Effects of heparin on hepatic regeneration and function after partial hepatectomy in rats

Abstract

AIM: To investigate the effects of heparin on the regenerative rate and serum alanine aminotransferase level in partial-hepatectomized (two- thirds) rats.

METHOD: Three different doses of heparin (100, 500 or 1000 U/kg) were given after partial hepatectomy through the tail vein at a 12 h interval for 2 or 3 d. After drug treatment, rats were killed and the remnant livers were weighed for the assessment of regenerative rate. Blood samples were also collected to measure serum alanine aminotransferase levels.

RESULTS: Heparin given in the present dosages for 2 or 3 d neither stimulated the regeneration of the liver nor improved the hepatic function as indicated by an insignificant change both on remnant liver weight and serum alanine aminotransferase activity as compared with the control.

CONCLUSION: Heparin alone has no beneficial effects on the regeneration of livers and improvement of hepatic function in hepatectomized rats.

Key Words: heparin, hepatectomy, liver regeneration, alanine aminotransferase, rats

INTRODUCTION

It is known that liver regenerates quickly in response to tissue damage. Hepatocyte growth factor (HGF) has been implicated in the regulation of liver growth after partial hepatectomy[1,2]. It has been reported that heparin is a potent inducer of HGF production in various types of cells, including human embryonic lung and skin dermal fibroblasts, and promyelocytic leukemic and umbilical vein endothelial cells as well[3]. Furthermore, it was also demonstrated that heparin, when given to partially hepatectomized rats, could increase the plasma HGF level and stimulate DNA synthesis in hepatocytes of the remnant livers during the early stage of liver regeneration. However, this indirect indicator of tissue growth in the early phase of liver regeneration can not extrapolate and ascertain whether heparin is a true hepatotrophic factor for liver regeneration at the later stage of the regenerative process. Also, there was no clear indication that heparin can indeed restore the liver function along with its regenerative capacity.

The purpose of this study is to investigate the effects of heparin on liver regeneration and function by measuring the regenerative rate and serum alanine aminotransferase level, respectively in partially hepatectomized rats. The results of this study could be useful for assessing the therapeutic implication of heparin as a hepatotrophic agent in man.

MATERIALS AND METHODS

Animals

Male Sprague-Dawley rats (180-200 g) purchased from the Laboratory Animal Unit, the University of Hong Kong, were reared on a standard laboratory diet (Ralston Purina Co., USA), and given tap water. They were kept in a room where the temperature (22 °C ± 1 °C), humidity (65%-70%), and day:night cycle (12:12 light:dark) were controlled.

Drug

Heparin (Sigma, St. Louse, MO, USA; sodium salt, 174 USP units/mg) was prepare d in 0.9% w/v NaCl (British Drug House, UK) solution (normal saline) in a concentration series of 50, 250, and 500 U/mL for intravenous injection. Different doses of heparin 100, 500 and 1000 U/kg, i.e. 0.2 mL/100 g body weight were given, respectively. Similar volume of normal saline was injected through the same route as the control.

Partial hepatectomy, drug treatment and hepatic regeneration assessment

Partial (two-thirds) hepatectomy was performed by excision of the median and left hepatic lobes according to the method of Higgins and Anderson[4] under pentobarbitone anaesthesia. The heparin at doses of 100, 500, and 1000 U/kg or its vehicle (normal saline) were administered intravenously through tail vein every 12 h starting 6 h after operation for 2 (4 injections) or 3 (7 injections) d. The rats were killed 3 h after the last dose. Blood samples were collected for the measurement of serum alanine aminotransferase level. At the time of killing, the remnant livers were removed and weighed to assess the hepatic regeneration. The hepatic regenerative rate in each postoperative rat was calculated from the wet weight of the remaining liver divided by the estimated preoperative weight of the whole liver times 100[5].

Measurement of serum alanine aminotran-sferase level

The serum alanine aminotransferase activity was measured by the method used in our laboratory with modifications[6]. Lactate dehydrogenase (LDH), L-ala nine, reduced nicotinamide adenine dinucleotide (NADH) and α-ketog lutarate were prepared at concentrations of 20 U/mL, 0.8 M, 2 mM and 0.1 mM, respectively. Serum (0.1 mL) was added to 0.1 mL NADH, 0.1 mL alanine, 0.1 mL α-ketoglutarate and 0.5 mL of 0.2 M phosphate buffer mixture. The final solution was incubated at 37 °C for 1 min. LDH in 0.1 mL was then added and the absorbance of the mixture was measured at 340 nm for 3 min with a spectrophotometer (Beckman, DU650, USA), using mixture without LDH as the blank. The rate of decrease in absorbance was determined and the amount of alanine aminotransferase present was calculated with a formula. Serum alanine aminotransferase levels were expressed as U/mL.

