Danqi Tablet () Regulates Energy Metabolism in Ischemic Heart Rat Model through AMPK/SIRT1-PGC-1α Pathway / 中国结合医学杂志

OBJECTIVE@#To investigate the cardioprotective effect of Danqi Tablet (DQT, ) on ischemic heart model rats and the regulative effect on energy metabolism through peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α).@*METHODS@#Rat ischemic heart model was induced by ligation of left anterior descending coronary artery. Totally 40 Sprague-Dawley rats were randomly divided into sham group, model group, DQT group (1.5 mg/kg daily) and trimetazidine (TMZ) group (6.3 mg/kg daily) according to a random number table, 10 rats in each group. Twenty-eight days after continuous administration, cardiac function was assessed by echocardiography and the structures of myocardial cells were observed by hematoxylin-eosin staining. The level of adenosine triphosphate (ATP) in myocardial cells was measured by ATP assay kit. Expressions level of key transcriptional regulators, including PGC-1α, Sirtuin 1 (SIRT1), AMP-activated protein kinase (AMPK), and downstream targets of PGC-1α, such as mitofusin 1 (MFN1), mitofusin 2 (MFN2) and superoxide dismutase 2 (SOD2) were measured by Western blot. Expression level of PGC-1α was examined by immunohistochemical staining.@*RESULTS@#The rat ischemic heart model was successfully induced and the heart function in model group was compromised. Compared with the model group, DQT exerted cardioprotective effects, up-regulated the ATP production in myocardial cells and inhibited the infiltration of inflammatory cells in the margin area of infarction of the myocardial tissues (P<0.01). The expressions of PGC-1α, SIRT1 and AMPK were increased in the DQT group (all P<0.05). Furthermore, the downstream targets, including MFN1, MFN2 and SOD2 were up-regulated (P<0.05 or P<0.01). Compared with the TMZ group, the expression levels of PGC-1α, MFN1 and SOD2 were increased by DQT treatment (P<0.05 or P<0.01).@*CONCLUSION@#DQT regulated energy metabolism in rats with ischemic heart model through AMPK/SIRT1 -PGC-1α pathway. PGC-1α might serve as a promising target in the treatment of ischemic heart disease.

Mechanisms of Fufang Danshen tablets on treating heart failure via regulating AT₁-mediated PLA2-COX2/5-LOX metabolic pathway / 中国中药杂志

Myocardial fibrosis (MF) is an important pathological change involved in the progress from myocardial infarction (MI) to heart failure(HF). Metabolic disorder of arachidonic acid (AA) in cardiomyocytes plays an important role in process of MF. Fufang Danshen tablets is a traditional Chinese medicine (TCM), which showed significant effect on coronary heart diseases and anti-MF. However, the underlying mechanism of anti-MF remains unclear. In this study, HF animal model of myocardial infarction was established by ligation of left anterior descending coronary artery. The heart function of rats in each group was evaluated by echocardiography and hemodynamic measurement. Histological examination, TUNEL and Western blot were used to detect the levels of MF and proteins related to AA metabolism. As a result, MI significantly decreased the levels of ejection fraction (EF), ejection fraction (FS) and left ventricular systolic pressure (LVSP), and these decreases were significantly improved by the treatment of Fufang Danshen tablets. Besides, Fufang Danshen tablets treatment down-regulated the levels of creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH) in serum. HE, Masson and TUNEL staining results showed that Fufang Danshen tablets treatment could inhibit the inflammatory cells infiltration and attenuate the fibrosis and apoptosis to exert cardioprotective effect. Western blot indicated that Fufang Danshen tablets treatment down-regulated the expressions of AT₁, MMP2, MM9, while up-regulated the expression of AT₂ to inhibit MF. Further mechanism study indicated that Fufang Danshen tablets inhibited MF by down-regulated the expressions of AA metabolism, such as PLA2, P450, COX2 and 5-LOX. In summary, Fufang Danshen tablets can effectively inhibit MF in the ischemic area after MI in rats. The mechanism is related to the regulation of AT₁-mediated PLA2-COX2 metabolic pathway.

