The Expression And Significance Of Nuclear Factor-κB In Thioacetamide-induced Hepatic Fibrosis In Beagles

Backgrouds:Nuclear factor-κB(NF-κB), a transcription factor, is retained in the cytoplasm in an inactive form. NF-κB plays a critical role in many aspects, such as the regulation of immune and inflammatory responses, apoptosis and tumorigenesis. Recent studies have found that NF-κB is involved in the formation and development of hepatic fibrosis, which the mechanism underlying fibrogenesis includes the activation and proliferation of cells in liver, such as hepatocyte, hepatic stellate cell(HSC) and Kupffer cell(KC). Our previous studies, in vitro and in vivo, have showed that NF-κB signaling pathway plays an important role in hepatic fibrosis.Animal models can provide experimental basis for studies of the occurrence, prevention and treatment of human diseases. Small mammals such as rats, mice, etc, are previously chosen as experimental models of hepatic fibrosis, which have a very important value for our understanding of the pathophysiology of the disease process. Studies should be based on large animals experiments before being translated into clinical researches. Monkeys, dogs and miniature pigs can be used as experimental large animals. Compared with monkeys and other non-human primates, dogs have the following advanstages: accessibility and low prices. Miniature pigs are mainly used to cardiovascular research. Beagles, four-way ovoss and mexican hairless dogs can be used in medical research. Beagles have many traits, including a docile temperament and genetic stability, sensitivities to the drug and less difference. With so many advantages, we chose beagles as our experimental subjects.Thioacetamide(TAA) is a hepatotoxic chemical substance. The modeling method is widely used in the study of small animals and has a high success rate. It is much similar to humans in the basic pathological features, hemodynamic and biochemical changes. But can TAA induce hepatic fibrosis in large animals? Is NF-κB signaling pathway involved in the process? There have been very few reports and we herein did this study. Objective:To investigate whether TAA can successfully induce hepatic fibrosis in beagles and to explore whether NF-κB signaling pathway is involved in the process. Methods:Twelve healthy male beagles, 8 months old, weighing 10±1 kilogrammes, were randomly divided into two groups: Control group and TAA group. Each group had six beagles. Beagles had a free access to food and water in control group for twelve weeks. In TAA group beagles were given subcutaneous injection of TAA(12 mg/kg, two times per week lasting for twelve weeks). Beagles were sacrificed humanely by 1.5% sodium pentobarbital anesthesia 12 weeks after TAA-treatment. Then we removed the same part of the liver tissue of all beagles. The liver tissues were fixed in 10% neutral formalin and embeded in paraffin wax. The morphological changes of liver tissue were observed by Hematoxylin and eosin(HE) staining. Masson trichrome staining aimed to evaluate the changes of content of collagen fibers. The expression of NF-κB p65, alpha-smooth muscle actin(α-SMA) and CollagenⅠ were tested by immunohistochernistry(IHC) staining. Results:1. The HE staining showed that hepatic cells arranged radially around the central vein in control group. Hepatic cord structure was clear and cytoplasm was full. While in TAA group hepatic cord structure disappeared, hepatic cells presented degeneration and necrosis, a large number of inflammatory cells infiltrated and fibrous cord formed.2. The Masson trichrome staining showed that filamentous collagen fibers expressed in the vascular wall of portal area and the central vein in control group. In TAA group, the expression of collagen fibers significantly increased. The fibers extended into the lobule. The liver tissue has a tendency to form false lobules.3. The results of the immunohistochernistry(IHC) staining are as follows:Brown yellow particles or dark brown particles were the positive expression.(1) Brown yellow particles were the positive expression of NF-κB p65 and were observed in the cytoplasm of individual hepatocytes around the central vein in control group. While the expression of NF-κB p65 increased and dark brown particles were observed in the cytoplasm and nucleus of hepatocytes in TAA group. Dark brown particles were particularly obvious in the nucleus of hepatocytes around the fibrous septa.(2) In control group, the expression of α-SMA was mild in the vessel wall. While in TAA group, α-SMA expression was increased in the fiber septa, the central vein, portal area and hepatic sinusoid.(3) We observed that a small amount of CollagenⅠ was expressed in the central vein and blood vessel wall in control group. While in TAA group, the expression of CollagenⅠ enhanced obviously in portal area, fiber septa and hepatic sinusoid.4. Image analysis of Masson trichrome staining showed that the expression of collagen fibers was significantly increased in TAA group compared with control group and the difference was statistically significant(1.54±0.11 vs 7.43±0.17, P<0.01).5. Image analysis of immunohistochemical staining showed that the expressions of NF-κB p65, α-SMA and CollagenⅠ were significantly enhanced in TAA group compared with control group and the difference was statistically significant(0.16±0.03 vs 5.17±0.02, P<0.01; 0.56±0.03 vs 5.39±0.17, P<0.01; 0.15±0.01 vs 4.41±0.11, P<0.01). Conclusions:NF-κB signaling pathway may be involved in TAA-induced hepatic fibrosis in Beagles.