Long non-coding RNA MALAT1 promotes angiogenesis and immunosuppressive properties of HCC cells by sponging miR-140.

Hepatocellular carcinoma (HCC) is the most prevalent primary liver cancer in adults. Previous studies in our laboratory found that long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) was upregulated in HCC cells, which could affect the metastasis and invasion of HCC. However, the underlying mechanism remains unknown. Herein, we studied the interaction between MALAT1 and miR-140 on the regulation of angiogenesis and immunosuppressive properties. We revealed that the expression of MALAT1 and VEGF-A was significantly increased in HCC cells. Knockdown of MALAT1 in HCC cells suppressed the production of VEGF-A, impaired the angiogenesis of HUVECs, and facilitated the polarization of macrophage toward the M1 subset. Mechanistically, the interaction between MALAT1 and miR-140 or between miR-140 and VEGF-A was confirmed by multiple assays. Besides, a negative correlation between MALAT1 and miR-140 was found in HCC tissues. Furthermore, miR-140 inhibition significantly increased VEGF-A expression, promoted angiogenesis of HUVECs, and redirected the polarization of macrophages toward the M2 subset. In addition, in vivo studies also verified the regulatory network of the MALAT1/miR-140 axis on VEGF-A in HCC progression. In summary, this study revealed the mechanism that MALAT1 worked as a putative HCC promotor via inhibiting miR-140. Therefore, targeting MALAT1 or miR-140 might alleviate the progression of HCC in the future.

Therapeutic plasma exchange: A prospective randomized trial to evaluate 2 strategies in patients with liver failure.

OBJECTIVE: To compare two means of performing therapeutic plasma exchange (TPE) in patients with liver failure. METHOD: This open-label monocentric randomized trial, conducted in a single prestigious general healthcare facility, recruited liver failure patients with an indication to receive artificial liver support therapy for TPE. All patients underwent TPE procedures and were administered in a random sequence: heparin-free or systemic heparinization with unfractionated heparin. The primary endpoint was completion of TPE sessions, and the secondary endpoints included the safety and efficacy. RESULTS: In the period of the studying, there were 164 patients being recruited in and underwent total of 398 randomized TPEs: 168 with unfractionated heparin and 230 with heparin-free. In unfractionated heparin group, there were 3 cases (1.79%) being interrupted due to uncontrollable intraoperative pulmonary hemorrhages and gastrointestinal bleeding. In heparin-free group, 228 (99.13%) were completed successfully and 2 of them (0.87%) were switched from heparin-free to unfractionated heparin eventually. No significant differences were found between the two groups for either RRs or IRs (P > 0.05). CONCLUSION: Heparin-free regimen is feasible and safer than systemic heparinization with unfractionated heparin in the process of TPEs in patients with liver failure.

Serum levels of miRNA in patients with hepatitis B virus-associated acute-on-chronic liver failure.

BACKGROUND: Hepatitis B virus (HBV)-associated acute-on-chronic liver failure (HBV-ACLF) is a life-threatening condition and its exact pathophysiology and progression remain unclear. The present study aimed to assess the role of serum miRNAs in the evaluation of HBV-ACLF and to develop a model to predict the outcomes for ACLF. METHODS: Serum was collected from 41 chronic hepatitis B and 55 HBV-ACLF patients in addition to 30 chronic asymptomatic HBV carriers as controls. The miRNAs expressions were measured by real-time quantitative PCR (q-PCR). Statistical analyses were conducted to assess the ability of differentially expressed miRNAs and other prognostic factors in identifying ACLF prognosis and to develop a new predictive model. RESULTS: Real-time q-PCR indicated that serum miR-146a-5p, miR-122-3p and miR-328-3p levels were significantly upregulated in ACLF patients compared to chronic hepatitis B and chronic asymptomatic HBV carriers patients. In addition, multivariate regression analyses indicated that Na+, INR, gastrointestinal bleeding and miR-122-3p are all independent factors that are reliable and sensitive to the prognosis of HBV-ACLF. Therefore, we developed a new model for the prediction of HBV-ACLF disease state: Y = 0.402 × Na+ - 1.72 × INR - 4.963 × gastrointestinal bleeding (Yes = 0; No = 1)-0.278 × (miR-122-3p) + 50.449. The predictive accuracy of the model was 95.3% and the area under the receiver operating characteristic curve (AUROC) was 0.847. CONCLUSIONS: Expression levels of these miRNAs (miR-146a-5p, miR-122-3p and miR-328-3p) positively correlate with the severity of liver inflammation in patients with ACLF and may be useful to predict HBV-ACLF severity.

