MiR-340 mediates the involvement of high mobility group box 1 in the pathogenesis of liver fibrosis / 中华肝脏病杂志

Objective: To explore the pathogenic mechanism of the miR-340/high mobility group box 1 (HMGB1) axis in the formation of liver fibrosis. Methods: A rat liver fibrosis model was established by injecting CCl(4) intraperitoneally. miRNAs targeting and validating HMGB1 were selected with gene microarrays after screening the differentially expressed miRNAs in rats with normal and hepatic fibrosis. The effect of miRNA expressional changes on HMGB1 levels was detected by qPCR. Dual luciferase gene reporter assays (LUC) was used to verify the targeting relationship between miR-340 and HMGB1. The proliferative activity of the hepatic stellate cell line HSC-T6 was detected by thiazolyl blue tetrazolium bromide (MTT) assay after co-transfection of miRNA mimics and HMGB1 overexpression vector, and the expression of extracellular matrix (ECM) proteins type I collagen and α-smooth muscle actin (SMA) was detected by western blot. Statistical analysis was performed by analysis of variance and the LSD-t test. Results: Hematoxylin-eosin and Masson staining results showed that the rat model of liver fibrosis was successfully established. Gene microarray analysis and bioinformatics prediction had detected eight miRNAs possibly targeting HMGB1, and animal model validation had detected miR-340. qPCR detection results showed that miR-340 had inhibited the expression of HMGB1, and a luciferase complementation assay suggested that miR-340 had targeted HMGB1. Functional experiments results showed that HMGB1 overexpression had enhanced cell proliferation activity and the expression of type I collagen and α-SMA, while miR-340 mimics had not only inhibited cell proliferation activity and the expression of HMGB1, type I collagen, and α-SMA, but also partially reversed the promoting effect of HMGB1 on cell proliferation and ECM synthesis. Conclusion: miR-340 targets HMGB1 to inhibit the proliferation and ECM deposition in hepatic stellate cells and plays a protective role during the process of liver fibrosis.

Antimicrobial resistance and molecular epidemiological characteristics of clinical isolates of Staphylococcus aureus in Changsha area / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Increasing prevalence of Staphylococcus aureus (S. aureus), particularly methicillin-resistant S. aureus (MRSA) has been reported in China. In this study, we investigated the drug resistance characteristic, genetic background, and molecular epidemiological characteristic of S. aureus in Changsha.</p><p><b>METHODS</b>Between January 2006 and December 2008, 293 clinical isolates of S. aureus were collected from 11 hospitals in Changsha and identified by the Vitek-2 system. All the isolates were verified as MRSA by PCR amplification of both femA and mecA genes. K-B disk method was used to test drug sensitivity of S. aureus to antibiotics. Pulsed-field gel electrophoresis (PFGE) was performed for genotypic and homologous analysis of 115 isolates randomly selected from the original 293 clinical S. aureus isolates.</p><p><b>RESULTS</b>S. aureus was highly resistant to penicillin, ampicillin, erythromycin, and clindamycin with resistant rates of 96.6%, 96.6%, 77.1%, and 67.2% respectively. All the isolates were susceptible to tecoplanin, vancomycin, and linezolid. MRSA accounted for 64.8% (190/293) of all the S. aureus strains. The 115 S. aureus isolates were clustered into 39 PFGE types by PFGE typing, with 13 predominant patterns (designated types A to M) accounting for 89 isolates. The most prevalent PFGE type was type A (n = 56, 48.7%) and 100.0% of type A strains were MRSA. PFGE type A included 13 subtypes, and the most prevalent subtype was subtype A1 (46.4%, 26/56). Strains with PFGE type A were isolated from eight hospitals (8/11), and both subtypes A1 and A4 strains were isolated in a university hospital.</p><p><b>CONCLUSIONS</b>Clinical isolates of S. aureus in Changsha were resistant to multiple traditional antibiotics. There was an outbreak of PFGE type A MRSA in this area and the A1 subtype was the predominant epidemic clone. Dissemination of the same clone was an important reason for the wide spread of MRSA.</p>

