Zimmermann H, Kurzen P, Klossner W, Renner E L, Marti U
Department of Clinical Pharmacology, University of Berne, Switzerland.
J Hepatol. 1996 Oct;25(4):567-73. doi: 10.1016/s0168-8278(96)80218-2.
BACKGROUND/AIMS: Inducible nitric oxide synthase (iNOS) in the liver has been shown to increase after stimulation by different compounds. Its exact role in cirrhosis is unclear, but it is thought to be an important factor in the development of the hyperdynamic state. In contrast, little is known about the constitutive form of nitric oxide synthase (cNOS) in the liver, and in particular in liver disease. We therefore investigated immunohistochemical expression of endothelial NOS (cNOS) and, in addition, of macrophage NOS (iNOS), a constitutive and inducible form of NOS, in secondary biliary fibrosis and after reversing these changes by biliodigestive anastomosis.
Sprague-Dawley rats were allocated to one of seven groups: sham-operated controls (CTR), bile duct ligation for 3 weeks (BDL) and BDL followed by Roux-en-Y choledochojejunostomy (RY); the latter group was studied after 1, 2, 3, 7 and 45 days (RY1, RY2, RY3, RY7, and RY45). cNOS-positive hepatocytes were stained with a monoclonal antibody specifically recognizing this isoenzyme, and quantitated stereologically.
Virtually all hepatocytes were positive for cNOS in CTR (99.9 +/- 0.2%); this was markedly reduced in BDL (73.7 +/- 9.8%; p < 0.05). After RY, cNOS positive hepatocytes increased to reach normal levels (99.7 +/- 0.5%) in RY45. In contrast to cNOS, there was no iNOS positive staining with a monoclonal macrophage NOS antibody for this isoenzyme, which was confirmed by determining total iNOS protein concentration by Western blot. Serum nitrite/nitrate concentrations mirrored these findings and decreased from 7.9 +/- 3.7 mol/l in CTR to 2.1 +/- 0.4 mol/l in BDL. After RY, nitrite/nitrate concentration increased to 22.7 +/- 30.1, 32.4 +/- 21.4 and 44.6 +/- 32.7 mol/l in RY1, RY2 and RY3, respectively; in RY45, serum nitrite/nitrate concentration was normal averaging 6.1 +/- 1.2 mol/l.
The present investigation shows that, in secondary biliary fibrosis, endothelial nitric oxide synthase is decreased in hepatocytes, macrophage nitric oxide synthase is not increased, and that-in contrast to current thinking-nitrate/nitrate production is diminished.
背景/目的：肝脏中的诱导型一氧化氮合酶（iNOS）已被证明在受到不同化合物刺激后会增加。其在肝硬化中的确切作用尚不清楚，但被认为是高动力状态发展的一个重要因素。相比之下，关于肝脏中一氧化氮合酶的组成型形式（cNOS），尤其是在肝脏疾病中的情况，人们了解甚少。因此，我们研究了继发性胆汁性肝纤维化以及通过胆肠吻合术逆转这些变化后，内皮型一氧化氮合酶（cNOS）以及巨噬细胞一氧化氮合酶（iNOS，一氧化氮合酶的组成型和诱导型形式）的免疫组化表达。
将Sprague-Dawley大鼠分为七组之一：假手术对照组（CTR）、胆管结扎3周（BDL）以及胆管结扎后行Roux-en-Y胆总管空肠吻合术（RY）；对后一组在术后1、2、3、7和45天进行研究（RY1、RY2、RY3、RY7和RY45）。用特异性识别该同工酶的单克隆抗体对cNOS阳性肝细胞进行染色，并进行体视学定量分析。
在CTR组中，几乎所有肝细胞cNOS呈阳性（99.9±0.2%）；在BDL组中这一比例显著降低（73.7±9.8%；p<0.05）。RY术后，cNOS阳性肝细胞数量增加，在RY45组达到正常水平（99.7±0.5%）。与cNOS不同，用单克隆巨噬细胞NOS抗体对该同工酶进行染色未发现iNOS阳性，通过蛋白质免疫印迹法测定总iNOS蛋白浓度也证实了这一点。血清亚硝酸盐/硝酸盐浓度反映了这些结果，从CTR组的7.9±3.7μmol/l降至BDL组的2.1±0.4μmol/l。RY术后，亚硝酸盐/硝酸盐浓度在RY1、RY2和RY3组分别升至22.7±30.1、32.4±21.4和44.6±32.7μmol/l；在RY45组，血清亚硝酸盐/硝酸盐浓度正常，平均为6.1±1.2μmol/l。
本研究表明，在继发性胆汁性肝纤维化中，肝细胞内的内皮型一氧化氮合酶减少，巨噬细胞一氧化氮合酶未增加，并且与目前的认识相反，亚硝酸盐/硝酸盐的产生减少。