Novel HBV mutations and their value in predicting efficacy of conventional interferon.

BACKGROUND: Accumulating studies assessing the impacts of hot spot mutations on conventional interferon (IFN) efficacy come to discrepant conclusions; studies regarding the mutations in S and RT regions are also unclear. The present study aimed to evaluate the impacts of HBV mutations on the efficacy of conventional IFN. METHODS: A total of 126 patients who received conventional IFN treatment for 48 weeks were enrolled. Biochemical and serological parameters were routinely tested. The sequences of HBV from 78 serum samples were amplified by nested-PCR; mutations were identified with sequence scanner V1.0 after ABI 3730xl direct sequencing, HBV genotypes were determined according to RT gene sequences utilizing NCBI Genotyping Tool which was based on phylogenetic analysis. RESULTS: The baseline DNA levels of virological response (VR) group were significantly lower than those of no VR group [7.13+/-0.76 vs 7.69+/-0.56 lg (copies/mL), P=0.001]. The baseline ALT levels were significantly higher in the HBeAg clearance group (204.72+/-88.65 vs 162.80+/-85.81 IU/L, P<0.05) and HBeAg seroconversion group (204.89+/-95.68 vs 166.75+/-84.43 IU/L, P<0.05). Females and lower BMI levels (20.01+/-2.33 vs 21.65+/-3.66 kg/m2, P<0.05) were prone to acquired biochemical response (BR). PC-W28STOP (ntG1896A) was significantly higher in the combined response (CR) group than that in the no CR group (91.7% vs 39.7%, P=0.001). Multivariate logistic regression analysis showed that baseline DNA, PC-P159T (ntC2288A), BCP-N118T (ntA1726C) and BCP-L134L (ntA1775C/G/T) influenced VR independently. PC-G182C (ntG2357T), PC-S64A/T (ntT2003G/A) and BMI were independent influence factors for HBeAg clearance, HBeAg seroconversion and BR, respectively. The new predicting model concluded that baseline DNA and new mutations for VR were established successfully, and ROC analysis showed that AUC was 0.842 (P<0.001) with a sensitivity of 0.652 and a specificity of 0.933. CONCLUSIONS: PC-P159T (ntC2288A), BCP-N118T (ntA1726C), BCP-L134L (ntA1775C/G/T), PC-G182C (ntG2357T) and PC-S64A/T (ntT2003G/A) were novel identified mutations that impacted IFN therapeutic efficacy. These novel mutations could serve as important predictors before conventional IFN treatment.

A novel prognostic score for acute-on-chronic hepatitis B liver failure.

Patients with acute-on-chronic hepatitis B liver failure (HBV-ACLF) show high morbidity and mortality. Independent prognostic predictors of short-term HBV-ACLF mortality include the Child-Turcotte-Pugh (CTP) score, the model for end-stage liver disease (MELD) score, other MELD-based indices and the dynamic changes in these indices. The aims of this study were to evaluate the existing prognostic scores in a large cohort of HBV-ACLF patients and create a new predictive model. We retrospectively reviewed 392 HBV-ACLF patients from December 2008 to November 2011 and evaluated their 3-month survival. The predictive accuracy of CTP, MELD and MELD-based indices and the dynamic changes in the MELD-related scores (Δ scoring systems) upon admission and after two weeks of treatment were compared using the area under the receiver operating characteristic (ROC) curve method. Life-threatening factors and a series of bio-clinical parameters were studied by univariate and multivariate analyses. Among the existing scores, MELD had the best predictive ability. However, our new regression model provided an area under the curve of 0.930 ± 0.0161 (95% CI: 0.869 to 0.943), which was significantly larger than that obtained with the MELD score at admission and after two weeks of treatment as well as with the dynamic changes of the MELD score (0.819, 0.921, and 0.826, respectively) (Z=3.542, P=0.0004). In a large cohort of patients retrospectively reviewed for this study, our prognostic model was superior to the MELD score and is, therefore, a promising predictor of short-term survival in patients with HBV-ACLF.

[Effect of hepatitis B virus X protein on suppressor of cytokine signaling-1 expression].

