Combined effects of caspase inhibitor GS-9450 and lithospermate B on the inhibition of hepatic fibrosis in rats

Abstract

Chronic liver diseases progress from mild inflammation, to more severeinflammation, to fibrosis, and finally to cirrhosis. Dysregulated apoptosis is known to be a main cause of liver injury. Oxidative injury is also a possible mechanism of chronic liver damage. GS-9450, a pan-caspase inhibitor was developed and its anti-fibrogenic efficacy had been demonstrated in animal experiment. Magnesium lithospermate B (LAB), an anti-oxidant extracted from Salvia miltiorrhiza, has been give attention for its anti-fibrogenic effect. The purpose of this study was to investigate whether an additive or synergistic effect of anti-fibrosis can occur when two different drugs (GS-9450 and LAB) were used, compared to the individual administration of each agent in thioacetamide (TAA)-induced hepatic fibrosis rat model. To observe the antifibrotic effect of GS-9450, LAB, and GS-9450+LAB in theaspect of fibrosis-prevention and fibrosis-treatment, two parts of experimentswere separately performed. In the first part, male Spraque-Dawley (SD) rats weretreated with intraperitoneal TAA for 10 weeks and concomitantly with oralGS-9450 (or LAB or GS-9450+LAB) for 12 weeks. The rats were divided into 6groups: normal control group (n=5), normal+LAB+GS-9450 group (n=5), TAAcontrol group (n=5), TAA+LAB group (n=5), TAA+GS-9450 group (n=5),TAA+LAB+GS-9450 group (n=5). In the second part, SD rats were treated withoral GS-9450 (or LAB or GS-9450+LAB) for 12 weeks after establishment ofliver cirrhosis with TAA injection for 10 weeks. The rats were divided into 4groups: TAA control group (n=5), TAA+GS-9450 group (n=5), TAA+LAB group(n=5), TAA+GS-9450+LAB group (n=5). Histological assessment with H&Estain, Masson’s trichrome, Sirius red stain, and immunostaining for α-smoothmuscle actin (α–SMA), malondialdehyde (MDA) and 4-hydroxy-2 nonenal(4-HNE) were performed. Measurement of fibrotic area and seurm biochemicalanalysis such as aspartate aminotransferase (AST) were done. Reversetranscription-polymerase chain reaction (PCR) and quantitative real-time PCRwere carried out for the transcription of genes involved in liver fibrosis, that is,type I collagen α1, transforming growth factor β, and α–SMA. Apoptosis wasstudied by TUNEL assay. In fibrosis-prevention experiment (part I), the fibrosisarea in group ‘TAA’ was 16.5%, whereas the fibrotic area in group ‘TAA+LAB’,‘TAA+GS-9450’ was 6.3% and 6.7%, respectively (p < 0.001). The area tended tobe less in group ‘TAA+GS-9450+LAB’ compared to other groups. This result wassimilarly observed in fibrosis-treatment experiment (part II). The administration ofGS-9450 or LAB attenuated the increased expression of type I collagen α1induced by TAA injection. Co-administration of GS-9450+LAB further reducedthis fibrosis-related gene expression in part I (p < 0.05). TUNEL assasy showedthat apoptosis significantly decreased in rats treated with GS-9450 compared tothose with LAB. In both experiments of fibrosis prevention and treatment, thedegree of decrease tended to be more in rats treated with ‘GS-9450+LAB’compared with those with only ‘GS-9450. The anti-oxidant effect of LAB was also poteniated by co-administration ofGS-9450. In conculusion, our data suggest that treatment with GS-9450 andLAB, which have different mechanism of action, increased the fibrosis-preventiveand fibrosis-reversal effect in TAA-induced cirrhotic rat model.