PubMed2005
OBJECTIVE: To investigate the signal transduction mechanism underlying the effects of angiotensin II (AngII) and aldosterone (Aldo) on nuclear factor-kappaB (NF-kappaB) DNA binding activity. METHODS: Sixty male Wistar rats were randomly divided into 4 groups: model group (Mo group), injected with CCl(4) subcutaneously twice a week to establish a model of hepatic fibrosis; perindopril group (Pe group), injected with CCl(4) subcutaneously twice a week and perfused with perindopril once a day; losartan group (Lo group), injected with CCl(4) subcutaneously twice a week and perfused with losartan once a day; and control group (Nc group), injected with olive oil subcutaneously. The rats were killed in batches respectively 4 and 6 weeks after and their livers were collected to undergo Masson staining and be observed by light microscope. Electrophoretic gel mobility shift assay (EMSA) was used to detect the NF-kappaB DNA binding activity in the liver tissues. Western blotting was used to detect the expression of IkappaBalpha in the plasma protein. Hepatic stellate cells (HSCs)-T6 were cultured and preincubated for 1 h or not with U0126 (an inhibitor of MAPK/ERK kinase MEK), irbesartan (an AT-1 receptor blocker), and N-acetylcysteine (NAC, an antioxidant), angiotensin-converting enzyme inhibitor (ACEI), or tumor necrosis factor alpha (TNFalpha) prior to exposure to AngII or Aldo for 0.5 h, 1 h, 2 h, and 4 h respectively. The binding activities of NF-kappaB DNA were observed by EMSA. The expression of IkappaBalpha protein was detected with Western blotting. Histochemistry was used to detect the expression of NF-kappaB p65. RT-PCR was used to detect the expression of TNFalpha mRNA in HSC-T6 cells. RESULTS: The binding activity to NF-kappaB of the liver tissues was the strongest in the Mo group, followed by the Pe and Lo groups and Nc group. The IkappaBalpha expressions in liver tissues 4 and 6 weeks after the beginning of experiment in the Pe and Lo groups were significantly stronger than that in the Mo group (both P < 0.05). 0.5 hour after the intervention of AngII the DNA binding activity of the HSCs began to increase and peaked 1 hour later and then gradually decreased. The increase of NF-kappaB activity induced by AngII could be inhibited by irbesartan, ACEI and NAC pretreatment and could not be inhibited by U0126 pretreatment. Combined action of AngII and TNFalpha significantly increased the NF-kappaB DNA binding activity. The IkappaBalpha expression began to decrease 0.5 hour after the intervention of AngII and reached the lowest value 2 hours after. The expression of IkappaBalpha protein was increased by ACEI (P < 0.05), irbesartan and NAC (both P < 0.01). EMSA showed that 0.5 hour after the intervention of Aldo the DNA binding activity began to be increased and peaked by 1 hour and then began to be decreased. NAC, but not U0126 partly inhibited the increased of NF-kappaB activity induced by Aldo. Combined action of Aldo and TNFalpha significantly increased the NF-kappaB activity. Aldo increased the expression of IkappaBalpha protein in the HSCs at different time points (all P < 0.05). 0.5 hour after the AngII intervention the IkappaBalpha protein expression began to decrease and reach the lowest value 1 hour later and then began to increase 2 hours later. the IkappaBalpha protein expression was significantly decreased in the NAC and NAC+ Aldo intervention groups (both P < 0.05). There was no significant difference in IkappaBalpha protein expression between the Aldo intervention group and U0126 + Aldo, TNFalpha, and Aldo + TNFalpha treatment groups (all P > 0.05). Before stimulation, NF-kappaB was expressed in the plasma of HSCs, however, after the stimulation of AngII or Aldo for 1 hour it was expressed in the nuclei, and then transferred from the nuclei to the plasma 4 hours after the stimulation. However, little nuclear transfer was observed after pretreatment of NAC followed by AngII or Aldo intervention. The TNFalpha mRNA expression was significantly increased in the AngII and Aldo treatment groups in comparison with the control group (both P < 0.05). The TNFalpha mRNA expression was significantly weaker in the irbesartan + AngII, NAC + AngII, and ACEI groups in comparison with the AngII group (all P < 0.05). CONCLUSION: Stimulation of NF-kappaB activity mediates hepatic fibrosis induced by intrahepatic renin-angiotensin-aldosterone system (RAAS).