Dissertation
Dissertation > Medicine, health > Internal Medicine > Digestive and abdominal diseases > Liver and gall bladder disease > Cirrhosis

Mechanism of the Preventive Effect in Liver Fibrosis of Rats after Local Blockage of Renin-angiptensin-aldosterone System at Different Levels

Author JiaoZhanJiang
Tutor ZhouJie
School Southern Medical University,
Course Hepatobiliary Surgery
Keywords Liver fibrosis renin-angiotensin-aldosterone system transforminggrowth factor connective tissue growth factor tumor necrosis factor-α AngiotensinⅡ Angiotensinl-7
CLC R575.2
Type PhD thesis
Year 2013
Downloads 172
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Background and objectveLiver fibrosis is a common pathological change of several chronic liver diseases, it always exists in these chronic liver disease and liver damage which is caused by a variety of pathogens. Liver fibrosis is a necessary procedure when several liver diseases develop into cirrhosis and it is also a risk factor of hepatocellular carcinoma. In these liver fibrosis patients about25%-40%of them will develop into cirrhosis or even cancer. Therefore, the liver fibrosis which as a worldwide disease is serious for human health. Because the liver fibrosis is a dynamic and reversible pathological process. So it will have comprehensive economic value and social effect along with applications prospect for us to make depth research of the pathogenesis of liver fibrosis, look for an effective therapeutic targets and reverse liver fibrosis actively and effectively in the prevention and treatment of cirrhosis and liver cancer.As an endocrine system, Renin-Angiotensin-Aldosterone System(RAAS) has a comprehensive biological effect and works on several organ. RAAS plays an important role in liver fibrosis. In addition to the circulatory system, RAAS exists in many organs, such as heart, kidney, lung and pancreas; and it also takes part in tissue fibrosis and/or the reconstruction process. RAAS still exists in the some part of liver, participating in the development of liver fibrosis by paracrine and autocrine effect. In the past, as the classic RAAS axis, ACE-Ang Ⅱ-AT1receptor axis is the only system of biological relevance which can promote liver fibrosis. Recent study found that ACE2-Ang (1-7)-Mas receptor axis, namely the RAAS role axis, was the reverse adjustment shaft of ACE-AngⅡ-AT1receptor axis and could prevent from liver fibrosis. Ang Ⅱ, as the main functional components of the RAAS, play an important role in promoting the development of liver fibrosis; whereas ACE2can split AngⅡ into Ang1-7which has a negative regulatory effect for AngⅡ and protective effect on liver fibrosis.Cytokines plays a key role in the process of liver fibrosis. They are a collection of proteins or small molecule peptides which have immunomodulatory and effector functions along with information-transition between cells. They play a local biological effect through acting on target receptors with paracrine or autocrine effect. In the development of liver fibrosis, TGF-β1, CTGF and TNF-α have an unbalance expression, which has a dramatic significance in the occurrence and development of liver fibrosis.In the past10years, the angiotensin-converting enzyme inhibitors, angiotensin Ⅱ receptor blockers and aldosterone receptor antagonists which are all included in RAAS blockers were confirmed by a lot of evidence-based medicine in the field of cardiovascular disease, diabetes and kidney disease. Whereas the information that RAAS blockers applied in liver fibrosis were mainly from retrospective study, non-control prospective study or treatment experience of other diseases which have the same pathogenesis, therefore the patients without cardiovascular disease, diabetes and kidney disease were still not recommended to take. In recent years, the potential of RAAS blockers in the treatment of liver fibrosis was gradually being confirmed by experiments.Based on the preliminary studies, this research induced rat liver fibrosis model using CCL4and selected the angiotensin-converting enzyme inhibitor(Captopril), angiotensin II receptor blocker (Losartan) and aldosterone receptor antagonist(Spironolactone) to work different part of the RAAS respectively; then we observed the histopathology change of liver, the influence of HA, LN, PCIII and CIV in serum, ACE2, AngⅡ, Ang-(1-7) as RAAS components and some cytokines such as TGF-β1, CTGF and TNF-a in the liver tissue to find out which link of RAAS we intervened can prevent rats from liver fibrosis best, and to explore the possible mechanism which can provide experimental evidences for the prevention and treatment of liver fibrosis.MethodLiver fibrosis was induced by intraperitoneal injection of chronic carbon tetrachloride in rats. Fifty SD rats aged6weeks were randomized into five groups, normal control group, model group, captopril group, losartan group and spironolactone group, with10rats in each. Except rats in the normal control group, all were received the first