Objective: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 the liver injury in the rats with alcohol-induced liver disease (ALD).Methods:Twenty eight Wistar rats were divided into two groups: ethanol-fed group(group E) and control group (group C). Group E were fed with ethanol (dose of 5～12g/kg/d) and group C received dextrose instead of ethanol. Rats of the two groups were sacrificed at 4 wk and 8 wk. KCs were isolated and incubated.TLR 4 protein in KCs was determined by laser scanning confocal microscopy (LSCM).TLR 4 mRNA in KCs was determined by the reverse transcription polymerase chain reaction (RT-PCR).Plasma endotoxin levels were measured using the Limulus Amebocyte Lysate test kit.The liver pathology was observed under light and electronic microscopy.Results:The fluorescence intensity of TLR 4 protein in KCs by LSCM was stronger in group E than those in group C. Ethanol administration led to a significant increase in TLR 4 mRNA expression compared with group C ( P

During the process of teaching in clinical practice, there objectively exist the contradiction between the obligation of teaching and the invasion of the right to informed consent, and the contradiction between the growth of medical students and the invasion of patients' right to privacy. Health reforms, such as the regulation of "patient selecting doctor", bring some side effects to the teaching in clinical practice. In order to maintain the right to informed consent and privacy, to benefit the teaching in clinical practice, to develop the doctor-patient relationship, and to decrease medical disputes, the art to deal with the doctor-patient relationship must be explored. The laws and regulations of the teaching in clinical practice and of the right of doctors and patients must be developed too.

Humans ; Non-alcoholic Fatty Liver Disease

Objective To study the feasibility of the Fe3O4-loaded lipid perfluorooctylbromide nanoparticles (Fe3O4-PFOB) for enhanced ultrasound imaging.Methods The Fe3O4-PFOB nanoparticles,incubated with RAW264.7 macrophage cells,were monitored by microscope and ultrasound.Twelve SD rats were randomized into two groups,Fe3O4-PFOB group and PFOB group.Ultrasound imaging of rats' liver was performed before and after intravenous injection of the contrast agents.The liver echogenic intensity was quantified by DFY ultrasound quantified system analysis.Results Incubation of the Fe3O4-PFOB nanoparticles with macrophages resulted in the uptake of Fe3O4-PFOB by macrophages.Macrophages loaded with Fe3O4-PFOB exhibited enhanced echogenicity in vitro.In in vivo imaging,Fe3O4-PFOB produced better and prolonged ultrasound enhancement of rats' liver compared to PFOB nanoparticles.Conclusions Fe3O4-PFOB nanoparticles could enhance ultrasound imaging and may potentially serve as a multimodal probe for ultrasound,CT and MR imaging.

Objective To observe the effects of oxaliplatin on proliferation in human hepatoma cell lines QGY in vitro and investigate the mechanism. To provide the theory foundation whether it can be used for the chemotherapy of hepatocellular carcinoma. Methods The inhibition of proliferation in QGY cell was estimated by MTT-test. Morphologic changes were observed under light microscope and electronic microscope. Distribution of cell cycle and apoptosis was analyzed using flow cytometry. The expression of cell cycle protein and apoptosis-associated gene protein was detected with immunohistochemical technique. Results Oxaliplatin could inhibit the proliferation of QGY cells and the inhibition depended on the exposure time and dose. The cells showed morphologic changes at the early stage of apoptosis under the light microscope: the shrunk and round cells, condensed cytoplasma and pycnosis nucleus. Apoptotic cells and apoptotic body could be found under the transmission electronic microscope. The analysis of cell cycle indicated that oxaliplatin blocked cells at S and G_2/M phases and the cells of G_0/G_1 phase reduced. When treated with oxaliplatin for 72 h, the expression of cyclin A and Bax were up-regulated, mutant type P53, Bcl-2 and Myc down-regulated, and Fas was not changed. Conclusion Oxaliplatin could inhibit proliferation of the hepatoma cell lines. Cell cycle blocked at S and G_2/M phase. The apoptosis were related to the up-regulation of Bax and down-regulation of mutant type P53, Bcl-2 and Myc. It could not induce apoptosis through the Fas approach.

OBJECTIVETo explore the effect of microRNA-30a-5p (miRNA-30a-5p) on the biological behavior of human hepatoma cells.

METHODSThe liver cancer cell line SMCC-7721 cells and the normal liver cell line L02 cells (control) were transiently transfected with miRNA-30a-5p mimics and an miRNA-30a-5p inhibitor by Lipofectamine 2000 (Life Technologies). miR-30a-5p mRNA expression was detected by quantitative real-time (q)PCR. Cell proliferation was evaluated using the Cell Counting Kit-8 (CCK-8) assay and apoptosis was assessed by flow cytometry.Invasion and migration were measured by transwell chamber assays. The SMCC-7721 cells was injected subcutaneously into nude mice to establish a tumor animal model.