Genomics of hepatitis B virus-related hepatocellular carcinoma and adjacent noncancerous tissues with cDNA microarray / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Hepatocellular carcinoma (HCC) is a common primary cancer frequently associated with hepatitis B virus (HBV) infection. However, whether these identified genes are particularly associated with HBV-related HCC remains unknown. The aim of this study was to investigate the differential gene expression between HBV-related HCC tissues and adjacent noncancerous tissues.</p><p><b>METHODS</b>cDNA microarray was used to detect the differential gene expression profile in the HBV-related HCC tissues and adjacent noncancerous tissues, and reverse transcription-polymerase chain reaction (RT-PCR) was performed to verify the differential expression of candidate genes obtained from cDNA microarray experiment.</p><p><b>RESULTS</b>In this study, 1369 genes or expressed sequence tags (ESTs) including 121 genes or ESTs with at least two-fold expression alterations between cancerous and noncancerous tissues were identified. Special AT-rich sequence binding protein 1 (SATB-1) expression was positive in 73% (16/22) of cancerous tissues and negative (0/22) in all noncancerous tissues of HBV-related HCC patients. Transmembrane 4 superfamily member 1 (TM4SF-1) expression was positive in 86% (19/22) of cancerous tissues and negative (0/22) in all noncancerous tissues. Suppression of tumorigenicity 14 (ST-14) expression was positive in 73% (16/22) of noncancerous tissues in patients with HBV-related HCC and negative in all HCC tissues (0/22).</p><p><b>CONCLUSION</b>This study provided the gene expression profile of HBV-related HCC and presented differential expression patterns of SATB-1, TM4SF-1 and ST-14 between cancerous and noncancerous tissues in patients with HBV-related HCC.</p>

Effect of HMGB1-siRNA on proliferation and apoptosis of HepG2 cells / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the effect of decreased expression of high mobility group Box-1 on the proliferation and apoptosis of HepG2 cells.</p><p><b>METHODS</b>Three specific siRNAs of HMGB1 were designed and synthesized, and were transiently transfected into HepG2 cells by Lipofectamine 2000. The HMGB1 expression in HepG2 cells was detected by RT-PCR and Western blotting respectively. The proliferation activity in vitro was assessed by MTT assay. In situ apoptosis was evaluated by terminal deoxynucleotidyl transferase-deoxyuridine triphosphate nick end labeling (TUNEL) assay.</p><p><b>RESULTS</b>All of these specific HMGB1-siRNAs (1, 2, 3) efficiently and specifically inhibited the expression of the HMGB1 gene, and the levels of HMGB1 mRNA were 1.147+/-0.024, 1.014+/-0.042, 0.435+/-0.055, respectively, in HMGB1-siRNAs transfection group, which were significantly lower than that in Lipofectamine 2000 alone group (1.411+/-0.065, P < 0.01). Correspondingly, all of these specific HMGB1-siRNAs (1, 2, 3) could efficiently and specifically inhibit the expression of the HMGB1 protein, and the levels of HMGB1 protein were 0.369+/-0.035, 0.340+/-0.028, 0.097+/-0.020, respectively, in HMGB1-siRNAs transfection group, which were significantly lower than that in Lipofectamine 2000 alone group (0.553+/-0.051, P < 0.01). Of the 3 specific HMGB1-siRNAs, HMGB1-siRNA-3 (siRNAH3) had the highest inhibition rate (80%). The proliferation of HepG2 cells was markedly inhibited by siRNAH3 transfection. Compared to mock-transfection, siRNAH3 transfection dramatically suppressed the proliferation of HepG2 cells (P < 0.01). Moreover, siRNAH3 can induce apoptosis (P < 0.01).</p><p><b>CONCLUSION</b>siRNA targeting HMGB1 mRNA can specifically reduce HMGB1 gene and protein expression. siRNAH3 can effectively suppress the proliferation and induce apoptosis of HepG2 cells.</p>

Effect of suppressive oligodeoxynucleotides on the functional differentiation in CD4(+) Th1 lymphocytes in mice in vitro / 中南大学学报(医学版)

OBJECTIVE@#To explore the effect of suppressive oligodeoxynucleotides (Sup ODN) on the Th1 differentiation of CD4(+)T splenetic lymphocytes in mice.@*METHODS@#The splenetic lymphocytes of BALB/c mice were separated, and then CD4(+) cells were purified with immune magnetic CD4(+) microbeads (positive selection). The purification was examined by fluorescence-activated cell sorter. CD4(+) cells, anti-CD3epsilon, anti-CD28, IL-12 and Sup ODN or control oligodeoxynucleotides (Con ODN) were co-incubated for 72 h. IFN-gamma and IL-4 in the supernatant were detected using enzyme-linked immunosorbent assay. The expression of T-bet mRNA in CD4(+) cells was tested by reverse transcription polymerase chain reaction.@*RESULTS@#Sup ODN could significantly inhibit the release of INF-gamma and increase IL-4 production respectively (P<0.01). T-bet mRNA of CD4(+) lymphocytes was remarkably inhibited by Sup ODN as well (P<0.01).@*CONCLUSION@#In the presence of pro-Th1-cytokines, Sup ODN may affect the differentiation of CD4(+) T lymphocytes in vitro. Sup ODN can promote CD4(+) T cells to differentiate into Th2, and suppress them into Th1.

