The Expression and Mechanism of NF-κ B and IL-6 after 90% Portal Vein Ligation in Rats
|School||Third Military Medical University|
|Course||Human Anatomy and Embryology|
|Keywords||Liver regeneration Portal vein branch Ligation Hyperplasia Apoptosis Rats NF-κB IL-6|
BackgroundThe partial hepatectomy is still the preferred and most effective treatment of liver cancer and hepatic hilar cholangiocarcinoma,the residual liver function after a large number of liver tissue was removed is the most important surgical problems. The small-for-size syndrome and receptor and donor liver decompensation after over-harvesting of donor liver tissue also hindered the wide range of living donor liver transplantation.The liver has a powerful regenerative capacity.With the continuous development of biological experimental techniques,the molecular mechanism of liver regeneration and regulation mechanisms of a lot of research has carried out, transgenic and gene knockout mice animal models of liver regeneration applications enable the molecular a breakthrough in biological research.PH models mostly used in the research of liver regeneration. The liver volume is restored in about 1 week after 70% PH. The proliferation of liver regeneration depends on the remaining liver tissue differentiation and maturation of cells re-activation, access to proliferative activity, start the follow-up of DNA replication, cell division and proliferation. Different types of non-parenchymal cells can secrete various cytokines acting on the liver cells and control of liver regeneration precisely.The portal vein ligation model have used in the last 20 years by researchers,it is not attracted widespread attention until the end of last century due to radical resection for get a large number of liver tissue removed in hilar cholangiocarcinoma. The current results show that the peak of liver regeneration is reached at the ligation of part portal vein branch after 2 weeks.This shows that liver regeneration is closely related to the changes in portal vein pressure.The process of liver regeneration is regulated by many cytokines and growth factors. NF-κB is the convergence point of a variety of signal transduction pathways. Interleukin -6 (IL-6) which induced by NF-κB plays a key role in a variety of gene regulation of inflammation, proliferation.In recent years, a great deal of NF-κB and IL-6 research have done in the rat PH model by domestic and foreign scholars. Injury can stimulate the rapid release of TNF-αand other cytokines to activate a nuclear transfer of NF-κB, the subsequent downstream gene activation after PH.The IL-6 expression is increased as IL-6 gene are mainly three cis-binding sites:NF-κB, C / EBPβ, AP-1. IL-6 and its receptor complex (gp130/IL-6Rα) combination, activated JAK kinases, STAT1 and STAT3 to phosphorylated, activated STAT3 and other transcription factors (C / EBPβ, AP-1) increased cell number and the proliferation factor expression, such as c-myc, c-fos, cooperative growth factor (such as HGF, EGF, etc.) to promote liver cell mitosis, for proliferation, also activated downstream of MAPK signaling pathway, and the pathway can promote and growth, differentiation, splitting the gene expression and mediated liver regeneration.Although a lot of research and exploration have been carried out from different angles and have made some preliminary conclusions, but most studies have based on the model of hepatectomy, or in the main body of liver cell proliferation, the results and conclusions have some limitations. This study discuss the mechanism of NF-κB, and IL-6 in liver regeneration after the limit of portal vein branch ligation by the establishment of the 90% PBL model in rats.Purpose1. To establish 90% limit of portal vein branch ligation animal model, provide a theoretical basis for the pre-processing of advanced liver cancer and intrahepatic large bile duct stones prior to surgery.2. To discuss the mechanism of NF-κB and IL-6 in the 90% limit of portal vein branch ligation in rat liver regeneration.Methodology96 male Sprague-Dawley rats were provided by the Experimental Animal Center of Sichuan University. Their body mass are (220±20) g.The rats were randomly divided into 2 groups: experimental group and control group of 48 rats, each group was randomly divided into eight sub-groups of 6 rats, respectively, POD0.5, POD1, POD3, POD5 , POD7, POD14, POD21 and POD28.Experimental group:1. Animal Model:With 1.5% sodium pentobarbital intraperitoneal injection anesthesia. Reference to literature method, from the median