Cytochrome c modulates the mitochondrial signaling pathway and polymorphonuclear neutrophil apoptosis in bile duct-ligated rats

Abstract

It has been observed that polymorphonuclear neutrophils (PMN) increase in number and function during obstructive jaundice (OJ). However, the precise mechanisms underlying PMN apoptosis during OJ remain poorly understood. The aim of the present study was to investigate the modulation of cytochrome c (Cytc) on the mitochondrial signaling pathway in bile duct-ligated (BDL) rats and the effect on PMN apoptosis following the intravenous administration of Cytc. Rats were randomly divided into four groups: A control group, a sham group, a BDL group and a BDL + Cytc group (rats with common bile duct ligation as well as Cytc intravenous injection). Blood samples were collected from the inferior vein cava for biochemical analysis and separation of the PMN. PMN apoptosis was evaluated using flow cytometry. The mitochondrial membrane potential (ΔΨm) of PMN was detected by rhodamine-123 staining. The Cytc protein expression levels were examined using western blotting. PMN mitochondria were observed using transmission electron microscopy. The results of the present study revealed that the PMN apoptosis rate in rats decreased gradually from 12 to 72 h following BDL to levels that were significantly lower than those of the control group and the sham group. Compared with the corresponding time point of the BDL group, the BDL + Cytc group showed a significantly increased PMN apoptosis rate. The mean fluorescence intensity (MFI) of ΔΨm decreased from 12 to 72 h following BDL, and was significantly increased compared with the control and sham groups. MFI in the BDL + Cytc group was higher compared with that in the BDL group. Cytc expression levels increased in the mitochondria and decreased in the cytoplasm from the 12 to 72 h in the BDL group, which was significantly different from that in the control and sham groups at the corresponding time points. Compared with the BDL group, Cytc expression levels in the cytoplasm for the BDL + Cytc group tended to gradually and significantly increase. Morphological changes in PMN mitochondria were marginal in BDL rats and marked in the BDL + Cytc group. In the BDL rats, PMN apoptosis was inhibited, a process induced by the mitochondrial apoptotic signaling pathway in which Cytc has an important role. High ΔΨm in the mitochondria and decreased Cytc expression levels in the cytoplasm result in PMN apoptosis inhibition. Intravenous injection of Cytc may help compensate for the lack of Cytc proteins in the cytoplasm, inducing PMN apoptosis following BDL.