Wang H, Tanihata T, Fukumoto S, Hirai K
Department of Medical Zoology, Faculty of Medicine, Tottori University, Yonago, Japan.
Int J Parasitol. 1997 Apr;27(4):367-75. doi: 10.1016/s0020-7519(96)00197-x.
In this study, we observed the level of normal murine hepatocyte inducible NOS (iNOS) mRNA by semi-quantitative polymerase chain reaction (SQ-PCR) analysis after stimulation with ES products (ESP) and/or ESP fractions from the plerocercoids. We found that ESP are able to induce the expression of iNOS gene in a dose-dependent fashion. Treatment of ESP with polymyxin B did not affect their ability to induce the expression of iNOS gene, suggesting that bacterial lipopolysaccharide (LPS) is not involved. The iNOS-inducing factor (a) is soluble, and may be a component whose molecular mass exceeds 94 kDa as analyzed by a combination of SDS-PAGE and SQ-PCR. The peak of iNOS mRNA level was detected 3 h after stimulation with ESP; the mRNA level decreased sharply from 9 h. Dexamethasone inhibited the induction of mRNA for hepatocyte iNOS. In contrast, cycloheximide stimulated the induction; this suggests that de nova protein synthesis is important in the regulation of the ESP-induced expression of iNOS mRNA. Actinomycin D blocked the induction. In addition, the results of Northern blot analysis showed that ESP suppressed the LPS (10 micrograms/ml) and interferon-gamma (IFN-gamma, 100 U/ml)-induced hepatocyte iNOS mRNA expression in a dose-dependent fashion and the suppressing effect was more marked when hepatocytes were exposed to ESP 3 h prior to LPS and IFN-gamma. These results demonstrate that the soluble factor(s) of ESP is capable of inducing murine iNOS gene expression in hepatocytes in the absence of added cytokines.
在本研究中，我们通过半定量聚合酶链反应（SQ-PCR）分析，观察了正常小鼠肝细胞诱导型一氧化氮合酶（iNOS）mRNA在用来自裂头蚴的ES产物（ESP）和/或ESP组分刺激后的水平。我们发现ESP能够以剂量依赖的方式诱导iNOS基因的表达。用多粘菌素B处理ESP并不影响其诱导iNOS基因表达的能力，这表明细菌脂多糖（LPS）不参与其中。诱导iNOS的因子（a）是可溶的，通过SDS-PAGE和SQ-PCR相结合分析，其可能是一种分子量超过94 kDa的成分。在用ESP刺激3小时后检测到iNOS mRNA水平的峰值；从9小时起mRNA水平急剧下降。地塞米松抑制肝细胞iNOS mRNA的诱导。相反，放线菌酮刺激诱导作用；这表明从头合成蛋白质在ESP诱导的iNOS mRNA表达的调节中很重要。放线菌素D阻断诱导作用。此外，Northern印迹分析结果表明，ESP以剂量依赖的方式抑制LPS（10微克/毫升）和干扰素-γ（IFN-γ，100单位/毫升）诱导的肝细胞iNOS mRNA表达，并且当肝细胞在LPS和IFN-γ之前3小时暴露于ESP时，抑制作用更明显。这些结果表明，在不添加细胞因子的情况下，ESP的可溶性因子能够诱导小鼠肝细胞中iNOS基因的表达。
