Sophocarpine Protects Mice from ConA-Induced Hepatitis via Inhibition of the IFN-Gamma/STAT1 Pathway.

Sophocarpine is the major pharmacologically active compound of the traditional Chinese herbal medicine Radix Sophorae Subprostratae which has been used in treating hepatitis for years in China. It has been demonstrated that Sophocarpine exerts an activity in immune modulation and significantly decreases the production of inflammatory cytokines. However, the protective effects of Sophocarpine in T cell-dependent immune hepatitis remained unknown. The aim of this study was to determine the protective effects and pharmacological mechanisms of Sophocarpine on Concanavalin A (ConA)-induced hepatitis, an experimental model of T cell-mediated liver injury. BALB/C mice were pretreated with Sophocarpine or Bicyclol for five consecutive days. Thirty minutes after the final administration, the mice were injected with 15 mg⋅kg-1 of ConA intravenously. The results indicated that pretreatment with Sophocarpine significantly ameliorated liver inflammation and injury as evidenced by both biochemical and histopathological observations. Moreover, in Sophocarpine-pretreated mice, liver messenger RNA expression levels of chemokines and adhesion molecules, such as macrophage inflammatory protein-1α, CXC chemokine ligand 10, and Intercellular adhesion molecule-1, were markedly reduced. Further studies revealed that Sophocarpine significantly downregulated the expression of T-bet via inhibition of signal transducers and activators of transcription1 (STAT1) activation and overexpression of suppressor of cytokine signaling1, inhibiting the activation of Th1 cells and the expression of Interferon-γ (IFN-γ). Altogether, these results suggest new opportunities to use Sophocarpine in the treatment of T cell-mediated liver disease. In summary, Sophocarpine could attenuate ConA-induced liver injury, and the protective effect of Sophocarpine was associated with its inhibition effect of pro-inflammatory cytokines, chemokines, and the IFN-γ/STAT1 signaling pathway.

Anti-hepatitis B virus effect of matrine-type alkaloid and involvement of p38 mitogen-activated protein kinase and tumor necrosis factor receptor-associated factor 6.

The matrine-type alkaloid, oxymatrine inhibits hepatitis B virus (HBV) replication but very little is known about these effects in other matrine-type alkaloids, including sophoridine and sophocarpine. Therefore, we compared the in vitro anti-HBV effects of matrine, oxymatrine, sophocarpine, and sophoridine by treating an HBV-transfected cell line (HepG2.2.15) with 0.4-1.6mM of the compounds for 24 or 72h. The levels of the HBV surface antigen (HBsAg) and e antigen (HBeAg) in the culture medium, as well as the intracellular and extracellular HBV DNA levels, were determined. Metabolomic analysis and detection of the mRNA level of p38 mitogen-activated protein kinase (MAPK), tumor necrosis factor receptor-associated factor (TRAF) 6, extracellular signal-regulated kinase (ERK) 1, NOD-like receptor family pyrin domain containing 10 (NLRP10), and caspase-1 were conducted in sophoridine-treated HepG2.2.15 cells. HepG2.2.15 cell exposure to 0.4-1.6mM sophocarpine or sophoridine for 24h reduced the HBsAg level of the medium more effectively than exposure to matrine and oxymatrine did, and reduced the HBeAg levels more effectively than these compounds did at 1.6mM. Sophoridine (0.4-1.6mM) reduced the cell medium HBV DNA levels more than the same concentrations of matrine, oxymatrine, or sophocarpine did. After 72h, 0.4 and 0.8mM sophoridine reduced HBsAg and intracellular HBV DNA levels more potently than matrine, oxymatrine, or sophocarpine did. Furthermore, sophoridine (0.8mM) potently reduced the cell medium HBeAg levels while the metabolomic analyses revealed that HepG2.2.15 cells exposed to 0.8mM sophoridine for 72h exhibited reduced cycloleucine and phytosphingosine levels. In addition, the mRNA expression analyses revealed that HepG2.2.15 cells exposed to 0.8mM sophoridine showed reduced levels of p38 MAPK, TRAF6, ERK1, NLRP10, and caspase-1. Sophoridine produced more potent anti-HBV effects than matrine, oxymatrine, and sophocarpine did. These effects may be related to the sophoridine-mediated reduction of p38 MAPK and TRAF6 levels.