Molecular cloning, characterization and expression analysis of woodchuck retinoic acid-inducible gene I / 华中科技大学学报（医学）（英德文版）

Cytosolic retinoic acid-inducible gene I (RIG-I) is an important innate immune RNA sensor and can induce antiviral cytokines, e.g., interferon-β (IFN-β). Innate immune response to hepatitis B virus (HBV) plays a pivotal role in viral clearance and persistence. However, knowledge of the role that RIG-I plays in HBV infection is limited. The woodchuck is a valuable model for studying HBV infection. To characterize the molecular basis of woodchuck RIG-I (wRIG-I), we analyzed the complete coding sequences (CDSs) of wRIG-I, containing 2778 base pairs that encode 925 amino acids. The deduced wRIG-I protein was 106.847 kD with a theoretical isoelectric point (pI) of 6.07, and contained three important functional structures [caspase activation and recruitment domains (CARDs), DExD/H-box helicases, and a repressor domain (RD)]. In woodchuck fibroblastoma cell line (WH12/6), wRIG-I-targeted small interfering RNA (siRNA) down-regulated RIG-I and its downstrean effector-IFN-β transcripts under RIG-I' ligand, 5'-ppp double stranded RNA (dsRNA) stimulation. We also measured mRNA levels of wRIG-I in different tissues from healthy woodchucks and in the livers from woodchuck hepatitis virus (WHV)-infected woodchucks. The basal expression levels of wRIG-I were abundant in the kidney and liver. Importantly, wRIG-I was significantly up-regulated in acutely infected woodchuck livers, suggesting that RIG-I might be involved in WHV infection. These results may characterize RIG-I in the woodchuck model, providing a strong basis for further study on RIG-I-mediated innate immunity in HBV infection.

CLINICAL CURATIVE EFFECT OF BIOLOGICAL FEEDBACK THERAPY ON TWO KINDS OF FUNCTIONAL CONSTIPATION / 现代医院

Objective To explore the clinical curative effect of biofeedback therapy on two kinds of functional constipation . Methods 50 cases of patients with functional constipation treated in our hospital from May 2013 to May 2014 were selected as the re-search objects.The clinical data and effects of patients before and after treatment were compared and analyzed .Results Through time monitoring of patients'colons, 72-hour discharge numbers of markers after treatment were improved significantly , with a signifi-cant difference (p<0.05).The quality of life score after treatment was significantly increased, with a significant difference (p<0.05 ) .Results of patients before and after treatment showed that the symptoms was significantly relieved after treatment , with a signif-icant difference (p<0.05).Conclusion The biological feedback therapy treated for two types of functional constipation can effec-tively enhance the therapeutic effect, improve the patients'ability of regulation and control of their own unreasonably physiological ac-tivities.Besides, for its high security, it is worthy of recommendation.

Clinical research of acupuncture at stellate ganglion in the treatment of posterior circulation ischemia and its impacts on blood pressure / 中国针灸