intraperitoneal injection of40%CCl4(6ml/kg), then the second intraperitoneal injection of40%CCl4(3ml/kg, injection once every three days).The rats in the normal control group received subcutaneous injection of the same dosage of olive oil. Next day, rats of captopril group were given captopril (60mg·kg-1·d-1), rats of losartan group were given losartan (10mg·kg-1·d-1), rats of captopril group were given captopril (60mg·kg-1-·-1), rats of spironolactone group were given spironolactone (100mg·kg-1·d-1) by gastric canal, all drugs diluted with10ml of saline. The normal control group and model group received the same saline everyday. Each group was sacrificed at the end of8weeks, blood samples and liver tissue were collected. Histopathological study of liver tissue was done with hematoxylin-eosin staining masson staining. Enzyme-linked immuno sorbent assay was used to determine concentration of Hyaluronidase (HA), Laminin (LN) Collagen Type Ⅳ(CⅣ), Procollagen Ⅲ (PCⅢ), ACE2, AngⅡ, Ang(1-7) in serum and the concentration of ACE2, AngⅡ, Ang(1-7) in liver tissue, the expression of transforming growth factor β1, connective tissue growth factor and Tumor necrosis factor-a were assessed by realtime-PCR and western blot analyses.Statistical methodApply the statisticals soft system of SPSS for windows to analyze the materials, the measurement data indicated by the mean value and standard deviation. Different between treatment group compares the single factor analysis of variance (ANOVA) variance when using multiple comparison together-t method inspection, LSD variance not neat, the use of approximate variance analysis, multiple comparison Welch method using Dunnett T3method inspection. P<0.05for with a statistical significance.Result1. The body weight of ratsOne-way ANOVA shows, the body weight of each groups is significant difference (F=50.649, P=0.000). Multiple comparison between groups, model group, captopril group, losartan group and spironolactone group compared with normal control group show the significant difference (P<0.05). Captopril group, losartan group and spironolactone group compared with model group show the significant difference (P<0.05). Losartan group and spironolactone group compared with captopril group show no significant difference (P>0.05)2. The liver weight of ratsOne-way ANOVA shows, the liver weight of each groups is significant difference(F=36.137, P=0.000). Multiple comparison between groups, normal control group, captopril group, losartan group and spironolactone group compared with model group show the significant difference (P<0.05). Captopril group, losartan group and spironolactone group compared with normal control group show no significant difference (P>0.05). Losartan group and spironolactone group compared with captopril group show no significant difference (P>0.05).3. The result of hepatic fibrosis evaluated by hematoxylin-eosin stainingLight microscope to observe the pathological changes of the liver tissue, normal lobular architecture can be seen in the normal group, the liver cells to the central venous radially arranged around the center, while fibroblasts rarely seen. Model group show liver cell cord arranged in a disordered, hepatocyte steatosis appears enlarged portal area, the pseudolobules can be seen. Compared with the model group, captopril group, losartan group, spironolactone group periportal area slightly expanded, no pseudolobules.4. The result of hepatic fibrosis evaluated by masson stainingExpression of a small amount of collagen fibers in the perivascular in the normal group; the model group show a large number of collagen fibers, pseudolobules can be seen; compared with the model group, captopril group, losartan group, spironolactone group show collagen fiber significantly reduced, no pseudolobules.5. The results of semi-quantitative scoring of liver fibrosis of ratsThe results of semi-quantitative scoring of liver fibrosis of each groups is significant difference(welch=249.665, P=0.000). Multiple comparison between groups, model group, captopril group, losartan group and spironolactone group compared with normal control group show the significant difference(P=0.000、0.000、0.000、0.000). Captopril group, losartan group and spironolactone group compared with model group show the significant difference (P=0.000、0.000、0.000). Losartan group and spironolactone group compared with captopril group show no significant difference (P=1.000、0.980)6. The percentage size of liver fibrosis of ratsThe results of semi-quantitative scoring of liver fibrosis of each groups is significant difference(welch=243.047, P=0.000). Multiple comparison between groups, normal control group, captopril group, losartan group and spironolactone group compared with model group show the significant difference (P<0.05). Captopril group, losartan group and spironolactone group compared with model control group show the significant difference (P<0.05). Losartan group and spironolactone group compared with captopril group show