[Thymopolypeptides combined with matrine type alkaloids suppress HBV replication].

To investigate the antiviral effect of thymopolypeptides combined with 4 kinds of matrine type alkaloids on HepG2.2.15 cells, oxymatrine, sophocarpidine, sophocarpine, and sophoridine (at concentration of 0.2 mmol•L⁻¹ respectively) were respectively combined with thymopolypeptides (0.025, 0.1 g•L⁻¹), and after 48 h and 72 h treatment on HepG2.2.15 cells, the cells and supernatants were collected. The cells activity in various groups was determined by CCK-8 method to evaluate the toxic effects of the drugs on HepG2.2.15 cells. Enzyme linked immunosorbent assay (ELISA) was used to determine HBeAg and HBsAg levels in cellular supernatants. HBV DNA levels in cellular supernatants andcells were quantified with fluorogenic quantitative PCR method; and the expression level of IFN-α in supernatants was detected with CBA method. The results indicated that single thymopolypeptides at 0.025-0.4 g•L⁻¹ had no toxicity to cells. Thymopolypeptides in this concentration range combined with 0.2 mmol•L⁻¹ matrine type alkaloids also had no toxicity to cells. Anti-HBV activity of drug combination was better than that of alkali or thymopolypeptides alone. Thymopolypeptides at 0.025 g•L⁻¹ had better inhibitory effect than thymopolypeptides at 0.1 g•L⁻¹ on intracellular HBV DNA expression, but the inhibitory effect on supernatant HBeAg level was on the contrary. Anti-HBV activity was similar between alkaloids combined with 0.1 g•L⁻¹ and alkaloids combined with 0.025 g•L⁻¹. There was no statistical difference in anti-HBV effect between various combined groups (P<0.05). In general, 72 h anti-HBV effect was better than 48 h anti-HBV effect (P<0.05). The expression of IFN-α was increased after drug combination, with positive correlation to the changes of other four indicators (P<0.05). In conclusion, oxymatrine, sophocarpidine, sophocarpine and sophoridine combined with thymopolypeptides could inhibit HBsAg and HBeAg secretion in HepG2.2.15 cells and HBV DNA replication, and further promote the antiviral effect by promoting the expression of IFN-α.

Efficacy of Compound Kushen Injection in Relieving Cancer-Related Pain: A Systematic Review and Meta-Analysis.

Despite widespread popular use of complementary and alternative medicine (CAM) therapies, a rigorous evidence based on the efficacy of compound kushen injection (CKI) for cancer-related pain is lacking. In this study, we evaluated the efficacy and safety of compound kushen injection and provided information for current or future research and clinical application. Sixteen trials were identified with a total of 1564 patients. The total pain relief rate of CKI plus chemotherapy is better than chemotherapy except for colorectal cancer. The treatment groups achieved a reduction in the incidences of leukopenia and gastrointestinal, hepatic, and renal functional lesion. However, there is paucity of multi-institutional RCTs evaluating compound kushen injection for cancer pain with adequate power, duration, and sham control. The quantity and quality of RCTs are lower so that we still have to boost the research level through scientific design and normative report.

Serum soluble CD40 is associated with liver injury in patients with chronic hepatitis B.