<p><b>OBJECTIVE</b>To compare the difference in the clinical efficacy on posterior circulation ischemia between acupuncture at stellate ganglion and conventional acupuncture as well as the impacts on blood pressure.</p><p><b>METHODS</b>Eighty cases of posterior circulation ischemia were randomized into an observation group (40 cases) and a control group (40 cases). In the observation group, acupuncture was applied to the bilateral stellate ganglions on the neck, stimulated with reinforcing technique by rotating the needles. In the control group, the acupuncture of reducing technique was applied to Fengchi (GB 20), Baihui (GV 20), Neiguan (PC 6) and Taichong (LR 3) in the excess syndrome. The even needling or reinforcing technique was applied to Fengchi (GB 20), Baihui (GV 20), Ganshu (BL 18), Shenshu (BL 23) and Zusanli (ST 36) for the deficiency syndrome. The treatment was given once every 3 days and 4 treatments were required totally in the two groups. The changes in total syndrome score, peak Systolic blood flow velocity (Vp) of vertebral artery and basilar artery, systolic and diastolic blood pressures were compared before and after treatment in the two groups. The clinical efficacy was compared between the two groups.</p><p><b>RESULTS</b>The total syndrome score was reduced apparently after treatment compared with that before treatment in the two groups (P < 0.01), and the reducing was more obvious in the observation group as compared with that in the control group (P < 0.01). The total effective rate was 87.5% (35/40) in the observation group, higher than 67.5% (27/40, P < 0.05) in the control group. After treatment, the reduced Vp of vertebral artery was not improved apparently as compared with that before treatment in the control group, Vp in blood velocity abnormality (including vascular spasm, stenosis or reduced velocity) of vertebral artery and basilar artery was all improved as compared with that before treatment in the two groups (P < 0.01), and the improvements in the observation group were more obvious than those in the control group (P < 0.01). After treatment, the systolic and diastolic pressures were reduced as compared with those before treatment in the two groups, and the reduced systolic and diastolic pressures in the observation group were more apparent than those in the control group (P < 0.01).</p><p><b>CONCLUSION</b>Acupuncture at stellate ganglion achieves the satisfactory efficacy in the treatment of posterior circulation ischemia and the significant efficacy of reducing blood pressure, more advanced than the conventional acupuncture.</p>

Establishment and application of hepatitis B virus persistent replication model in IFNAR(-/-) mouse / 华中科技大学学报（医学）（英德文版）

The type I interferon and IFNAR play an important role in hepatitis B virus (HBV) infection and anti-HBV therapy. However, its mechanism of action is still poorly understood. To gain more insights into the role of type I interferon and type I interferon receptor (IFNAR) in HBV infection, we established an HBV persistent replication IFNAR knockout (IFNAR(-/-)) mouse model and preliminarily applied this model. At first, the progeny of IFNAR(-/-) mouse was reproduced. Then hydrodynamic injection with pAAV/HBV1.2 plasmid was conducted to establish the persistent HBV replication IFNAR(-/-) mouse model. At last, we applied this model to evaluate the effect of nucleoside analogues entecavir (ETV) on HBV replication. It was found that there was no difference in the serum HBsAg and HBeAg levels and HBcAg expression in the liver tissue between the ETV treated groups and normal saline (NS) treated group, but the serum HBV DNA levels were significantly suppressed 10, 25, 40 and 55 days after the ETV treatment [P=0.035, P=0.00, P=0.149 and P=0.084, IFNAR knockout (KO) control group vs. C57BL/6 ETV groups, respectively; P=0.081, P=0.001, P=0.243 and P=0.147, IFNAR KO control group vs. IFNAR KO ETV groups, respectively]. Interestingly, there was no difference in serum HBV DNA levels between the ETV treated IFNAR(-/-) and C57BL/6 mice. This result suggests that HBV suppression during ETV treatments doesn't depend on type I interferon and IFNAR. Collectively, persistent HBV replication IFNAR(-/-) mouse model that we established is a useful and convenient tool to detect the function of the type I interferon and IFNAR in HBV infection and anti-HBV treatments.

Establishment and application of hepatitis B virus persistent replication model in IFNAR(-/-) mouse / 华中科技大学学报（医学）（英德文版）

