Inducible nitric oxide synthase inhibitor 1 400W suppresses endoplasmic reticulum stress and alleviates ischemia-reperfusion injury in human intrahepatic bile duct epithelial cells

Abstract

Objective To explore the role and mechanism of inducible nitric oxide synthase inhibitor 1 400W in alleviating ischemia-reperfusion injury of human intrahepatic bile duct epithelial cells. Methods Human intrahepatic bile duct epithelial cells (HIBEC) in logarithmic phase were inoculated into culture plate at an appropriate density. The samples were randomly divided into control group (group C), ischemia-reperfusion group (group I/R) and ischemia-reperfusion + 1 400W group (group I/R+ 1 400W). Group C was cultured routinely; cells in I/R and I/R+ 1 400W groups were placed in a three-gas incubator for 12h for simulating ischemia and then normal culture for 6h for simulating reperfusion. The I/R+ 1 400W group had a final concentration of 100 μmol/L of 1 400W before ischemia and hypoxia. After reperfusion, cells and culture medium were collected, CCK 8 was used for detecting cell vitality, microplate method for detecting the content of lactate dehydrogenase (LDH) in culture medium, AnnexinV-FITC/PI double stain for detecting apoptosis level, Western blot for analyzing the expressions of endoplasmic reticulum stress (ERS) related protein cysteinyl aspartic acid protease 12 (caspase-12), glucose regulatory protein 78 (GRP78) C/EBP homologous protein (CHOP) and inducible nitric oxide synthase (iNOS). Results As compared with group C, cell viability significantly decreased in I/R and I/R+ 1 400W groups (53.8%±2.3% vs.100%, 66.5%±2.8% vs.100%) (P<0.05) while LDH increased markedly in cell culture medium (287.4±9.0U/L vs 120.2±8.7U/L, 212.0±8.3U/L vs 120.2±8.7U/L) (P<0.05). Apoptosis accelerated markedly (41.5%±2.3% vs 5.2%±0.5%, 32.7%±1.8% vs 5.2%±0.5%) (P<0.05) and the expressions of caspase-12, GRP78, CHOP and iNOS spiked (P<0.05); as compared with I/R group, cell viability of I/R+ 1 400W group rose while LDH, apoptosis level, caspase-12, GRP78 and CHOP declined in cell culture medium (P<0.05). Conclusions 1 400W may alleviate ischemia-reperfusion injury of human intrahepatic bile duct epithelial cells and its mechanism may be correlated with a suppression of endoplasmic reticulum stress. Key words: Ischemia-reperfusion injury; Bile duct epithelial cells; Nitric oxide synthase