Calcitriol inhibits hepatocyte apoptosis in rat allograft by regulating apoptosis-associated genes

Abstract

Calcitriol, the active form of vitamin D, exerts important immunoregulatory effects. After rat liver allografting, calcitriol suppresses acute rejection. The aim of this study was to investigate whether calcitriol regulates hepatocyte apoptosis, in parallel with its inhibition of acute rejection in rat liver allografts. Liver allografts were transplanted in a high responder strain combination (SD to Wistar rats) and calcitriol was administered to the recipients, while control recipients received no immunosuppressant. Graft specimens were harvested on postoperative days 1, 3, 5 and 7 for histological analysis and protein assay. Hepatocyte apoptosis was assessed by the TUNEL method. Levels of intragraft Bcl-2, Bcl-xL, Bax, TNF-α and IFN-γ proteins were measured by Western blot analysis. Expression of Fas, Fas ligand and caspase-3 was determined by immunohistochemical analysis. Calcitriol markedly inhibited hepatocyte apoptosis. In the calcitriol-treated allografts, Bcl-2 and Bcl-xL levels increased while Bax and caspase-3 levels significantly decreased. The expression of Fas ligand was clearly reduced while Fas remained unchanged. TNF-α and IFN-γ proteins were also significantly decreased in the presence of calcitriol. These results show that calcitriol acts as a promoter of the anti-apoptosis genes Bcl-2 and Bcl-xL and an inhibitor of the pro-apoptosis genes Bax and caspase-3. These effects may be related to its suppression of the Fas/Fas ligand pathway and its inhibition of cytotoxic T lymphocyte products.

Introduction

Liver allograft rejection results from the immune response to allogeneic antigens, in which cytotoxic T cells play a major role in the cytolysis of allogeneic target cells. The incidence of apoptotic hepatocyte death in rat liver allografts increases steadily over time, but the same pattern is not detected in tolerated graft livers [1], [2]. This indicates that apoptosis is an important mechanism leading to hepatocyte death in liver allografts during rejection. Regulators of hepatocyte apoptosis include Fas/Fas ligand, TNF-α/TNF-α receptor, and IFN-γ/IFN-γ receptor [3], [4], [5], [6]. Furthermore, the signals induced by these molecules on the cell membrane of target cells probably interact with regulators such as caspase and the Bcl-2 family located in the cytosol or on the nuclear membrane of cells [7], [8], [9]. Although the precise mechanism underlying apoptosis during rejection remains unclear, these pathways are probably involved.

Accumulating evidence shows that calcitriol (1alpha, 25-dihydroxyvitamin D3) inhibits the activation of immunoactive cells [10], [11], [12]. In our previous study, we confirmed that calcitriol inhibits acute rejection and prolongs recipient survival after rat liver allografting [13], [14], [15]. The present study investigated whether calcitriol regulates hepatocyte apoptosis, in parallel with its inhibition of acute rejection of the allograft.