The type I interferon and IFNAR play an important role in hepatitis B virus (HBV) infection and anti-HBV therapy. However, its mechanism of action is still poorly understood. To gain more insights into the role of type I interferon and type I interferon receptor (IFNAR) in HBV infection, we established an HBV persistent replication IFNAR knockout (IFNAR(-/-)) mouse model and preliminarily applied this model. At first, the progeny of IFNAR(-/-) mouse was reproduced. Then hydrodynamic injection with pAAV/HBV1.2 plasmid was conducted to establish the persistent HBV replication IFNAR(-/-) mouse model. At last, we applied this model to evaluate the effect of nucleoside analogues entecavir (ETV) on HBV replication. It was found that there was no difference in the serum HBsAg and HBeAg levels and HBcAg expression in the liver tissue between the ETV treated groups and normal saline (NS) treated group, but the serum HBV DNA levels were significantly suppressed 10, 25, 40 and 55 days after the ETV treatment [P=0.035, P=0.00, P=0.149 and P=0.084, IFNAR knockout (KO) control group vs. C57BL/6 ETV groups, respectively; P=0.081, P=0.001, P=0.243 and P=0.147, IFNAR KO control group vs. IFNAR KO ETV groups, respectively]. Interestingly, there was no difference in serum HBV DNA levels between the ETV treated IFNAR(-/-) and C57BL/6 mice. This result suggests that HBV suppression during ETV treatments doesn't depend on type I interferon and IFNAR. Collectively, persistent HBV replication IFNAR(-/-) mouse model that we established is a useful and convenient tool to detect the function of the type I interferon and IFNAR in HBV infection and anti-HBV treatments.

Toll-like receptor dependent innate immune responses by primary mouse hepatocytes and its control of HBV replication / 中华肝脏病杂志

<p><b>OBJECTIVE</b>This report aims to investigate the Toll-like receptor (TLR) signaling pathways and induced antiviral activity in hepatocytes.</p><p><b>METHODS</b>We isolated primary hepatocytes from wild-type C57BL/6 mice and examined the expression of TLR by realtime RT-PCR. Hepatocytes were stimulated with TLR 1-9 agonists and the supernatants were harvested. The secretion of cytokines were tested by ELISA. The antiviral effectors in supernatants were assayed via virus protection assay (in EMCV system) and the control of HBV replication were assessed via Southern blotting (in HBV system).</p><p><b>RESULTS</b>We demonstrated that hepatocytes expressed TLR1-9. In accordance with these TLR expression profiles, hepatocytes responded to all TLR ligands by producing inflammatory cytokines (TNF-α or IL-6), to TLR -1,-3,-7 and -9 ligands by producing type I IFN (IFN-α or IFN-β). Only TLR 3 and TLR 7 agonists could stimulate the production of high amounts of antiviral mediators by hepatocytes in virus protection assay. By contrast, supernatants from TLR1, -3 and -4 directly stimulated hepatocytes and TLR 3, -7 and -9 transfected hepatocytes were able to potently suppress HBV replication.</p><p><b>CONCLUSION</b>Primary hepatocytes display a unique TLR signaling pathway and can control HBV replication after stimulation by TLR agonists in mice. It may be helpful for the development of TLR-based therapeutic approaches against hepatotropic virus.</p>

The influence of HBV and its antigens on the expressions of JAK-STAT signal transduction pathway molecules and antiviral proteins of IFN alpha / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the possible influence of HBV and its antigens on the expressions of JAK-STAT signal transduction pathway molecules and the antiviral proteins of IFN alpha.</p><p><b>METHODS</b>The HepG2 cells were transfected with pSM2, pHBS2-S and pHBc-EGFP plasmids which express HBV whole particles or S-antigen, Pre-S antigen and core antigens. The infectious supernatant from HepG2.2.15 cells and the pured HBV proteins which contained the S, Pre-S antigens were used to treat the HepG2 cells. Northern blot and RT-PCR were applied to analyse the expressions of the antiviral proteins MxA, 2' -5' OAS, 9-27 and the JAK-STAT signal transduction pathway molecules STAT1 in HepG2 cells responded to the IFN alpha treatment.</p><p><b>RESULTS</b>The HepG2 cells transfected with pSM2, pHBS2-S and pHBc-EGFP plasmids could express whole HBV particles and HBsAg, Pre-S antigen and HBcAg. The quantitation of expressed HBV particles and antigens increased significantly during the course of transfection. Northern blot hybridization analysis indicated that the HepG2 cells expressed IFN alpha antiviral proteins MxA, 2' -5' OAS and 9-27. When transfected with pHBV-dimer, pHBS2-S, pHBc-EGFP plasmids, the IFN/A antiviral proteins MxA, 2' -5' OAS and 9-27 in transfected cells were reduced greatly as compared to the un-transfected HepG2 cells, and the expressed antiviral proteins decreased sharply with the development of transfection time. Furthermore, the expression of IFN alpha JAK-STAT signal transduction pathway molecule STAT1 was also inhibited with the expression of HBV particles and HBV antigens in transfected HepG2 cells.</p><p><b>CONCLUSIONS</b>The HBV and its antigens influence the expressions of IFN alpha JAK-STAT signal transduction pathway molecules and antiviral proteins in the hepatocellular models in vitro. It is indicated that HBV might possess the activity to antagonise or counteract the IFN alpha antiviral action.</p>