vertical incision abdominal surgery under a microscope with 4-0 silk ligature supply of right lobe of liver and caudate lobe of the right portal vein branch and supply of the left lateral lobe and the middle of the portal vein branch, leaving only a small leading to two leaves of the mastoid branch of portal vein without ligation, hepatic artery and bile ducts remained intact. Was ligated side of the liver accounts for about 90% of the total hepatic mass.2. Determination of PVP:The total mesenteric vein to portal vein portal vein pressure measured after intubation, and immediately executed accordingly rat liver tissue obtained detection and observation.3. Index:(1) Observe the color and texture of the liver, measured using electronic scales rats in each group and retention of the overall quality of side-lobe liver mass, calculate the percentage of retention side lobe;(2) To fixed-embedded and sliced the retain side lobe of conventional, HE staining of pathological changes in the structure;(3) To detected the retain side lobe of liver cell proliferation (PCNA) by the immunohistochemical S-P method and analyzed to calculate the average positive rate.(4) To detected the retain side lobe of liver cell apoptosis using in situ end labeling (TUNEL) on the retention side lobe of liver and calculate the average positive rate.(5) To detected the retain side lobe of liver NF-κB, IL-6 expression by the immunohistochemical detection of S-P method to retain side-lobe liver and to determine an average cumulative optical density (IOD).Control group:sham operation group, only free to go out vein branch ligation that is not closed abdomen, the other experiment the same experimental group.Results(1) 95.8% rats survived from the ligation of 90% portal branch. (2) Hepatic lobes at the ligated side diminished progressively after ligation,whereas the lobes of the unligated side underwent compensatory regeneration. The ratio of non-ligated lobes weight to the whole liver increased slowly within 24h, speeded up significantly during 24h-5d period, increased slowly after POD5,and got "the plateau stage"at POD7. The total mass of the whole liver only slightly lower than the 1d-3d after the first normal levels, the remaining time points were close to normal levels, the difference was not statistically different.(3) Portal vein pressure increased immediately after the operation, it reached the peak point at 12h(P<0.01), and dropped to normal at POD7.(4) The control group showed the hepatocells arranged radially centered of the central vein .The experiment group showed that liver cells are swelling mildly,the Boundary of portal areas, interlobular blood vessels and bile duct are clearly after 12h.The hepatocells started to proliferation obviously at POD1,there are many mitotic-phases, accompanied by the nuclear increased periportal areas, interlobular blood vessel dilatation and congestion. The hepatocells nuclei divided actively at POD3-5. The inflammatory cells area infiltrated in periportal area. There are more mitotic-phases existed at POD7-14 ,and the hepatocells are larger than before.Little inflammatory cells are infiltrated in interstitial area at POD21-28, and the small bile duct is hyperplasia, liver cord structure, and sinusoidal is clear.(5) PCNA index were markedly increased within POD0.5 -3(P<0.01).It reached the peak at POD5 and decreased slightly at POD7,but still higher than pre-operation level(P<0.01),then gradually return to normal lately.(6) It is hard to find any apoptotic cells from the pre-operation liver and the non-ligated lobes after 90% PBL at any time point.(7) The expression of NF-κB and IL-6 of the non-ligated liver lobes were reached the peak at POD1and POD0.5, then gradually return to normal lately.(8) The expressions of PCNA in non-ligated lobes and portal vein pressure(PVP) had a positive correlation at POD1,3,5 and a negative correlation at POD14(P <0. 05). The PCNA and NF-κB in liver had a positive correlation at POD0.5,1,14,21,28.The expressions of PCNA and IL-6 had a positive correlation at POD1,14,21.The expressions of NF-κB and IL-6 had a positive correlation at POD1,7,21,28. ConclusionLigation of 90% portal branch can induce active regeneration of hepatic cell of non-ligated liver lobes in rats and restore to previous liver weight. 90% PBL of rat is a model of studing liver regeneration. The portal pressure may has played a pivotal role in liver regeneration.The expression of NF-κB and IL-6 may has played a pivotal role in liver regeneration after 90% portal branch ligation in rats.