【作者】 刘波；

【导师】 吴小翎；

【作者基本信息】 重庆医科大学 ， 内科学， 2012， 博士

【摘要】 背景：血管紧张素（Angiotensin，Ang）Ⅱ作为肾素血管紧张素系统（Renin-angiotensin system，RAS）的主要功能组分，扮演着促肝纤维化发展的角色；血管紧张素转换酶（Angiotensin-converting enzyme，ACE）是促进AngⅡ生成的关键酶，而ACE2可裂解AngⅡ生成Ang-(1-7)，负向调节AngⅡ的作用；AngⅡ可促进瘦素和抑制脂联素的生成，而瘦素和脂联素分别具有促和抗纤维化的作用。目的：（1）本研究利用高脂饮食诱导非酒精性脂肪性肝炎（Nonalcoholic steatohepatitis，NASH）伴肝纤维化大鼠模型，探讨ACE抑制剂福辛普利对肝纤维化的作用。（2）探讨福辛普利对NASH伴肝纤维化大鼠ACE2，瘦素和脂联素表达的影响。方法：40只雄性SD大鼠，随机分成4组，每组10只：正常对照组（NC组，普通饮食+生理盐水1mL灌胃）、模型组（HC组，高脂饮食+生理盐水1mL灌胃）、药物对照组（NF组，普通饮食+福辛普利3.6mg/kg/d灌胃）和药物干预组（HF组，高脂饮食+福辛普利3.6mg/kg/d灌胃）。24周后处死所有大鼠。（1）测体重和肝湿重，并计算肝湿重/体重比值；（2）肝组织病理学形态检查，分别做HE染色和Masson染色，行非酒精性脂肪性肝病活动度积分（NAFLD activityscore，NAS）和肝纤维化分期；（3）检测肝功，血脂和空腹血糖（fastingblood glucose，FBG），采用放射免疫法测定血清胰岛素浓度，并计算胰岛素抵抗指数；（4）采用酶联免疫吸附测定法（Enzyme-linked immunosorbent assay，ELISA）测定血清ACE2、AngII、Ang-(1-7)、瘦素、脂联素、透明质酸（Hyaluronic acid，HA）和转化生长因子（Transforminggrowth factor，TGF）-β1浓度。（5）RT-PCR检测肝组织中ACE2、瘦素和脂联素mRNA转录水平以及白色脂肪组织中的瘦素和脂联素mRNA转录水平。（6）Western blotting检测肝组织ACE2、胶原蛋白I和α-平滑肌动蛋白（α-SMA）蛋白的表达。结果：（1）与HC组比较， HF组大鼠肝湿重（32.61±8.65vs23.42±4.28，P <0.05）和肝湿重/体重比值（0.050±0.004vs0.042±0.004，P <0.05）显著降低，体重变化无显著差异（643.60±134.34vs553.00±63.41，P>0.05）。（2）与HC组比较，HF组大鼠肝细胞脂肪变性（2.85±0.24vs1.40±0.52）、炎症浸润（2.25±0.64vs1.20±0.26）、NAS（5.85±1.31vs3.00±0.82）和纤维化分期（3.80±0.75vs2.05±0.60）评分显著降低，P值均小于0.05，气球样变差异无显著性（0.75±0.59vs0.40±0.39，P>0.05）。（3）与HC组比较，HF组大鼠血清ALT（91.30±30.71vs50.20±12.13）、ALP（243.00±77.23vs161.30±37.16）、TG（2.25±0.31vs1.09±0.34）、TCHO（3.12±0.50vs2.48±0.36）、LDL（1.53±0.31vs1.16±0.13）、FFA（0.96±0.06vs0.56±0.13）、胰岛素水平（21.62±5.78vs14.13±3.26）和胰岛素抵抗指数（11.29±3.67vs5.77±1.76）显著降低，P值均小于0.05；而GGT、TBA和FBG变化无显著差异，P值均大于0.05。（4）HC组大鼠血清中ACE2、AngⅡ和Ang-(1-7)的水平均明显高于NC组，P值均小于0.05；与HC组比较，HF组大鼠血清AngⅡ（0.39±0.10vs0.12±0.13）的水平显著降低，血清ACE2（12.15±4.83vs22.65±5.65）和Ang-(1-7)（0.67±0.18vs1.58±72）水平显著升高，P值均小于0.05。（5）与NC组比较，HC组大鼠血清瘦素和瘦素/脂联素比值（L/A）比值显著升高（P <0.05），而脂联素水平显著降低（P <0.05）；与HC组比较，HF组大鼠血清瘦素水平（130.61±35.56vs31.74±36.35）和L/A（25.07±13.41vs1.67±2.07）显著降低，脂联素水平（5.97±2.19vs21.60±8.43）显著升高，P值均小于0.05。（6）HC组大鼠血清HA和TGF-β1显著高于NC组，P值均小于0.05；与HC组比较， HF组大鼠血清HA（165.72±29.13vs90.13±27.42）和TGF-β1（125.95±8.23vs72.55±23.25）水平显著降低，P值均小于0.05。（7）RT-PCR检测，HC组大鼠肝组织ACE2mRNA表达显著高于NC组（P<0.05）；与HC组比较，HF组大鼠肝组织ACE2mRNA表达水平显著升高（P<0.05）。（8）与NC组比较，HC组肝组织瘦素mRNA表达水平显著升高，脂联素表达水平显著降低，P值均小于0.05；与HC组比较，HF组大鼠肝组织瘦素mRNA表达水平显著降低，脂联素mRNA表达水平显著升高，P值均小于0.05。（9）与NC组比较，HC组白色脂肪组织瘦素mRNA表达水平显著升高，脂联素表达水平显著降低，P值均小于0.05；与HC组比较，HF组大鼠白色脂肪组织脂联素mRNA表达水平显著升高（P <0.05），瘦素表达差异无显著意义（P>0.05）。（10）Western blotting检测，与NC组比较，HC组大鼠肝组织ACE2、胶原蛋白I和α-SMA蛋白表达水平都显著升高，P值均小于0.05；与HC组比较，HF组大鼠肝组织ACE2蛋白的表达水平显著升高，胶原蛋白I和α-SMA蛋白表达水平显著降低，P值均小于0.05。结论：（1）本研究通过高脂饮食，成功构建NASH伴肝纤维化的大鼠模型。（2）福辛普利可显著改善NASH大鼠胰岛素抵抗、肝脂肪变性和肝纤维化。（3）福辛普利可升高NASH大鼠肝ACE2的表达。福辛普利可能通过增加NASH大鼠血清和肝ACE2的表达，降低AngⅡ的生成，进一步下调瘦素和上调脂联素的表达，从而延缓肝纤维化的发展。更多还原

