干预肾素—血管紧张素—醛固酮系统不同环节抗大鼠肝纤维化的机制研究
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摘要
一、背景及目的
肝纤维化是多种慢性肝病共同的病理改变,各种病因引起的慢性肝病或损害绝大多数都存在肝纤维化。肝纤维化是诸多肝病发展为肝硬化的必由之路,也是肝细胞肝癌的危险因素之一,其中25%-40%最终发展为肝硬化甚至肝癌。因此,肝纤维化是一类严重危害人类健康的世界性疾病。肝纤维化是一个动态的可逆性的病变过程。所以,深入研究肝纤维化的发病机制,寻找有效的治疗靶点,积极有效的逆转肝脏纤维化,对防治肝硬化与肝癌具有重大的广泛的经济价值、社会意义和应用前景。
肾素-血管紧张素-醛固酮系统(renin-angiotensin-aldosterone system, RAAS)为一内分泌系统,具有广泛的生物学效应,作用于全身多个器官。RAAS在器官纤维化中起着重要的作用。除了循环系统中存在RAAS,诸多器官,心,肾,肺,胰腺都存在局部或器官内的RAAS,并且参与了组织器官纤维化和/或结构重构过程。肝脏局部也存在RAAS,以旁分泌、自分泌的方式发挥作用,参与肝纤维化的发展。这提示我们,RAAS可能是抗肝纤维化治疗的潜在靶点。以往认为,经典的肾素血管紧张素醛固酮系统作用轴ACE-Angll-AT1受体轴是其生物关联性的唯一体系,对肝纤维化的形成具有促进作用。最新研究发现,ACE2-Ang(1-7)-Mas受体轴亦为RAAS作用轴,是ACE-Angll-ATl受体轴的反向调节轴,对肝纤维化的发生具有保护作用。AngⅡ作为RAAS的主要功能成分,具有促进肝纤维化发展的重要作；而ACE2可裂解AngⅡ,生成Angl-7, Ang-(1-7)对AngⅡ具有负向调节,对肝纤维化具有保护作用。
另一方面,在肝纤维化形成的过程中,细胞因子起了关键性的作用。细胞因子是一类能在细胞间传递信息、具有免疫调节和效应功能的蛋白质或小分子多肽,它们通过旁分泌或者自分泌的方式作用于靶细胞特定受体,发挥着局部生物学效应。在肝纤维化的形成过程中,转化生长因子β1(Transforming growth factor, TGF-β1)、结缔组织生长因子(Connective tissue growth factor, CTGF)、肿瘤坏死因子-α (Tumor necrosis factor-α, TNF-α)存在表达的失衡,对肝纤维化的发生和发展起了重要的促进作用。
当前,肝纤维化的治疗仍以药物为主,过去的10余年,RAAS阻滞剂中的血管紧张素转化酶抑制剂、血管紧张素Ⅱ受体阻滞剂、醛固酮受体拮抗剂在心血管疾病、糖尿病、肾病等领域的地位被大量的循证医学证据所证实,而关于RAAS阻滞剂在肝病领域中的应用,多来源于回顾性、非对照前瞻性研究或者是有类似发病机制的其他疾病的治疗经验,对不合并心血管疾病、糖尿病、肾病的肝病患者仍不推荐应用RAAS抑制剂抗肝纤维化治疗,然而,对能耐受RAAS抑制剂的肝病患者,其积极作用也不能忽视,值得进一步研究。
因此,本课题在前期研究工作的基础上,采用(Carbon tetrachloride, CCl4)诱导制备大鼠肝纤维化模型,选用血管紧张素转化酶抑制剂卡托普利、血管紧张素Ⅱ受体阻滞剂氯沙坦、醛固酮受体拮抗剂螺内酯,分别干预RAAS不同环节,观察其对大鼠肝组织病理形态学的影响,对血清透明质酸(Hyaluronidase, HA)、层粘连蛋白(Laminin, LN)、Ⅲ型前胶原(ProcollagenⅢ, PCⅢ)、 Ⅳ型胶原(Collagen Type Ⅳ, CⅣ), RAAS组分ACE2、AngⅡ、Ang (1-7),细胞因子TGF-β1、CTGF、TNF-α的影响,以期明确干预RAAS哪个环节抗大鼠肝纤维化作用最佳,并探讨其可能的作用机制,为肝纤维化的防治提供实验依据。
二、方法
取6周龄SD (Sprague Dawley, SD)大鼠50只,随机分为正常组、模型组、卡托普利组、氯沙坦组和螺内酯组,每组各10只。正常组皮下注射橄榄油,3ml/kg;其余大鼠予40%CCl4腹壁皮下注射,3ml/kg,首剂加倍,1次/3d,制备大鼠肝纤维化模型；次日,卡托普利组用血管紧张素转换酶抑制剂卡托普利60mg/kg、氯沙坦组用血管紧张素Ⅱ的Ⅰ型受体阻断剂氯沙坦10mg/kg、螺内酯组用醛固酮受体拮抗剂螺内酯100mg/kg,均用10ml生理盐水稀释后灌胃,模型组和正常组用等量生理盐水灌胃,1次/d。于第8周处死各组大鼠,收集血液及肝组织标本。
采用HE染色和Masson染色,光镜观察肝组织的病理形态学的变化；酶联免疫吸附测定法测定血清HA、LN、PCⅢ、CⅣ及RAAS组分ACE2、AngⅡ、 Ang(1-7)的浓度；应用Real time-PCR检测肝组织TGF-β1mRNA、CTGFmRNA、 TNF-α mRNA的表达；Western blotting检测肝组织TGF-β1蛋白、CTGF蛋白、TNF-α蛋白的表达。
三、数据分析
数据用SPSS11.5软件进行分析,计量资料用均数±标准差(x±s)表示。方差齐时,组间比较采用单因素方差分析(one-way ANOVA)进行统计分析,多重比较采用LSD-t法检验；方差不齐时,用近似方差分析的Welch法,多重比较采用Dunnett T3法检验,P<0.05为有统计学意义。
四、结果