OBJECTIVE: To investigate the effect of hepatitis B virus-encoded X protein (HBx) on the expression of host-encoded suppressor of cytokine signaling-1 (SOCS-1) and to explore the possibility of an underlying mechanism involving modulation of CpG island methylation in the SOCS-1 gene promoter. METHODS: The immortalized human derived non-tumor liver cell line QSG7701 was transfected with a recombinant HBx plasmid (pcDNA-X) or an empty vector control plasmid (pcDNA3.0) and stably transfected clones were selected by G418 resistance screening. Untransfected cells served as negative controls. Expression of SOCS-1 mRNA and protein was detected by real-time quantitative PCR and western blotting. The methylation status of SOCS-1 was detected by methylation-specific PCR (MSP). The significance of intergroup differences was analyzed by one-way ANOVA or pairwise comparison with post-hoc LSD test. RESULTS: SOCS-1 mRNA level was significantly lower in the pcDNA-X/QSG7701 cells compared to that in the pcDNA3.0/QSG7701 and untransfected cells (0.3249+/-0.0536 vs. 1.0543+/-0.1937 and 1.00; F = 19.6, P = 0.042). SOCS-1 protein level was similarly lower in the pcDNA-X/QSG7701 cells (0.1496+/-0.0106 vs. 0.1984+/-0.0438 and 0.2152+/-0.0816; F = 19.4, P = 0.048). The SOCS-1 promoter region showed methylation only in the pcDNA-X/QSG7701 cells. CONCLUSION: HBx-expressing human hepatocytes have down-regulated SOCS-1 expression, both at the mRNA and protein levels, and this effect corresponds to increased methylation in the SOCS-1 promoter region harboring CpG islands.

Randomized controlled study of plasma exchange combined with molecular adsorbent re-circulating system for the treatment of liver failure complicated with hepatic encephalopathy.

BACKGROUND/AIMS: To evaluate the use of plasma exchange (PE) combined with the molecular adsorbent re-circulating system (MARS) for the treatment of liver failure complicated with hepatic encephalopathy. METHODOLOGY: A prospective randomized controlled study was conducted to compare the therapeutic effect of MARS treatment (MARS group, n=60) with that of PE combined with MARS treatment (PE+MARS group, n=60) in patients with liver failure complicated with hepatic encephalopathy. RESULTS: The serum total bilirubin and blood ammonia levels were significantly decreased compared with pretreatment levels after 3 days of both the MARS treatment (p=0.0001, p<0.001) and PE+MARS treatment (both p<0.0001) and the Glasgow coma scale score was significantly increased (both p<0.0001). The 30-day mortality rate was 10.0% (6/60) in the MARS group and 11.7% (7/60) in the PE + MARS group. The per capita cost of treatment was significantly lower in the PE + MARS group than in the MARS group (p=0.0003). CONCLUSIONS: Both MARS and PE + MARS therapy can safely and effectively be used to treat liver failure complicated with hepatic encephalopathy, but PE + MARS therapy reduces serum total bilirubin level more effectively and is more cost-effective.

[Effect of microRNA on proliferation caused by mutant HBx in human hepatocytes].