【Abstract】 Background: Angiotensin (Ang)II, a major functional component ofthe renin-angiotensin system (RAS), plays an important role in thedevelopment of liver fibrosis. Angiotensin-converting enzyme (ACE) is thekey enzyme of AngII generation. ACE2degrades AngII to Ang-(1-7) andcounter-regulates AngII. AngII promotes leptin production and suppressesadiponectin production. Leptin and adiponectin have profibrotic andantifibrotic effects, respectively.Objective:(1) The aim of this research was to evaluate the effect of theangiotensin converting enzyme (ACE) inhibitor fosinopril on liver fibrosisin rats with high-fat diet (HFD)-induced nonalcoholic steatohepatitis(NASH).(2) To evaluate the effect of fosinopril on ACE2, leptin andadiponectin expression in rats with NASH.Methods: Forty male Sprague-Dawley rats were randomized into fourexperimental groups (n=10each): NC (control, normal diet), HC (HFD),NF (normal diet+fosinopril), and HF (HFD+fosinopril). Rats in the NFand HF groups were fed fosinopril (3.6mg/kg body weight/day by gavage) in addition to their respective diet. All rats were euthanized after24weekstreatment.(1) Body and liver weight were weighed. Ratio of liver/bodyweight also were counted.(2) Hematoxylin and eosin staining was used toassess the degree of hepatic steatosis, lobular inflammation, hepatocyteballooning, for counting Non-alcoholic fatty liver disease (NAFLD)activity score (NAS). Masson’s trichrome-staining was also used to assessthe stage of hepatic fibrosis.(3) Liver function, blood lipid and fastingblood glucose (FBG) were determined. Radioimmunoassay was used todetermine concentration of serum insulin. Homeostasis model assessmentof insulin resistance (HOMA-IR) was used to count insulin resistance index(IRI).(4) Enzyme-linked immuno sorbent assay (ELISA) was used todetermine concentration of serum ACE2, AngII, Ang-(1-7), leptin,adiponectin, Hyaluronic acid (HA) and Transforming growth factor(TGF)-β1.(5) Reverse transcription-polymerase chain reaction (RT-PCR)was used to determine the expression of ACE2, leptin and adiponectin inliver. White adipose tissue leptin and adiponectin expression also weredetermined.(6) Western blotting analysis was used to determine hepaticACE2, collagen I and α-smooth muscle actin (α-SMA) protein expression.Results:(1) Treatment with fosinopril significantly decreased the liverweight (32.61±8.65vs23.42±4.28, P <0.05) and ratio of liver/bodyweight (0.050±0.004vs0.042±0.004, P <0.05), but had no effect onbody weight (643.60±134.34vs553.00±63.41, P>0.05) in rats with NASH. (2) Treatment with fosinopril significantly decreased the score of steatosis(2.85±0.24vs1.40±0.52), lobular inflammation (2.85±0.24vs1.40±0.52),NAS (5.85±1.31vs3.00±0.82) and stage of liver fibrosis (3.80±0.75vs2.05±0.60; P <0.05, respectively), whereas no significant improvement wasobserved in hepatocyte ballooning (0.75±0.59vs0.40±0.39, P>0.05).(3)Treatment with fosinopril significantly decreased the concentrations ofserun ALT (91.30±30.715vs0.20±12.13), ALP (243.00±77.23vs161.30±37.16), TG (2.25±0.31vs1.09±0.34), TCHO (3.12±0.50vs2.48±0.36), LDL (1.53±0.31vs1.16±0.13), FFA (0.96±0.06vs0.56±0.13),insulin (21.62±5.78vs14.13±3.26) and IRI (11.29±3.67vs5.77±1.76; P<0.05, respectively), whereas had no effect on GGT, TBA, and FBG(P>0.05).