所有大鼠无一脱失,均进入实验结果分析,无皮下脓肿,无自噬及舔咬。
1.各组大鼠体重
正常对照组、模型组、卡托普利组、氯沙坦组、螺内酯组,各组体重差异有显著性意义(F=50.649,P=0.000)。组间多重比较,模型组、卡托普利组、氯沙坦组、螺内酯组,体重低于正常对照组,差异有显著性意义(P<0.05)；卡托普利组、氯沙坦组、螺内酯组,体重高于模型组,差异有显著性意义(P<0.01)。氯沙坦组、螺内酯组体重与卡托普利组相比差异无显著性意义(P>0.05)。
2.各组大鼠肝湿重
正常对照组、模型组、卡托普利组、氯沙坦组、螺内酯组,各组肝湿重差异有显著性意义(F=36.137,P=0.000)。组间多重比较,正常对照组、卡托普利组、氯沙坦组、螺内酯组,肝湿重低于模型组,差异有显著性意义(P<0.05)；卡托普利组、氯沙坦组、螺内酯组肝湿重与正常对照组相比较,差异无显著性意义(P>0.05)。氯沙坦组、螺内酯组肝湿重与卡托普利组相比,差异无显著性意义(P>0.05)。
3.HE染色结果
光镜下观察肝脏组织病理变化,正常组肝小叶结构正常,见肝细胞以中央静脉为中心向周围呈放射状排列,结构完整,肝细胞无变性坏死,成纤维细胞极少见到。模型组显示肝细胞索排列较紊乱,肝细胞出现脂肪变性,汇管区扩大,见假小叶形成。与模型组相比较,卡托普利组、氯沙坦组、螺内酯组汇管区稍微扩大,纤维间隔形成减少,未见假小叶形成。
4. Masson染色结果
正常组胶原纤维在血管周围有少量的表达；模型组胶原纤维增生明显,纤维间隔形成,可见弓形纤维,形成假小叶；与模型组相比较,卡托普利组、氯沙坦组、螺内酯组的胶原纤维明显减少,形成的纤维间隔亦明显减少,未见假小叶形成。
5.各组大鼠肝纤维化半定量评分
正常对照组、模型组、卡托普利组、氯沙坦组、螺内酯组,各组肝纤维化半定量评分差异有显著性意义(welch值=354.152,P=0.000)。组间多重比较,模型组、卡托普利组、氯沙坦组、螺内酯组肝纤维化半定量评分高于正常对照组,差异有显著性意义(P<0.05)；卡托普利组、氯沙坦组、螺内酯组肝纤维化半定量评分低于模型组,差异有显著性意义(P<0.05)。氯沙坦组、螺内酯组肝纤维化半定量评分与卡托普利组相比,差异无显著性意义(P>0.05)。
6.各组大鼠肝纤维化面积百分比
正常对照组、模型组、卡托普利组、氯沙坦组、螺内酯组,各组肝纤维面积百分比差异有显著性意义(welch值=243.047,P=0.000)。组间多重比较,模型组、卡托普利组、氯沙坦组、螺内酯组肝纤维面积百分比高于正常对照组,差异有显著性意义(P<0.05)；卡托普利组、氯沙坦组、螺内酯组肝纤维面积百分比低于与模型组,差异有显著性意义(P<0.05)。氯沙坦组、螺内酯组肝纤维面积百分比与卡托普利组相比,差异无显著性意义(P>0.05)。
7各组大鼠血清中HA. LN. PCⅢ、CIV水平
7.1HA：正常对照组、模型组、卡托普利组、氯沙坦组、螺内酯组,各组血清HA水平差异有显著性意义(F=121.629,P=0.000)。组间多重比较，模型组、卡托普利组、氯沙坦组、螺内酯组血清HA水平高于正常对照组,差异有显著性意义(P<0.05)；卡托普利组、氯沙坦组、螺内酯组,血清HA水平低于模型组,差异有显著性意义(P<0.05)；氯沙坦组、螺内酯组血清HA水平与卡托普利组相比,差异无显著性意义(P>0.05)。
7.2LN：：正常对照组、模型组、卡托普利组、氯沙坦组、螺内酯组,各组血清LN水平差异有显著性意义(F=42.972,P=0.000)。组间多重比较,模型组、卡托普利组、氯沙坦组、螺内酯组,血清LN水平高于正常对照组,差异有显著性意义(P<0.05)；卡托普利组、氯沙坦组、螺内酯组,血清LN水平低于模型组,差异有显著性意义(P<0.05)；氯沙坦组、螺内酯组血清LN水平与卡托普利组相比,差异无显著性意义(P>0.05)。
7.3PCⅢ:正常对照组、模型组、卡托普利组、氯沙坦组、螺内酯组,各组血清PCⅢ水平差异有显著性意义(F=213.310,P=0.000)。组间多重比较,模型组、卡托普利组、氯沙坦组、螺内酯组,血清PCⅢ水平高于正常对照组,差异有显著性意义((P<0.05))；卡托普利组、氯沙坦组、螺内酯组,血清PCⅢ水平低于模型组,差异有显著性意义((P<0.05)；氯沙坦组、螺内酯组血清PCⅢ水平与卡托普利组相比,差异无显著性意义(P>0.05)。
7.4CIV：正常对照组、模型组、卡托普利组、氯沙坦组、螺内酯组,各组血清CIV水平差异有显著性意义(F=238.215,P=0.000)。组间多重比较,模型组、卡托普利组、氯沙坦组、螺内酯组,血清CIV水平高于正常对照组,差异有显著性意义(P<0.05)；卡托普利组、氯沙坦组、螺内酯组,血清CIV水平低于模型组,差异有显著性意义(P<0.05)；氯沙坦组、螺内酯组血清CIV水平与卡托普利组相比,差异无显著性意义(P>0.05)。
8各组大鼠血清ACE2、AngⅡ和Ang (1-7)水平
8.1ACE2：正常对照组、模型组、卡托普利组、氯沙坦组、螺内酯组,各组血清ACE2水平差异有显著性意义(welch值=310.273,P=0.000)。组间多重比较,模型组、卡托普利组、氯沙坦组、螺内酯组,血清ACE2水平高于正常对照组,差异有显著性意义(P<0.05)；卡托普利组、氯沙坦组、螺内酯组,血清ACE2水平高于模型组,差异有显著性意义(P<0.05)；氯沙坦组、螺内酯组ACE2水平与卡托普利组相比,差异无显著性意义(P>0.05)。
