Hong Shan Wei, Ding Guo Li, Han Ming Lu, Yu Tao Zhan, Zhi Rong Wang, Xin Huang, Jing Zhang, Ji Lin Cheng and Qin Fang Xu  Department of Gastroenterology, Xinhua Hospital, Shanghai Second Medical Univers ity, Shanghai 200092, China
Dr Hong Shan Wei, male, born on 1965-12-06 in Shandong Province, graduated f rom Jining Medical Collage and earned doctoral degree from Shanghai Second Medic al University, majoring in hepatic fibrosis, having 15 papers published.
Correspondence to: Dr Hong-Shan Wei, Department of Gastroenterology, Xinhua Hospital, Shanghai S econd Medical University, Shanghai 200092, China
Telephone: +86-21-65790000, Ext. 5319
Received: 2000-02-01 Accepted: 2000-03-04

Subject headings: liver cirrhosis/drug therapy; renin-angiotensin system; angiotensin Ⅱ type 1 receptor antagonist; losartan

Wei HS, Li DG, Lu HM, Zhan YT, Wang ZR, Huang X, Zhang J, Cheng JL, Xu QF. Effects of AT1 receptor antagonist, losartan, 
on rat hepatic fibrosis induced by CCl4. World J Gastroentero, 2000;6(4):540-545

Abstract
AIM: To investigate effect o f losartan, an AT1 receptor antagonist, on hepatic fibrosis induced by CCl₄; a nd to determine whether or not AT1 receptors are expressed on hepatic stellat e cells.

METHODS AND RESULTS: Fifty male Sprague-Dawley rats, weighing (180 ±20) g, were randomized into five groups (control group, model group, and three los artan treated groups), in which all rats were given the subcutaneous injection o f 40% CCl₄ (every 3 days for 6 weeks) except for rats of control group. Rats of losartan-treated groups were treated with losartan (20mg/kg, 10mg/kg, 5mg/ kg, daily gavage). After 6 weeks liver tissue and serum samples of all rats were examined. Serum hyaluronic acid (HA), procollagen type Ⅲ (PC Ⅲ) were detected by radioimmunoassays. van Giesion collagen staining was used to evaluate the extracellular matrix of rats with liver fibrosis. The e xpression of AT1 receptors, transforming growth factor-beta (TGF-β), and alpha-smooth muscle actin (α-SMA) in liver tissue were d et ermined by immunohistochemical techniques. Compared with model group, serum ALT and AST of losartan-treated groups were significantly reduced (t=4.20, P <0.01 and t=4.57, P<0.01). Serum HA and PC Ⅲ als o had significant differences (t=3.53, P<0.01 and t=2.20, P<0.05). The degree of fibrosis was improved by losartan and correlated with the expressions of AT1 receptors, TGF-β, and α-SMA in liver tissue.

CONCLUSION: AT1 receptor antagonist, losartan, could limit the p rogression of the hepatic fibrosis induced by CCl₄. The mechanism may be relat ed to the decrease in the expression of AT1 receptors and TGF-β, a meliorating the injury of hepatocytes; activation of local renin-angiotensin system mi ght relate to hepatic fibrosis; and during progression of fibrosis, activated hepatic stellate cells might express AT1 receptors.