To study the effect of micro (mi)RNA on cellular proliferation induced by hepatitis B x protein, HBx, in human liver cells and to investigate the underlying molecular mechanism of this cancer-related effect. The human L02 hepatocyte cell line was stably transfected with HBx (L02/HBx) or an HBx mutant (L02/HBx-d382) that induces higher levels of cellular proliferation. The differential miRNA expression profiles were determined by microarray analysis and confirmed by real-time PCR. Two miRNAs, miR-338-3p and miR-551b, that were found to be significantly down-regulated in the L02/HBx-d382 cells were selected for further study and transfected individually into cells using the lipofectamine procedure. The cell survival rate was analyzed by MTT assay, and cell cycles were assessed by flow cytometry. Expressions of cyclinD1, cyclinG1, and E2F1 were assessed by real-time PCR and Western blotting. Compared with the microarray miRNA profile of L02/pcDNA3.0 cells, six miRNAs were up-regulated and five miRNAs were down-regulated in the L02/HBx-d382 cells, while four miRNAs were up-regulated and 12 were down-regulated in the L02/HBx cells. The microarray results were consistent with real-time PCR results. Transfection of miR-338-3p and miR-551b significantly inhibited the cell survival rates (P less than 0.001) and induced G0/G1 phase cycle arrest. According to MTT results: for L02/HBx-d382 cells, compared with lipofectamine or non-transfected (NC) controls, the t value of miR-338-3p was 10.402, 9.133 and the t value of miR-551b was 8.763, 7.403; for L02/HBx cells, compared with lipofectamine or NC controls, the t value of miR-338-3p was 9.105, 8.074 and the t value of miR-551b was 7.673, 7.52. According to flow cytometry results: for L02/HBx-d382 cells, compared with lipofectamine or NC controls, the t value of miR-338-3p was 12.173, 11.107 and the t value of miR-551b was 15.364, 13.377; for L02/HBx cells, compared with lipofectamine or NC controls, the t value of miR-338-3p was 15.416, 13.378, and the t value of miR-551b was 13.276, 13.109. The protein levels of cyclinD1, cyclinG1, and E2F1 were significantly reduced by both miR-338-3p and miR-551b ( P less than 0.001). For L02/HBx-d382 cells, compared with lipofectamine or NC controls: E2F1 had t = 11.132, 10.031 and 12.017, 10.973, respectively; cyclinD1 had t = 15.654, 15.013 and 15.447, 14.733, respectively; cyclinG1 had t = 8.017, 7.661 and 7.402, 7.417, respectively. For L02/HBx cells, compared with lipofectamine or NC controls: E2F1 had t = 14.244, 13.331 and 15.022, 14.468, respectively; cyclinD1 had t = 8.695, 8.137 and 7.877, 7.503, respectively; cyclinG1 had t = 7.73, 7.471 and 7.596, 7.41, respectively. In contrast, the mRNA levels for E2F1, cyclinD1, and cylcinG1 showed no significant differences between the miRNA transfected cells and controls. Wild-type HBx and the high proliferation-inducing mutant HBx can influence the miRNA expression profile of L02 cells. HBx down-regulates miR-338-3p and miR-551b in L02 cells, and the high proliferation-inducing mutant has a more robust effect. The mechanism of miR-338-3p- or miR-551b-mediated cell growth inhibition appears to be related to the direct modulation of cyclinD1, cyclinG1, and E2F1.

The strategy and efficacy of prophylaxis against hepatitis B virus recurrence after liver transplantation for HBV-related diseases in the era of potent nucleos(t)ide analogues: A meta-analysis.

OBJECTIVES: This meta-analysis aimed to evaluate the clinical outcome of liver transplant (LT) recipients under potent nucleoside or nucleotide analogue (NA)-based regimens and investigate different prophylactic schemes. METHODS: We followed PRISMA statement to conduct this study. Two reviewers independently searched relevant literature via PubMed, Embase, Ovid MEDLINE, Web of Science and Insightmeme. Studies were included if they evaluated hepatitis B virus (HBV) recurrence under potent NA-based regimens in patients who received HBV-related LT. Primary and secondary outcomes were HBV recurrence, hepatocellular carcinoma (HCC) recurrence, all-cause and HBV recurrence-related mortality. Incidences with 95% confidence intervals were calculated and assessed by fixed and random effects models. Subgroup analyses were used to examine the impact of different treatment strategies. RESULTS: Altogether 25 studies (N = 2327) were included, with a pooled HBV recurrence rate of 1.01% (95% CI 0.53%-1.59%). HBV viremia or hepatitis D virus superinfection did not influence HBV recurrence significantly (P = 0.23 and 0.71, respectively). The recurrence rate under an indefinite combination of potent NA and hepatitis B immunoglobulin (HBIG) was lower than that under potent NA monotherapy (P = 0.000) and similar to that under NA plus a finite course of HBIG (P = 0.48). The pooled HCC recurrence rate was 5.34% (95% CI 0.78%-12.48%). HBV recurrence-related mortality and all-cause mortality were 0% and 6.95% (95% CI 4.30%-10.08%), respectively. CONCLUSIONS: Potent NA-based regimens provide satisfactory HBV antiviral prophylaxis and improve long-term outcomes for LT recipients. A finite combination of potent NA and HBIG is an alternative to life-long dual therapy.

[The roles of CD4+CD25+ T regulatory cells and Foxp3 mRNA expression in the subjects with and without responses to hepatitis B virus vaccination].