RESULTSThe SMCC-7721 cells transfected with miRNA-30a-5p mimics showed significantly higher miRNA-30a-5p mRNA expression than the non-transfected SMCC-7721 cells and the transfected control L02 cells (P<0.01). The miRNA-30a-5p mRNA expression was significantly lower in the SMCC-7721 cells transfected with the miRNA-30a-5p inhibitor than the non-transfected SMCC-7721 cells the control L02 cells (P<0.01). The overexpression of miRNA-30a-5p inhibited the viability, colony formation rate, and invasion and migration abilities, as shown in the cells transfected with the miRNA-30a-5p mimics (P<0.05); in addition, the miRNA-30a-5p promoted proliferation of cells (P<0.05), as shown by more S phase cells detected by flow cytometry. SMCC-7122 cells transfected with miRNA-30a-5p mimics produced tumors with significantly higher average weight than tumors produced by SMCC-7122 cells that were untransfected or transfected with empty vector (both P<0.01).

CONCLUSIONOverexpression ofmiR-30a-5p had an inhibitory effect on cell proliferation, induced apoptosis, increased the number of cells in S phase, and markedly inhibited invasion and migration of SMCC-7721 HCC cells in vitro and in vivo.

Animals ; Apoptosis ; Carcinoma, Hepatocellular ; Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Gene Expression Regulation, Neoplastic ; Humans ; Liver Neoplasms ; Mice ; Mice, Nude ; MicroRNAs ; Neoplasm Invasiveness ; Transfection

Antineoplastic Agents ; pharmacology ; Carcinoma, Hepatocellular ; drug therapy ; pathology ; Cell Division ; drug effects ; Dose-Response Relationship, Drug ; Drug Synergism ; Fluorouracil ; pharmacology ; Humans ; Liver Neoplasms ; drug therapy ; pathology ; Tumor Cells, Cultured ; drug effects

OBJECTIVETo observe the expression of lipopolysaccharide binding protein (LBP) and CD14 mRNA in alcohol-induced liver disease (ALD) and evaluate the relationship between the expression of LBP and CD14 mRNA and the severity of liver injury in alcoholic-fed rats.

METHODSTwenty Wistar rats were divided into two groups: ethanol-fed group and control group. Ethanol-fed group were fed ethanol (by intragastric infusion of 500 ml/L ethanol orally, dose of 5~12 g/kg/d) and control group received dextrose instead of ethanol. Rats of both groups were sacrificed at 4 weeks and 8 weeks, respectively. Levels of endotoxin and alanine transaminase (ALT) in blood were measured, and liver pathology was observed by light and electronic microscopy. Expression of LBP and CD14 mRNA in liver tissues were determined with the reverse transcription polymerase chain reaction (RT-PCR) analysis.

RESULTSPlasma endotoxin levels were increased significantly in ethanol-fed rats [(129 21) pg/ml and (187 35) pg/ml at 4 weeks and 8 weeks] than in control rats [(48 9) pg/ml and (53 11) pg/ml, respectively, t=11.2, 11.6, P<0.05]. Mean values for plasma ALT levels were increased dramatically in ethanol-fed rats after 4 weeks and 8 weeks [(112 15) U/L and (147 22) U/L, respectively] than in the control animals [(31 12)U/L and (33 9)U/L, respectively, t=5.9, 20.6, P<0.05]. In liver sections from ethanol-fed rats, there was marked pathological changes (steatosis, cell infiltration and necrosis). In the control rats, there was no significant difference in the levels of LBP and CD14 mRNA at the two time points. In ethanol-fed rats, ethanol administration led to a significant increase in LBP and CD14 mRNA levels as compared with the control group (P<0.05).

CONCLUSIONSEthanol administration lead to a significant increase in endotoxin levels of the serum and LBP and CD14 mRNA expression in liver tissues in ethanol- fed rats when compared with the control rats. Increase of LBP and CD14 mRNA expression may result in greater sensitivity to endotoxin and thus lead to liver injury.