INTRODUCTION
Hepatic fibrosis, which may ultimately lead to cirrhosis, is associated with mos t chronic liver diseases, and is characterized by the net accumulation of extrac ellular matrix (ECM), including collagen, glycoproteins, and proteoglycans^{[ 1,2]}. Many reports have suggested that hepatic stellate cells (HSCs) are the major producers of ECM in liver injury, and play a prominent role in liver fibro sis^[3-7]. Tissue repair after acute liver damage involves “activatio n” of “quiescent” HSCs to myofibroblast-like cells^[8-12]. Transform ing growth factor-beta (TGF-β) is a pleiotropic cytokine that has been assigned a key role in epithelial repair, and HSCs were shown to its main source^[13-16]. In cultured HSCs, TGF-β-mediated up-regul ation of collagen and other ECM components mRNA was time and dose-dependent^[17,18]. In the past years, significant progress has been made in our understanding of this pathologic mechanism, however, few effective drugs can slo w the progression of the fibrosis^[19,20].
       Over the past decade, preventing the formation of angiotensin Ⅱ by angiotensin-converting enzyme (ACE) inhibitors has revolutionized the therap y of hypertension and especially of congestive heart failure^[21]. Recen tly, a number of studies demonstr ated that ACE inhibitors also effectively limit ed the progression of cardiac, renal and pulmonary interstitial fibrosis^{[22 - 26]}. Recent work has shown that angiotensin Ⅱ type 1 (AT1) recep tor antagonist, losartan, can also ameliorate the renal and cardiac fibrosis^[27,28]. The prevailing hypothesis for the main mechanism was suppressing the expression of TGF-β in kidney and heart, rather than its dynamic effects^[29-32]. Based on this and other information, we hypothesized that the AT1 receptor antagonist, losartan, could also limit the progression of hepatic fibrosis. To explore our speculation, the present study was designed to investigate the effect of losartan on rat’s hepatic fibrosis induced by CCl₄, and determine whether or not there was expression of AT1 receptor on hepatic s tellate cells.

MATERIALS AND METHODS
Animals and reagents
Fifty male Sprague-Dawley rats, weighing 180±20 g, were purchased from Animal Center of Shanghai Medical University (Shanghai, China). Losartan was obtained f rom MSD Co. (England). Polyclonal rabbit antibody to rat TGF-β was purchased from Boster Biotech Co. (Wuhan, China). Monoclonal antibody of alpha -sm ooth muscle actin (α-SMA) was purchased from Maixin Biotech Co. (F uzhou, China). Hyaluronic acid (HA) and procollagen type Ⅲ (PC Ⅲ) radioimmunoassays kits were purchased from Navy Shanghai Medical In stitute (Shanghai, China).

Serum function tests
Fifty rats were randomized into five groups (control group, model group and thre e losartan-treated groups) in which all rats were given subcutaneous inject ion of 40% CCl₄ (0.3 mL/100 g, every 3 days for 6 weeks) except for rats o f co ntrol group (only given injection of same dose of olive oil). In an initial exp eriment, rats of losartan-treated groups were treated with losartan (20 mg/kg, 10 mg/kg, 5 mg/kg by daily gavage). After six weeks, all rats were sacrificed. S erum was collected and stored at -20℃ for analysis of aspartate transaminase (AST) and alanine transaminase (ALT) activity by standard enzymatic methods. The serum levels of PC Ⅲ and HA was determined by radioimmunoassays.

Immunohistochemical detections and histological data
The liver sections were fixed in a 10% solution of formaldehyde in 0.1 mol/L pho sphate-buffered saline (pH 7.4), and embedded in paraffin. Five-micrometer sli des were prepared. van Giesion collagen staining was used to evaluate the ECM o f rats. According to van Giesion collagen staining, the degree of fibrosis was di vided into five grades (0-5). Specimens were scored blindly by the histologist and were also ranked blindly for severity of fibrosis. The expression of AT1 re ceptor (anti-rat rabbit polyclonal antibody was the product of Santa Cruz Biote ch Co), TGF-β and α-SMA were detected by immunohisto chemical techniques.

Statistics
Data are presented as x-±s x-. Comparision between two gro ups was made using Student’s t test. Difference of fibrosis between model and losartan-treated groups was compared using Ridit analysis. The test was con sidered significant at P<0.05.

RESULTS
Serum function tests
Compared with control group, ALT and AST increased significantly in fibrot ic rats in the model group, but only marginally in losartan treated rats. ALT and AST activities were significantly lower in 20 mg losartan-treated group than i n mod el group of CCl₄ rats. The effects were associated with doses of losartan (Tab le 1).