The expression of fetuin-A and its pathological significance in fulminant hepatic failure in mice / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To explore the dynamic changes of fetuin-A expression and the influences of the changes on liver damage, hepatocyte apoptosis and inflammation in a mouse fulminant hepatic failure (FHF) model.</p><p><b>METHODS</b>The changes of fetuin-A expression were investigated by semi-quantitative RT-PCR and Western blot. Immunohistochemical staining was used in TNFa and fetuin-A detection. Hepatocyte apoptosis was detected by TUNEL.</p><p><b>RESULTS</b>Fetuin-A mRNA expression decreased after the FHF model was established for 3 hours (compared with the normal group, P less than 0.01), while the protein expression decreased after nine hours (compared with the normal group, P less than 0.01). Fetuin-A expressions were negatively correlated with the liver pathological scores and TNFa levels.</p><p><b>CONCLUSION</b>In our mouse FHF model, fetuin-A is a possible protective factor for liver damage.</p>

A two-year animal experimental study on the pathological effects of Helicobacter pylori on liver tissues / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To observe whether H. pylori administered orally in mice could arrive in their livers after a long-term infection, leading to active inflammation and even causing HCC as an independent etiological factor.</p><p><b>METHODS</b>Twenty C57BL/6 mice were orally administered H. pylori SS1 and kept for 24 months (experimental group) along with 13 mice which served as blank controls (control group). H. pylori colonization and pathologic consequences were studied in the livers and gastric tissues of the mice. The bacterial DNA extracted from liver tissues was examined by nested PCR for H. pylori 16S rRNA genes. 16S rRNA PCR amplicons were sequenced and compared with sequencing results of 16S rRNA PCR amplicons of the bacteria cultured from gastric mucosa and compared with that of the inoculated H. pylori SS1.</p><p><b>RESULTS</b>Of the 20 mice in the experimental group, H. pylori was found in the gastric mucosa of 12, and in 11 of them pathological gastric lesions were found, including one with gastric lymphoma. H. pylori were found in the livers of 7 mice. Liver lesions, one with mild inflammation, 3 with inflammation and fibrosis, 2 with inflammation, fibrosis and hepatocyte hyperplasia with atypia were found in 6 of them. No liver lesions were found in the mice of the control group. In the mice of the experimental group no liver lesions were found in those mice with no H. pylori in their gastric mucosae. Sequencing results of 16S rRNA PCR products of the liver showed 100% homogeneity with the cultured H. pylori from gastric mucosa and the administered H. pylori SS1.</p><p><b>CONCLUSION</b>Two years after oral administration of H. pylori to C57BL/6 mice, gastric mucosal lesions and liver lesions, including inflammation, cirrhosis and hepatocyte hyperplasia with atypia were found in those animals.</p>

Serum level of HMGB1 in patients with hepatitis B and its clinical significance / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate whether there is a possible role of pro-inflammatory cytokine high mobility group box protein 1 (HMGB1) causing liver failure in severe hepatitis B patients.</p><p><b>METHODS</b>Serum HMGB1 levels of chronic hepatitis B (CHB) patients with different clinical conditions were measured and the correlations between HMGB1 and TBil or PTA were analyzed. (1) 54 chronic hepatitis B patients in different clinical conditions were enrolled in our study. Their serum TBil and PTA levels were detected by routine methods. (2) Their serum HMGB1 levels were also detected. 100 KD super-filtration columns were used to get rid of large proteins in the serum and 10 KD columns were used to condense the protein. Western blot was used to determine HMGB1 levels, and correlations between HMGB1 and TBil or PTA were analyzed.</p><p><b>RESULTS</b>The detection rates of serum HMGB1 were 100% (23/23), 90% (9/10), and 55% (6/11) in 23 patients with hepatic failure, 10 patients with chronic severe hepatitis B, and 11 patients with chronic moderate hepatitis B respectively. The concentration of serum HMGB1 levels in these three groups was (83.4+/-21.3), (78.1+/-19.5) and (60.3+/-14.3) microg/L respectively. Serum HMGB1 was not detected in normal healthy controls and hardly detected in convalescent and mild hepatitis patients. There were positive correlations between HMGB1 and TBil and negative correlations between HMGB1 and PTA.</p><p><b>CONCLUSION</b>HMGB1 levels in serum were closely associated with disease severity in chronic hepatitis B patients. HMGB1 may play a key role in the pathogenesis of chronic severe hepatitis B and liver failure.</p>