OBJECTIVES: To investigate the internal links between immune responses and Tregs and cytokine by the expression of T regulatory cells (Tregs), Foxp3 mRNA of different response groups and the detection of cytokine secretion after hepatitis B vaccination. METHODS: Blood samples were collected in different response groups. Real-time fluorescence quantitative PCR was used to detect the expression of Foxp3 mRNA of peripheral blood mononuclear cells; The surface markers CD4 and CD25 in peripheral-blood mononuclear cells were determined by flow cytometry; ELISA tests were used to detect the production level of phytohemagglutinin (PHA) in peripheral blood mononuclear cells, IL -4, IL-12, IL-18 stimulated by HBsAg and (IFN) gamma. RESULTS: (1) Foxp3 expressions in response group and non-response group were higher before or after PHA and HBsAg were stimulated. Differences were statistically significant (P value less than 0.05) ; (2) In peripheral blood, the percentage of CD4+CD25+ Treg of CD4+ T cells in response group (0.59%+/-0.46%) was obviously lower than those in control group (1.30%+/-1.44%) ; (3) Peripheral blood mononuclear cells stimulated by PHA and HbsAg in each group, the concentration of IFNgamma in non-response group [(11.00+/-9.03) IU/ml] was markedly lower than those in response group [(38.88+/-28.16) IU/ml],and differences were statistically significant (P value less than 0.01); (4) In PHA- or HBsAg-stimulated peripheral-blood mononuclear cells, the concentrations of IL-18, IL-4 and IL-12 had no significant difference. CONCLUSIONS: To some extent, CD4+CD25+Foxp3+ Treg cells may be involved in negative regulation of the immune responses to HBV vaccination. Immune non-response to HBV vaccination may be connected to insufficient secretion of IFNgamma; There was no correlation between the titer of anti-HBs and the expressions of IFNgamma and CD4+CD25+ Foxp3.

Hepatitis B virus X protein promotes proliferation and upregulates TGF-beta1 and CTGF in human hepatic stellate cell line, LX-2.

BACKGROUND: Chronic hepatitis B virus (HBV) infection is a major cause of liver fibrosis, but the mechanisms underlying HBV-related fibrogenesis are still unknown. Although the roles of HBV X protein (HBx) remain poorly understood, it is thought to play an important role in the regulation of cellular growth and hepatocarcinogenesis. The aim of this study was to determine the role of HBx in liver fibrogenesis by studying the effect of HBx on the proliferation and expression of fibrosis-related molecules in the human hepatic stellate cell line, LX-2. METHODS: We established an in vitro co-culture system with LX-2 cells and a stable QSG7701-HBx cell line which had been transfected with the HBx gene. 3H-TdR incorporation and flow cytometry were used to determine the effects of HBx on the proliferation of LX-2 cells. Alpha-smooth muscle actin (alpha-SMA), transforming growth factor-beta1 (TGF-beta1), transforming growth factor-beta receptor II (TGF-betaRII), and connective tissue growth factor (CTGF) in LX-2 cells were analyzed by Western blotting. In addition, the expression levels of collagen type I (ColI) from the co-cultured media were measured by ELISA. RESULTS: 3H-TdR incorporation increased significantly in LX-2 cells co-cultured with QSG7701-HBx cells compared to those cultured with QSG7701-pcDNA3 and QSG7701 (non-tumorigenic human liver cell line). Cell cycle results revealed that HBx accelerated the progression of G1 to S in LX-2 cells. The expressions of alpha-SMA, TGF-beta1, TGF-betaRII, CTGF and ColI were significantly increased in the co-cultures of LX-2 cells with stable QSG7701-HBx cells. CONCLUSION: These results suggest that HBx may facilitate liver fibrosis by promoting hepatic stellate cell proliferation and upregulating the expression of fibrosis-related molecules.

[Study on tumor formation of hepatocyte transformed by hepatitis B virus X gene in nude mice.].

OBJECTIVES: To investigate whether hepatitis B virus X gene alone is sufficient to transform the non-transformed immortalized human liver cell line QSG7701 and induce hepatocellular carcinoma in vivo. METHODS: pCMVX/QSG7701 cells were transplanted into subcutaneous tissue of nude mice. pRcCMV2/QSG7701 and QSG7701 cells were used as control. Tumor formation was checked within 5 weeks after transplantation. The activity and food intake of the nude mice were recorded. The texture, volume and metastasis of transplantation tumor were observed grossly, and the HE stained transplantation tumor tissues were observed under optical microscope. RESULTS: The transplantation tumor occurred in all of the six nude mice inoculated with pCMVX/QSG7701 cells at the second week after inoculation. No metastatic tumor was found in other organs. Transplant tumor was not formed in all of the negative control groups. The activity, eating and drinking of the nude mice transplanted with pCMVX/QSG7701 cells were normal, while their weights were increased gradually in the first 3 weeks. Since the 4th week after transplantation with pCMVX/QSG7701 cells, the activity of the mice was decreased and their body weight was no longer increased. It is interesting that the mental state and eating of those nude mice inoculated with pRcCMV2/QSG7701and QSG7701 cells were normal, and the weight was increasing all the time after inoculation. HE staining analysis confirmed that the transplanted tumor was hepatocellular carcinoma. CONCLUSION: HBx alone is sufficient to transform the non-transformed immortalized human liver cell line QSG7701 and induce hepatocellular carcinoma in vivo.