8.2AngⅡ:正常对照组、模型组、卡托普利组、氯沙坦组、螺内酯组,各组血清AngⅡ水平差异有显著性意义(welch值=33.916,P=0.000)。组间多重比较,模型组、卡托普利组、氯沙坦组、螺内酯组,血清AngⅡ水平高于正常对照组,差异有显著性意义(P<0.05)；卡托普利组、氯沙坦组、螺内酯组,血清AngⅡ水平低于模型组,差异有显著性意义(P<0.05)；氯沙坦组、螺内酯组血清AngⅡ水平与卡托普利组相比,差异无显著性意义(P>0.05)。
8.3Ang (1-7):正常对照组、模型组、卡托普利组、氯沙坦组、螺内酯组,各组血清Ang(1-7)水平差异有显著性意义(welch值=732.022,P=0.000)。组间多重比较,模型组、卡托普利组、氯沙坦组、螺内酯组,血清Ang(1-7)水平高于正常对照组,差异有显著性意义(P<0.05)；卡托普利组、氯沙坦组、螺内酯组,血清Ang(1-7)水平高于模型组,差异有显著性意义(P<0.05)；氯沙坦组、螺内酯组,血清Ang(1-7)水平与卡托普利组相比,差异无显著性意义(P>0.05)。
9各组大鼠肝组织ACE2、AngⅡ和Ang (1-7)水平
9.1ACE2：正常对照组、模型组、卡托普利组、氯沙坦组、螺内酯组,各组ACE2水平差异有显著性意义(F=229.378,P=0.000)。组间多重比较,模型组、卡托普利组、氯沙坦组、螺内酯组,ACE2水平高于正常对照组,差异有显著性意义(P<0.05)；卡托普利组、氯沙坦组、螺内酯组,ACE2水平高于模型组,差异有显著性意义(P<0.05)；氯沙坦组、螺内酯组ACE2水平与卡托普利组相比,差异无显著性意义(P>0.05)。
9.2AngⅡ:正常对照组、模型组、卡托普利组、氯沙坦组、螺内酯组,各组AngⅡ水平差异有显著性意义(F=52.695,P=0.000)。组间多重比较,模型组、卡托普利组、氯沙坦组、螺内酯组,AngⅡ水平高于正常对照线，组差异有显著性意义(P<0.05)；卡托普利组、氯沙坦组、螺内酯组AngⅡ水平低于模型组,差异有显著性意义(P<0.05)；氯沙坦组、螺内酯组AngⅡ水平与卡托普利组相比,差异无显著性意义(P>0.05)。9.3Ang (1-7):正常对照组、模型组、卡托普利组、氯沙坦组、螺内酯组,各组Ang(1-7)水平差异有显著性意义(F=210.254,P=0.000)。组向多重比较,模型组、卡托普利组、氯沙坦组、螺内酯组,Ang(1-7)水平高于正常对照组,差异有显著性意义(P<0.05)；卡托普利组、氯沙坦组、螺内酯组,Ang(1-7)水平高于模型组,差异有显著性意义(P<0.05)；氯沙坦组、螺内酯组Ang (1-7)水平与卡托普利组相比,差异无显著性意义(P>0.05)。
10各组大鼠肝组织TGF-β1mRNA、CTGF mRNA、TNF-αmRNA的表达
10.1TGF-β1mRNA:正常对照组、模型组、卡托普利组、氯沙坦组、螺内酯组,各组TGF-β1mRNA表达差异有显著性意义(welch值=162.836,P=0.000)。组间多重比较,模型组、卡托普利组、氯沙坦组、螺内酯组,TGF-β1mRNA表达高于正常对照组,差异有显著性意义(P<0.05)；卡托普利组、氯沙坦组、螺内酯组,TGF-β1mRNA表达低于模型组,差异有显著性意义(P<0.05)；氯沙坦组、螺内酯组TGF-β1mRNA的表达与卡托普利组相比,差异无显著性意义(P>0.05)。
10.2CTGF mRNA:正常对照组、模型组、卡托普利组、氯沙坦组、螺内酯组,各组CTGF mRNA表达差异有显著性意义(welch值=41.175,P=0.000)。组间多重比较,模型组、卡托普利组、氯沙坦组、螺内酯组,CTGF mRNA表达高于正常对照组,差异有显著性意义(P<0.05)；卡托普利组、氯沙坦组、螺内酯组,CTGF mRNA表达低于模型组,差异有显著性意义(P<0.05)；氯沙坦组、螺内酯组CTGF mRNA表达与卡托普利组相比,差异无显著性意义(P>0.05)。
10.3TNF-αmRNA:正常对照组、模型组、卡托普利组、氯沙坦组、螺内酯组,各组TNF-α mRNA表达差异有显著性意义(welch值=90.987,P=0.000)。组间多重比较,模型组、卡托普利组、氯沙坦组、螺内酯组,TNF-α mRNA表达高于正常对照组,差异有显著性意义(P<0.05)；卡托普利组、氯沙坦组、螺内酯组,TNF-α mRNA表达低于模型组,差异有显著性意义(P<0.05)；氯沙坦组、螺内酯组。TNF-α mRNA表达与卡托普利组相比,差异无显著性意义(P>0.05)。
11各组大鼠肝组织TGF-β1、CTGF、TNF-α蛋白的表达
11.1TGF-β1蛋白：正常对照组、模型组、卡托普利组、氯沙坦组、螺内酯组,各组TGF-β1蛋白的表达,差异有显著性意义(F=53.288,P=0.000)。组间多重比较,模型组、卡托普利组、氯沙坦组、螺内酯组TGF-β1蛋白表达高于正常对照组,差异有显著性意义(P<0.05)；卡托普利组、氯沙坦组、螺内酯组,TGF-β1蛋白表达低于模型组,差异有显著性意义(P<0.05)；氯沙坦组、螺内酯组TGF-β1蛋白表达与卡托普利组相比,差异无显著性意义(P>0.05)。