Establishment of 2-dimensional electrophoresis maps of peripheral blood mononuclear cells / 中南大学学报(医学版)

OBJECTIVE@#To establish the 2-dimensional electrophoresis(2-DE) profiles of peripheral blood mononuclear cells(PBMC) in patients with hepatocellular carcinoma(HCC) and health adults.@*METHODS@#The total proteins from PBMC in patients with HCC and healthy adult were separated by immobilized pH gradient-based 2-DE. The differential expression proteins were analyzed by PDQuest analysis software.@*RESULTS@#The well-resolved, reproducible 2-DE patterns of PBMC in patients with HCC and healthy adults were obtained. For HCC, the average spots of 2-DE maps were 1 206 +/- 48, and the average matching rate was 90.8%. For normal adults, the average spots were 1 123 +/- 37, and the average matching rate was 92.6%.@*CONCLUSION@#The well-resolved, reproducible 2-DE patterns of PBMC in patients with HCC and healthy adults are established. These proteomic analysis methods are useful to screen the potential biomarkers in the early diagnosis, treatment and prognosis monitor in patients with malignant tumor.

Inhibition of hepatitis B virus replication and expression by RNA interference in vivo / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To evaluate the inhibitory effect of small interfering RNA (siRNA) targeting HBV C gene region on hepatitis B virus (HBV) in vivo.</p><p><b>METHODS</b>An animal model of HBV infection was developed hydrodynamically, and pcDNA3.1-HBV and siRNA were together injected into the tail vein of the BALB/c mice. HBsAg was analyzed by time-resolved immunofluorometric assay, HBV DNA was analyzed by fluorogenic quantitative PCR (FQ-PCR), HBV C-mRNA was detected by semi-quantitative RT-PCR, and viral specific proteins (HBsAg and HBcAg) in the mice livers were assayed using immunohistochemical staining.</p><p><b>RESULTS</b>In the mice, the siRNA effectively inhibited HBV replication and expression compared with the controls. The inhibitive effect of siRNA on HBV lasted at least 3 days.</p><p><b>CONCLUSION</b>These results demonstrate that RNAi can substantially inhibit HBV replication and expression in vivo.</p>

Toll-like receptor 9 in cpG oligodeoxynucleotides-induced species-specific immune responses / 中南大学学报(医学版)

OBJECTIVE@#To determine the role of Toll-like receptor 9 (TLR9) in synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG dinucleotides (CpG-ODN)-induced species-specific immune responses.@*METHODS@#CpG-ODN was co-cultured with peripheral blood mononuclear cells (PBMC) of humans, macaques and mice. The IFN-alpha in the supernatant was measured by ELISA. The reverse transcription PCR was used to analyze the expression levels of TLR9 mRNA in PBMC.@*RESULTS@#CpG-ODN induced high amounts of IFN-alpha in human PBMC, but had no effect on macaques and mice. The expression of TLR9 mRNA was observed in all human PBMC, and the levels of TLR9 mRNA were significantly up-regulated with the stimulation of CpG-ODN. We did not observe any expression of TLR9 mRNA in PBMC in macaques.@*CONCLUSION@#TLR9 underlies the molecular foundation of CpG-ODN-induced species specificity.

Sensitivity of nanoparticlized cefazolin sodium to the bacteria in vitro / 中南大学学报(医学版)

OBJECTIVE@#To investigate whether the in vitro sensitivity of cefazolin sodium to the bacteria was altered after nanoparticlization.@*METHODS@#The minimal inhibitory concentrations (MIC) of cefazolin sodium before and after nanoparticlization to S. aureaus and E. coli. were determined by microdilution.@*RESULTS@#The MIC of nanoparticlized cefazolin sodium to S. aureaus and E. coli. had no significant change compared with that of non-nanoparticlized one.@*CONCLUSION@#Nanoparticlization will not decrease the sensitivity of cefazolin sodium to the bacteria.