no significant difference (P>0.05).7. The HA level in serum of ratsOne-way ANOVA shows, The HA levels in serum of each groups is significant difference(F=121.629, P=0.000). Multiple comparison between groups, model group, captopril group, losartan group and spironolactone group compared with normal control group show the significant difference (P<0.05). Captopril group, losartan group and spironolactone group compared with model group show the significant difference (P<0.05). Losartan group and spironolactone group compared with captopril group show no significant difference (P>0.05).8. The LN level in serum of ratsOne-way ANOVA shows, The LN levels in serum of each groups is significant difference (F=42.972, P=0.000). Multiple comparison between groups, model group, captopril group, losartan group and spironolactone group compared with normal control group show the significant difference (P<0.05). Captopril group, losartan group and spironolactone group compared with model group show the significant difference (P<0.05). Losartan group and spironolactone group compared with captopril group show no significant difference (P>0.05). 9. The PCIII levels in serum of rats One-way ANOVA shows, The PCIII levels in serum of each groups is significant difference (F=213.310, P=0.000). Multiple comparison between groups, model group, captopril group, losartan group and spironolactone group compared with normal control group show the significant difference (P<0.05). Captopril group, losartan group and spironolactone group compared with model group show the significant difference (P<0.05). Losartan group and spironolactone group compared with captopril group show no significant difference (P>0.05)10. The CIV levels in serum of ratsOne-way ANOVA shows, The CIV levels in serum of each groups is significant difference(F=238.215, P=0.000). Multiple comparison between groups, model group, captopril group, losartan group and spironolactone group compared with normal control group show the significant difference (P<0.05). Captopril group, losartan group and spironolactone group compared with model group show the significant difference (P<0.05). Losartan group and spironolactone group compared with captopril group show no significant difference (P>0.05)11. The ACE2levels in serum of ratsThe ACE2levels in serum of each groups is significant difference (welch=310.273, P=0.000). Multiple comparison between groups, model group, captopril group, losartan group and spironolactone group compared with normal control group show the significant difference (P<0.05). Captopril group, losartan group and spironolactone group compared with model group show the significant difference (P<0.05). Losartan group and spironolactone group compared with captopril group show no significant difference (P>0.05)12. The AngⅡ levels in serum of ratsThe AngⅡ levels in serum of each groups is significant difference (welch=33.916, P=0.000). Multiple comparison between groups, model group, captopril group, losartan group and spironolactone group compared with normal control group show the significant difference (P<0.05). Captopril group, losartan group and spironolactone group compared with model group show the significant difference (P<0.05). Losartan group and spironolactone group compared with captopril group show no significant difference (P>0.05).13. The Ang (1-7) levels in serum of ratsThe Ang (1-7) levels in serum of each groups is significant difference (welch=732.022, P=0.000). Multiple comparison between groups, model group, captopril group, losartan group and spironolactone group compared with normal control group show the significant difference (P<0.05). Captopril group, losartan group and spironolactone group compared with model group show the significant difference (P<0.05). Losartan group and spironolactone group compared with captopril group show no significant difference (P>0.05).14. The ACE2levels in liver tissue of ratsOne-way ANOVA shows, The ACE2levels in liver tissue of each groups is significant difference (F=229.378, P=0.000). Multiple comparison between groups, model group, captopril group, losartan group and spironolactone group compared with normal control group show the significant difference (P<0.05). Captopril group, losartan group and spironolactone group compared with model group show the significant difference (P<0.05). Losartan group and spironolactone group compared with captopril group show no significant difference (P>0.05).15.The AngⅡ levels in liver tissue of ratsOne-way ANOVA shows, The AngⅡ levels in liver tissue of each groups is significant difference (F=52.695, P=0.000). Multiple comparison between groups, model group, captopril group, losartan group and spironolactone group compared with normal control group show the significant difference (P<0.05). Captopril group, losartan group and spironolactone group compared with model group show the significant difference (P<0.05). Losartan group and spironolactone group compared with captopril group