Soluble cluster of differentiation 40 (sCD40) is proteolytically cleaved from membrane-bound CD40 and binds to CD154, thereby inhibiting CD40-CD154-mediated immune responses. The aim of the present study was to clarify the role of sCD40 in chronic hepatitis B (CHB). The sCD40 levels in sera from 132 patients with CHB and 33 healthy individuals were retrospectively measured. sCD40 concentrations in patients with CHB were higher than those in healthy controls, and sCD40 levels correlated positively with serum levels of the liver dysfunction biomarkers alanine transaminase (ALT) and aspartate transaminase (AST). sCD40 concentrations increased with a rise in the severity of liver necroinflammation and fibrosis. Patients with >75% liver tissue staining positive for hepatitis B virus (HBV) antigen expression showed significantly lower sCD40 levels than those who stained negative for the HBV antigen. The area under the receiver operating characteristic curve of sCD40 was greater than that of ALT and AST; thus, sCD40 levels have a high diagnostic accuracy for detecting severe liver inflammation in patients with CHB, and could serve as an immunological marker of hepatic tissue injury.

[Analysis of hemagglutination inhibition antibody level in patients with influenza A H1N1].

OBJECTIVE: To understand the hemagglutination inhibition antibody level in patients with influenza A H1N1. METHODS: Sera from 28 patients with influenza A H1N1 at different time points after illness onset were collected and measured by hemagglutination inhibition assay. RESULTS: The serum hemagglutination inhibition antibody titers at 1, 5, 15, 22, 37, 49 and 58 days after illness onset were 5.36, 9.39, 39.02, 57.99, 137.92, 55.19 and 57.99 respectively. The top geometric mean titer of hemagglutination inhibition antibody was 148.55. The antibody seroconversion rate and seroprotection rate were occurred in 96.4% (27/28) of patients. CONCLUSION: The patients with influenza A H1N1 have effective immune response.

[Construction and identification of attenuated Salmonella which harboring enterovirus 71 VP1 gene].

OBJECTIVE: To develop attenuated Salmonella which harboring enterovirus 71 (EV71) VP1 gene. METHODS: The plasmid which expressed VP1 protein of EV71 was constructed by gene recombination. Cellular expression was assessed by Western Blot analysis. The recombinant plasmid was then transformed into attenuated Salmonella SL7207. RESULTS: EV71 VP1 gene sequence was inserted into a eukaryotic expression plasmid VR1012. VP1 protein was detected by Western Blot analysis in the culture supernatant. And the attenuated Salmonella harbored the plasmid stable. CONCLUSION: The plasmid was constructed successfully and it can express effectively in vitro. The bacteria which harboring the plasmid were constructed successfully. This has provided a basis for further research of an oral EV71 vaccine.

[Construction and identification of a vector inserted with gene of T7 RNA polymerase].

OBJECTIVE: To develop a system to rescue virus by intracellular expression of T7 RNA Polymerase. METHODS: The gene of T7 RNA Polymerase was amplified and cloned to VR1012 by molecular biological technology. The expression plasmid VR-1a was then identified. VR-1a and EV71 infectious plasmid were co-transfected in Vero cell. CPE was observed and viral gene viral antigen were detected. RESULTS: The gene of T7 RNA Polymerase was successfully cloned into vector VR1012. Vero cell developed to CPE after being transfected VR-1a and EV71 infectious plasmid. EV71 gene was amplified by RT-PCR from the culture. EV71 antigen was also detected by ELISA. CONCLUSION: The method can be used to rescue virus. It could apply to immunologic research of EV71 DNA vaccine.

Immunologic changes during Pandemic (H1N1) 2009, China.

We analyzed changes in immunologic values over time for 28 hospitalized patients with pandemic (H1N1) 2009. Levels of interleukin-6, interferon-y, and interleukin-10 increased 1 day after illness onset and then decreased to baseline levels. Levels of virus-specific antibody were undetectable 1 day after illness onset and peaked 36 days later.

[Construction and expression of a coxsackievirus A16 VP1 gene plasmid which delivered by live attenuated Salmonella].

AIM: To develop a coxsackievirus A16 (Cox A16) VP1 gene plasmid which delivered by live attenuated Salmonella. METHODS: The plasmid which expressed VP1 protein of CoxA16 was constructed by gene recombination. Cellular expression was assessed by Western bloten analysis. Then the recombinant attenuated Salmonella which harboring the plasmid were constructed by electro transformation. RESULTS: CoxA16 VP1 gene sequence was inserted into a eukaryotic expression plasmid. VP1 protein was detected in the culture supernatant. CONCLUSION: The plasmid is constructed successfully and it can be expressed effectively in vitro. The recombinant bacteria are constructed successfully. This has provided a basis for further research of an oral CoxA16 vaccine.