Alanine Transaminase ; metabolism ; Animals ; Disease Models, Animal ; Female ; Lipopolysaccharide Receptors ; biosynthesis ; genetics ; Liver Diseases, Alcoholic ; enzymology ; metabolism ; pathology ; RNA, Messenger ; biosynthesis ; Rats ; Rats, Wistar

Objective:To evaluate the role of ErbB2 interacting protein (Erbin) in sepsis-associated encephalopathy (SAE) in mice and the relationship with nod-like receptor thermoprotein domain associated protein 3 (NLRP3) inflammasomes.Methods:Sixty SPF-grade healthy male wild-type C57BL/6 mice and 60 Erbin (-/-)C57BL/6 mice, aged 8-10 weeks, weighing 20-25 g, were divided into 4 groups ( n=30 each) by a random number table method: wild-type sham operation group (WT+ Sham group), wild-type SAE group (WT+ SAE group), Erbin (-/-) sham operation group (EKO+ Sham group) and Erbin (-/-) plus SAE group (EKO+ SAE group). The model of SAE was established by cecal ligation and perforation in anesthetized mice.Open field test (total distance moved) was performed at 7 days after establishing the model, new object recognition test (recognition index) was performed at 8 days after establishing the model, and Morris water maze test (time of staying at target quadrant) was performed at 10 days after establishing the model.The mice were sacrificed, and hippocampal tissues were removed for microscopic examination of pathologic changes (by HE staining) and for determination of neuron count, expression of NLRP3, caspase-1 and apoptosis-associated speck-like protein containing a CARD (ASC) (by Western blot), the number of NLRP3 positive cells (by immunohistochemistry), and contents of interleukin-1beta (IL-1β), tumor necrosis factor-alpha (TNF-α) and IL-18 (by enzyme-linked immunosorbent assay). The cell survival rate was calculated. Results:Compared with group WT+ Sham, the time of staying at target quadrant was significantly shortened, the recognition index and cell survival rate were decreased, the contents of IL-1β, IL-18 and TNF-α and the number of NLRP3 positive cells were increased, and the expression of NLRP3, caspase-1 and ASC was up-regulated in group WT+ SAE ( P<0.05). Compared with group EKO+ Sham, the time of staying at target quadrant was significantly shortened, the recognition index and cell survival rate were decreased, the contents of IL-1β, IL-18 and TNF-α and the number of NLRP3 positive cells were increased, and the expression of NLRP3, caspase-1 and ASC was up-regulated in group EKO+ SAE ( P<0.05). Compared with group WT+ SAE, the time of staying at target quadrant was significantly shortened, the recognition index and cell survival rate were decreased, the contents of IL-1β, IL-18 and TNF-α and the number of NLRP3 positive cells were increased, and the expression of NLRP3, caspase-1 and ASC was up-regulated in group EKO+ SAE ( P<0.05). There was no significant difference in total distance moved between the four groups ( P>0.05). Conclusion:Erbin can exert endogenous protection by inhibiting the activation of NLRP3 inflammasomes in mice with SAE.

Objective:To evaluate the relationship between Erbin and Bax/Bcl-xL-mediated cell apoptosis during sepsis-induced acute kidney injury in mice.Methods:Thirty-two SPF male wild type C57BL/6 mice, 32 SPF male Erbin (-/-) C57BL/6 mice, aged 6-8 weeks, weighing 20-30 g, were divided into 2 groups ( n=16 each) using the random number table method: wild type sham operation group (WT+ Sham group), wild type sepsis group (WT+ S group), Erbin(-/-) sham operation group (EKO+ Sham group), and Erbin(-/-) sepsis group (EKO+ S group). The sepsis model was established using the moderate cecal ligation and puncture (CLP) in anesthetized animals.The survival rates within 7 days after CLP were recorded.The serum concentrations of tumor necrosis factor-alpha (TNF-α), interleukin-10 (IL-10), IL-1β, creatinine (Cr), blood urea nitrogen (BUN) and lactic dehydrogenase (LDH) were determined at 24 h after CLP.Then the renal tissues were taken for assessment of renal injury which was scored and for determination of the apoptosis rate (by TUNEL) and expression of cleaved-caspase-3, Bcl-xL and Bax (by Western blot). Results:Compared with sham operation groups, the survival rates were significantly decreased, the serum concentrations of IL-1β, IL-10, TNF-α, Cr, BUN and LDH, renal injury score and apoptosis rate were increased, the expression of Bax and cleaved-caspase-3 was up-regulated, and the expression of Bcl-xL was down-regulated in sepsis groups ( P<0.05). Compared with WT+ S group, the survival rates were significantly decreased, the serum concentrations of IL-1β, LDH, TNF-α, Cr and BUN and renal injury score were increased, the serum concentration of IL-10 was decreased, the apoptosis rate of renal tissues was increased, the expression of Bax and cleaved-caspase-3 was up-regulated, and the expression of Bcl-xL was down-regulated in EKO+ S group ( P<0.05). Conclusions:Erbin can inhibit Bax/Bcl-xL-mediated cell apoptosis and is involved in endogenous protective mechanism against sepsis-induced acute kidney injury in mice.