ACE2-Ang-（1-7）-Mas轴对肝星状细胞胶原合成的影响及其信号转导机制研究
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摘要
研究背景
肝纤维化是各种原因引起的慢性肝损伤结果,是肝硬化形成的病理基础。探讨肝纤维化的发生机制及防治方法具有重要的社会价值和经济意义。尽管现已认为肝纤维化在一定情况下可被逆转,但目前尚未有公认有效的逆转肝纤维化药物,因此肝纤维化治疗一直是慢性肝病研究的重点和难点。
本课题组前期研究及近年来国内外研究表明,肝内肾素-血管紧张素系统(renin-angiotensin system, RAS)与肝纤维化形成密切相关。慢性肝损伤导致肝内RAS的各组分上调,进一步可导致氧应激、炎症细胞聚集和肝纤维化形成。血管紧张素Ⅱ(angiotensinⅡ, AngⅡ)是这个系统的主要效应成员,它能刺激肝星状细胞(hepatic stellate cells, HSC)增殖与活化,并能释放炎症因子、生长因子以及促纤维化因子等,诱导HSC合成细胞外基质(extracellular matrix, ECM)及抑制ECM降解,导致ECM在肝内过度沉积,促使肝纤维化发生发展。近年来越来越多的证据表明,应用RAS的阻断剂可以减轻肝损伤、抑制肝纤维化形成和降低门静脉高压。
近年来,ACE2-Ang-(1-7)-Mas轴的发现,引起了许多研究者的浓厚兴趣。血管紧张素转换酶2 (angiotensin converting enzyme, ACE2)降解AngⅡ并同时产生Ang-(1-7). Ang-(1-7)在体内外能拮抗AngⅡ的活性,具有舒张血管、降低血压、利尿、抑制成纤维细胞增殖等作用。此外,Ang-(1-7)还可以竞争性地与AngⅡ的受体AT1R结合,因此被认为是一种内源性AngⅡ阻断剂。由于ACE2-Ang-(1-7)-Mas轴具有与经典RAS (ACE-AngⅡ-AT1R轴)相反的作用,因而被假设为具有抗纤维化作用,有可能成为肝纤维化治疗的新策略。
然而ACE2-Ang-(1-7)-Mas轴在肝纤维化中的研究少有报道。目前的研究报告发现,慢性肝损伤反应促发ACE2-Ang-(1-7)-Mas轴在肝内表达增强,而阻断这条轴的作用则会导致肝损害加重。然而,ACE2-Ang-(1-7)-Mas轴影响肝纤维化的诸多环节尚未得到充分阐述,具体机制尚不清楚。肝纤维化的特点是ECM的合成分泌增加和降解减少,导致ECM大量沉积致纤维结缔组织增生。胶原是ECM的主要成分。HSC是产生ECM的主要细胞,HSC活化、增殖、转型和分泌胶原在肝纤维化的发生发展中起关键作用。近来对纤维化信号机制的研究表明,Smad和Rho/ROCK信号通路是新发现与RAS作用相关的两个重要途径,与胶原合成密切相关。因此,本研究将观察ACE2-Ang-(1-7)-Mas轴在肝纤维化动物模型和培养细胞株HSC中的表达,探讨ACE2-Ang-(1-7)-Mas轴对HSC胶原合成及Smad和Rho/ROCK信号转导途径的影响,这对于阐明ACE2-Ang-(1-7)-Mas轴在肝纤维化中的作用及其机制有重要意义。
方法
1、利用四氯化碳(CCl4)造成肝中毒损伤,构建肝纤维化大鼠模型,通过HE染色法观察肝脏组织病理的改变,免疫组化方法观察ACE2在肝组织中的表达及分布,RT-PCR法检测ACE2和Mas受体mRNA的表达水平,Western blot法检测ACE2蛋白表达水平；以HSC为体外实验刺激对象,通过RT-PCR法和Western blot法,检测不同浓度AngⅡ干预时ACE2和Mas受体的表达水平。
2、应用AngⅡ与Ang-(1-7)干预HSC,及利用信号通路抑制剂阻断方法,通过Western blot法检测Smad3磷酸化表达水平,免疫荧光法观察Smad4核转位,应用地高辛标记的凝胶阻滞实验检测Smad-DNA结合活性；通过Western blot法检测RhoA蛋白表达水平,RT-PCR法检测ROCK2 mRNA水平。
3、应用慢病毒载体介导的RNA干扰技术,构建含针对ACE2基因的RNA干扰序列的慢病毒敲除载体,并感染HSC细胞株,real time PCR法和Western blot法检测干扰效率；通过Western blot、RT-PCR、基因定量(QuantiGene)等方法,检测AngⅡ和Ang-(1-7)干预及ACE2基因沉默前后HSC中COL1和CTGF表达的变化,同时利用信号通路抑制剂阻断方法,比较分析ACE2-Ang-(1-7)对HSC胶原合成的影响。
结果
1肝纤维化大鼠肝内ACE2和Mas表达增高
在给予大鼠皮下注射CCl44周后,HE染色见肝小叶结构破坏,肝纤维结节形成；免疫组化表明,肝脏ACE2表达水平明显增加,而且广泛分布；RT-PCR结果显示,与对照组相比,CCl4注射2周后大鼠肝内ACE2 mRNA水平轻度增加,但CCl4累积到4周时,ACE2水平显著增加(0.509±0.105 vs 0.114±0.044,P=0.000)。Western blot结果显示,ACE2蛋白表达水平变化与ACE2 mRNA表达水平相一致。Mas基因表达水平与ACE2的表达变化基本平行。此外,应用perindopril(一种ACE抑制剂(ACE inhibitor, ACEI))可促使肝损伤大鼠ACE2水平上调,提前出现ACE2增高,且增高水平较单独CCl4处理组显著(P=0.000)。
2 AngⅡ处理后HSC中ACE2和Mas表达增高
AngⅡ干预处理HSC 24hr后,ACE2及Mas mRNA的表达水平增加,且与不同浓度干预的AngⅡ呈剂量依赖性效应关系。当AngⅡ升至1μmol/L (μM)时两者表达与对照组比较均有显著性意义差别(分别为对照的1.553±0.090和1.536±0.053倍,均P<0.01)。Western blot分析进一步证实不同浓度AngⅡ处理后,HSC中ACE2蛋白水平呈剂量依赖性地增高。此外ACEI预处理组可使ACE2表达及Mas mRNA水平较AngⅡ处理组进一步增加(均P<0.01)。
3 Ang-(1-7)抑制AngⅡ激活HSC中Smad信号通路
