Growth induction of hepatic stimulator substance in hepatocytes through its regulation on EGF receptors

The cytosolic liver-specific growth factor-hepatic stimulator substance (HSS) has been shown to be able to amplify the rat hepatocyte proliferation responded to EGF. In order to get more insight into the mechanism, the regulatory effect of HSS on EGF-receptor(EGF-R) and the receptor phosphorylation at molecular level was studied. HSS partially purified from weanling rat liver was given to cultured hepatocytes and its influence on EGF-R specific binding and internalization as well as mRNA expression were investigated. The results showed that preincubation of hepatocytes with HSS could lead to an increase in [125I]-EGF binding to its receptors and inhibit EGFinduced receptor down-regulation. Furthermore, the overexpression of EGF-R mRNA stimulated by HSS was seen during 2-12 h after the incubation. Additionally, it was demonstrated with human hepatoma sMMC-7721 cells in Western blot that the EGF-R expression and the receptor autophosphorylation were increased with dose/timedependency after HSS treatment. These results strongly suggest that the mechanism of HSS action on hepatocyte growth might be related to its modulation on EGF-R and receptor-mediated signaling transduction.

QUANTITATIVE ANALYSIS ON CHANGES OF HEPATIC HEMODYNAMICS AFTER TRANSCUTANEOUS ELECTRIC PULSE STIMULATION OF LOCAL POINTS BY USING COLOR DOPPLER ULTRASOUND DIAGNOSTIC APPARATUS

Objective: To evaluate the effect of transcutaneous electric pulse stimulation (TEPS) on hepatic blood flow and parenchymal microcirculation in patients with fatty liver. Methods: A total of 31 fatty liver volunteer patients were observed in this study. Changes of color Doppler energy (CDE) images before and after TEPS of local points nearby the liver were recorded by using color Doppler ultrasound diagnostic apparatus (ACUSON 128XP/10C). Sum of color pixel area (SCPA), average of color value (ACV) and SCPA×ACV (integral) of the hepatic flow images were analyzed by an image processing system, single blind method and paired t-test. Programmed TEPS (0.5- 150 Hz / 2 000 Hz , 10- 25 V ) was applied to the right Qimen (期门 LR 14)-Jingmen (京门 GB 25), Fuai (腹哀 SP 16)-Ganshu (肝俞 BL 18) respectively for 15 min. Results: Compared with basic values of pretreatment, SCPA, ACV and SCPA×ACV increased significantly (t=2.71, P<0.02; t=3.42, P<0.01; and t=8.15, P<0.001) after TEPS, meaning improvement of hepatic blood flow supply. Conclusion: TEPS of acupoints near the liver can improve hepatic blood flow and hepatic parenchymal microcirculation in patients with fatty liver.

Insights on augmenter of liver regeneration cloning and function

Hepatic stimulator substance （HSS） has been referred to as a liver-specific but species non-specific growth factor. Gradient purification and sequence analysis of HSS protein indicated that it contained the augmenter of liver regeneration （ALR）, also known as hepatopoietin （HPO）. ALR, acting as a hepatotrophic growth factor, specifically stimulated proliferation of cultured hepatocytes as well as hepatoma cells in vitro, promoted liver regeneration and recovery of damaged hepatocytes and rescued acute hepatic failure in vivo. ALR belongs to the new Erv1/Alr protein family, members of which are found in lower and higher eukaryotes from yeast to man and even in some double-stranded DNA viruses. The present review article focuses on the molecular biology of ALR, examining the ALR gene and its expression from yeast to man and the biological function of ALR protein. ALR protein seems to be non-liver-specific as was previously believed, increasing the necessity to extend research on mammalian ALR protein in different tissues, organs and developmental stages in conditions of normal and abnormal cellular growth.