[Construction and identification of a vector inserted with complete genome of poliovirus strain Sabin I].

OBJECTIVE: To develop a vector inserted with complete genome of poliovirus strain Sabin I. METHODS: The 3 fragments of the complete genome of Sabin I was amplified and cloned to pEASY-T3 by molecular biological technology. These cloned pEASY-T3 were then digested by Restriction enzymes and ligated to pWSK29 step by step and identified. RESULTS: The complete genome of poliovirus strain Sabin I was successfully cloned into vector pWSK29 with 9 nucleotide mutations. CONCLUSION: The complete genome plasmid was constructed and it provided a basis for further research of the function of Sabin I.

[Estimation of liver inflammation and necrosis in patients with chronic hepatitis B through different biochemical items].

OBJECTIVES: To establish a new grading system to evaluate liver inflammation and necrosis in patients with chronic hepatitis B through clinical biochemical assays. METHODS: Clinical and pathological data were collected from 255 cases with chronic hepatitis B. 19 biochemical items were analyzed and 5 items were selected for our grading system. Each of the five items was scored 0 to 4 based on the different values. The extent of liver inflammation and necrosis was evaluated according to the total score. RESULTS: ALT, AST, ChE, GGT and TBA were selected for our grading system. The grade of liver inflammation and necrosis was considered less than 2.0 if total score lower than 6, and higher grade was considered with higher total score. The estimated results shared an identity of 82.8% with the real grades of liver inflammation and necrosis. When this grading system was applied to patients with liver inflammation and necrosis equal to or higher than grade 2.0, it exhibited a sensitivity of 83.8%, a specificity of 81.2%, a positive prediction value of 88.6%, a negative prediction value of 74.2% in all cases, and 88.0%, 84.7%, 90.6%, 82.4% respectively in patients older than 12 years. CONCLUSION: Our data suggest that the grading system can be used to evaluate the extent of liver inflammation and necrosis in patients with chronic hepatitis B.

[Construction and expression of hepatitis B virus envelope protein combined with core protein with two multiple cloning sites vector].

OBJECTIVE: To develop a coexpression plasmid which expressing envelope protein and nucleoprotein of hepatitis B virus and know of its expressing efficiency. METHODS: The plasmid coexpressing envelope protein and nucleoprotein of hepatitis B virus under the CMV promoter respectively was constructed by gene recombination. Cellular expression was assessed by ELISA. RESULTS: Multiple cloning site was inserted into expression vector contain hepatitis B virus PreS2-S gene. And expression unit containing hepatitis B virus PreC-C was cloned into it. HBsAg and HBeAg was detected both in the culture supernatant and in the cells. CONCLUSION: The coexpressing plasmid was constructed successfully and it can express effectively in vitro. This has provided a basis for further research of the therapeutic HBV DNA vaccine.

[Construction and antigenic evaluation of a recombinant MVA virus-like particle expressing HBV C gene].

OBJECTIVE: To construct the virus-like parcel expressing hepatitis B virus (HBV) C gene and identify its immunogenicity. METHODS: HBV C gene was cloned into the shuttle vector pSC11, and the resulted plasmid pSC11-C was transfected into modified vaccinia virus Ankara (MVA). RESULTS: pSC11-C was correctly constructed as verified by sequence analysis and PCR, and the recombinant virus-like parcel possessed good immunogenicity. CONCLUSION: The MVA-C expressing HBV C gene has been successfully constructed to provide important basis for gene therapy research of chronic HBV infection.

[Level and clinical significance of soluble CD40 in patients with chronic hepatitis B].

