| It has been known that renin-angiotensin-aldosterone system (RAAS) plays a key role in the fibrogenesis of local tissue. Angiotensin II (Ang II) and Aldosterone (Aldo), the principal effector molecules of the RAAS, exert local effects on cell growth and fibrogenesis. In recent years, much attention has been focused on the novel relationship between activation of RAAS and fibrosis of liver. Ang II can stimulate hepatic stellate cell (HSC) and increase the production of extracelluar matrix (ECM) by interaction with Angiotensin II type1 receptor (AT-1 receptor), leading to hepatic fibrosis. However, the singnal transduction mechanisms underlying effects of AngIIand Aldo on hepatic fibrogenesis remain fully be elucidated. There are a few reports about the investigation of this field can be found so far.The present study aims to investigate the signal transduction mechanism underlying effects of Ang II and Aldo on the signal passageway of NF-K. AP-1 and EGR-1 in HSC. In addition, the current study was undertaken to confirm the existence of AT-1 receptor in Kupffer cell.In vivo, the model of hepatic fibrosis was established by subcutaneously injecting rats with CCl4. Simultaneously, the rats were administered with perindopril and losartan. After 4,6 weeks, morphological examination was based on microscopy. The protein expressions of AT-1-5-receptor, TGF-pl and PDGF-BB in liver tissue were examined by Western blot. The activity of matrix metalloproteinase-2, 9 (MMP-2,9) was assessed by zymography. Serum laminin (LN) and hyaluronic acid (HA) were measured by using radioimmunoassays. Electrophoretic gel mobility shift assay ( EMS A ) was utilized to detect NF-KB DNA binding activity in liver tissues. The protein expression of IicBa was examined by Western blot. Apoptosis of hepatocyte was examined by TUNEL, and apoptosis of HSC was detected by dual staining for TUNEL and a-SMA. The results show that there was a up-regulation in AT-1 receptor expression in model group compared with control group. Both of perindopril and losartan treatment significantly reduced mean fibrosis score; protein levels of ATI receptor, TGF-pl and PDGF-BB; DNA binding activity of NF-icB; serum levels of HA and LN; and MMP-2,9 activity. Perindopril and losartan treatment increased protein expression of IicBa. More over, perindopril and losartan treatment reduced apoptosis index of hepatocyte, and have no change on apoptosis index of HSC compared with model group.In vitro, HSCs-T6 cell line were preincubated for 1 h or not with UO126 (an inhibitor of the MAPK/ERK kinase MEK), irbesartan (an AT-1 receptor blocker) and JV-acetylcysteine (anantioxidant) prior to exposure to Angllor Aldo for the indicated times.Protein expression of Phospho-P42/44, IxBa, and PDGF-BB were measure by Western blot. DNA biding activity of NF-KB, AP-1 and EGR-1 were analyzed by EMS A. By means of RT-PCR, expression of TNFamRNA and al ( I) procollagen mRNA were detected. Protein expression of NF-icB p65, COX-2 and PDGF-BB were detected by immunohistochemistry in HSC. AT-1 receptor was confirmed in Kupffer cell by immunohistochemistry.These results showed: l.Anglland Aldo stimulate HSC via extracellular signal-regulated kinase (ERK) pathway. Time course experiments showed that Anglland Aldo induced Phospho-P42/44 expression, which can be abrogated by U0126 or irbesartan, reaching a maximum at 10 minutes, and then declined progressively. ACEI and NAC can not inhibit Phospho-P42/44 expression.2. Gel shift and supershift studies showed that stimulation of HSC by Ang II and Aldo increased NF-KB p65 DNA binding activity, which can be inhibited by irbesartan and ACEI. NAC markedly attenuated NF-KB DNA binding activity induced by Ang II, and partly decreased it induced by Aldo. U0126 could not inhibit NF-KB activity.More over, Ang and Aldo attenuated IicBa level in cytoplasm of HSC, which can be increased by irbesartan, ACEI and NAC.On the contrary, AngII and Aldo up-regulated TNFa mRNA expression and COX-2 protein level; TNFa can be inhibited by irbesartan, ACEI and NAC...
|
PDF Full Text Request