Characterization of HBsAbs in occult hepatitis B virus infection / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the properties of HBsAb in occult hepatitis B virus infection and its affinity to different serotypes of hepatitis B virus surface antigen (HBsAg).</p><p><b>METHODS</b>Long-term follow-up was conducted in 2 HBsAb positive patients with occult hepatitis B virus infection. HBsAg was detected using multiple diagnostic kits and the HBsAb subtype was determined by performing neutralization experiments with different serotypes of HBsAg. The viral S gene was PCR-amplified and mutation analysis was conducted. Plasmids expressing HBsAgs were constructed by inserting these PCR products into an eukaryotic expression vector and were then transfected into HepG2 cells. The cell culture supernatant and cellular extracts were detected for HBsAg respectively. Neutralization experiments were carried out in the cell culture supernatant from HBsAg plasmids transfected HepG2 cells and serum samples from these patients and others who had been confirmed to be positive for HBsAb.</p><p><b>RESULTS</b>Multiple tests using various diagnostic kits showed that the 2 patients were negative for HBsAg and the three different serotypes of HBsAg (adr, adw, ay) could neutralize 82.1%-100% of HBsAb existed in the 2 patients. Sequence analysis of S gene cloned from these patients revealed that the homology to reference strain were 95.13%-97.79% and 92.04%-95.58% respectively at the nucleotide and amino acid levels. Quantitation of HBsAg showed that the expression levels of HBsAg from the two patients were 41.1% and 22.6% respectively of that of control HBsAg in cell culture supernatant and 48.1% and 59.3% respectively in cellular extract, and the supernatant/cell lysate ratios were 0.85 and 0.38 respectively. In neutralization experiments, HBsAg could be totally absorbed by control serum, whereas could only be partially neutralized by HBsAbs from the two patients (F = 353.6 and 645.2, P is less than 0.01).</p><p><b>CONCLUSION</b>Both the antigenicity and the ability of HBsAg secreted outside of the cells are decreased in these HBsAb-positive patients with occult HBV infection. The HBsAbs are mainly specific for common epitopes among different serotypes of HBsAg and are probably different as compared with those produced by vaccine inoculation.</p>

Influence of excessive complement activation on pathological process of acute graft versus host disease in mice / 中国实验血液学杂志

This study was aimed to explore the influence of excessive complement activation on the pathological process of acute graft-versus-host disease (aGVHD) in mice. A murine model with aGVHD was established by injecting cell mixture containing splenocytes and bone marrow cells at 2:1 ratio from donor C57BL/6(H-2K(b)) mice into recipient BALB/c (H-2K(d)) mice within 4-6 hours after 8 Gy (60)Co γ-ray total body irradiation. The mice received syngeneic bone marrow transplantation were used as control group. After transplantation, the mice were monitored daily for body weight and mortality. At day 14, all mice were sacrificed and each liver was freshly dissociated for histological analysis. The hepatic mRNA abundance for complement components C3a and C5a as well as receptors for these two anaphylatoxin were tested by real-time quantitative PCR method. And the levels of C3a and C5a production in liver were detected by ELISA. The deposition of complement C3 in liver was determined by immunofluorescence staining using frozen section. The results indicated that as compared with syngeneic bone-marrow transplantation control group, experimental animals underwent aGVHD characterized by weight loss, depilation, diarrhea and lassitude. Interestingly, the hepatic mRNA expression for complement anaphylatoxin family member C3a and C5a as well as their receptors C3aR and C5aR1 in mice with aGVHD were significantly up-regulated in comparison with control group (p < 0.05). Consistently, the content of C3a and C5a in liver increased markedly in mice with aGVHD (p < 0.01). For animals ongoing aGVHD, complement component C3 depositions were observed in hepatic portal areas, around which massive inflammatory cell infiltration was also observed. It is concluded that in aGVHD animals, excessive complement activation occurs, and the activated complement components participate in pathological process of the aGVHD.