ERK1/2 contributes negative regulation to STAT3 activity in HSS-transfected HepG2 cells

Signal transducer and activator of transcription 3 (STAT3) is a recently characterized transcription factor which is essential to liver regeneration. We have previously reported that hepatic stimulator substance (HSS), a novel growthpromoting substance, phosphorylated the epidermal growth factor (EGF) receptors and activated downstream RasMAP kinase (extracellular signal-regulated kinases, ERK1/2) cascade. However, whether HSS signal is related to STAT3pathway remains unclear. The present study is aiming to explore the regulatory effect of activation of ERK1/2 evoked by HSS on STAT3 phosphorylation and STAT3 signaling. Human hepatoma cell line HepG2 was stably transfected with HSS cDNA and HSS expression was measured by Northern blot. The results showed that the transfection of HSS into HepG2 resulted in remarkable increase in cellular proliferation as compared with the non-transfected cells, and it was further proved that the cellular proliferation in the HSS-transfected cells was related to ERK1/2 activation. Treatment of the cells with 50 μM of PD98059, an ERK1/2 specific upstream inhibitor, resulted in ERK1/2 inactivation completely.Inhibition of ERK1/2 allowed the tyrosine of STAT3 to be phosphorylated in a dose-dependent manner to PD98059.Furthermore, transient transfection of STAT3 mutant (STAT3S727A) into HSS-bearing cells could remarkably reverse the inhibitory effect of ERK1/2 on STAT3 phosphorylation. Based upon these results, it is concluded that ERK1/2negatively modulates STAT3 phosphorylation and this function is dependent on residual serine-727 (S727) of STAT3.

Verification and Improvement of the MTT Method for in vitro HSS Bioactivity Activity Determination

To optimize the experimental conditions of MTT colorimetric assay for HSS bioactivity in vitro,we studied the optimal combination of the major conditions of the MTT assay by orthogonal test and other experiments,and compared HSS bioactivity in vitro measured by the improved MTT protocol and published MTT assay at serial protein doses.Results showed that the absorbance value(A value)of the MTT assay directly correlated with the number of human hepatoma cell lines SMMC7721.The result of orthogonal test was the number of 5×104 SMMC7721 cells/ml,culture period 6 h before adding HSS,concentration of HSS 100μg/ml,incubation time with HSS 36 h.Additionally,several experiments demonstrated the optimal combination of other conditions was 50μg MTT,incubation time for MTT 6 h,DMSO was used to dissolve the MTT formazan crystals and measured with ELISA scanner at 570 nm.The result of determining HSS bio-activity in vitro by optimized MTT protocol showed that sHSS bio-activity increased with the growth of protein dose,but decreased when it beyond a certain dose.The optimized MTT protocol was a sensitive,convenient and stable quantitative method to evaluate HSS bio-activity.

A pilot study on the combined therapy of granulocyte-macrophage colony-stimulating factor and hepatitis B vaccine on chronic hepatitis B virus carrier children

Objective To observe the efficacy of treating intrauterine infected chronic hepatitis B virus (HBV) carrier children with a combination of granulocyte macrophage colony stimulating factor (GM CSF) or hepatitis B immunoglobulin (HBIG) plus recombinant hepatitis B vaccine (rHBvac) Methods A total of 27 chronic HBV infected children, who were born to HBV carrier mothers and received hepatitis B immunoprophylaxis at birth, were randomized into 2 groups: one receiving a combined therapy of 50 μg of GM CSF plus 10 μg of rHBvac injected intramuscularly at the same location (GM CSF group, 14 children) or 200 IU HBIG and 10 μg rHBvac in different muscles (HBIG group, 13 children) on a monthly four dose schedule HBV DNA quantification and other HBV serological markers were tested before and after the four dose therapy Results Twelve children in each group completed the study Of them, 3 children in the GM CSF group and 4 in the HBIG group had elevated serum alanine transaminase (ALT) before the trial, and then 2 in each group became ALT normal after the treatment Before the therapy, hepatitis B e antigen (HBeAg) positivity was found in nine children in the GM CSF group and 10 in the HBIG group One from each group had an HBeAg/anti HBe seroconversion after the treatment The quantity of HBV DNA was significantly lower after the treatment ( P =0 023) in GM CSF group, but was not significantly reduced in HBIG group No subjects were found to be negative for hepatitis B surface antigen (HBsAg) after the treatment, and no serious adverse events occurred in either group Conclusion Combined GM CSF and rHBvac therapy inhibit HBV replication in carrier children who were not protected after treatment with immunoprophylaxis