(4) The concentrations of serumACE2, AngII and Ang-(1-7)were significantly higher in the HC group compared with the NC group (P<0.05, respectively); Treatment with fosinopril significantly decreased theconcentrations of serum AngII (0.39±0.10vs0.12±0.13), and increasedserum ACE2(12.15±4.83vs22.65±5.65) and Ang-(1-7)(0.67±0.18vs1.58±72) concentrations (P <0.05, respectively).(5) The concentrations ofserum leptin and the ratio of leptin/adiponectin (L/A) were significantlyhigher in the HC group compared with the NC group, whereas adiponectinwas significantly lower (P <0.05, respectively). Treatment with fosinoprilsignificantly decreased the concentrations of serum leptin (130.61±35.56vs31.74±36.35) and the L/A (25.07±13.41vs1.67±2.07); and increased serum adiponectin concentration significantly (5.97±2.19vs21.60±8.43; P <0.05,respectively).(6) Treatment with fosinopril significantly decreased theconcentrations of serum HA (165.72±29.10vs90.13±27.42) and TGF-β1(125.95±8.23vs72.55±23.25) in rats with NASH (P <0.05, respectively).(7) The hepatic ACE2mRNA expression were singnificantly higher in theHC group compared with the NC group (P <0.05, respectively). Treatmentwith fosinopril significantly increased ACE2mRNA expression in the liverof rats with NASH (P <0.05).(8) The mRNA expression of leptin wassignificantly higher (P <0.05), whereas adiponectin was singnificantlylower (P <0.05) in the liver of the HC group compared with the NC group.Treatment with fosinopril significantly decreased lepatin mRNA expression(P <0.05), and increased adiponectin mRNA expression in the liver of ratswith NASH (P <0.05).(9) In the rats white adipose tissue of the HC group,the mRNA expression of leptin was significantly higher (P <0.05), whereasadiponectin was singnificantly lower (P <0.05) compared with the NCgroup. Treatment with fosinopril significantly increased adiponectin mRNAexpression (P <0.05), but had no effect on lepatin mRNA expression(P>0.05) in the white adipose tissue of rats with NASH.(10) The hepaticprotein expression of ACE2, collagen I and α-SMA were singnificantlyhigher in the HC group compared with the NC group (P <0.05,respectively). Treatment with fosinopril significantly increased ACE2protein expression (P>0.05), and significantly decreased collagen I (P>0.05) and α-SMA (P>0.05) protein expression in the liver of rats withNASH.Conclusions:(1) In the study, HFD could induce SD rat with NASHsuccessfully.(2) ACEI fosinopril improves insulin resistance，favorablymodulates hepatic steatosis and liver fibrosis and leads to improvements inrats with NASH.(3) Fosinopril improves liver fibrosis by upregulating theexpression of hepatic ACE2. Furthermore, it is possible that fosinoprilregulates the progression of liver fibrosis through the downregulation ofleptin and the upregulation of adiponectin by upregulating of ACE2.更多还原