Expression of enhancer of zeste homolog 2 in transitional cell carcinoma of the bladder cell lines and tissues / 中华泌尿外科杂志

Objective To study the expression of enhancer of zeste homolog 2 (EZH2) gene in transitional cell carcinoma (TCC) of the bladder celt lines, carcinoma tissues and normal bladder tis-sues and to evaluate the roles of EZH2 in the development and progression of bladder carcinoma. Methods RT-PCR, Western-blot and immunocytochemistry were used to analyze the expression of EZH2 of the bladder cell lines (T24, EJ, MGH-U1, BIU-87). The prostate cancer cell line PC-3M was used as an EZH2-positive cell line. EZH2 gene expressions in 45 cases of bladder carcinoma and 12 cases of normal bladder mueosa were detected by RT-PCR. Of cancer cases, 31 were superficial tumors and 14 were invasive tumors; 13 were G1, 21 were G2 and 11 were G3. Results EZH2 was detected in the 4 TCC cell lines. The EZH2 expression rate of TCC (82.2%) was significantly higher than that of normal bladder tissues (8.3%, P<0.05). The expression rate in superficial tumors was 74.2% and in invasive tumors was 100.0%, but there was no significant difference (P>0.05). The expression rates increased with tumor cell grade increase, but there was no significant difference (P> 0.05). Conclusions EZH2 could play an important role in the development and progression of blad-der carcinoma. It could be used as a potential gene therapy target of bladder cancer.

Comparison between auditory steady-state response and 40 Hz auditory event related potential in the evaluation of hearing thresholds / 法医学杂志

OBJECTIVE@#To compare the results between auditory steady-state response (ASSR) and 40 Hz auditory event related potential (AERP), and explore the accuracy of hearing thresholds by using ASSR and AERP and the clinic forensic value.@*METHODS@#Thirty seven ears were tested with pure-tone audiometer, 40Hz AERP and ASSR, respectively. All the volunteers in our study were awake during 40 Hz AERP test and ASSR test.@*RESULTS@#Thresholds acquired with ASSR and 40Hz AERP test had a close correlativity and showed higher than those acquired with PTA test. There was no significant difference between the accuracy of ASSR and 40Hz AERP in estimating pure-tone thresholds.@*CONCLUSION@#After determining the correct value, ASSR can be used directly to evaluate hearing loss objectively.

Staged treatment of the severe hypospadias in adult / 中华整形外科杂志

<p><b>OBJECTIVE</b>To report the treatment of serious hypospadias in adults with free graft of tubed mouth mucosa and scrotal fascia flaps.</p><p><b>METHODS</b>The tubed mouth mucosa was free grafted to fabricate the distal segment of urethra. It was anastomosed to the urethra at the second stage. The scrotal fascia flap was used to cover the penile wound. The biggest flap was 3 cm in width and 6.5 cm in length.</p><p><b>RESULTS</b>From Jan. 2002 to Dec. 2007, 76 adults with severe hypospadias were treated. Infection happened in 4 cases. 2 cases had urethral fistula due to the partial flap necrosis which was healed automatically within 2-4 weeks. All the other patients healed primarily.</p><p><b>CONCLUSIONS</b>It is a good method for the treatment of serious hypospadias in adults with scrotal fascia flaps and free graft of tubed mouth mucosa which is anastomosed to the urethra at the second stage.</p>