在AngⅡ干预HSC 5min后,即可检测到磷酸化Smad3 (pSmad3)增加,与对照组相比差异有统计学意义(为对照的2.183±0.222倍,P=0.003),15 min时pSmad3蛋白水平达到高峰,之后pSmad3逐渐降低。AngⅡ干预0-120min内各时间点非磷酸化Smad3蛋白表达水平无明显变化。AngⅡ处理HSC 20 min后,免疫荧光检测可见Smad4蛋白明显的核转位；凝胶阻滞实验(EMSA)显示Smad DNA-结合活性较对照组显著增加(为对照的9.007±1.007倍,P=0.000)。单独Ang-(1-7)干预HSC与对照组相比无显著变化(均P>0.05)。Irbesartan (AT1R阻断剂)或Ang-(1-7)预处理HSC可抑制AngⅡ诱导的Smad3磷酸化(均P<0.01)、Smad4核转位以及Smad DNA-结合活性增加(均P<0.05),Mas受体阻断剂A779则明显逆转Ang-(1-7)对AngⅡ诱导Smad3磷酸化和Smad DNA结合活性增加(均P<0.01),提示AngⅡ通过AT1R途径迅速激活Smad通路,Ang-(1-7)则通过Mas受体发挥拮抗AngⅡ作用。SB203580 (p38 MAPK的抑制剂)预处理亦可显著抑制AngⅡ诱导Smad3磷酸化(P=0.000),而PD98059 (p42/44 MAPK或称ERK 1/2的抑制剂)预处理组则否(P=0.575)。提示AngⅡ快速激活Smad通路依赖于p38 MAPK的活化。
4 Ang-(1-7)抑制AngⅡ激活HSC中Rho/ROCK通路
AngⅡ干预处理HSC 24hr后,RhoA蛋白表达及ROCK2 mRNA水平较正常对照显著增高(均P=0.000), Irbesartan、Ang-(1-7)以及Y27632 (ROCK抑制剂)预处理组RhoA蛋白与ROCK2 mRNA表达均显著低于AngⅡ处理组(均P<0.01)。A779可以逆转Ang-(1-7)抑制AngⅡ诱导RhoA与ROCK2表达的作用(均P<0.01)。实验提示AngⅡ通过AT1R途径激活Rho/ROCK通路,Ang-(1-7)则通过Mas受体拮抗此作用。
5 ACE2-Ang-(1-7)抑制AngⅡ诱导HSC胶原合成
设计shRNA并构建慢病毒载体,验证HSC感染细胞株中针对ACE2基因的干扰效率,Western blot检测基因敲除后ACE2蛋白水平约为正常对照的2/3(0.154±0.018 vs 0.446±0.026,P=0.000),命名为Lenti-SiACE2。
分别利用RT-PCR检测Ⅰ型胶原(COL1) mRNA、Western blot检测结缔组织生长因子(CTGF)蛋白以及QuantiGene Plex方法分析COL1和CTGF mRNA的表达水平,结果提示AngⅡ作用24hr后,可诱导HSC中COL1和CTGF表达增加,与正常对照比较差异有统计学意义(均P<0.01)。Lenti-SiACE2感染细胞后COL1和CTGF表达与正常HSC对照无差别(P=1.000),但加入AngⅡ刺激后,它们的表达显著增强,较AngⅡ干预正常HSC细胞株更加明显(均P<0.01),提示ACE2基因干扰后AngⅡ促HSC胶原合成作用放大。Ang-(1-7)预处理可抑制AngⅡ诱导的HSC COL1和CTGF高表达(均P=0.000),此作用可被其Mas受体的阻断剂A779逆转(均P=0.000). QuantiGene Plex检测数据还提示,Ang-(1-7)对AngⅡ诱导COL1和CTGF mRNA表达的抑制程度较SB203580、PD98059以及Y27632对AngⅡ的这种抑制更加明显(与Y27632组CTGF表达比较P<0.05,其余均P<0.01)。提示HSC胶原合成有多种信号通路参与调节,Ang-(1-7)可通过全面阻断AngⅡ的活性而抑制HSC胶原合成。
结论
1、随着肝纤维化进展大鼠肝内ACE2和Mas受体表达增强,可能是机体对肝损伤应激的一种自我保护机制。在体外HSC中ACE2和Mas受体表达随AngⅡ浓度增加而增高,推测这是HSC中RAS维持系统平衡的反馈调节机制,即对过多AngⅡ的拮抗反应。此结果支持ACE2-Ang-(1-7)-Mas轴对肝纤维化起保护作用的假设。ACEI上调ACE2的表达,可能有延缓肝纤维化进展的作用。
2、AngⅡ可通过其受体ATlR激活HSC中Smad和Rho/ROCK信号通路,这可能是AngⅡ促肝纤维化的重要机制。Ang-(1-7)可通过拮抗AngⅡ的活性而抑制AngⅡ对这两个通路的激活。另外,p38 MAPK的抑制剂可以减少AngⅡ诱导的Smad3磷酸化,提示AngⅡ可以经非TGF-β依赖途径直接激活Smad信号通路。
3、AngⅡ刺激可显著增加HSC中COL1和CTGF的表达,ACE2基因沉默后加重AngⅡ对这两者的诱导表达作用,而Ang-(1-7)则可抑制AngⅡ对它们的诱导表达,且其抑制程度较p38 MAPK, ERK 1/2和Rho/ROCK的阻断剂对AngⅡ诱导COL1和CTGF表达的抑制作用更加明显。结果提示ACE2-Ang-(1-7)可通过拮抗AngⅡ的作用而明显抑制HSC中胶原的合成,起着重要的抗纤维化作用,这对于扩展肝纤维化治疗新策略具有重要意义。
Background & Objectives
It is well known that liver fibrosis is a result of chronic hepatocellular damage due to a variety of liver diseases, and it usually progresses to liver cirrhosis. Liver fibrosis has traditionally been regarded as an irreversible process. However, increasing evidence indicates that even advanced fibrosis may, in fact, be a reversible condition. Yet till now there is no effective treatment for liver fibrosis. Therefore, it is quite important and valuable to investigate underlying mechanisms and to develop more effective preventive and therapeutic interventions for liver fibrosis.