Experimental study on the pathological effect of Helicobacter pylori on liver tissues / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To observe whether H. pylori inoculated by oral route could arrive in livers and cause liver inflammation as an independent etiological factor.</p><p><b>METHODS</b>C57BL/6 mice were orally inoculated with H. pylori SS1 strains and fed for 8 months. H. pylori colonization and pathologic consequences were studied in the liver and gallbladder tissues of the mice; the blood, liver tissue and gastric mucosa were obtained and cultured for H. pylori growth; The bacterial DNA extracted from the liver, bile and blood was examined by nested PCR for H. pylori genes. 16S rRNA PCR amplicons were sequenced and compared with the sequencing results of 16S rRNA PCR amplicons of the bacteria cultured from gastric mucosa and the inoculated H. pylori SS1.</p><p><b>RESULTS</b>The bacterial DNA extracted from the liver, bile and blood of the infected mice was detected for H. pylori genes by nested PCR. Six of the 15 samples were positive (40%) in the liver, 6 of 10 samples in the bile (60%), and 2 of 10 samples in the blood (20%). Sequencing results of 16S rRNA PCR products of the livers showed 100% homogeneity when compared with the cultured H. pylori from gastric mucosa and inoculated H. pylori SS1. H. pylori was found in 4 liver tissues of the 15 infected mice (26.7%) and 6 in the gallbladders (40%). Infiltrations of lymphocyte cells along hepatic sinusoids and a lower degree infiltration around interlobular arteries and veins were observed; ballooning degeneration was also observed in some hepatocytes.</p><p><b>CONCLUSION</b>H. pylori inoculated by oral route could arrive in the liver and cause inflammation as an independent etiological factor. The routes which the microorganisms took to reach the livers may involve hematogenous and/or biliary system dissemination.</p>

Inhibition of hepatitis B virus replication by RNA interference in vitro / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To design pSilencer3.1-H1hygro plasmid expressing short interfering RNAs (siRNA) that targets HBV core gene region, and to evaluate inhibitory effect of this siRNA on HBV in vitro.</p><p><b>METHODS</b>HepG2 2.2.15 was used as target cells. The plasmid and liposome metafectene were cotransfected into the cultured cells, HBV DNA were analyzed by fluorogenic quantitative PCR (FQ-PCR), HBV C-mRNA was detected by semi-quantitative RT-PCR.</p><p><b>RESULTS</b>The plasmid expressing siRNA was successfully constructed. The two constructed siRNAs could effectively inhibit HBV replication, and their inhibitive effect on HBV was dose-dependent.</p><p><b>CONCLUSION</b>These results showed that siRNA could substantially inhibit HBV replication in the infected cells</p>

Preliminary study on proteomic patterns in hepatic tissue to identify HBV related hepatocellular carcinoma / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To identify proteomic patterns in hepatic tissues for diagnosing early HBV related HCC.</p><p><b>METHODS</b>Proteomic spectra were generated by two-dimensional gel electrophoresis (2-DE), A preliminary "raining" set of spectra derived from analysis of 14 cancer tissues and 14 non-cancer tissues, a proteomic patterns that completely discriminated cancer from non-cancer was identified. The discovered pattern was then used to classify an independent set of 48 masked samples: 24 from cancer tissues, and 24 from non-cancer tissues.</p><p><b>RESULTS</b>The discriminatory pattern correctly identified all cancer tissues and non-cancer tissues in the masked set. This result yielded a sensitivity of 100%, specificity of 100%.</p><p><b>CONCLUSION</b>Further analysis on these proteins in the proteomic pattern will be helpful to screen tumor markers for HBV related HCC. These findings justify a prospective assessment of proteomic pattern technology as a screening tool for cancer in high-risk and general populations.</p>

Inhibition of Hepatitis B virus replication by small interfering RNA in vivo / 中华传染病杂志

Objective To evaluate the inhibitory effect of the small interfering RNA(siRNA) on hepatitis B virus(HBV)in vivo which targets HBV S gene region.Methods An animal model of HBV infection was developed hydrodynamically by injecting pcDNA3.1-HBV together with siRNA through the tail vein of Balb/c.HBsAg was analyzed by time resolved immunofluorometric assay, HBV DNA was analyzed by fluorogenic quantitative PCR(FQ-PCR),HBV S-mRNA was detected by semi-quantitative RT-PCR,and viral specific proteins(HBsAg and HBcAg)in the liver were assayed by immunohistochemical staining.Results In the mice,the siRNA could effectively inhibit the secre- tion of HBsAg,reduce the titers of HBV DNA,and immunohistochemical results also indicated that the number of HBsAg and HBcAg positive cells was reduced.The inhibitory effect of siRNA on HBV lasted 3 clays at least.Conclusion These results demonstrate that the siRNA targeting HBV S gene region can substantially and specifically inhibit HBV replication and expression in vivo.