11.2CTGF蛋白：正常对照组、模型组、卡托普利组、氯沙坦组、螺内酯组,各组CTGF蛋白表达差异有显著性意义(welch值=36.304,P=0.000)。组间多重比较,模型组、卡托普利组、氯沙坦组、螺内酯组CTGF蛋白的表达高于正常对照组,差异有显著性意义(P<0.05)；卡托普利组、氯沙坦组、螺内酯组,CTGF蛋白表达低于模型组,差异有显著性意义(P<0.05)；氯沙坦组、螺内酯组CTGF蛋白表达与卡托普利组相比,差异无显著性意义(P>0.05)。
11.3TNF-α蛋白：正常对照组、模型组、卡托普利组、氯沙坦组、螺内酯组,各组TNF-α蛋白表达差异有显著性意义(welch值=41.812,P=0.000)。组间多重比较,模型组、卡托普利组、氯沙坦组、螺内酯组,TNF-α蛋白表达高于正常对照组,差异有显著性意义(P<0.05)；卡托普利组、氯沙坦组、螺内酯组,TNF-α蛋白表达低于模型组,差异有显著性意义(P<0.05)；氯沙坦组、螺内酯组TNF-α蛋白表达与卡托普利组相比,差异无显著性意义(P>0.05)。
结论
1.本研究通过CCl4诱导,成功构建了的大鼠肝纤维化模型。
2.干预RAAS不同环节均能够改善实验性肝纤维化大鼠的一般状态。
3.干预RAAS不同环节均能改善大鼠肝脏组织病理学改变,降低大鼠血清HA、 LN、PCⅢ、CIV的水平,改善实验性肝纤维化大鼠的肝脏纤维化程度,且效果无优劣之分。
4.干预RAAS不同环节抗肝纤维化的作用机制可能是通过升高循环和肝脏组织中ACE2、Ang (1-7)的水平,降低Angll的生成；并且下调肝组织中TGF-β1、 CTGF、TNF-α的表达有关。
Background and objectve
Liver 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.
Method
Liver 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 method
Apply 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.
Result
1. The body weight of rats
One-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 rats
One-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 staining
Light 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 staining
Expression 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 rats
The 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 rats
The 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 rats
One-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 rats
One-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 rats
One-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 rats
The 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 rats
The 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 rats
The 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 rats
One-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 rats
One-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 rats
One-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 rats
The 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 rats
The 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 rats
The 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 rats
One-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 rats
The 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 rats
The 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).