Recent reports and our previous studies have shown that intrahepatic renin-angiotensin system (RAS) is closely related with liver fibrosis. Experimental evidence indicates that there is marked upregulation of intrahepatic RAS components in experimental liver injury, which will further lead to oxidative stress, accumulation of inflammatory cells, and finally, development of liver fibrosis. Angiotensin II (Ang II) is the most important effector of this system. It induces the activation and proliferation of hepatic stellate cells (HSC), releases some inflammatory cytokines, growth factors and pro-fibrosis factors, and stimulates extracellular matrix (ECM) production and inhibits its degradation, leading to excessive deposition of ECM in liver, thus liver fibrosis progression occurs. Recent evidence has shown that blocking of RAS may reduce liver damage, alleviate fibrogenesis and relieve portal hypertension.
More recently, the newly found ACE2-Ang-(1-7)-Mas axis has aroused great interest among many researchers. Angiotensin-converting enzyme 2 (ACE2) degrades AngⅡand simultaneously produces Ang-(1-7).Ang-(1-7) counterworks the effects of AngⅡby dilation of blood vessels, down-regulation of blood pressure, diuresis, and inhibition of fibroblast proliferation, and other roles. In addition, Ang-(1-7) may also competitively bind to the AngⅡtype 1 receptor (AT1R). Therefore Ang-(1-7) has been considered as an endogenous antagonist of AngⅡ. As the new ACE2-Ang-(1-7)-Mas axis may counter-regulate the classical RAS (ACE-AngⅡ-AT1R axis), it is presumed to have a role in reducing fibrosis, and is expected to be a new potential target for treatment of liver fibrosis.
However, the role of the ACE2-Ang-(1-7)-Mas axis in liver fibrosis has been rarely reported. Recent several reports demonstrated that the intrahepatic ACE2-Ang-(1-7)-Mas axis was up-regulated during liver injury, and that blocking of this axis worsened liver damage. However, the direct effects of the ACE2-Ang-(1-7)-Mas axis in hepatic fibrosis and the underlying mechanisms have not been elucidated. Liver fibrosis is characterized by activation of hepatic stellate cells, which are then involved in synthesis of matrix proteins and in regulating matrix degradation. It has been widely believed that HSC are the main cells producing ECM and collagen is the main component of ECM. Recent studies suggest that two newly discovered signaling pathways, Smad and Rho/ROCK pathways, are strongly related with the profibrogenic role of the RAS, and also closely associated with collagen synthesis. In our study, we observe the expression of the ACE2-Ang-(1-7)-Mas axis in animal models and in cultured HSC, and then evaluate the effects of this novel axis on Smad and Rho/ROCK signal transduction pathways, as well as on collagen synthesis in HSC. We hope that these investigations will help to understand the roles and mechanisms of the ACE2-Ang-(1-7)-Mas axis in liver fibrosis.
Materials and Methods
1. Liver fibrosis was induced in rats by repeated injections of 40% CCl4 (2.5mL/kg, twice per week). The pathological changes of rat livers were evaluated by HE staining. ACE2 immunoreactivity was assessed by immunohistochemical staining, ACE2 protein expression was detected by Western blot, ACE2 and Mas mRNA expression was determined by reverse transcription-polymerase chain reaction (RT-PCR). As an in vitro study, HSC-T6 were treated with AngⅡat concentrations ranging from 0.1 to 10×10-6mol/L. Western blot and RT-PCR were used to measure ACE2 expression and Mas mRNA levels in HSC.
2. HSC-LX-2 were treated with AngⅡor Ang-(1-7) alone or together. Some cells were pretreated with Irbesartan (AT1R antagonist), A779 (Mas antagonist), SB203580 (p38 MAPK inhibitor), PD98059 (ERK inhibitor), or Y27632 (ROCK inhibitor) before AngⅡtreatment. The phosphorylated-Smad3 protein expression level was analyzed by Western blot, nuclear translocation of Smad4 was observed by immunofluorescence microscopy, and Smad DNA binding activity was assessed by electrophoretic mobility shift assay (EMSA) using digoxin labeled double-stranded oligonucleotide containing Smad consensus sequence. Western blot and RT-PCR were used to examine RhoA protein and ROCK2 mRNA expression levels, respectively.
3. A lentiviral vector encoding a short hairpin RNA specifically targeting human ACE2 was constructed, and the silencing efficiency was determined by real time PCR and Western blot in HSC-LX-2 after transduction with the lentiviral vector. RT-PCR, Western blot and Gene quantification (QuantiGene Plex) were performed to analyze the expression of collagen type 1 (COL1) and connective tissue growth factor (CTGF) in HSC treated with AngⅡ, Ang-(1-7) or after knock-down of ACE2 gene. Inhibitors of p38 MAPK, ERK or ROCK were employed to assess the effects of these signaling pathways on the expression of COL1 and CTGF mRNA in HSC.
Results
1. The expression of ACE2 and Mas was increased in CCl4-intoxicated rat liver.
Hematoxylin-eosin (HE) staining revealed that, in rat livers after subcutaneous injection of CCl4 for 4 weeks, the normal structure of hepatic lobules was destroyed and the regenerative nodules were separated by dense fibrous septa. Immunohisto-chemical staining showed that intrahepatic ACE2 expression was increased and redistribution after 4 weeks of CCl4 exposure. RT-PCR showed that ACE2 mRNA expression levels were mildly increased after 2 weeks of CCl4 administration, and markedly increased at the 4th week (0.509±0.105 vs 0.114±0.044, P=0.000). ACE2 protein expression as determined by Western blotting mirrored the changes observed in ACE2 mRNA levels. In addition, in rats treated with both CCl4 and perindopril, ACE2 expression were significantly increased by 2 weeks as compared with rats receiving CCl4 alone (P<0.01), and showed a further slight increase by week 4. Mas gene expression followed a similar pattern to that seen for ACE2 in all three groups of rats.
2. The expression of ACE2 and Mas was increased in AngⅡ-treated HSC.
Incubation of HSC with different concentrations of AngⅡfor 24 hr led to a dose-dependent increase in ACE2 and Mas mRNA expression. Although these increases were only slight following treatment with 0.1μmol/L Angll and did not reach statistical significance, they were marked following treatment of HSC with 1μM AngⅡ(1.553±0.090 and 1.536±0.053 fold over control, respectively, both P< 0.01), and increased further at 10μM. Western blot analysis showed similar changes in ACE2 protein. Besides, ACE2 and Mas expression was significantly increased in HSC simultaneously administered with AngⅡand ACEI as compared with cells treated with AngⅡalone (P<0.01).
