compared to control rats (4.45%, P<0.01). The expression of CD(14) mRNA in LSECs was stronger than that in control rats. The levels of TNF-alpha were significantly increased in group of LPS (54.49 +/- 6.02 pg/ml, 84.65 +/- 10.16 pg/ml, 206.54 +/- 23.55 pg/ml, 349.87 +/- 39.47 pg/ml, and 365.76 +/- 40.31 pg/ml) than those in group of anti-CD(14) blockade (55.93 +/- 6.95 pg/ml, 63.32 +/- 7.81 pg/ml, 85.34 +/- 9.72 pg/ml, 112.75 +/- 13.54 pg/ml, and 198.66 +/- 21.54 pg/ml) (P<0.01). The levels of IL-6 also increased significantly in group of LPS (103.34 +/- 12.52 pg/ml, 187.39 +/- 20.31 pg/ml, 243.87 +/- 27.83 pg/ml, 289.51 +/- 30.15 pg/ml, and 298.53 +/- 31.94 pg/ml) than those in group of anti-CD(14) blockade (104.37 +/- 11.49 pg/ml, 125.02 +/- 13.58 pg/ml, 164.59 +/- 19.47 pg/ml, 183.47 +/- 20.17 pg/ml, and 221.76 +/- 26.43pg/ml) (P<0.01).</p><p><b>CONCLUSIONS</b>LSEC can synthesize CD(14) protein and express CD(14) gene during endotoxemia. Anti CD(14) antibody can inhibit the production of TNF-alpha and IL-6 in LSECs induced by LPS. The expression of CD(14) protein may take an important part in the activation of LSECs induced by LPS.</p>