[Clinical experience of successful management of a human case with A/H5N1 infection in Hunan Province].

OBJECTIVE: To study the clinical features and successful management of a survived human case with A/H5N1 infection. METHODS: The data of a confirmed case of human case with A/H5N1 infection in Hunan province were collected and analyzed. RESULTS: This patient with A/H5N1 infection in Hunan province was confirmed by reverse-transcription polymerase chain reaction (RT-PCR) for A/H5N1 in airway secretions. The main clinical presentations included fever, cough and dyspnea. An extensive pulmonary infiltration developed quickly, followed by multi-organ dysfunction. Early administration of oseltamivir, early protection of organ function and extensive support were effective for the cure of the disease in this case. CONCLUSION: Early administration of oseltamivir, early protection of organ function and adequate support therapy may be useful for the treatment of human A/H5N1 infection.

miR-183-5p enhances the radioresistance of colorectal cancer by directly targeting ATG5.

Radioresistance is a material obstacle for effective treatment of colorectal cancer (CRC). Thus, the discovery of a novel biomarker for determining the CRC radiosensitivity is necessary. Recent studies have confirmed that miR-183-3p regulates cell phenotypes and tumor growth in various cancers. However, the role and mechanism of this micro-ribonucleic acid in CRC radiosensitivity remains unclear. Here, the abundances of miR-183-5p and ATG5 mRNAwere detected by a real-time quantitative reverse transcription polymerase chain reaction. Kaplan-Meier survival analysis was carried out to explore the correlation between miR-183-5p and patient prognosis. Cell viability was evaluated by the MTT assay. Survival fraction analysis through colony formation was performed to assess the cell radiation response. Bioinformatic, luciferase and western blot assays were employed to verify the targeted interaction between miR-183-5p and ATG5. The results showed that an elevated abundance of miR-183-5p and a reduced ATG5 level in CRC were associated with the poor prognosis. The knockdown of miR-183-5p enhanced the sensitivity of CRC cells to radiation, inflected by the decreased cell viability and survival fraction. Mechanically, ATG5 was targeted by miR-183-5p. The addition of ATG5 conferred the radiosensitivity of the CRC cells, which was revered by miR-183-5p restoration. Furthermore, miR-183-5p knockdown hindered the tumor growth by repressing ATG5 in vivo after radiation treatment. In summary, the output data indicated that miR-183-5p heightened the radiation response of the CRC cells by targeting ATG5, promising a novel therapeutic target for CRC patients with radioresistance.

Mycophenolate mofetil prevents lethal acute liver failure in mice induced by bacille Calmette-Guérin and lipopolysaccharide.

BACKGROUND/AIMS: To investigate the effect of mycophenolate mofetil (MMF) on acute liver injury induced by bacille Calmette-Guérin (BCG) and lipopolysaccharide (LPS). METHODS: Acute liver failure was induced in male Kunming strain mice by injecting the animals with BCG 2.5 mg per mouse, and LPS 10 microg per mouse 10 days later. The mice in the treatment groups were given MMF 2 h before, simultaneous with, or 2 h after administration of LPS, and the mice in the control group were given the same dose of saline. The 24-h survival rate, serum alanine aminotransferase (ALT), and aspartate aminotransferase (AST) levels were compared. Serum levels of tumor necrosis factor alpha (TNF-alpha), interferon gamma (IFN-gamma), and interleukin 6 (IL-6) were measured and the expressions of TNF-alpha, IFN-gamma, and IL-6 mRNA in the liver tissue were determined by reverse transcription-polymerase chain reaction (RT-PCR). Concanavalin A (Con A)-induced splenocyte proliferation were determined by methods of methyl thiazolyl tetrazolium. RESULTS: Injecting a small dose of LPS into BCG-primed mice caused a lethal hepatic injury mimicking acute hepatitis, from which 16 of the 20 mice died within 24 h (20% survival rate). Massive necrosis of parenchymal hepatocytes with marked inflammatory cell infiltration was observed by histological examination. In parallel, serum ALT and TNF-alpha, IFN-gamma, and IL-6 levels were increased. Expression of TNF-alpha, IFN-gamma, and IL-6 mRNA in the liver were significantly increased also. Treatment with MMF markedly reduced the death rate in a dose-dependent manner. It reached its maximal effect at the dosage of 150 mg per kg of body weight when pretreated 2 h before LPS injection, with improvement of histological feather and survival rate (84.2%, 16/19). MMF significantly inhibited serum levels of TNF-alpha, IFN-gamma, and IL-6, and significantly reduced TNF-alpha, IFN-gamma, and IL-6 expression in the liver, which increased after BCG and LPS injection. Moreover, splenocyte proliferation response induced by Con A was also inhibited by MMF treatment. CONCLUSIONS: Treatment with MMF has a protective effect on endotoxin-induced fatal liver failure by regulating the production of inflammatory cytokines and T-cell proliferation.