3. Ang-(1-7) inhibited AngⅡ-induced activation of Smad pathway in HSC.
The results of Western blot showed that AngⅡinduced phosphorylation of Smad3 (pSmac3) in HSC in a time-dependent manner. The levels of pSmad3 were rapidly increased at 5 min (2.183±0.222 fold of control, P=0.003), and reached with a peak at 15 min, then gradually declined. No change was observed in the levels of non-phosphorylation of Smad3 protein expression. Immunofluorescence assay showed that AngⅡinduced translocation of Smad4 from cytoplasm to nuclear, while gel shift assay (EMSA) showed that Ang II elevated Smad DNA binding activity (9.007±1.007 fold over control, P=0.000). Ang-(1-7) alone showed little impact on the Smad pathway in HSC. However, it inhibited AngⅡ-induced phosphorylation of Smad3 (P<0.01), nuclear translocation of Smad4 and elevation of Smad DNA-binding activity (P<0.05) in HSC. Mas receptor antagonist A779 significantly reversed the inhibiting effect of Ang-(1-7) on AngⅡinduced pSmad3 expression and Smad DNA-binding activity (P<0.01). Irbesartan (AT1R blocker) also potently restrained the activation of Smad pathways induced by AngⅡ. These results indicated that AngⅡactivates the Smad pathway through AT1R, and Ang-(1-7) plays an opposite role in Ang II-induced Smad pathway through Mas receptor. In addition, the levels of AngⅡ-induced pSmad3 were significantly lowered by SB203580 (p38 MAPK inhibitor) (P=0.000), but not by PD98059 (ERK inhibitor) (P=0.575), indicating that Ang II activates Smad signaling pathway at least partly via the p38 MAPK pathway.
4. Ang-(1-7) inhibited AngⅡ-induced activation of Rho/ROCK pathway in HSC.
The expression levels of RhoA protein and ROCK2 mRNA were significantly increased in HSC after treated with Ang II for 24 hr, as compared with controls (both P=0.000). Irbesartan, Ang-(1-7) and Y27632 (ROCK inhibitor) markedly abrogated the effect of Ang II on RhoA protein and ROCK2 mRNA expression (all P<0.01). A779 reversed the inhibition of Ang-(1-7) on AngⅡ-induced activation of RhoA and ROCK2 (both P<0.01). These results suggested that AngⅡactivates Rho/ROCK pathway through AT1R, and Ang-(1-7) antagonizes this effect of AngⅡthrough Mas receptor.
5. ACE2-Ang-(1-7) inhibited AngⅡ-induced collagen synthesis in HSC.
A lentiviral vector encoding a shRNA directed against ACE2 was designed and constructed, and then transferred into HSC. The know-down efficiency was near 70% as determined by real-time PCR at the mRNA levels and Western blot at protein levels (0.154±0.018 vs 0.446±0.026, P=0.000). This vector was named as Lenti-SiACE2.
The expression of COL1 mRNA and CTGF protein were evaluated by RT-PCR and Western blot respectively, and their mRNA expression levels were further analyzed by QuantiGene Plex assay. The results showed that AngⅡstimulated significant increase of COL1 and CTGF expression levels in HSC as compared with controls (both P<0.01). The expression levels of COL1 and CTGF were little changed in cells transferred with Lenti-SiACE2 (P=1.000), but they were significantly increased following AngⅡstimulation, as compared with normal HSC treated with AngⅡ(both P<0.01). This result indicated that knock-down of ACE2 prolongs AngⅡ-stimulated collagen synthesis. Ang-(1-7) abolished AngⅡ-induced COL1 and CTGF expression (both P<0.01), which was reversed by Mas receptor antagonist A779 (both P<0.01). Besides, QuantiGene assay showed that the degree of inhibition of Ang-(1-7) on AngⅡ-induced upregulation of COL1 and CTGF mRNA was more pronounced than that of SB203580, PD98059 and Y27632 (all P< 0.01, except P<0.05 compared with the CTGF mRNA level in HSC pre-treated with Y27632). These data indicated that collagen synthesis in HSC may involve a variety of pathways, yet it can be dramatically inhibited by Ang-(1-7) through directly against AngⅡ.
Conclusions
1. In the present study, we observed an increase in hepatic ACE2 and Mas receptor expression with the progression of liver fibrosis induced by CCl4 in rats. Furthermore, in our in vitro experiments, we found that AngⅡupregulated ACE2 and Mas expression in a dose-dependent manner in HSC. The changes in ACE2 and Mas expression indicate that it may be a resistance response to the augmented levels of AngⅡ, and may further represent a negative feedback mechanism involved in maintaining equilibrium in the intrahepatic RAS. Therefore, our results support the previous hypothesis that the ACE2-Ang-(1-7)-Mas axis may play a protective role in liver fibrosis. Besides, our study showed that ACEI is able to upregulate ACE2 expression under conditions of liver injury, both in vivo and in vitro, which may in turn contribute to additional benefits of ACEI on liver fibrosis.
2. Our investigation showed that AngⅡactivated the Smad and Rho/ROCK signaling pathways in HSC through interacting with its receptor AT1R, which may be important mechanisms of the pro-fibrosis effect of AngⅡ. Ang-(1-7) inhibited the activation of these two pathways through counteracting with AngⅡ, which may be attributed to its anti-fibrosis function in liver. In addition, p38 MAPK inhibitor reduced the AngⅡ-mediated phosphorylation of Smad3, suggesting that Ang II can directly activate the Smad signaling pathway in a TGF-β-independent manner.
3. We also found that AngⅡstimulation significantly increased COL1 and CTGF expression in HSC. Silencing of ACE2 further enhanced AngⅡ-induced overexpression of these two effectors. Ang-(1-7) abated the activation of COL1 and CTGF caused by AngⅡ, and this inhibiting effect was stronger than that of p38 MAPK, ERK 1/2 or Rho/ROCK inhibitors. These results indicated that ACE2 and Ang-(1-7) can suppress collagen synthesis in HSC through antagonizing the effect of Ang II. Therefore, our study provides experimental evidence that the new ACE2-Ang-(1-7)-Mas axis has a protective role in liver, and thus, it can be a promising target for the therapy of liver fibrosis.
