| PartⅠProtective effects of Angiotensin (1-7) on5/6nephrectomized ratsObjective:ACE2-Angiotensin (1-7)-Mas axis have characteristics on antagonizing Rennin-Angiotensin system, anti-proliferation and anti-fibrosis. This study aimed to observe the effects of Angiotensin (1-7) or combined with ARB on5/6nephrectomized rats.Methods:1. Male Sprague-Dawley rats at the beginning of the experiments underwent sham and5/6nephrectomy. Groups:1) Sham (n=6);2)5/6nephrectomy(Nx, n=6);3) Angiotensin (1-7)(Ang (1-7),24μg/kg/h, subcutaneously, n=6);4) Losartan (10mg/kg/d, intragastric administration, n=6);5) Ang (1-7) combined with Losartan (A+L, n=6). Systolic blood pressure and serum creatinine was tested8weeks later, and24h urine samples were collected every4weeks. The levels of plasma Ang Ⅱand Ang (1-7) were measured using RIA.2. Phathological changes were observed by PAS staining; the expression of fibrosis index, such as fibronectin, Collagen Ⅰ, were tested by immuohistochemistric staining; The expression of ROS production was measured by DCFDA.3. The gene expression of Mas and PAI-1were detected by Real-time PCR while the protein expression of FN, Angiopoietins-Tie-2, p47-phox and p67-phox were observed by western blot.Results:Compared with Nx group, the serum creatinine, proteinuria and fibrosis index, such as FN, PAI-1and Col-1were ameliorated, the plasma levels of Ang (1-7) were upregulated, the expression of ROS production and the activity of NADPH oxidase were downregulated in these three goups of treatment. The ratio of Ang-1and Ang-2have no significant difference in the groups of Ang (1-7) and Losartan compared with the group of Sham, wheras Ang-1/Ang-2was significantly upregulated in group of A+L.Conclusion:Ang (1-7) ameliorate the renal injury of rats underwent5/6nephrectomy through resistance of Ang Ⅱ, the combination of Ang (1-7) with Losartan exert better protective effects on postponing chronic kidney diseases. Part ⅡThe effects of Angiotensin (1-7) on Ang Ⅱ-induced fibronectin production in mouse mesangial cellsObjective:It’s uncertainty that Ang (1-7) exerts anti-fibrosis effect in glomerular, such as mesangial cells. This study aimed to observe the effects of Ang (1-7) on Ang Ⅱ-induced fibronectin production in mouse mesangial cells (MMCs).Methods:1. MMCs were treated with Ang (1-7) after induced by Ang Ⅱ, or combined with Losartan without Ang Ⅱ induced. The expression of FN was evaluated by Western blot2. MMCs cultured to50%confluence in six-well plates were transiently transfected with500nM Mas siRNA or Vehicle siRNA. After24h, MMCs were treated with Ang (1-7), Ang Ⅱ and DX-600, the expression of FN was detected by Western blot and Real-time PCR.3. After MMCs were transfected with500nM Mas siRNA or Vehicle siRNA, the expressions of ATI and Mas were detected and the interaction of them was evaluated by Co-immunoprecipitation and Western blot.Results:1. There’s no significant difference in expression of FN when the MMCs were treated with Ang (1-7) after induced by Ang Ⅱ compare with induced by Ang Ⅱ only.2. The expression of FN was significantly upregulated when cells were transfected with Mas siRNA or treated with DX-600(P<0.05).3. The expression of FN was remarkably downregulated when cells were treated with Ang (1-7) combined with Losartan(P<0.05).4. Compound was formed between AT1receptor and Mas receptor.Conclusion:There’s no significant difference in expression of FN when the MMCs were treated with Ang (1-7) after induced by Ang Ⅱ compare with induced by Ang Ⅱ only, it’s probably related to the compound of AT1-Mas. Part IIIThe effects of Angiotensin (1-7) on glomerular angiopoietins-Tie-2system and its potential mechanismObjective:Angiopoietins-Tie-2is a specific angiogenesis system in glomerular cells, such as endothelial cells and podocytes. Though Ang (1-7) inhibits endothelial cell tub formation in vitro in human umbilical vein endothelial cells, there’s no report in glomerlar angiogensis inhibition. This study aimed to observe the effects of Ang (1-7) on glomerular angiopoietins-Tie-2system and its potential mechanism.Methods:1. The mouse glomerular endothelial cells (MGECs) were cultured. The ability of endothelial cells tube formation was observed by Matrigel.2. The MGECs and podocytes were grown. The gene expressions of Ang-1in podocytes and Ang-2in MGECs treated with different dose of Ang (1-7) and Ang Ⅱ were detected by Real-time PCR.3. The MGECs and podocytes were grown. In the condition with/without exogenous Ang Ⅱ, these cells were treated with Ang (1-7), Losartan, A-779and PD98059. The expressions of Ang-1, Ang-2and Tie-2were detected by Western blot, and the levels of phosphorylate eNOS and ERK1/2were tested either.Results:1. In the condition without exogenous Ang Ⅱ, the synergism of Losartan with Ang (1-7) was existed in inhibition the expressions of aniopoietins-Tie-2, while in the condition with exogenous Ang II, the expressions of angiopoietin-Tie-2were upregulated significantly when the cells were treated with Ang (1-7)(P<0.05). However, the inhibitory action was reversed by Losartan, A-779and PD98059(P <0.05).2. The eNOS pathway was activated by Ang (1-7) itself(P<0.05). However, in the condition with exogenous Ang Ⅱ, the phosphorylate eNOS was downregulated wereas the phosphorylated ERK1/2was upregulated, but this elevation was reversed by L-NAME(P<0.05).Conclusion:Ang (1-7) inhibited angiogenesis itself, and resisted the effect of growth promotion of Ang Ⅱ. In the condition with exogenous Ang Ⅱ, Ang (1-7) inhibited the expression of angiopoietins-Tie-2remarkably via downregulating eNOS and then activating ERK1/2pathway, those effects were reversed by Losartan. |
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