Table 1  Serum function tests

^aP<0.05 vs control; ^cP<0.05 vs model; ^dP<0.01 vs model

Serum components of ECM
As expected, serum levels of HA and PCⅢ increased in rats of model group. Serum HA levels were approximately three times higher in rats of model g roup than rats of control group. There was a tendency towards a decrease in HA a nd PC Ⅲ levels in losartan treated group (Table 2).

Table 2 Serum component of ECM

^aP<0.05 vs control; ^cP<0.05 vs model; ^dP<0.01 vs model

Histological data
Piecemeal and lobular necrosis was obvious in the CCl₄ model compared to rats in control group. The lobular necrosis was significantly decreased by losartan i n three treated groups. There was an increase in the area of fibrosis in model r ats compared with rats in control group. There was a significant decrease in the losartan treated rats (Table 3, Figure 1).

Table 3 Degree of fibrosis

^aP<0.05 vs model

Figure 1 Collagen expression in liver section of model rat (v an Gieson staining, ×200) 
(A), and of losarta n treated group (van Giesion staining, ×200). Significantly less fibrosis can b e noted compared to that in the model group 
(B).

Expression of AT1 receptors, TGF-β, and α-SMA
Compared with normal liver tissue, in which AT1 receptors mainly locate in vasculature, in the fibrotic liver tissue, the expression of AT1 receptors significan tly enhanced, and scattered in fibrotic areas. The express ion of AT1 receptors was markedly reduced by losartan (Figure 2). The expressio n of α-SMA was a marker of HSC activation. Immnohistochemical dete ction demonstrated that vascular smooth muscle cells and pericytes were positive for α-SMA in control livers, whereas HSCs strongly positive of α-SMA were observed in rats of model group, and they were scattered along the sinusoidal walls. Many α-SMA-positive HSCs were detecte d in the area of centrilobular and periportal fibrotic bands in rats of model gr oup. Compared with model group, liver of rats treated with losartan, showed mark edly reduced numbers of α-SMApositive HSC. At same time, its serum levels of PC Ⅲ and LN were also significantly decreased (P<0.05).
      Similar to the α-SMA, TGF-β was also strongly expre ssed in areas of periportal fibrotic bands in rats of model group. In contrast, livers of rats treated with losartan showed significantly reduced numbers of TGF -β-positive cells (Figure 3).

Figure 2 AT1 receptor expression in liver section of model rat (DAB staining, ×200). AT1 receptor is seen mainly scattered in fibrotic areas and vascular wall 
(A), and of los artan treated group (DAB staining, ×200). Note significantly less AT1 expression than that seen in the model group
(B).

DISCUSSION
Our study firstly demonstrated that AT1 receptor antagonist, losartan, could slo w the progression of hepatic fibrosis induced by CCl₄. Activated HSC might express AT1 receptors in fibrotic liver tissue.
      Over the last decade, many lines of evidence have demonstrated that local RAS ac tivation was the major mechanism of cardiac and renal interstitial fibrosis ^[33-37]. In vivo studies have shown that ACE inhibitors and AT1 receptor s antagonists can limit the progression of cardiac, renal, and pulmonary fibrosi s^[23,38-41], and the mechanism is independent of their dynamic effects. This is based on several in vitro and in vivo findings. First, all cardi ac and renal fibroblasts express AT1 receptors; secondly, Ang Ⅱ ind uces mitogenic response, increases protein synthesis, production of collagen, and TGF-β in fibroblasts in a dose-dependent manner^[29,42,43]. These results support the notion that Ang Ⅱ can both directly act on fibroblasts and/or enhance the expression of TGF-β.
      In the present study, the three different doses (20 mg/kg, 10 mg/kg, 5 mg/kg) of losartan were given to the rats. As the results shown, losartan could limit the progression of the hepatic fibrosis in a dose-dependent manner. At the dose not influencing systolic blood pressure (10 mg/kg) in the normotensive rats^{[4 4]}, losartan could attenuate the fibrosis, but even at a very low dose (5 mg/ kg) , had a weak but significant effect. In agreement with this study, Boffa et a l ^[45]also reported that losartan completely prevented collagen I gene activation and attenuated the degree of fibrosis without influencing the systoli c pressure in the kidneys of transgenic mice. This suggested that Ang Ⅱ might act as an important regulator in the progression of hepatic fibrosis induced by CCl₄.