show no significant difference (P>0.05).16. The Ang (1-7) levels in liver tissue of ratsOne-way ANOVA shows, The Ang (1-7) levels in liver tissue of each groups is significant difference (F=210.254, P=0.000). Multiple comparison between groups, model group, captopril group, losartan group and spironolactone group compared with normal control group show the significant difference (P<0.05). Captopril group, losartan group and spironolactone group compared with model group show the significant difference (P<0.05). Losartan group and spironolactone group compared with captopril group show no significant difference (P>0.05).17. The expression of TGF-β1mRNA in liver tissue of ratsThe expression of TGF-β1mRNA in liver tissue of each groups is significant difference (welch=162.836, P=0.000). Multiple comparison between groups, model group, captopril group, losartan group and spironolactone group compared with normal control group show the significant difference (P<0.05). Captopril group, losartan group and spironolactone group compared with model group show the significant difference (P<0.05). Losartan group and spironolactone group compared with captopril group show no significant difference (P>0.05).18. The expression of CTGFmRNA in liver tissue of ratsThe expression of CTGFmRNA in liver tissue of each groups is significant difference (welch=47.175, P=0.000). Multiple comparison between groups, model group, captopril group, losartan group and spironolactone group compared with normal control group show the significant difference (P<0.05). Captopril group, losartan group and spironolactone group compared with model group show the significant difference (P<0.05). Losartan group and spironolactone group compared with captopril group show no significant difference (P>0.05).19. The expression of TNF-a mRNA in liver tissue of ratsThe expression of CTGFmRNA in liver tissue of each groups is significant difference (welch=90.987, P=0.000). Multiple comparison between groups, model group, captopril group, losartan group and spironolactone group compared with normal control group show the significant difference (P<0.05). Captopril group, losartan group and spironolactone group compared with model group show the significant difference (P<0.05). Losartan group and spironolactone group compared with captopril group show no significant difference (P>0.05).20. The expression of TGF-β1protein in liver tissue of ratsOne-way ANOVA shows, The expression of TGF-β1protein in liver tissue of each groups is significant difference (F=53.288, P=0.000). Multiple comparison between groups, model group, captopril group, losartan group and spironolactone group compared with normal control group show the significant difference (P<0.05) Captopril group, losartan group and spironolactone group compared with model group show the significant difference (P<0.05). Losartan group and spironolactone group compared with captopril group show no significant difference (P>0.05).21. The expression of CTGF protein in liver tissue of ratsThe expression of CTGF protein in liver tissue of each groups is significant difference (welch=36.304, P=0.000). Multiple comparison between groups, model group, captopril group, losartan group and spironolactone group compared with normal control group show the significant difference (P<0.05). Captopril group, losartan group and spironolactone group compared with normal control group show the significant difference (P<0.05). Losartan group and spironolactone group compared with captopril group show no significant difference (P>0.05). 22. The expression of TNF-a protein in liver tissue of ratsThe expression of TNF-a protein in liver tissue of each groups is significant difference (welch=41.812, P=0.000). Multiple comparison between groups, model group, captopril group, losartan group and spironolactone group compared with normal control group show the significant difference (P<0.05). Captopril group, losartan group and spironolactone group compared with normal control group show the significant difference (P<0.05). Losartan group and spironolactone group compared with captopril group show no significant difference (P>0.05).Conclusion1. The study constructed rat liver fibrosis model by CCl4induction successfully.2. The intervention of different links by Captopril, Losartan and Spironolactone could better the general behavior of the experimental liver fibrosis rats.3. The intervention of different links by Captopril, Losartan and Spironolactone could improve histopathological changes of liver fibrosis rats, decrease HA, LN, PCⅢ and CⅣ in serum and better the degree of liver fibrosis in rats, and they were same in anti-liver fibrosis in rats.4. The mechanism of intervention of different links by Captopril, Losartan and Spironolactone may be related to increasing of ACE2and Ang (1-7) and decreasing of AngⅡ in serum and liver tissue along with reducing expression of TGF-β1, CTGF and TNF-a in liver tissue.

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