Jun Shi, Jing-Hua Hao, Wan-Hua Ren, Ju-Ren Zhu, Center for Liver Diseases, Shandong Provincial Hospital, Jinan 250021, Shandong Province, China
Supported by Research Grant of Shandong Provincial Health Committee. No. 2001CA2CKA2
Correspondence to: Dr. Jun Shi, Center for Liver Diseases, Shandong Provincial Hospital, 342 Jing Wu Wei Qi Road, Jinan 250021, Shandong Province, China.  sdshij@yahoo.com.cn
Telephone: +86-531-7938911-2450
Received: 2002-11-29    Accepted: 2003-03-02

Abstract
AIM: To evaluate the effects of heparin on liver fibrosis in patients with chronic hepatitis B.

METHODS: Fifty-two cases under study were divided into two groups, group A and group B. The two groups were given regular treatment and heparin/low molecular weight heparin (LMWH) treatment respectively. Hepatic functions, serum hyaluronic acid (HA) and type IV collagen levels were measured before and after the treatment, and six cases were taken liver biopsy twice.

RESULTS: After treatment, hepatic functions became significantly better in both groups. Serum HA and type IV collagen levels in group B compared with group A, decreased significantly after treatment. Collagen proliferation also decreased in group B after treatment.

CONCLUSION: Heparin/LMWH can inhibit collagen proliferation in liver tissues with hepatitis B.

Shi J, Hao JH, Ren WH, Zhu JR. Effects of heparin on liver fibrosis in patients with chronic hepatitis B. World J Gastroenterol  2003; 9(7): 1611-1614
http://www.wjgnet.com/1007-9327/9/1611.asp

INTRODUCTION
The treatment of liver cirrhosis is always a problem in the clinical practice. To control and stop liver fibrosis towards liver cirrhosis is of utmost importance. A recent trial indicated that heparin could inhibit the growth of Ito cells effectively in vitro^[1], which suggested that heparin might act as an antifibrosis drug. In this study, we aimed to seek a safe and effective antifibrosis drug in 52 patients with chronic hepatitis B.

MATERIALS AND METHODS
Materials
Fifty-two patients were treated in Shandong Provincial Hospital from 1999 to 2002. There were 39 males and 13 females, age ranged from 14 to 70 years, diagnosis was made by clinical manifestations and serum hepatitis B viral markers.

Experimental design
These 52 cases were divided into two groups randomly. The treatment regime of each group is listed in Table 1.

Table 1  Treatment regimes in group A and B

Note: In group B, 18 cases were treated with heparin and 16 with low molecular weight heparin (LMWH). The LMWH was FLUX manufactured by ALFA WASSERMANN S.P.A (Italy).

      All cases were treated for a course of 3 weeks. Serum alanine  transaminase (ALT), prothrombin time (PT), total bilirubin (TBIL), hyaluronic acid (HA) and type IV collagen (IV-C) were measured before and after treatment. The liver tissue specimens were obtained by percutaneous needle biopsy. Ten cases in group A and sixteen in group B had liver biopsies before treatment. Six cases in group B had a second biopsy at 30-60 days after treatment.

Determination of serum HA and IV-C level
Serum HA and IV-C level was determined with radioimmunoassay. The procedures were strictly in accordance with the instructions.

Light microscopic examination
Part of the liver tissues were fixed in 10 % formalin, embedded in paraffin, and then cut into slices. The sections were stained with hematoxylin and eosin for histological study and Masson trichrome for collagen stained green.

Electron microscopic examination
Small liver blocks were fixed in 2.5 % glutaraldehyde, postfixed in 1 % OsO₄, dehydrated with ethanol, and embedded in epoxy resin. Ultrathin sections were stained with uranyl acetate and lead citrate and examined with H-800 transmitted electron microscope (Tokyo, Japan).

RESULTS
Changes of serum/plasma indexes before and after treatment
As shown in Table 2, the levels of ALT and TBIL decreased significantly after treatment, while the level of PT changed slightly only. The level of HA and IV-C in group B decreased markedly, while those in group A were elevated.

Table 2  Changes of the serum/plasma indexes before and after treatment in group A and B ( )

^aP<0.05, vs before treatment.