Statistical analysis

The results were expressed as mean ± S.E. (mean ± SD) and statistical analysis was performed with an analysis of variance (ANOVA) followed by a Dunnett t test. A value of P < 0.05 was considered to be statistic ally significant.

RESULTS

Liver regeneration

The remaining liver tissue regenerated along with the time after partial hepatectomy. Two or three days after hepatectomy the regenerative rates were 51% or 84%, respectively in the vehicle treatment groups. Heparin had no significant effect on hepatic regeneration. The regenerative rates in all three doses of heparin treated for 2 or 3 d, except for the highest dose in the 3 d group, remained similar to those of the control. The highest dose of heparin treated for 3 d, however, significantly attenuated the liver regeneration when compared with control group (Figure 1).

Figure 1 Effect of different doses of heparin treated for 2 or 3 d on liver regeneration in partially hepatectomized rats. Open columns represent groups with normal saline treatment. Hatched, solid, or cross-hatched columns stand for groups treated with heparin at doses of 100, 500, or 1000 U/kg, respectively. Each column represents the mean ± S.E.M. of 5 animals. ^aP < 0.05 vs corresponding group with normal saline treatment.

Serum alanine aminotransferase level

Serum alanine aminotransferase activity was significantly increased after partial hepatectomy at day 2 when compared with the enzyme level of normal rats. These levels seemed to be recovered back to normal level at the 3^rd day after operation. Heparin at all three doses treated for 2 or 3 d did not significantly affect the serum alanine aminotransferase activity in partially hepatecto-mized animals (Figure 2).

Figure 2 Effect of different doses of heparin treated for 2 or 3 d on serum alanine aminotransferase level in partially hepatectomized rats. Open columns represent groups with normal saline treatment. Hatched, solid, or cross-hatched columns stand for groups treated with hepari n at doses of 100, 500, or 1000 U/kg, respectively. Each column represent s the mean ± S.E.M. of 5 animals. ^aP < 0.05, ^bP < 0.001 vs 0 day with normal saline treatment.

DISCUSSION

The present study showed that partial hepatectomy stimulated the remnant liver to regenerate in a time dependent manner which was comparable with the previous study[5]. Liver regeneration is a compensative mechanism to restore the organ function after injury or related diseases. After partial hepatectomy, liver mass doubled within 48 h and completely recovered in 7-10 d. This process was relatively well synchronized with the peaks of DNA synthesis and mitosis occurring at approximately 24 and 30 h, respectively[4,7]. It has been re ported that heparin could stimulate endogenous HGF production and consequently enhance DNA synthesis in hepatocytes in vivo after hepatic injury. The latter effect was significant within36 h after hepatectomy, but the same effect was not examined thereafter. More importantly, the actual liver regeneration and function had not been assessed[8].

Although the early phase of liver regeneration is important for liver growth, the later stage of liver development and maturation of hepatocytes is the final determinant for a normal functional liver. The present study determined the effects of heparin on liver regeneration and its function 48 and 72 h after hepatectomy. Our results demonstrated that heparin at all doses accelerated no hepatic regeneration. The highest dose instead significantly retarded liver regeneration in the 3^rd day. This finding suggested that higher dose and longer period of heparin treatment might be harmful rather than beneficial to liver regeneration.

Increased liver regeneration is reflected by improvement of hepatic functions. Serum alanine aminotransferase level is one of the indicators for hepatic functions in clinical practice. In the present study, partial hepatectomy induced a significant increase in serum enzyme activity and tended to recover along with the natural liver regeneration at the 3^rd day after hepatectomy. These findings were in accord with the previous study[5]. Our present data also indicated that heparin could not accelerate the recovery of alanine aminotransferase level as liver regenerated on the 2^nd and 3^rd after hepatectomy. Although heparin could stimulate DNA synthesis of hepatocytes at the first day after partial hepatectomy[8], it could not improve the deteriorated action on the liver due to hepatectomy beyond that period of time. Taken together, heparin is not an ideal hepatotrophic drug when it is used alone in liver injury or related diseases.

ACKNOWLEDGMENTS

The project is supported in part by the CRCG and RGC grants from the University of Hong Kong and the Hong Kong Research Grant Council, respectively.