[Polymorphism of hepatitis B virus x gene in hepatocellular carcinoma patients and the biological effects of HBx gene with deletion varations on QSG7701 cells].

OBJECTIVE: To investigate whether there were particular HBx gene mutations associated with hepatocellular carcinoma (HCC) development in patients. METHODS: The HBx genes were examined in 51 paraffin-embedded tumor tissue samples from patients with HCC and 25 serum samples from HBV carriers from southern China by nested polymerase chain reaction (PCR), single-stranded conformational polymorphism analysis, heteroduplex analysis and DNA sequencing. The HBx genes with deletion variations (HBx-d382, HBx-d431) from tumor tissues were cloned and transfected into QSG7701 cells. Then, the biological characteristics of the transfected cells were analyzed in nude mice by MTT assay, soft agar colony formation assay, flow cytometry and xenografting. RESULTS: Deletion mutation and point mutation were found in the HBx genes of HCC tumor tissues, and there were some differences between the HBx gene mutations in genotype B and those in genotype C. More mutations were found in genotype C than those in genotype B (t=-2.522, P < 0.05), but the deletion variations (HBx-d382, HBx-d431) were detected in genotype B HBV from HCC liver tissues. The HBx genes with deletion variations (HBx-d382, HBx-d431) were recombinant with pcDNA3 and transfected into QSG7701 cell lines successfully, which established four permanent transfected QSG7701 cell lines, including pcDNA3/HBx-d382/QSG7701, pcDNA3/HBx-d431/QSG7701, pcDNA3/HBx/QSG7701, and pcDNA3/QSG7701. pcDNA3/HBx-d382/QSG7701 and pcDNA3/HBx-d431/QSG7701 grew faster and had more potential colony formative activity than those of pcDNA3/QSG7701. Moreover, pcDNA3/HBx-d382/QSG7701 and pcDNA3/HBx-431/QSG7701 cells inoculated in nude mice produced tumors more rapidly than those of pcDNA3/HBx/QSG7701, and pcDNA3/QSG7701. The volumes of the tumors in nude mice were also obviously larger in pcDNA3/HBx-d382 and pcDNA3/HBx-d431 groups than those in pcDNA3/HBx/QSG7701 and pcDNA3/QSG7701 groups. CONCLUSION: Our results suggest that HBx gene mutations occur frequently in HCC tissues, and the deletion at nt382-400 of the HBx gene might play a role in carcinogenesis of HCC in southern China.

[Heat-shock protein 27 linked to multi-drug resistance in human hepatic cancer cell lines].

OBJECTIVE: A proteomics approach was applied for finding multi-drug resistance (MDR) related proteins in hepatic cancer cell lines. METHODS: Two-dimensional electrophoresis (2-DE) was used to separate the total proteins of vincristine-resistant hepatic cancer cell line HepG2/VCR and its counterpart HepG2. The differential expression proteins between the two cell lines were identified by both MALDI-TOF-MS and ESI-Q-TOF-MS. Heat-shock protein 27 (HSP27), one of the differential expression proteins in the development of MDR of HepG2/VCR, was analyzed by HSP27 antisense oligonucleotides (ASOs). RESULTS: As a high expression protein in HepG2/VCR, HSP27 was identified, and the suppression of HSP27 expression by HSP27 ASO enhanced the vincristine chemosensitivity of HepG2/VCR (P less than 0.05). CONCLUSION: HSP27 is linked to MDR in human hepatic cancer cell line HepG2/VCR.