Histologic changes before and after treatment with heparin/LMWH
Hematoxylin and eosin staining  Hepatocytes swelled and appeared balloon-like before treatment. Inflammatory cells penetrated into the interstitium. Red blood cells congregated in the sinusoids. After treatment with heparin/LMWH, the swollen hepatocytes alleviated , and the sinusoids became clearly seen (Figure 1,2).
Masson trichrome staining  Collagens could be seen evidently before treatment. Some sinusoids had been compressed by collagens. After treatment with heparin/LMWH, the collagen fibers decreased significantly (Figure 3,4).

Figure 1  The liver tissue before treatment with heparin. H&E staining. ×200.
Figure 2  The liver tissue after treatment with heparin. H&E staining. ×200.
Figure 3  The liver tissue before treatment with heparin. Masson staining. ×200.
Figure 4  The liver tissue after treatment with heparin. Masson staining. ×200.

Electron microscopic observation  Before treatment, hepatocytes were enlarged and cytoplasm appeared dissolved with swollen mitochondria. Base membrane was seen under the hepatic sinusoidal endothelial cells with collagen deposited in the Disse's space. The Ito cells simulated fibroblasts. The edge of membrane looked uneven, saw-like in severe cases. The number of fat drops decreased markedly. There was microfilament-like structure in the cytoplasm, fibrils were seen around the Ito cells. After treatment, the swollen hepatocytes decreased , so did the base membrane and the depositioning collagen in the Disse's space. The edge of Ito cells turned smooth. Several fat drops could be seen in the cytoplasm of Ito cells (Figure 5-8).

Figure 5  The hepatocyte before treatment heparin. ×6000.
Figure 6  The hepatocyte after treatment with with heparin. ×3500.
Figure 7  The Ito cell before treatment with heparin. ×5000.
Figure 8  The Ito cell after treatment with with heparin. ×5000.

DISCUSSION
Liver fibrosis is caused by the deposition of extracellular matrix(ECM)^[2-3]. All cells in the liver can synthesize and secrete ECM, which regulates the proliferation, differentiation and metabolism of liver cells. The abnormal metabolism and deposition of ECM lead to liver fibrosis. It has been recognized that Ito cells have intimate relationships with liver fibrosis^[4], which have been postulated to play critical roles in the development of fibrosis of the liver from viral infection, alcohol and many drugs^[5,6].Ito cells are relatively inactive fibroblasts in the liver lobules. During liver fibrogenesis, cytokines such as TGF-b₁, PDGF can activate Ito cells^[7-9] to acquire a myofibroblast-like phenotype characterized by increased proliferation and synthesis of ECM component^[10-27].
      It has been proved in animal studies that heparin can inhibit the growth of Ito cells and the expression of b-actin, types I and IV procollagen in vitro^[1]. Our studies showed that heparin/LMWH could decrease serum HA and IV-C levels in patients with chronic hepatitis B. After treatment, the collagen fibrils in the liver tissues decreased significantly and Ito cells turned oval and fatty drops reappeared in the cytoplasm. The above results indicate that heparin/LMWH act on Ito cells.
     The liver functions were improved in both group A and B after treatment. HA and IV-C levels decreased significantly in group B, in contrast, they were elevated in group A. These results suggest that the routine liver function tests could not reflect the fibrosis completely. Kopke-Aguiar et al^[28] also proved that serum hyaluronic acid was a good marker for hepatic fibrosis at the initial phase.
     Wanless et al^[29] have studied hepatic veins of medium size(0.2 to 3 mm in diameter) in 61 cirrhotic livers. Intimal fibrosis with at least 10 % luminal narrowing was found in 70 % of cirrhotic livers. They considered that multiple layers of intimal fibrosis in some livers suggested the presence of recurrent thrombosis. In other words, thrombosis was related to intimal fibrosis and even caused  obstruction of the veins. Our previous studies^[30] also showed that as an anticoagulant agent, heparin could improve hepatic microcirculation significantly and lessen sinusoidal capillarization. IV-C is considered an important marker of the development of hepatic sinusoidal capillarization and may appear basal-like membrane^[31,32]. Therefore, decrease of the IV-C concentration can not only reflect the improvement of hepatic microcirculation, but also  inhibit the  fibrosis. It can be used as antifibrosis drug together with antiviral drugs.
     Heparin is cheap and safe. LMWH has a weaker effect on thrombin than heparin, but has stronger effect on Xa. 90 % of LMWH can be absorbed hypodermically and its anti-Xa effect can last for 24 hours and therefore, LMWH can be used once a day. One needs not measure the activated coagulation time(ACT) during the procedure^[33]. As to the mechanisms of its antifibrosis effect ,further studies are necessary.