Conclusion
1. 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.
肝纤维化是多种慢性肝病共同的病理改变,各种病因引起的慢性肝病或损害绝大多数都存在肝纤维化。肝纤维化是诸多肝病发展为肝硬化的必由之路,也是肝细胞肝癌的危险因素之一,其中25%-40%最终发展为肝硬化甚至肝癌。因此,肝纤维化是一类严重危害人类健康的世界性疾病。肝纤维化是一个动态的可逆性的病变过程。所以,深入研究肝纤维化的发病机制,寻找有效的治疗靶点,积极有效的逆转肝脏纤维化,对防治肝硬化与肝癌具有重大的广泛的经济价值、社会意义和应用前景。
肾素-血管紧张素-醛固酮系统(renin-angiotensin-aldosterone system, RAAS)为一内分泌系统,具有广泛的生物学效应,作用于全身多个器官。RAAS在器官纤维化中起着重要的作用。除了循环系统中存在RAAS,诸多器官,心,肾,肺,胰腺都存在局部或器官内的RAAS,并且参与了组织器官纤维化和/或结构重构过程。肝脏局部也存在RAAS,以旁分泌、自分泌的方式发挥作用,参与肝纤维化的发展。这提示我们,RAAS可能是抗肝纤维化治疗的潜在靶点。以往认为,经典的肾素血管紧张素醛固酮系统作用轴ACE-Angll-AT1受体轴是其生物关联性的唯一体系,对肝纤维化的形成具有促进作用。最新研究发现,ACE2-Ang(1-7)-Mas受体轴亦为RAAS作用轴,是ACE-Angll-ATl受体轴的反向调节轴,对肝纤维化的发生具有保护作用。AngⅡ作为RAAS的主要功能成分,具有促进肝纤维化发展的重要作；而ACE2可裂解AngⅡ,生成Angl-7, Ang-(1-7)对AngⅡ具有负向调节,对肝纤维化具有保护作用。
另一方面,在肝纤维化形成的过程中,细胞因子起了关键性的作用。细胞因子是一类能在细胞间传递信息、具有免疫调节和效应功能的蛋白质或小分子多肽,它们通过旁分泌或者自分泌的方式作用于靶细胞特定受体,发挥着局部生物学效应。在肝纤维化的形成过程中,转化生长因子β1(Transforming growth factor, TGF-β1)、结缔组织生长因子(Connective tissue growth factor, CTGF)、肿瘤坏死因子-α (Tumor necrosis factor-α, TNF-α)存在表达的失衡,对肝纤维化的发生和发展起了重要的促进作用。
当前,肝纤维化的治疗仍以药物为主,过去的10余年,RAAS阻滞剂中的血管紧张素转化酶抑制剂、血管紧张素Ⅱ受体阻滞剂、醛固酮受体拮抗剂在心血管疾病、糖尿病、肾病等领域的地位被大量的循证医学证据所证实,而关于RAAS阻滞剂在肝病领域中的应用,多来源于回顾性、非对照前瞻性研究或者是有类似发病机制的其他疾病的治疗经验,对不合并心血管疾病、糖尿病、肾病的肝病患者仍不推荐应用RAAS抑制剂抗肝纤维化治疗,然而,对能耐受RAAS抑制剂的肝病患者,其积极作用也不能忽视,值得进一步研究。
因此,本课题在前期研究工作的基础上,采用(Carbon tetrachloride, CCl4)诱导制备大鼠肝纤维化模型,选用血管紧张素转化酶抑制剂卡托普利、血管紧张素Ⅱ受体阻滞剂氯沙坦、醛固酮受体拮抗剂螺内酯,分别干预RAAS不同环节,观察其对大鼠肝组织病理形态学的影响,对血清透明质酸(Hyaluronidase, HA)、层粘连蛋白(Laminin, LN)、Ⅲ型前胶原(ProcollagenⅢ, PCⅢ)、 Ⅳ型胶原(Collagen Type Ⅳ, CⅣ), RAAS组分ACE2、AngⅡ、Ang (1-7),细胞因子TGF-β1、CTGF、TNF-α的影响,以期明确干预RAAS哪个环节抗大鼠肝纤维化作用最佳,并探讨其可能的作用机制,为肝纤维化的防治提供实验依据。
二、方法
取6周龄SD (Sprague Dawley, SD)大鼠50只,随机分为正常组、模型组、卡托普利组、氯沙坦组和螺内酯组,每组各10只。正常组皮下注射橄榄油,3ml/kg;其余大鼠予40%CCl4腹壁皮下注射,3ml/kg,首剂加倍,1次/3d,制备大鼠肝纤维化模型；次日,卡托普利组用血管紧张素转换酶抑制剂卡托普利60mg/kg、氯沙坦组用血管紧张素Ⅱ的Ⅰ型受体阻断剂氯沙坦10mg/kg、螺内酯组用醛固酮受体拮抗剂螺内酯100mg/kg,均用10ml生理盐水稀释后灌胃,模型组和正常组用等量生理盐水灌胃,1次/d。于第8周处死各组大鼠,收集血液及肝组织标本。
采用HE染色和Masson染色,光镜观察肝组织的病理形态学的变化；酶联免疫吸附测定法测定血清HA、LN、PCⅢ、CⅣ及RAAS组分ACE2、AngⅡ、 Ang(1-7)的浓度；应用Real time-PCR检测肝组织TGF-β1mRNA、CTGFmRNA、 TNF-α mRNA的表达；Western blotting检测肝组织TGF-β1蛋白、CTGF蛋白、TNF-α蛋白的表达。
三、数据分析
数据用SPSS11.5软件进行分析,计量资料用均数±标准差(x±s)表示。方差齐时,组间比较采用单因素方差分析(one-way ANOVA)进行统计分析,多重比较采用LSD-t法检验；方差不齐时,用近似方差分析的Welch法,多重比较采用Dunnett T3法检验,P<0.05为有统计学意义。
四、结果
所有大鼠无一脱失,均进入实验结果分析,无皮下脓肿,无自噬及舔咬。
1.各组大鼠体重