[Clinical observation on auxiliary treatment with suanzaoren decoction for chronic severe hepatitis].

OBJECTIVE: To investigate the curative effect and safety of auxiliary treatment with Suanzaoren Decoction (SZRD) on patients with chronic severe hepatitis (CSH). METHODS: Sixty patients, with the diagnosis in accordance with the diagnostic criterion of CSH, were assigned to the treated group and the control group, 30 in each group. Patients in the control group were treated with comprehensive therapy including symptomatic supportive treatment, anti-infective therapy and artificial liver plasmapheresis etc., while those in the treated group were orally taken SZRD additionally. Patients' condition of sleeping and changes of total bilirubin (TBIL), prothrombin activity (PTA), tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1) were observed before and after treatment, and the adverse reactions were observed as well. RESULTS: The sleeping status were significantly improved in the treated group after treatment, and the serum levels of TBIL, TNF-alpha and IL-1 were significantly decreased. The improvement rate was 66.7% (20/30) and significantly higher than that (40.0%, 12/30) in the control group. CONCLUSION: SZRD can significantly improve the sleeping status of CSH patients, alleviate the hepato-cellular injury by inflammatory cytokines and without obvious adverse reaction.

[The degree of HBV suppression with 24 week telbivudine- or lamivudine-treatment in hepatitis B patients predicts the efficacy of the treatment at week 52].

OBJECTIVES: To investigate the possibilities of an association between the degrees of HBV suppression with nucleoside treatments at week 24 and week 52 in hepatitis B patients and to find a useful predictor for treatment efficacy. METHODS: In this phase III, double-blind, multicenter trial, we compared the efficacy of telbivudine treatment with lamivudine treatment in 332 Chinese compensated chronic hepatitis B patients. The patients were randomly assigned to a daily 600 mg telbivudine treatment group or daily 100 mg lamivudine group for 24 weeks. They were then categorized into 4 groups according to their serum HBV DNA levels (copies/ml) at week 24: a PCR-undetectable group (< 300 copies/ml); a QL- < 10(3) copies/ml group; a 10(3)-<10(4) copies/ml group; and a > or = 10(4) copies/ml group. The treatments were continued as they previously had been for another 28 weeks and the patients serum HBV DNA levels were examined again. RESULTS: At week 52, mean reductions of serum HBV DNA were significantly greater in the telbivudine-treated patients than in the lamivudine-treated group (6.2 log10 vs 5.4 log10, t = 3.6, P < 0.01). Viral resistance was twice as common in lamivudine-treated patients compared to those receiving telbivudine. Telbivudine was well-tolerated with an adverse event profile similar to that of lamivudine. The lower the HBV DNA level achieved at week 24, the higher HBV DNA non-detectable by PCR. ALT normalization and HBeAg seroconversion achieved at week 52, and viral resistance at week 48 decreased parallel to the degree of HBV DNA inhibition. CONCLUSION: HBV DNA PCR-undetectable at week 24 in nucleoside-treated hepatitis B patients suggests a better efficacy at week 52 and lower viral resistance at week 48. The degree of suppression of HBV at week 24 may be used as a predictor of 1-year outcome.

[Effect of Suanzaoren decoction on acute hepatic failure in mice].

OBJECTIVE: To investigate the effect of Suanzao nacute hepatic failure in mice. METHOD: Acute liver failure was induced in male Kunming strain mice by enterocoelia injecting the animals with D-Gal-N and LPS. The mice in treatment groups were given corresponding drug 2 h before administration of D-Ga1-N and LPS, and the mice in control group were given the same dose of distilled water. The 24 h survival rate, serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) levels were compared. Serum the levels of TNF-alpha and IL-1 and the levels of SOD, MDA, GR, GSH, NO and NOS in the liver were determined. RESULT: Treatment with suanzaoren decoction could increase the survival rate and improve the liver histological feather. Suanzaoren decoction inhibited the serum the levels of ALT, AST, TNF-alpha and IL-1, and reduced the levels of MDA, NO and NOS and increased the levels of GR and SOD in the liver. CONCLUSION: Treatment with Suanzaoren decoction can suppress the D-Gal-N/LPS-induced acute hepatic failure. It may be the mechanism that Suanzaoren decocotion regulate the production of inflammatory cytokines and free radicals.