REFERENCES
1    Yuan TX, Zhang JS, Zhang YE, Chen Q. Culture of rat liver Ito cells and the observation of inhibitory effect of heparin 
      on Ito cells. Shanghai Yike Daxue Xuebao 1996; 23: 90-93
2    Bissell DM, Friedman SL, Maher JJ, Roll FJ. Connective tissue biology and hepatic fibrosis: report of a 
      conferrence. Hepatology  1990; 11: 488-498
3    Schuppan D. Structure of the extracellular matrix in normal and fibrotic liver:collagens and glycoproteins.Semin Liver 
      Dis 1990;10: 1-10
4    Friedman SL. Cellular source of collagen and regulation of collagen production in liver. Semin Liver Dis 
      1990; 10: 20-29
5    Bataller R, Brenner DA. Hepatic stellate cells as a target for the treatment of liver fibrosis. Semin Liver Dis 
      2001; 21: 437-451 
6    Dai WJ, Jiang HC. Advances in gene therapy of liver cirrhosis,a review. World J Gastroenterol 2001; 7: 1-8
7    Kinnman N, Goria O, Wendum D, Gendron MC, Rey C, Poupon R, Housset C. Hepatic stellate cell proliferation is an 
      early platelet-derived growth factor-mediated cellular event in rat cholestatic liver injury. Lab Invest 
      2001; 81: 1709-1716
8    Gandhi CR, Kuddus RH, Uemura T, Rao AS. Endothelin stimulates transforming growth facter-?1 and collagen 
      synthesis in stellate cells from control but not cirrhotic rat liver. Eur J Pharmacol 2000; 406: 311-318
9    Gabriel A, Kuddus RH, Rao AS, Gandhi CR. Down-regulation of endothelin receptor by transforming growth factorb1 
      in hepatic stellate cells. Hepatol 1999; 30: 440-450
10  Huang GC, Zhang JS, Zhang YE. Effects of retinoic acid on proliferation, phenotype and expression of cyclin-
      dependent kinase inhibitors in TGF-b1 stimulated rat hepatic stellate cells. World J Gastroenterol 2000; 6: 819-823
11  Chen PS, Zhai WR, Zhou XM, Zhang JS, Zhang YE, Ling YQ, Gu YH. Effects of hypoxia, hyperoxia on the regulation 
      of expression and activity of matrix metalloproteinase-2 in hepatic stellate cells. World J Gastroenterol 
      2001; 7: 647-651
12  Wang JY, Zhang QS, Guo JS, Hu MY. Effects of glycyrrhetinic acid on collagen metabolism of hepatic stellate cells 
      at different stages of liver fibrosis in rats. World J Gastroenterol  2001; 7: 115-119
13  Eng FJ, Friedman SL, Fibrogenesis I. New insight into hepatic stellate cell activation: the simple becomes complex. 
      Am J Phsiol  2000; 279: G7-G11
14  Friedman SL. Molecular regulation of hepatic fibrosis, an integrated cellular response to tissue injury. J Biol Chem 
      2000; 275: 2247-2250
15  Albanis E, Friedman SL. Hepatic fibrosis. Pathogenesis and principles of therapy. Clin Liver Dis 2001; 5: 315-334
16  Paradis V, Perlemuter G, Bonvoust F, Dargere D, Parfait B, Vidaud M, Conti M, Huet S, Ba N, Buffet C, Bedossa P. 
      High glucose and hyperinsulinemia stimulate connective tissue growth factor expression: a potential mechanism 
      involved in progression to fibrosis in nonalcoholic steatohepatitis. Hepatology 2001; 34: 738-744
17  Schneiderhan W, Schmid-Kotsas A, Zhao J, Grunert A, Nussler A, Weidenbach H, Menke A, Schmid RM, Adler G, 
      Bachem MG.Oxidized low-density lipoproteins bind to the scavenger receptor, CD36, of hepatic stellate cells and 
      stimulate extracellular matrix synthesis. Hepatology 2001; 34: 729-737
18  Benyon RC, Arthur MJ. Extracellular matrix degradation and the role of hepatic stellate cells. Semin Liver Dis 
      2001; 21: 373-384 
