Effect of inhibiting nuclear factor-kB activity on expression of inducible nitric oxide synthase in pulmonary intravascular macrophages of rats with hepatopulmonary syndrome

Abstract

Objective To investigate the effect of inhibiting nuclear factor kB（NF-kB） activity on expression of inducible nitric oxide synthase（iNOS） in pulmonary intravascular maerophages （PIM） of rats with hepatopulmonary syndrome （HPS）. Methods The SD rats were randomized into four groups： control, control＋ PDTC, CCl4, CCl4＋PDTC groups. The rats in the control group received no pretreatment. In CCl4 group, CCl4 （3 ml/kg ·4day） was given by intramuscular injection and repeated for l6 times. In CCI4 ＋PDTC group, PDTC was given by intraperitoneal injection （20mg/kg ·48 h） after given CCl4 for 4 times. In control＋PDTC group, only PDTC was given according to that of CCl4 ＋PDTC group. Arterial blood was collected for measurement of blood gas. Venous blood was sampled for determination of hepatic function and endotoxin level. The mesenteric lymph nodes were dissected for bacteriological analysis. Immunolocalization of macrophages was performed using monoclonal macrophages antibody ED1 on paraffin sections. Proteins of NF-kB and iNOS of lung tissue were examined with immunohistochemistry. The activity of NF-kB in lung tissues was determined u sing electrophoretic mobility shift assay （EMSA）. By real-time polymerase chain reaction （PCR） u-sing SYBR Green I, the mRNA expression of iNOS in lung tissues was detected. Results CCl4 group developed HPS with decreased PaO2 and PaCO2 , increased alveolar arterial oxygen difference （AaDO2）, abnormal hepatic function and increased endotoxin level, which were significantly different from those in the control and control ＋PDTC groups （P〈0.05）. Culture positive mesenteric lymph nodes were found in 62.5% （5/8） of CCl4 group and 66.7% （6/9） of CCl4 ＋ PDTC group （P = l. 000）. There were no culture-positive mesenteric lymph nodes in control and control ＋PDTC groups. All lungs from CCl4 and CCl4 ＋PDTC group showed accumulation of large mononuelear macrophagelike cells within the lumen of numerous small muscular and nonmuscular pulmonary vessels. The percentages of vessels with more than 10 adherent macrophages was 60.8% （292/480） in CCl4 group but only l9.6% （106/540） in CCl4＋PDTC group （P〈0.0l）. Immunohistochemical analysis showed that the protein expression of NF-kB and iNOS was localized to PIM in CCl4 group. Pulmonary NF-kB activity in CCl4 group was significantly higher than that in control and control＋ PDTC group （P〈0.05）. The NF-kB activity in CCl4 ＋PDTC group was significantly lower than that in CCl4 group （P〈0.05） and was not markedly different from that in control and control＋PDTC group（P〉0.05）. The mRNA expression of iNOS in CCL group was increased significantly when compared with that in control and control＋PDTC group （P〈0.05）. The mRNA expression of iNOS in CCl4 ＋PDTC group was significantly lower than that in CCl4 group （P〈0.05） but not remarkably different from that in control and control＋PDTC group（P〈0.05）. Conclusion The iNOS expression in PIM induced by NF-kB plays an important role in HPS. The inhibitor of NF-kB PDTC can repress PIM activation and decrease the expression of iNOS. Subsequently, HPS severity is reduced. Key words: Hepatopulmonary syndrome; Pulmonary intravascular macrophage; Nuclearfactor-kB; Inducible nitric oxide synthase