OBJECTIVE: To understand the level and clinical significance of soluble CD40 in patients with chronic hepatitis B. METHODS: Detecting the concentration of sCD40 from 176 cases with chronic hepatitis B by ELISA and analyzing its relationship with different grades of inflammation and necrosis in liver tissue. RESULTS: sCD40 from patients with chronic hepatitis B was significantly higher than those from healthy. And that the concentration of sCD40 was positive correlation with severe clinical disease and liver inflammation and necrosis. In patients whose ALT lower than 80 IU/L and sCD40 higher than 80 pg/ml, it showed that 65.85% cases have high grade of liver inflammation and necrosis, which was significantly higher than patients with sCD40 lower than 80 pg/ ml. CONCLUSION: The concentration of sCD40 is positively related with the grade of liver inflammation and necrosis. This information could help us to evaluate the status of chronic hepatitis B as an immunological index.

[Immune effects of mutated hepatitis B virus precore-core DNA vaccines in mice].

OBJECTIVE: To observe the immune effect of DNA vaccines encoding mutated HBV pre-c/c gene (VE2,VE4) in mice. METHODS: Three kinds of plasmid VEC(DNA vaccines encoding HBV pre-c/c gene), VE2 and VE4 were injected into the thigh muscles of different group of BALB/c mice.Blood and splenocytes from mice were isolated at 4 weeks after immunization. We also have mouse groups immunized with three of these plasmid combined with IFN-gamma gene plasmids. The anti-HBc and anti-HBe antibody in peripheral blood in mice were detected by enzyme linked immunosorbent assay (ELISA), antigen-specific cell immune responses were detected by CTL test and enzyme linked immunospot assay(ELISpot). RESULTS: We found that anti-HBe titers of VE2 and VE4 immunizing groups are higher than VEC group (P < 0.05). We also observed that VE2 and VE4 could induce stronger antigen-specific immune responses than VEC and when combined with IFN-gamma plasmid,the antigen-specific immune responses are stronger than those without combination immunization in mice (P < 0.05). CONCLUSIONS: The DNA vaccine VE2 and VE4 could induces stronger antigen-specific immune responses than VEC, and when combined with IFN-gamma plasmid,the antigen-specific immune responses are improved in mice.

[Expression, purification, and characterization of an anti-human RBC ScFv-HIV gp160 fusion protein for hemagglutination-based rapid detection of antibodies to HIV in whole blood].

OBJECTIVE: To construct and express anti-human RBC and HIVgp160 fusion protein for rapid detection of antibody to HIV. METHODS: The gene of the anti human RBC ScFv and HIV antigen were constructed together into expression vector. The fusion protein was expressed in E. coli. RESULTS: The fusion protein was proved to be able to bind both anti-RBC and HIVgp160. It could cause agglutination of human RBC when HIVgp160 was present. CONCLUSION: The fusion protein has the potential in rapid detection of HIV.

[Establishment and evaluation of the diagnostic kit for anti-HIV1/2 antibody and P24 antigen].

OBJECTIVE: To establish and evaluate an Enzyme Immunoassay diagnostic kit combined with anti-HIV1/2 antibody and P24 antigen for shortening the examination window period of HIV infection in HIV laboratory diagnosis. METHODS: The enzyme-linked reaction plates was coated by anti-HIV P24 monoclonal antibody and HIV 1/2 antigen. Labeling HIV1/2 antigen and anti-HIV P24 polyclonal antibody with horseradish peroxidase, setup an integrated ELISA kit for detecting anti-HIV-1/2 antibody and HIV P24 antigen, and evaluate the specificity and sensitivity of this kit. RESULTS: The sensitivity of testing P24 antigen was up to 0.2 ng/ml. 78 serum samples of patients with AIDS, 85 serum samples of healthy people were compared with Abbott EIA kit, the coincidence was 100%. 12 051 sera from normal persons and patients were examined, the sensitivity of 100 %and specificity of 99.62 %, respectively. CONCLUSION: The anti-HIV1/2 antibody and HIV P24 antigen can be measured at the same time using this EIA kit, while the examination window period of HIV infection is shortened. Thus, the method is suitable for laboratory diagnosis and epidemiological investigation.