cytochrome-c-t and Hyperammonemia

Hepatic failure (HF) is caused by several factors, which induce liver cell damage and dysfunction. However, the specific mechanism of HF remains to be fully elucidated. The present study aimed to investigate the underlying cause of hepatocyte injury and liver dysfunction. Liver cells were isolated from healthy female Sprague‑Dawley rats, aged between 6 and 8 weeks, weighing ~230 g. The liver cells were cultured in RPMI‑1640 medium containing 10% fetal bovine serum. An MTT assay was used to examine the inhibitory rate of liver growth in each group. Flow cytometric analysis was performed to detect liver cells undergoing apoptosis. The protein expression levels of poly (ADP‑ribose) polymerase (PARP) and cytochrome c (Cyt C) were detected by western blotting. The level of calmodulin‑dependent kinase (CaMK) was assessed using an ELISA. The results indicated that the growth inhibitory rate of rat liver cells was significantly increased following treatment with increasing concentrations of NH4Cl. The results of flow cytometric analysis demonstrated that the apoptotic rate in the BAPTA‑acetoxymethyl ester group was significantly lower compared with the NH4Cl group (P<0.05). Treatment with NH4Cl increased the protein expression levels of PARP and Cyt C in the liver cells. The mRNA expression of CaMK decreased gradually following treatment with increasing concentrations of NH4Cl for 6, 12 and 24 h. The results suggested that hepatocyte injury and liver dysfunction may be caused by inducing apoptosis via the PARP and Cyt C pathways. Additionally, downregulation of CaMK may be associated with the apoptosis observed in hepatocyte injury.

Topics: Ammonium Chloride; Animals; Apoptosis; Calcium-Calmodulin-Dependent Protein Kinases; Cell Proliferation; Cytochromes c; Disease Models, Animal; Female; Hepatocytes; Hyperammonemia; Liver Failure; Poly(ADP-ribose) Polymerases; Rats; Signal Transduction

Numerous pathological processes that affect liver function in patients with liver failure have been identified. Among them, hyperammonia is one of the most common phenomena.The purpose of this study was to determine whether hyperammonia could induced specific liver injury.. Hyperammonemic cells were established using NH4Cl. The cells were assessed by MTT, ELISA, and flow cytometric analyses. The expression levels of selected genes and proteins were confirmed by quantitative RT-PCR and western blot analyses.. The effects of 20 mM NH4Cl pretreatment on the cell proliferation and apoptosis of primary hepatocytes and other cells were performed by MTT assays and flow cytometric analyses. Significant increasing in cytotoxicity and apoptosis were only observed in hepatocytes. The cell damage was reduced after adding BAPTA-AM but unchanged after adding EGTA. The expression levels of caspase-3, cytochrome C, calmodulin, and inducible nitric oxide synthase were increased and that of bcl-2 was reduced. The Na+-K+-ATPase activities in hyperammonia liver cells was no signiaficant difference compaired with the control group, but was decreased in astrocytes. NH4Cl pretreatment of primary hepatocytes promoted the activation of mitochondrial permeability transition pores and the mitochondria swelled irregularly.. Hyperammonia induces specific liver injury through an intrinsic Ca2+-independent apoptosis pathway.

Topics: Apoptosis; Calcium; Calmodulin; Caspase 3; Cell Line, Tumor; Cell Membrane Permeability; Cell Proliferation; Cytochromes c; Gene Expression Profiling; Hep G2 Cells; Hepatocytes; Humans; Hyperammonemia; Liver Diseases; MCF-7 Cells; Mitochondria; Nitric Oxide Synthase Type II; Proto-Oncogene Proteins c-bcl-2; Real-Time Polymerase Chain Reaction; RNA, Messenger; Sodium-Potassium-Exchanging ATPase