正常对照组、模型组、卡托普利组、氯沙坦组、螺内酯组,各组体重差异有显著性意义(F=50.649,P=0.000)。组间多重比较,模型组、卡托普利组、氯沙坦组、螺内酯组,体重低于正常对照组,差异有显著性意义(P<0.05)；卡托普利组、氯沙坦组、螺内酯组,体重高于模型组,差异有显著性意义(P<0.01)。氯沙坦组、螺内酯组体重与卡托普利组相比差异无显著性意义(P>0.05)。
2.各组大鼠肝湿重
正常对照组、模型组、卡托普利组、氯沙坦组、螺内酯组,各组肝湿重差异有显著性意义(F=36.137,P=0.000)。组间多重比较,正常对照组、卡托普利组、氯沙坦组、螺内酯组,肝湿重低于模型组,差异有显著性意义(P<0.05)；卡托普利组、氯沙坦组、螺内酯组肝湿重与正常对照组相比较,差异无显著性意义(P>0.05)。氯沙坦组、螺内酯组肝湿重与卡托普利组相比,差异无显著性意义(P>0.05)。
3.HE染色结果
光镜下观察肝脏组织病理变化,正常组肝小叶结构正常,见肝细胞以中央静脉为中心向周围呈放射状排列,结构完整,肝细胞无变性坏死,成纤维细胞极少见到。模型组显示肝细胞索排列较紊乱,肝细胞出现脂肪变性,汇管区扩大,见假小叶形成。与模型组相比较,卡托普利组、氯沙坦组、螺内酯组汇管区稍微扩大,纤维间隔形成减少,未见假小叶形成。
4. Masson染色结果
正常组胶原纤维在血管周围有少量的表达；模型组胶原纤维增生明显,纤维间隔形成,可见弓形纤维,形成假小叶；与模型组相比较,卡托普利组、氯沙坦组、螺内酯组的胶原纤维明显减少,形成的纤维间隔亦明显减少,未见假小叶形成。
5.各组大鼠肝纤维化半定量评分
正常对照组、模型组、卡托普利组、氯沙坦组、螺内酯组,各组肝纤维化半定量评分差异有显著性意义(welch值=354.152,P=0.000)。组间多重比较,模型组、卡托普利组、氯沙坦组、螺内酯组肝纤维化半定量评分高于正常对照组,差异有显著性意义(P<0.05)；卡托普利组、氯沙坦组、螺内酯组肝纤维化半定量评分低于模型组,差异有显著性意义(P<0.05)。氯沙坦组、螺内酯组肝纤维化半定量评分与卡托普利组相比,差异无显著性意义(P>0.05)。
6.各组大鼠肝纤维化面积百分比
正常对照组、模型组、卡托普利组、氯沙坦组、螺内酯组,各组肝纤维面积百分比差异有显著性意义(welch值=243.047,P=0.000)。组间多重比较,模型组、卡托普利组、氯沙坦组、螺内酯组肝纤维面积百分比高于正常对照组,差异有显著性意义(P<0.05)；卡托普利组、氯沙坦组、螺内酯组肝纤维面积百分比低于与模型组,差异有显著性意义(P<0.05)。氯沙坦组、螺内酯组肝纤维面积百分比与卡托普利组相比,差异无显著性意义(P>0.05)。
7各组大鼠血清中HA. LN. PCⅢ、CIV水平
7.1HA：正常对照组、模型组、卡托普利组、氯沙坦组、螺内酯组,各组血清HA水平差异有显著性意义(F=121.629,P=0.000)。组间多重比较，模型组、卡托普利组、氯沙坦组、螺内酯组血清HA水平高于正常对照组,差异有显著性意义(P<0.05)；卡托普利组、氯沙坦组、螺内酯组,血清HA水平低于模型组,差异有显著性意义(P<0.05)；氯沙坦组、螺内酯组血清HA水平与卡托普利组相比,差异无显著性意义(P>0.05)。
7.2LN：：正常对照组、模型组、卡托普利组、氯沙坦组、螺内酯组,各组血清LN水平差异有显著性意义(F=42.972,P=0.000)。组间多重比较,模型组、卡托普利组、氯沙坦组、螺内酯组,血清LN水平高于正常对照组,差异有显著性意义(P<0.05)；卡托普利组、氯沙坦组、螺内酯组,血清LN水平低于模型组,差异有显著性意义(P<0.05)；氯沙坦组、螺内酯组血清LN水平与卡托普利组相比,差异无显著性意义(P>0.05)。
7.3PCⅢ:正常对照组、模型组、卡托普利组、氯沙坦组、螺内酯组,各组血清PCⅢ水平差异有显著性意义(F=213.310,P=0.000)。组间多重比较,模型组、卡托普利组、氯沙坦组、螺内酯组,血清PCⅢ水平高于正常对照组,差异有显著性意义((P<0.05))；卡托普利组、氯沙坦组、螺内酯组,血清PCⅢ水平低于模型组,差异有显著性意义((P<0.05)；氯沙坦组、螺内酯组血清PCⅢ水平与卡托普利组相比,差异无显著性意义(P>0.05)。
7.4CIV：正常对照组、模型组、卡托普利组、氯沙坦组、螺内酯组,各组血清CIV水平差异有显著性意义(F=238.215,P=0.000)。组间多重比较,模型组、卡托普利组、氯沙坦组、螺内酯组,血清CIV水平高于正常对照组,差异有显著性意义(P<0.05)；卡托普利组、氯沙坦组、螺内酯组,血清CIV水平低于模型组,差异有显著性意义(P<0.05)；氯沙坦组、螺内酯组血清CIV水平与卡托普利组相比,差异无显著性意义(P>0.05)。
8各组大鼠血清ACE2、AngⅡ和Ang (1-7)水平
8.1ACE2：正常对照组、模型组、卡托普利组、氯沙坦组、螺内酯组,各组血清ACE2水平差异有显著性意义(welch值=310.273,P=0.000)。组间多重比较,模型组、卡托普利组、氯沙坦组、螺内酯组,血清ACE2水平高于正常对照组,差异有显著性意义(P<0.05)；卡托普利组、氯沙坦组、螺内酯组,血清ACE2水平高于模型组,差异有显著性意义(P<0.05)；氯沙坦组、螺内酯组ACE2水平与卡托普利组相比,差异无显著性意义(P>0.05)。