肝纤维化是各种原因引起的慢性肝损伤结果,是肝硬化形成的病理基础。探讨肝纤维化的发生机制及防治方法具有重要的社会价值和经济意义。尽管现已认为肝纤维化在一定情况下可被逆转,但目前尚未有公认有效的逆转肝纤维化药物,因此肝纤维化治疗一直是慢性肝病研究的重点和难点。
本课题组前期研究及近年来国内外研究表明,肝内肾素-血管紧张素系统(renin-angiotensin system, RAS)与肝纤维化形成密切相关。慢性肝损伤导致肝内RAS的各组分上调,进一步可导致氧应激、炎症细胞聚集和肝纤维化形成。血管紧张素Ⅱ(angiotensinⅡ, AngⅡ)是这个系统的主要效应成员,它能刺激肝星状细胞(hepatic stellate cells, HSC)增殖与活化,并能释放炎症因子、生长因子以及促纤维化因子等,诱导HSC合成细胞外基质(extracellular matrix, ECM)及抑制ECM降解,导致ECM在肝内过度沉积,促使肝纤维化发生发展。近年来越来越多的证据表明,应用RAS的阻断剂可以减轻肝损伤、抑制肝纤维化形成和降低门静脉高压。
近年来,ACE2-Ang-(1-7)-Mas轴的发现,引起了许多研究者的浓厚兴趣。血管紧张素转换酶2 (angiotensin converting enzyme, ACE2)降解AngⅡ并同时产生Ang-(1-7). Ang-(1-7)在体内外能拮抗AngⅡ的活性,具有舒张血管、降低血压、利尿、抑制成纤维细胞增殖等作用。此外,Ang-(1-7)还可以竞争性地与AngⅡ的受体AT1R结合,因此被认为是一种内源性AngⅡ阻断剂。由于ACE2-Ang-(1-7)-Mas轴具有与经典RAS (ACE-AngⅡ-AT1R轴)相反的作用,因而被假设为具有抗纤维化作用,有可能成为肝纤维化治疗的新策略。
然而ACE2-Ang-(1-7)-Mas轴在肝纤维化中的研究少有报道。目前的研究报告发现,慢性肝损伤反应促发ACE2-Ang-(1-7)-Mas轴在肝内表达增强,而阻断这条轴的作用则会导致肝损害加重。然而,ACE2-Ang-(1-7)-Mas轴影响肝纤维化的诸多环节尚未得到充分阐述,具体机制尚不清楚。肝纤维化的特点是ECM的合成分泌增加和降解减少,导致ECM大量沉积致纤维结缔组织增生。胶原是ECM的主要成分。HSC是产生ECM的主要细胞,HSC活化、增殖、转型和分泌胶原在肝纤维化的发生发展中起关键作用。近来对纤维化信号机制的研究表明,Smad和Rho/ROCK信号通路是新发现与RAS作用相关的两个重要途径,与胶原合成密切相关。因此,本研究将观察ACE2-Ang-(1-7)-Mas轴在肝纤维化动物模型和培养细胞株HSC中的表达,探讨ACE2-Ang-(1-7)-Mas轴对HSC胶原合成及Smad和Rho/ROCK信号转导途径的影响,这对于阐明ACE2-Ang-(1-7)-Mas轴在肝纤维化中的作用及其机制有重要意义。
方法
1、利用四氯化碳(CCl4)造成肝中毒损伤,构建肝纤维化大鼠模型,通过HE染色法观察肝脏组织病理的改变,免疫组化方法观察ACE2在肝组织中的表达及分布,RT-PCR法检测ACE2和Mas受体mRNA的表达水平,Western blot法检测ACE2蛋白表达水平；以HSC为体外实验刺激对象,通过RT-PCR法和Western blot法,检测不同浓度AngⅡ干预时ACE2和Mas受体的表达水平。
2、应用AngⅡ与Ang-(1-7)干预HSC,及利用信号通路抑制剂阻断方法,通过Western blot法检测Smad3磷酸化表达水平,免疫荧光法观察Smad4核转位,应用地高辛标记的凝胶阻滞实验检测Smad-DNA结合活性；通过Western blot法检测RhoA蛋白表达水平,RT-PCR法检测ROCK2 mRNA水平。
3、应用慢病毒载体介导的RNA干扰技术,构建含针对ACE2基因的RNA干扰序列的慢病毒敲除载体,并感染HSC细胞株,real time PCR法和Western blot法检测干扰效率；通过Western blot、RT-PCR、基因定量(QuantiGene)等方法,检测AngⅡ和Ang-(1-7)干预及ACE2基因沉默前后HSC中COL1和CTGF表达的变化,同时利用信号通路抑制剂阻断方法,比较分析ACE2-Ang-(1-7)对HSC胶原合成的影响。
结果
1肝纤维化大鼠肝内ACE2和Mas表达增高
在给予大鼠皮下注射CCl44周后,HE染色见肝小叶结构破坏,肝纤维结节形成；免疫组化表明,肝脏ACE2表达水平明显增加,而且广泛分布；RT-PCR结果显示,与对照组相比,CCl4注射2周后大鼠肝内ACE2 mRNA水平轻度增加,但CCl4累积到4周时,ACE2水平显著增加(0.509±0.105 vs 0.114±0.044,P=0.000)。Western blot结果显示,ACE2蛋白表达水平变化与ACE2 mRNA表达水平相一致。Mas基因表达水平与ACE2的表达变化基本平行。此外,应用perindopril(一种ACE抑制剂(ACE inhibitor, ACEI))可促使肝损伤大鼠ACE2水平上调,提前出现ACE2增高,且增高水平较单独CCl4处理组显著(P=0.000)。
2 AngⅡ处理后HSC中ACE2和Mas表达增高
AngⅡ干预处理HSC 24hr后,ACE2及Mas mRNA的表达水平增加,且与不同浓度干预的AngⅡ呈剂量依赖性效应关系。当AngⅡ升至1μmol/L (μM)时两者表达与对照组比较均有显著性意义差别(分别为对照的1.553±0.090和1.536±0.053倍,均P<0.01)。Western blot分析进一步证实不同浓度AngⅡ处理后,HSC中ACE2蛋白水平呈剂量依赖性地增高。此外ACEI预处理组可使ACE2表达及Mas mRNA水平较AngⅡ处理组进一步增加(均P<0.01)。
3 Ang-(1-7)抑制AngⅡ激活HSC中Smad信号通路