Establishment of reference sequences of hepatitis B virus genotype B and C in China / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To establish the reference sequences of genotype B and C of hepatitis B virus in China.</p><p><b>METHODS</b>Genome sequences of Hepatitis B virus isolated from different area in China were retrieved from GenBank. These genome sequences were alignmented and the most common sequences were regarded as reference sequences. The amino acid sequences were also alignmented.</p><p><b>RESULTS</b>The homology was 99.32% between Bc genome and Ba genome, and it was 95.52% between Bc genome and Bj genome. The S gene sequence homology was 99.71% between Bc and Ba, and it was 98.68% between Bc and Bj. The homology was 98.44% between Cc genome and C genome, and it was 93.97% between Cc genome and Caus genome. The S gene sequence homology was 99.27% between Cc and C, and it was 95.01% between Cc and Caus. There was significant difference at the sites of 1762, 1764, 1858 between genotype B and genotype C (P < 0.05). The amino acid sequences were also different between genotype B and genotype C.</p><p><b>CONCLUSION</b>Bc and Cc sequences of Hepatitis B virus can be regarded as the reference sequences of genotype B and C.</p>

Urethral Stricture Complicated with Infection An Analysis of 150 Patients Treated with Piperacillin and Tazobactam / 中华医院感染学杂志

0.05).CONCLUSIONS Piperacillin/tazobactam in the treatment of urethral stricture complicated with infection is significantly effective and safely.

Changes of Toll-like receptor (TLR) 2 and TLR4 on the peripheral blood mononuclear cells in patients with chronic hepatitis B and chronic severe hepatitis B / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To study the changes of TLR2 and TLR4 on peripheral blood mononuclear cells (PBMCs) and their role in the pathogenesis of chronic hepatitis B and chronic severe hepatitis B.</p><p><b>METHODS</b>The expressions of TLR2 and TLR4 on 10000 CD14+ PBMCs were determined by flow cytometry in 30 healthy controls, in 31 patients with chronic hepatitis B and in 30 patients with chronic severe hepatitis B. The level of serum tumor necrosis factor alpha (TNF alpha) was determined by ELISA. The differences of expression of TLR2 and TLR4 on PBMCs and serum TNFalpha among the three groups of study subjects were determined by Student-t test. The correlations between TLR2, TLR4 and TNF alpha were determined by linear correlation test.</p><p><b>RESULTS</b>The values of mean fluorescence intensity (MFI) of TLR2 on PBMCs of the healthy controls, patients with chronic hepatitis B and patients with chronic severe hepatitis B groups were 21.5+/-2.7, 39.0+/-4.1, and 47.7+/-21.4; TLR4 of those groups was 2.3+/-1.1, 3.7+/-2.3, and 6.9+/-4.1. The serum TNF alpha(ng/L) of the respective groups was 53.8+/-38.1, 164.3+/-89.9, and 359.8+/-140.0. There was a gradual increase of these values from the group of healthy controls to the group of patients with chronic hepatitis B and patients with chronic severe hepatitis B. No significant positive correlations between TLR2, TLR4 and serum TNFalpha were found.</p><p><b>CONCLUSION</b>TLR2 and TLR4 may have a role in the pathogenesis of chronic hepatitis B and chronic severe hepatitis B.</p>

Changes of RANTES levels in livers of patients with chronic hepatitis B: the clinical significance and the possible mechanisms / 中华肝脏病杂志