Figure 3 TGF-β expression in liver section of model rat ( DAB staining, ×200). TGF-β expression is seen scattered in the fibrotic areas and vascular wall 
(A), and of losart an treated group (DAB staining, ×200). Note significantly less TGF-β expression than that seen in the mod el group 
(B).

     Using the immunohistochemical methods, we observed the expression of AT1 recepto rs in liver tissue. As the results shown, this is the first report that confirms that the liver tissue expresses AT1 receptors. Compared with normal rats, wher e AT1 receptors are mainly located in the vasculature, the expression of AT1 rec eptors were significantly enhanced in rats of model group, which were mainly loc ated in fibrotic area, and correlated with the degree of fibrosis. The results s ugge sted that the expression of AT1 receptors might relate to hepatic fibrogenesis. Furthermore, immunostaining indicated that the distribution of AT1 receptor corr ela ted with the expression of TGF-β and α-SMA. As has b een recently reported, in a model of chronic cyclosporine (CsA), TGF-β played a role in CsA-induced tubulointerstitial fibrosis and arteriolopath y by stimulating ECM protein synthesis and inhibiting ECM degradation. Losartan resulted in decreasing expression of TGF-β and synthesis of ECM^[46]. Zhang et al^[28] have also reported that losartan is effe ctive in reducing the increasing expression of AT1 receptors and ECM protein in infarcted heart tissue. Based on the above data, we postulated that expression o f AT1 receptor might be related to the activation and ECM synthesis of HSCs.
      Another interesting finding in our study was that losartan could ameliorate the hepatocyte injury, as reflected by the release of liver enzymes. The explanatio n for this observation is not known, but it might relate to protection of the h epatocyte from free radical-mediated damage. Recently, Anthuber et al ^[47]demonstrated that non-thiol-containing ACE inhibitor, enalapril, coul d attenuate the hepatocyte injury induced by ischemia/reperfusion. The prevaili ng mechanism of action was considered to relate with modulation of the angiotens in, bradykinin, and prostacyclin metabolism. Whether losartan has some as-yet- unknown, specific, protective property, remains to be determined in future studi es.
      That transforming growth factor β (TGF-β) is a key m olecule responsible for tissue fibrosis, provides a basis for targeting TGF-β as an antifibrotic agent^[48-50]. Recently, Sun et al^[31]found that the early induction of TGF-β1 via the angiot ensin Ⅱ type 1 receptor played a major role in the development of cardiac fibrosis in infarcted heart. Shihab et al^[46]described that losartan reduced TGF-β overproduction in a dose-dependent manner , slowing the rate of renal interstitial fibrosis^[22].
      Overproduction of TGF-β and activation of HSC are key processes in the progression of hepatic fibrosis. Our results demonstrated that losartan could reduce the expression of TGF-β and α-SMA in li ver tissue and suppress fibrosis procession. It is suggested that RAS also parti cipates in the progression of hepatic fibrosis induced by CCl₄ in rats.
     In conclusion, our results demonstrated that (1) AT1 receptor antagonist, losar tan, could limit the progression of the hepatic fibrosis induced by CCl₄. Th e mechanism may be related to the decrease in the expression of AT1 receptors an d TGF-β, ameliorating the injury of hepatocyte; (2) activation of local renin-angiotensin system might relate to hepatic fibrogenesis; (3) in the progression of fibrosis, activated hepatic stellate cells might express AT1 re ceptor.

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