在AngⅡ干预HSC 5min后,即可检测到磷酸化Smad3 (pSmad3)增加,与对照组相比差异有统计学意义(为对照的2.183±0.222倍,P=0.003),15 min时pSmad3蛋白水平达到高峰,之后pSmad3逐渐降低。AngⅡ干预0-120min内各时间点非磷酸化Smad3蛋白表达水平无明显变化。AngⅡ处理HSC 20 min后,免疫荧光检测可见Smad4蛋白明显的核转位；凝胶阻滞实验(EMSA)显示Smad DNA-结合活性较对照组显著增加(为对照的9.007±1.007倍,P=0.000)。单独Ang-(1-7)干预HSC与对照组相比无显著变化(均P>0.05)。Irbesartan (AT1R阻断剂)或Ang-(1-7)预处理HSC可抑制AngⅡ诱导的Smad3磷酸化(均P<0.01)、Smad4核转位以及Smad DNA-结合活性增加(均P<0.05),Mas受体阻断剂A779则明显逆转Ang-(1-7)对AngⅡ诱导Smad3磷酸化和Smad DNA结合活性增加(均P<0.01),提示AngⅡ通过AT1R途径迅速激活Smad通路,Ang-(1-7)则通过Mas受体发挥拮抗AngⅡ作用。SB203580 (p38 MAPK的抑制剂)预处理亦可显著抑制AngⅡ诱导Smad3磷酸化(P=0.000),而PD98059 (p42/44 MAPK或称ERK 1/2的抑制剂)预处理组则否(P=0.575)。提示AngⅡ快速激活Smad通路依赖于p38 MAPK的活化。
4 Ang-(1-7)抑制AngⅡ激活HSC中Rho/ROCK通路
AngⅡ干预处理HSC 24hr后,RhoA蛋白表达及ROCK2 mRNA水平较正常对照显著增高(均P=0.000), Irbesartan、Ang-(1-7)以及Y27632 (ROCK抑制剂)预处理组RhoA蛋白与ROCK2 mRNA表达均显著低于AngⅡ处理组(均P<0.01)。A779可以逆转Ang-(1-7)抑制AngⅡ诱导RhoA与ROCK2表达的作用(均P<0.01)。实验提示AngⅡ通过AT1R途径激活Rho/ROCK通路,Ang-(1-7)则通过Mas受体拮抗此作用。
5 ACE2-Ang-(1-7)抑制AngⅡ诱导HSC胶原合成
设计shRNA并构建慢病毒载体,验证HSC感染细胞株中针对ACE2基因的干扰效率,Western blot检测基因敲除后ACE2蛋白水平约为正常对照的2/3(0.154±0.018 vs 0.446±0.026,P=0.000),命名为Lenti-SiACE2。
分别利用RT-PCR检测Ⅰ型胶原(COL1) mRNA、Western blot检测结缔组织生长因子(CTGF)蛋白以及QuantiGene Plex方法分析COL1和CTGF mRNA的表达水平,结果提示AngⅡ作用24hr后,可诱导HSC中COL1和CTGF表达增加,与正常对照比较差异有统计学意义(均P<0.01)。Lenti-SiACE2感染细胞后COL1和CTGF表达与正常HSC对照无差别(P=1.000),但加入AngⅡ刺激后,它们的表达显著增强,较AngⅡ干预正常HSC细胞株更加明显(均P<0.01),提示ACE2基因干扰后AngⅡ促HSC胶原合成作用放大。Ang-(1-7)预处理可抑制AngⅡ诱导的HSC COL1和CTGF高表达(均P=0.000),此作用可被其Mas受体的阻断剂A779逆转(均P=0.000). QuantiGene Plex检测数据还提示,Ang-(1-7)对AngⅡ诱导COL1和CTGF mRNA表达的抑制程度较SB203580、PD98059以及Y27632对AngⅡ的这种抑制更加明显(与Y27632组CTGF表达比较P<0.05,其余均P<0.01)。提示HSC胶原合成有多种信号通路参与调节,Ang-(1-7)可通过全面阻断AngⅡ的活性而抑制HSC胶原合成。
结论
1、随着肝纤维化进展大鼠肝内ACE2和Mas受体表达增强,可能是机体对肝损伤应激的一种自我保护机制。在体外HSC中ACE2和Mas受体表达随AngⅡ浓度增加而增高,推测这是HSC中RAS维持系统平衡的反馈调节机制,即对过多AngⅡ的拮抗反应。此结果支持ACE2-Ang-(1-7)-Mas轴对肝纤维化起保护作用的假设。ACEI上调ACE2的表达,可能有延缓肝纤维化进展的作用。
2、AngⅡ可通过其受体ATlR激活HSC中Smad和Rho/ROCK信号通路,这可能是AngⅡ促肝纤维化的重要机制。Ang-(1-7)可通过拮抗AngⅡ的活性而抑制AngⅡ对这两个通路的激活。另外,p38 MAPK的抑制剂可以减少AngⅡ诱导的Smad3磷酸化,提示AngⅡ可以经非TGF-β依赖途径直接激活Smad信号通路。
3、AngⅡ刺激可显著增加HSC中COL1和CTGF的表达,ACE2基因沉默后加重AngⅡ对这两者的诱导表达作用,而Ang-(1-7)则可抑制AngⅡ对它们的诱导表达,且其抑制程度较p38 MAPK, ERK 1/2和Rho/ROCK的阻断剂对AngⅡ诱导COL1和CTGF表达的抑制作用更加明显。结果提示ACE2-Ang-(1-7)可通过拮抗AngⅡ的作用而明显抑制HSC中胶原的合成,起着重要的抗纤维化作用,这对于扩展肝纤维化治疗新策略具有重要意义。
Background & Objectives
It is well known that liver fibrosis is a result of chronic hepatocellular damage due to a variety of liver diseases, and it usually progresses to liver cirrhosis. Liver fibrosis has traditionally been regarded as an irreversible process. However, increasing evidence indicates that even advanced fibrosis may, in fact, be a reversible condition. Yet till now there is no effective treatment for liver fibrosis. Therefore, it is quite important and valuable to investigate underlying mechanisms and to develop more effective preventive and therapeutic interventions for liver fibrosis.
