Effects Of Internal And External Biliary Drainage On Inducible Nitric Oxide Synthase Expression By Kupffer Cells And Serum Tumor Necrosis Factor-alpha In Rats With Obstructive Jaundice

[Background and Aims] Obstructive jaundice (OJ) is associated with a high postoperative complications and mortality. The patients with obstructive jaundice are often complicated with infection and septicemia, which suggested that the immune function of the patients is depressed in the presence of obstructive jaundice. The Kupffer cells, as the main composition of the important body defensive system, the reticuloendothelial system (RES), have become the focus of domestic and abroad scholars. Our previous study has found that Kupffer cells from rats with obstructive jaundice produce great amount of cytotoxic nitric oxide (NO) under the stimulation of endotoxin. Following relief of the obstructive jaundice, the changes can be reversed by internal biliary drainage, but not by external drainage. The mechanism that biliary drainage affects the production of NO by Kupffer cells is unclear yet. We propose a hypothesis that the effects of biliary drainage on the production of NO by Kupffer cells are based on the action of inducible nitric oxide synthase (iNOS) and related to tumor necrosis factor-alpha (TNF-a). In this experiment, we study the effects of internal and external biliary drainages on the expression of iNOS by Kupffer cells and the level of serum TNF-a in rats with obstructive jaundice, in order to explore the molecular biology basis of internal biliary drainage being superior to external drainage.[Methods] Male adult Sprague-Dawley rats were randomly assigned to four groups: obstructive jaundice (OJ; n=12), sham operation (SH; n=12), internal biliary drainage (ID; n = 12) and external drainage (ED; n = 12). Rat models were successfully established by twice operations. OJ was induced by common bile duct (CBD) ligation and division. SH was produced by separating CBD locally but not dividing. ED was performed by exteriorizing a drainage tube at the nape of the rat, while ID was performed by implanting a drainage tube between the dilated end of CBD and duodenum on the 7^th day after the first operation. On the 7^th dayafter the second operation, the rats were succumbed and specimen such as bloods and livers were taken. Kupffer cells were isolated by in situ hepatic perfusion and digestion with collagen IV, density gradient centrifuged by Percoll reagent and purified by cell culture attachment. Kupffer cells were identified by peroxidase staining with 3,3-diaminobenzidine tetrahydrochloride (DAB). The expression of iNOS mRNA by the Kupffer cells was detected by reverse transcription polymerase chain reaction (RT-PCR) and the serum TNF-alpha concentrations were measured with ELISA.[Results] Rats models were successfully established. rats in OJ group had poor appetite, and the mean weight development was lower than rats in SH group(P<0.01) . Meanwhile, the appetite and the weight development of ID rats were better than that of ED rats(P<0.05). The serum TNF-alpha level was increased in OJ rats (110.9±26.2 pg/ml), although it was not statistically significant compared to the level in SH rats (91.1±14.3 pg/ml). ED failed to reverse the elevation (118.6±22.7 pg/ml, P>0.05, ED vs OJ). The TNF-alpha level was decreased in ID group (89.8±28.3 pg/ml) and significantly lower than that in ED group (P<0.05). Expression of iNOS mRNA by Kupffer cells was stronger in OJ group(0.82±0.24) compared with SH group (0.38±0.35) (P<0.01). After relieving the OJ, the iNOS mRNA expression by the Kupffer cells from the ED group were not suppressed (0.98±0.48, P>0.05, ED vs OJ). On the contrary, the iNOS mRNA expression by the Kupffer cells from ID group (0.59±0.35) was depressed and significantly lower than that from ED group (P<0.05) and OJ group (P<0.01).[Conclusion ] Internal biliary drainage is superior to external drainage in terms of reversing the elevated serum TNF-alpha and in suppressing the iNOS mRNA expression by Kupffer cells in rats with obstructive jaundice. These findings indicate that the mechanism of internal biliary drainage is superior to external drainage in relief of obstructive jaundice seems to be based on the changes in the level of gene.