Angiotensin (1-7) inhibits angiotensin II-stimulated expression of connective tissue growth factor mRNA in hepatic stellate cells / 中华肝脏病杂志

To explore the angiotensin peptide [Ang (1-7)]-mediated inhibition of Ang II in human hepatic stellate cells (HSCs) and determine the involvement of the ACE2-Ang (1-7)-Mas axis. The human HSC line, LX2, was used in all experiments, and divided into control (unstimulated) and Ang II-stimulated (10-6 mol/L) groups. The Ang II-stimulated cells were further divided among several pre-treatment (prior to Ang II) groups: ROCK-inhibited (Y27632 blocking agent, 10-6 mol/L); irbesartan-inhibited (AT-1 receptor antagonist, 10-6 mol/L); and Mas receptor-inhibited (A779 Mas receptor antagonist, 10-6 mol/L). To explore the potential inhibitory effects of various Ang family members, the Ang II-stimulated and pre-treated LX2 cells were exposed to Ang (1-7) (10-6 mol/L) for 24 h. Western blot, reverse transcription-polymerase chain reaction (RT-PCR), and QuantiGene assay were used to assess changes in protein and mRNA expression levels of RhoA, ROCK, and connective tissue growth factor (CTGF). Compared with the control group, Ang II-stimulated cells showed significantly increased levels of RhoA protein (0.337+/-0.074 vs. 0.870+/-0.093), ROCK2 mRNA (0.747+/-0.061 vs. 0.368+/-0.023), and CTGF mRNA (0.262+/-0.007 vs. 0.578+/-0.028) (all, P less than 0.01). Pre-treatment with irbesartan or Y27632 eliminated these responses. Ang (1-7) inhibited the Ang II-stimulated up-regulation of RhoA, ROCK, and CTGF. Ang (1-7) can inhibit the Ang II-stimulated up-regulation of RhoA, ROCK and CTGF in hepatic stellate cells, indicating that the ACE2-Ang (1-7)-Mas axis, an important branch of the renin-Ang-aldosterone system is involved in the occurrence and development of liver fibrosis.

Effect of angiotensin1-7 on alpha-smooth muscle actin protein expression in rat hepatic stellate cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effect of angiotensin II and angiotensin1-7 on alpha-smooth muscle actin (alpha-SMA)-induced Ca(2+)-independent pathways mediated by Rho kinase2 in hepatic stellate cells (HSCs).</p><p><b>METHODS</b>HSC-T6 cells were treated with 10 micromol/L of AngII, Ang1-7, AngII +Ang1-7, and Ang1-7+A779. RT-PCR was used to detect the expression of Rho kinase2 (Rock2) in Ca(2+)-independent pathways, and alpha-SMA protein expression was detected by Western blotting.</p><p><b>RESULTS</b>The mRNA expression of Rock2 increased significantly in the cells after AngII treatment (P<0.01), but decreased following Ang1-7 treatment. Ang1-7 treatment significantly reduced alpha-SMA level in AngII-induced cells (P<0.01).</p><p><b>CONCLUSION</b>Ang1-7 can inhibit AngII-induced activation of Rock2 and reduce alpha-SMA expression in HSCs.</p>

