The prolyl 4-hydroxylase inhibitor HOE 077 prevents activation of Ito cells, reducing procollagen gene expression in rat liver fibrosis induced by choline-deficient L-amino acid-defined diet

Abstract

No effective therapy has yet developed for liver fibrosis by directory inhibiting the accumulation of extracellular matrix. The effect of a newly synthesized prolyl4-hydroxylase (PH) inhibitor, HOE 077 (pyridine-2, 4-di-carboxylic-di(2-methoxyethyl)amide), was examined using the model of choline-deficient L-amino acid (CDAA) defined diet-induced liver fibrosis in 16-week-old male Wistar rats. HOE 077 at doses up to 200 ppm prevented fibrosis in a dose-dependent manner, as indicated by reduced hydroxyproline content in liver as well as inhibition of increased serum fibrotic markers (PIIIP, 7S, hyaluronic acid). HOE 077 at 200 ppm reduced expression of type III procollagen alpha 1, messenger RNA (mRNA) in the liver, with a good correlation with serum PIIIP and hydroxyproline content of the liver. Histologically, HOE 077 at 200 ppm also reduced proliferation of myofibroblastlike cells (activated Ito cells). These results indicate that a PH inhibitor can prevent fibrosis by inhibiting not only the hydroxylation of proline but also the activation of Ito cells, which are considered the main collagen-producing cells, resulting in reduced expression of procollagen mRNA.

Fibrosis accelerates the development of enzyme-altered lesions in the rat liver

“…Five-micrometer thick sections of the right lobe of all rat livers were processed routinely for hematoxylin and eosin and Azan-Mallory staining, and were examined immunohistochemically for GSTP and ␣SMA by the advidinbiotin-peroxidase complex method, as described previously. 2,3 Rabbit anti-rat GSTP antibody (MBL Inc., Nagoya, Japan) and anti-␣SMA monoclonal antibody (DAKO Inc., Kyoto, Japan) were employed. Quantitative analysis of ␣SMA-positive cells and the number and size of the GSTP-positive lesions were carried out with an image analyzing system (Personal Image Analysis System LA-555, Pias Ltd., Osaka, Japan).…”

“…As vessels were stained by anti-␣SMA monoclonal antibody, the mean value of three specimens (6 ocular fields per one specimen) of untreated (CSAA diet alone) rat livers was subtracted from each experimental specimen and ␣SMA positive cells were expressed as a percentage of the total area of the specimen as described previously. 2,3 The number of GSTP-positive lesions was expressed as number/cm 2 .…”

“…The hydroxyproline content was determined by a modification of Kivirikko' s method, as reported previously. 3 Briefly, liver specimens were weighed, and 20 mg of freezedried sample was hydrolyzed in 6 mol/L HCl at 110°C in an autoclave at a pressure of 1.2 kgf/cm 2 for 24 hours. After centrifugation at 2,000 rpm at a temperature of 4°C for 5 minutes 2 mL of supernatant was mixed with 50 mL of 1% phenolphthalein and 8-N KOH to obtain a total volume of 5 mL liquid at pH 7 to 8 mol/L.…”

“…Because it induces preneoplastic lesions as a consequence of liver damage and regeneration. [1][2][3][4] These preneoplastic lesions can be detected by using an antibody to the placental form of glutathione S-transferase (GSTP). With regard to hepatocarcinogenesis induced by dietary choline deficiency in rats, induction of DNA lesions as well as lipid peroxidation of the inner nuclear, microsomal and mitochondrial membranes has been demonstrated.…”

“…13,14 Previously, we reported that the suppression of fibrosis by inhibiting stellate cell activation resulted in a reduction of preneoplastic lesions in rats fed a CDAA diet. 2,3 The present study was conducted to investigate the influence of pig serum-induced pre-existing fibrosis on the development of preneoplastic lesions in rats fed a CDAA diet.…”