8.2AngⅡ:正常对照组、模型组、卡托普利组、氯沙坦组、螺内酯组,各组血清AngⅡ水平差异有显著性意义(welch值=33.916,P=0.000)。组间多重比较,模型组、卡托普利组、氯沙坦组、螺内酯组,血清AngⅡ水平高于正常对照组,差异有显著性意义(P<0.05)；卡托普利组、氯沙坦组、螺内酯组,血清AngⅡ水平低于模型组,差异有显著性意义(P<0.05)；氯沙坦组、螺内酯组血清AngⅡ水平与卡托普利组相比,差异无显著性意义(P>0.05)。
8.3Ang (1-7):正常对照组、模型组、卡托普利组、氯沙坦组、螺内酯组,各组血清Ang(1-7)水平差异有显著性意义(welch值=732.022,P=0.000)。组间多重比较,模型组、卡托普利组、氯沙坦组、螺内酯组,血清Ang(1-7)水平高于正常对照组,差异有显著性意义(P<0.05)；卡托普利组、氯沙坦组、螺内酯组,血清Ang(1-7)水平高于模型组,差异有显著性意义(P<0.05)；氯沙坦组、螺内酯组,血清Ang(1-7)水平与卡托普利组相比,差异无显著性意义(P>0.05)。
9各组大鼠肝组织ACE2、AngⅡ和Ang (1-7)水平
9.1ACE2：正常对照组、模型组、卡托普利组、氯沙坦组、螺内酯组,各组ACE2水平差异有显著性意义(F=229.378,P=0.000)。组间多重比较,模型组、卡托普利组、氯沙坦组、螺内酯组,ACE2水平高于正常对照组,差异有显著性意义(P<0.05)；卡托普利组、氯沙坦组、螺内酯组,ACE2水平高于模型组,差异有显著性意义(P<0.05)；氯沙坦组、螺内酯组ACE2水平与卡托普利组相比,差异无显著性意义(P>0.05)。
9.2AngⅡ:正常对照组、模型组、卡托普利组、氯沙坦组、螺内酯组,各组AngⅡ水平差异有显著性意义(F=52.695,P=0.000)。组间多重比较,模型组、卡托普利组、氯沙坦组、螺内酯组,AngⅡ水平高于正常对照线，组差异有显著性意义(P<0.05)；卡托普利组、氯沙坦组、螺内酯组AngⅡ水平低于模型组,差异有显著性意义(P<0.05)；氯沙坦组、螺内酯组AngⅡ水平与卡托普利组相比,差异无显著性意义(P>0.05)。9.3Ang (1-7):正常对照组、模型组、卡托普利组、氯沙坦组、螺内酯组,各组Ang(1-7)水平差异有显著性意义(F=210.254,P=0.000)。组向多重比较,模型组、卡托普利组、氯沙坦组、螺内酯组,Ang(1-7)水平高于正常对照组,差异有显著性意义(P<0.05)；卡托普利组、氯沙坦组、螺内酯组,Ang(1-7)水平高于模型组,差异有显著性意义(P<0.05)；氯沙坦组、螺内酯组Ang (1-7)水平与卡托普利组相比,差异无显著性意义(P>0.05)。
10各组大鼠肝组织TGF-β1mRNA、CTGF mRNA、TNF-αmRNA的表达
10.1TGF-β1mRNA:正常对照组、模型组、卡托普利组、氯沙坦组、螺内酯组,各组TGF-β1mRNA表达差异有显著性意义(welch值=162.836,P=0.000)。组间多重比较,模型组、卡托普利组、氯沙坦组、螺内酯组,TGF-β1mRNA表达高于正常对照组,差异有显著性意义(P<0.05)；卡托普利组、氯沙坦组、螺内酯组,TGF-β1mRNA表达低于模型组,差异有显著性意义(P<0.05)；氯沙坦组、螺内酯组TGF-β1mRNA的表达与卡托普利组相比,差异无显著性意义(P>0.05)。
10.2CTGF mRNA:正常对照组、模型组、卡托普利组、氯沙坦组、螺内酯组,各组CTGF mRNA表达差异有显著性意义(welch值=41.175,P=0.000)。组间多重比较,模型组、卡托普利组、氯沙坦组、螺内酯组,CTGF mRNA表达高于正常对照组,差异有显著性意义(P<0.05)；卡托普利组、氯沙坦组、螺内酯组,CTGF mRNA表达低于模型组,差异有显著性意义(P<0.05)；氯沙坦组、螺内酯组CTGF mRNA表达与卡托普利组相比,差异无显著性意义(P>0.05)。
10.3TNF-αmRNA:正常对照组、模型组、卡托普利组、氯沙坦组、螺内酯组,各组TNF-α mRNA表达差异有显著性意义(welch值=90.987,P=0.000)。组间多重比较,模型组、卡托普利组、氯沙坦组、螺内酯组,TNF-α mRNA表达高于正常对照组,差异有显著性意义(P<0.05)；卡托普利组、氯沙坦组、螺内酯组,TNF-α mRNA表达低于模型组,差异有显著性意义(P<0.05)；氯沙坦组、螺内酯组。TNF-α mRNA表达与卡托普利组相比,差异无显著性意义(P>0.05)。
11各组大鼠肝组织TGF-β1、CTGF、TNF-α蛋白的表达
11.1TGF-β1蛋白：正常对照组、模型组、卡托普利组、氯沙坦组、螺内酯组,各组TGF-β1蛋白的表达,差异有显著性意义(F=53.288,P=0.000)。组间多重比较,模型组、卡托普利组、氯沙坦组、螺内酯组TGF-β1蛋白表达高于正常对照组,差异有显著性意义(P<0.05)；卡托普利组、氯沙坦组、螺内酯组,TGF-β1蛋白表达低于模型组,差异有显著性意义(P<0.05)；氯沙坦组、螺内酯组TGF-β1蛋白表达与卡托普利组相比,差异无显著性意义(P>0.05)。