Angiotensin II stimulates platelet-derived growth factor-B expression in hepatic stellate cells by activating EGR-1 / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the signal transduction mechanism underlying the effects of angiotensin II (AngII) on extracellular signal-regulated kinase 1/2 (ERK1/2), early growth response-1 (EGR-1) and platelet-derived growth factor-B (PDGF-B) in hepatic stellate cells (HSCs).</p><p><b>METHODS</b>HSC-T6 cells treated with AngII for 10 or 30 min were examined for phospho-P42/44 protein expression using Western blotting. In another experiment, the cells were preincubated for 1 h in the presence of U0126 (an inhibitor of the MAPK/ERK kinase), irbesartan (an AT-1 receptor blocker), or antioxidant-N-acetylcysteine (NAC) prior to AngII exposure, and the protein expression of phospho-P42/44 and PDGF-B were measured with Western blotting. The DNA binding activity of EGR-1 was analyzed using electrophoretic gel mobility shift assay (EMSA), and the expression of PDGF-B was detected immunohistochemically.</p><p><b>RESULTS</b>AngII induced phospho-P42/44 expression in HSC-T6, which was abrogated by U0126 or irbesartan. NAC did not inhibit phospho-P42/44 expression. EMSA showed that AngII exposure of the HSC cells markedly increased EGR-1 DNA binding activity, reaching the maximum after 60 min of exposure followed by progressive declination; irbesartan and U0126 significantly suppressed AngII-induced EGR-1 activity enhancement. ACEI at 1 micromol/L and 10 nmol/L inhibited EGR-1 activity, but ACEI at the concentration of 0.1 nmol/L resulted in enhanced EGR-1 activity. NAC showed no obvious effect in suppressing EGR-1 activity. AngII increased PDGF-B protein level in the HSCs, the effect of which was inhibited by irbesartan. U0126, NAC and ACEI did not attenuate PDGF-BB protein level in the HSCs.</p><p><b>CONCLUSION</b>Stimulation of the HSCs with AngII results in EGR-1 activation via the ERK1/2 pathway, leading to up-regulation of PDGF-B expression.</p>

Effect of angiotensin II on Rho-Rock pathway in rat hepatic stellate cell contraction / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the mechanisms of angiotonin II (AngII)-induced Ca(2+)-independent pathways mediated by Rho kinase in hepatic stellate cells (HSCs).</p><p><b>METHODS</b>HSC-T6 cells were treated with 1 micromol/L of AngII, and the subsequent cell contraction was directly observed with silicone rubber membrane culture method. The cells with 10 micromol/L AngII treatment were examined for myosin light chain (MLC) phosphorylation level using Western blotting, and the effects of irbesartan (a specific inhibitor of AngII 1- receptor) and Y27632 (a Rho kinase inhibitor) on AngII-induced MLC phosphorylation were evaluated. RT-PCR was used to detect the expression of Rock2 in Ca(2+)- independent pathways mediated by Rho kinase.</p><p><b>RESULTS</b>AngII induced HSC contraction and time-dependent MLC phosphorylation changes, which peaked 15 min after the treatment followed by gradual reduction. Irbesartan or Y27632 treatment significantly lowered MLC phosphorylation level in AngII-induced cells (P<0.01). The mRNA expression of Rock2 increased significantly after AngII treatment (P<0.01), but decreased following subsequent irbesartan or Y27632 treatment.</p><p><b>CONCLUSION</b>AngII induces HSC contraction through Ca(2+)-independent pathways mediated by Rho kinase.</p>

A preliminary investigation of nuclear factor kappa B in peripheral blood lymphocytes of patients with hepatitis B / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To explore the activity of nuclear factor kappa B (NF-kappaB) in peripheral blood lymphocytes (PBL) of patients with hepatitis B.</p><p><b>METHODS</b>The PBL of patients with different types of hepatitis B and healthy individuals were isolated and then the nuclear extract was prepared. Assessment of NF-kappaB DNA binding activity was performed by electrophoretic mobility shift assay (EMSA) using digoxin labeled double-stranded oligonucleotide containing kappa B consensus sequence.</p><p><b>RESULTS</b>Densitometric scanning of the EMSA bands showed that the mean optical densities (A) from the groups of normal control, acute hepatitis B, chronic hepatitis B and chronic severe hepatitis B were 20.18+/-2.16, 27.75+/-4.11, 13.90+/-3.20 and 8.02+/-2.65 respectively. Analysis of variance showed the F value was 26.112 and the difference among the groups was significant. The difference between two groups with Dunnett T3 analysis showed there are statistically difference between the groups of the normal group and the chronic severe hepatitis B group, the acute hepatitis B group and the chronic hepatitis B group, and the acute hepatitis B group and the chronic severe hepatitis B.</p><p><b>CONCLUSION</b>The activity of NF-kappaB in PBL of patients with hepatitis B is related with the different outcomes of hepatitis B virus (HBV) infection. Decreased activity of NF-kappaB may be an important cause for the dysfunction of PBL in chronic HBV-infected patients.</p>