19  Vaillant B, Chiaramonte MG, Cheever AW, Soloway PD, Wynn TA. Regulation of hepatic fibrosis and extracellular 
      matrix genes by the Th response: new insight into the role of tissue inhibitors of matrix metalloproteinases. J 
      Immunol 2001; 167: 7017-7026
20  Breitkopf K, Lahme B, Tag CG, Gressner AM. Expression and matrix deposition of latent transforming growth factor 
      beta binding proteins in normal and fibrotic rat liver and transdifferentiating hepatic stellate cells in culture. 
      Hepatology  2001; 33: 387-396
21  Bruck R, Genina O, Aeed H, Alexiev R, Nagler A, Avni Y, Pines M. Halofuginone to prevent and treat thioacetamide-
      induced liver fibrosis in rats. Hepatology 2001; 33: 379-386
22  Watanabe T, Niioka M, Hozawa S, Kameyama K, Hayashi T, Arai M, Ishikawa A, Maruyama K, Okazaki I. Gene 
      expression of interstitial collagenase in both progressive and recovery phase of rat liver fibrosis induced by 
      carbon tetrachloride. J Hepatol 2000; 33: 224-235
23  Nakamura T, Sakata R, Ueno T, Sata M, Ueno H. Inhibition of transforming growth factor beta prevents progression 
      of liver fibrosis and enhances hepatocyte regeneration in dimethylnitrosamine-treated rats. Hepatology 
      2000; 32: 247-255
24  Castera L, Hartmann DJ, Chapel F, Guettier C, Mall F, Lons T, Richardet JP, Grimbert S, Morassi O, Beaugrand M, 
      Trinchet JC.Serum laminin and type IV collagen are accurate markers of histologically severe alcoholic hepatitis in 
      patients with cirrhosis. J Hepatol 2000; 32: 412-418
25  Brenner DA, Waterboer T, Choi SK, Lindquist JN, Stefanovic B,Burchardt E, Yamauchi M, Gillan A, Rippe RA. New 
      aspects of hepatic fibrosis. J Hepatol 2000; 32(Suppl 1): 32-38
26  Benyon RC, Iredale JP. Is liver fibrosis reversible? Gut 2000; 46: 443-446
27  Friedman SL. Molecular regulation of hepatic fibrosis, an integrated cellular response to tissue injury. J Biol Chem 
      2000; 275: 2247-2250
28  Kopke-Aguiar LA, Martins JR, Passerotti CC, Toledo CF, Nader HB, Borges DR. Serum hyaluronic acid as a 
      comprehensive marker to assess severity of liver disease in schistosomiasis. Acta Trop 2002; 84: 117-126
29  Wanless IR, Wong F, Blendis LM, Greig P, Heathcote EJ, Levy G. Hepatic and portal vein thombosis in cirrhosis: 
      possible role in development of parenchymal extinction and portal hypertention. Hepatology 1995; 21: 1238-1247
30  Hao JH, Shi J, Ren WH, Han GQ, Wang WZ, Zhu JR, Wang SY, Xie YB. Usage of heparin in the patients with 
      chronic hepatitis B. Wei Xunhuan Xue Zazhi 2001; 11: 9-11
31  Matsumoto S, Yamamoto K, Nagano T, Okamoto R, Ibuki N, Tagashira M, Tsuji T. Immunohistochemical study 
      on phenotypical changes of hepatocytes in liver disease with reference to extracelluar matrix composition. Liver 
      1999; 19: 32-38
32  Marcato PS, Bettini G, Della Salda L, Galeotti M. Pretelangiectasis and telangiectasis of the bovine liver:a 
      morphological immunohistochemical and ultrastructural study. J comp Pathol 1998; 119:95-110
33  Khosla S, Kunjummen B, Guerrero M, Manda R, Razminia M, Trivedi A, Vidyarthi V, Elbazour M, Ahmed A, Lubell D. 
      Safety and efficacy of combined use of low molecular weight heparin (enoxaparin, lovenox) and glycoprotein 
      IIb/IIIa receptor antagonist (eptifibatide,integrelin)during nonemergent coronary and peripheral vascular 
      intervention. Am J Ther 2002; 9: 488-491

   Edited by Wu XN  

Reviews	Add
more>>