Recent reports and our previous studies have shown that intrahepatic renin-angiotensin system (RAS) is closely related with liver fibrosis. Experimental evidence indicates that there is marked upregulation of intrahepatic RAS components in experimental liver injury, which will further lead to oxidative stress, accumulation of inflammatory cells, and finally, development of liver fibrosis. Angiotensin II (Ang II) is the most important effector of this system. It induces the activation and proliferation of hepatic stellate cells (HSC), releases some inflammatory cytokines, growth factors and pro-fibrosis factors, and stimulates extracellular matrix (ECM) production and inhibits its degradation, leading to excessive deposition of ECM in liver, thus liver fibrosis progression occurs. Recent evidence has shown that blocking of RAS may reduce liver damage, alleviate fibrogenesis and relieve portal hypertension.
More recently, the newly found ACE2-Ang-(1-7)-Mas axis has aroused great interest among many researchers. Angiotensin-converting enzyme 2 (ACE2) degrades AngⅡand simultaneously produces Ang-(1-7).Ang-(1-7) counterworks the effects of AngⅡby dilation of blood vessels, down-regulation of blood pressure, diuresis, and inhibition of fibroblast proliferation, and other roles. In addition, Ang-(1-7) may also competitively bind to the AngⅡtype 1 receptor (AT1R). Therefore Ang-(1-7) has been considered as an endogenous antagonist of AngⅡ. As the new ACE2-Ang-(1-7)-Mas axis may counter-regulate the classical RAS (ACE-AngⅡ-AT1R axis), it is presumed to have a role in reducing fibrosis, and is expected to be a new potential target for treatment of liver fibrosis.
However, the role of the ACE2-Ang-(1-7)-Mas axis in liver fibrosis has been rarely reported. Recent several reports demonstrated that the intrahepatic ACE2-Ang-(1-7)-Mas axis was up-regulated during liver injury, and that blocking of this axis worsened liver damage. However, the direct effects of the ACE2-Ang-(1-7)-Mas axis in hepatic fibrosis and the underlying mechanisms have not been elucidated. Liver fibrosis is characterized by activation of hepatic stellate cells, which are then involved in synthesis of matrix proteins and in regulating matrix degradation. It has been widely believed that HSC are the main cells producing ECM and collagen is the main component of ECM. Recent studies suggest that two newly discovered signaling pathways, Smad and Rho/ROCK pathways, are strongly related with the profibrogenic role of the RAS, and also closely associated with collagen synthesis. In our study, we observe the expression of the ACE2-Ang-(1-7)-Mas axis in animal models and in cultured HSC, and then evaluate the effects of this novel axis on Smad and Rho/ROCK signal transduction pathways, as well as on collagen synthesis in HSC. We hope that these investigations will help to understand the roles and mechanisms of the ACE2-Ang-(1-7)-Mas axis in liver fibrosis.
Materials and Methods
1. Liver fibrosis was induced in rats by repeated injections of 40% CCl4 (2.5mL/kg, twice per week). The pathological changes of rat livers were evaluated by HE staining. ACE2 immunoreactivity was assessed by immunohistochemical staining, ACE2 protein expression was detected by Western blot, ACE2 and Mas mRNA expression was determined by reverse transcription-polymerase chain reaction (RT-PCR). As an in vitro study, HSC-T6 were treated with AngⅡat concentrations ranging from 0.1 to 10×10-6mol/L. Western blot and RT-PCR were used to measure ACE2 expression and Mas mRNA levels in HSC.
2. HSC-LX-2 were treated with AngⅡor Ang-(1-7) alone or together. Some cells were pretreated with Irbesartan (AT1R antagonist), A779 (Mas antagonist), SB203580 (p38 MAPK inhibitor), PD98059 (ERK inhibitor), or Y27632 (ROCK inhibitor) before AngⅡtreatment. The phosphorylated-Smad3 protein expression level was analyzed by Western blot, nuclear translocation of Smad4 was observed by immunofluorescence microscopy, and Smad DNA binding activity was assessed by electrophoretic mobility shift assay (EMSA) using digoxin labeled double-stranded oligonucleotide containing Smad consensus sequence. Western blot and RT-PCR were used to examine RhoA protein and ROCK2 mRNA expression levels, respectively.
3. A lentiviral vector encoding a short hairpin RNA specifically targeting human ACE2 was constructed, and the silencing efficiency was determined by real time PCR and Western blot in HSC-LX-2 after transduction with the lentiviral vector. RT-PCR, Western blot and Gene quantification (QuantiGene Plex) were performed to analyze the expression of collagen type 1 (COL1) and connective tissue growth factor (CTGF) in HSC treated with AngⅡ, Ang-(1-7) or after knock-down of ACE2 gene. Inhibitors of p38 MAPK, ERK or ROCK were employed to assess the effects of these signaling pathways on the expression of COL1 and CTGF mRNA in HSC.
Results
1. The expression of ACE2 and Mas was increased in CCl4-intoxicated rat liver.
Hematoxylin-eosin (HE) staining revealed that, in rat livers after subcutaneous injection of CCl4 for 4 weeks, the normal structure of hepatic lobules was destroyed and the regenerative nodules were separated by dense fibrous septa. Immunohisto-chemical staining showed that intrahepatic ACE2 expression was increased and redistribution after 4 weeks of CCl4 exposure. RT-PCR showed that ACE2 mRNA expression levels were mildly increased after 2 weeks of CCl4 administration, and markedly increased at the 4th week (0.509±0.105 vs 0.114±0.044, P=0.000). ACE2 protein expression as determined by Western blotting mirrored the changes observed in ACE2 mRNA levels. In addition, in rats treated with both CCl4 and perindopril, ACE2 expression were significantly increased by 2 weeks as compared with rats receiving CCl4 alone (P<0.01), and showed a further slight increase by week 4. Mas gene expression followed a similar pattern to that seen for ACE2 in all three groups of rats.
2. The expression of ACE2 and Mas was increased in AngⅡ-treated HSC.
Incubation of HSC with different concentrations of AngⅡfor 24 hr led to a dose-dependent increase in ACE2 and Mas mRNA expression. Although these increases were only slight following treatment with 0.1μmol/L Angll and did not reach statistical significance, they were marked following treatment of HSC with 1μM AngⅡ(1.553±0.090 and 1.536±0.053 fold over control, respectively, both P< 0.01), and increased further at 10μM. Western blot analysis showed similar changes in ACE2 protein. Besides, ACE2 and Mas expression was significantly increased in HSC simultaneously administered with AngⅡand ACEI as compared with cells treated with AngⅡalone (P<0.01).