11.2CTGF蛋白：正常对照组、模型组、卡托普利组、氯沙坦组、螺内酯组,各组CTGF蛋白表达差异有显著性意义(welch值=36.304,P=0.000)。组间多重比较,模型组、卡托普利组、氯沙坦组、螺内酯组CTGF蛋白的表达高于正常对照组,差异有显著性意义(P<0.05)；卡托普利组、氯沙坦组、螺内酯组,CTGF蛋白表达低于模型组,差异有显著性意义(P<0.05)；氯沙坦组、螺内酯组CTGF蛋白表达与卡托普利组相比,差异无显著性意义(P>0.05)。
11.3TNF-α蛋白：正常对照组、模型组、卡托普利组、氯沙坦组、螺内酯组,各组TNF-α蛋白表达差异有显著性意义(welch值=41.812,P=0.000)。组间多重比较,模型组、卡托普利组、氯沙坦组、螺内酯组,TNF-α蛋白表达高于正常对照组,差异有显著性意义(P<0.05)；卡托普利组、氯沙坦组、螺内酯组,TNF-α蛋白表达低于模型组,差异有显著性意义(P<0.05)；氯沙坦组、螺内酯组TNF-α蛋白表达与卡托普利组相比,差异无显著性意义(P>0.05)。
结论
1.本研究通过CCl4诱导,成功构建了的大鼠肝纤维化模型。
2.干预RAAS不同环节均能够改善实验性肝纤维化大鼠的一般状态。
3.干预RAAS不同环节均能改善大鼠肝脏组织病理学改变,降低大鼠血清HA、 LN、PCⅢ、CIV的水平,改善实验性肝纤维化大鼠的肝脏纤维化程度,且效果无优劣之分。
4.干预RAAS不同环节抗肝纤维化的作用机制可能是通过升高循环和肝脏组织中ACE2、Ang (1-7)的水平,降低Angll的生成；并且下调肝组织中TGF-β1、 CTGF、TNF-α的表达有关。
Background and objectve
Liver 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.
Method
Liver 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 method
Apply 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.
Result
1. The body weight of rats
One-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 rats
One-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 staining
Light 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 staining
Expression 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 rats
The 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 rats
The 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 rats
One-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 rats
One-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 rats
One-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 rats
The 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 rats
The 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 rats
The 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 rats
One-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 rats
One-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 rats
One-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 rats
The 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 rats
The 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 rats
The 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 rats
One-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 rats
The 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 rats
The 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).
Conclusion
1. 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.
引文
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