<p><b>OBJECTIVES</b>To study the relationship between intra-hepatic levels of regulated on activation, normal T-cell expressed and secreted (RANTES) and the disease severity and liver inflammatory degrees in patients with chronic hepatitis B and the possible mechanism of the changes of intra-hepatic levels of RANTES.</p><p><b>METHODS</b>The expression of RANTES of the livers was studied using immunohistochemical stainings and morphometric quantitative measurements in liver specimens from 10 normal subjects and 64 patients with chronic hepatitis B with different degrees of liver inflammation and different clinical severity. The expressions of RANTES protein and mRNA in cell line HepG2, HepG2.2.15 and HepG2 treated with 10 ng/ml TNFa at different times were quantified by ELISA and one-step RT-PCR.</p><p><b>RESULTS</b>The expression of RANTES of the livers in patients was significantly higher than that in the normal controls. Hepatic RANTES levels increased significantly and the increases were parallel to the increases of the severity of the hepatitis, from mild, moderate to severe hepatitis (the positive units were 3.7+/-1.5, 15.6+/-6.9, 24.0+/-4.0, 37.9+/-11.1, respectively) and from G0 degree to G4 degrees of liver inflammation (the positive units were 3.7+/-1.5, 15.0+/-5.7, 21.6+/-5.9, 30.3+/-8.2, 40.9+/-12.3, respectively). The expressions of RANTES protein and mRNA of HepG2.2.15 were higher than that of HepG2. RANTES protein and mRNA were induced in HepG2 by TNFa.</p><p><b>CONCLUSION</b>RANTES may have an important role in the pathogenesis of chronic hepatitis B. The elevation of hepatic RANTES may be caused by hepatitis B virus and TNFa.</p>

Common linear B-cell epitopes in human hepatitis B virus core protein and woodchuck hepatitis virus core protein / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To search for and verify some common B cell epitopes in the core proteins of woodchuck hepatitis virus and human hepatitis B virus.</p><p><b>METHODS</b>Monoclonal antibodies against both core proteins of woodchuck hepatitis virus (WHV) and human hepatitis B virus (HBV) were prepared by inoculating Balb/c mice with denatured recombination WHV and HBV core proteins. ELISA and immunoblotting assays for WHcAg and HBcAg were carried out by using these antibodies. Immunohistochemistry was carried out with liver tissue sections of both WHV-infected woodchucks and chronic HBV-infected patients. The epitopes were mapped with the mouse mAbs (6D1 and 1H4) by using a panel of 24 16mer overlapping peptides covering the entire WHcAg. The amino acid sequences of WHcAg and HBcAg were compared.</p><p><b>RESULTS</b>Cross-reactions were observed between mAbs (6D1 and 1H4) and WHcAg and between Mabs and HBcAg/HBcAg in ELISA and immunoblotting assay. Liver tissue sections of both WHV-infected woodchucks and chronic HBV-infected patients could be stained specifically by mAbs. The epitopes were mapped at aa1-8 (6D1) and aa125-140 (1H4) of the core proteins of both WHV and HBV by using ELISA assay. WHcAg and HBcAg share similar amino acids sequences at aa1-8 and aa125-140 respectively.</p><p><b>CONCLUSION</b>The core proteins of woodchuck hepatitis virus and human hepatitis B virus share common linear B cell epitopes which span aa1-8 and aa125-140 respectively.</p>

Significance of novel HBV expression vectors in selecting antiviral drugs in clinical therapy / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To establish a new method for rapidly selecting anti-hepatitis B virus drugs in clinical therapy.</p><p><b>METHODS</b>The full-length hepatitis B virus (HBV) genomes from 8 patients with chronic hepatitis B (CHB) were generated by polymerase chain reaction (PCR). All patients were resistant to lamivudine therapy. Their HBV DNA fragments were inserted into Sap I site of pHY106 eukaryotic expression vector separately. The recombinant plasmids containing 1.1 copies of HBV genome were transfected into Huh7 cell line; the levels of HBsAg, HBeAg and HBV DNA in supernatants of Huh7 cells were measured by ELISA and real-time quantitative PCR, and intracellular HBV replicative intermediates were detected by Southern blot. Antiviral effects of lamivudine and adefovir were evaluated in this vitro system.</p><p><b>RESULTS</b>The 8 recombinant plasmids containing a full-length genome of clinical HBV isolates could replicate and be expressed in Huh 7 cells. There were 6 isolates with polymerase YVDD mutations and 2 isolates with polymerase YIDD mutations. Adefovir, but not lamivudine, inhibited the HBV replication and gene expression in vitro. Furthermore, adefovir inhibited HBV replication in these CHB patients.</p><p><b>CONCLUSION</b>The method described here enables a rapid selection of anti-HBV drugs in clinical therapy and is very useful in antiviral therapy for CHB patients.</p>