3. Ang-(1-7) inhibited AngⅡ-induced activation of Smad pathway in HSC.
The results of Western blot showed that AngⅡinduced phosphorylation of Smad3 (pSmac3) in HSC in a time-dependent manner. The levels of pSmad3 were rapidly increased at 5 min (2.183±0.222 fold of control, P=0.003), and reached with a peak at 15 min, then gradually declined. No change was observed in the levels of non-phosphorylation of Smad3 protein expression. Immunofluorescence assay showed that AngⅡinduced translocation of Smad4 from cytoplasm to nuclear, while gel shift assay (EMSA) showed that Ang II elevated Smad DNA binding activity (9.007±1.007 fold over control, P=0.000). Ang-(1-7) alone showed little impact on the Smad pathway in HSC. However, it inhibited AngⅡ-induced phosphorylation of Smad3 (P<0.01), nuclear translocation of Smad4 and elevation of Smad DNA-binding activity (P<0.05) in HSC. Mas receptor antagonist A779 significantly reversed the inhibiting effect of Ang-(1-7) on AngⅡinduced pSmad3 expression and Smad DNA-binding activity (P<0.01). Irbesartan (AT1R blocker) also potently restrained the activation of Smad pathways induced by AngⅡ. These results indicated that AngⅡactivates the Smad pathway through AT1R, and Ang-(1-7) plays an opposite role in Ang II-induced Smad pathway through Mas receptor. In addition, the levels of AngⅡ-induced pSmad3 were significantly lowered by SB203580 (p38 MAPK inhibitor) (P=0.000), but not by PD98059 (ERK inhibitor) (P=0.575), indicating that Ang II activates Smad signaling pathway at least partly via the p38 MAPK pathway.
4. Ang-(1-7) inhibited AngⅡ-induced activation of Rho/ROCK pathway in HSC.
The expression levels of RhoA protein and ROCK2 mRNA were significantly increased in HSC after treated with Ang II for 24 hr, as compared with controls (both P=0.000). Irbesartan, Ang-(1-7) and Y27632 (ROCK inhibitor) markedly abrogated the effect of Ang II on RhoA protein and ROCK2 mRNA expression (all P<0.01). A779 reversed the inhibition of Ang-(1-7) on AngⅡ-induced activation of RhoA and ROCK2 (both P<0.01). These results suggested that AngⅡactivates Rho/ROCK pathway through AT1R, and Ang-(1-7) antagonizes this effect of AngⅡthrough Mas receptor.
5. ACE2-Ang-(1-7) inhibited AngⅡ-induced collagen synthesis in HSC.
A lentiviral vector encoding a shRNA directed against ACE2 was designed and constructed, and then transferred into HSC. The know-down efficiency was near 70% as determined by real-time PCR at the mRNA levels and Western blot at protein levels (0.154±0.018 vs 0.446±0.026, P=0.000). This vector was named as Lenti-SiACE2.
The expression of COL1 mRNA and CTGF protein were evaluated by RT-PCR and Western blot respectively, and their mRNA expression levels were further analyzed by QuantiGene Plex assay. The results showed that AngⅡstimulated significant increase of COL1 and CTGF expression levels in HSC as compared with controls (both P<0.01). The expression levels of COL1 and CTGF were little changed in cells transferred with Lenti-SiACE2 (P=1.000), but they were significantly increased following AngⅡstimulation, as compared with normal HSC treated with AngⅡ(both P<0.01). This result indicated that knock-down of ACE2 prolongs AngⅡ-stimulated collagen synthesis. Ang-(1-7) abolished AngⅡ-induced COL1 and CTGF expression (both P<0.01), which was reversed by Mas receptor antagonist A779 (both P<0.01). Besides, QuantiGene assay showed that the degree of inhibition of Ang-(1-7) on AngⅡ-induced upregulation of COL1 and CTGF mRNA was more pronounced than that of SB203580, PD98059 and Y27632 (all P< 0.01, except P<0.05 compared with the CTGF mRNA level in HSC pre-treated with Y27632). These data indicated that collagen synthesis in HSC may involve a variety of pathways, yet it can be dramatically inhibited by Ang-(1-7) through directly against AngⅡ.
Conclusions
1. In the present study, we observed an increase in hepatic ACE2 and Mas receptor expression with the progression of liver fibrosis induced by CCl4 in rats. Furthermore, in our in vitro experiments, we found that AngⅡupregulated ACE2 and Mas expression in a dose-dependent manner in HSC. The changes in ACE2 and Mas expression indicate that it may be a resistance response to the augmented levels of AngⅡ, and may further represent a negative feedback mechanism involved in maintaining equilibrium in the intrahepatic RAS. Therefore, our results support the previous hypothesis that the ACE2-Ang-(1-7)-Mas axis may play a protective role in liver fibrosis. Besides, our study showed that ACEI is able to upregulate ACE2 expression under conditions of liver injury, both in vivo and in vitro, which may in turn contribute to additional benefits of ACEI on liver fibrosis.
2. Our investigation showed that AngⅡactivated the Smad and Rho/ROCK signaling pathways in HSC through interacting with its receptor AT1R, which may be important mechanisms of the pro-fibrosis effect of AngⅡ. Ang-(1-7) inhibited the activation of these two pathways through counteracting with AngⅡ, which may be attributed to its anti-fibrosis function in liver. In addition, p38 MAPK inhibitor reduced the AngⅡ-mediated phosphorylation of Smad3, suggesting that Ang II can directly activate the Smad signaling pathway in a TGF-β-independent manner.
3. We also found that AngⅡstimulation significantly increased COL1 and CTGF expression in HSC. Silencing of ACE2 further enhanced AngⅡ-induced overexpression of these two effectors. Ang-(1-7) abated the activation of COL1 and CTGF caused by AngⅡ, and this inhibiting effect was stronger than that of p38 MAPK, ERK 1/2 or Rho/ROCK inhibitors. These results indicated that ACE2 and Ang-(1-7) can suppress collagen synthesis in HSC through antagonizing the effect of Ang II. Therefore, our study provides experimental evidence that the new ACE2-Ang-(1-7)-Mas axis has a protective role in liver, and thus, it can be a promising target for the therapy of liver fibrosis.
引文
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