The Effects Of TPA And UPA In Mouse Liver Fibrosis

Hepatic fibrosis is a major histological finding of chronic liver disease caused by excess extracellular matrix (ECM) production and reduced ECM degradation. ECM includes collagen, noncollagenous glycoproteins and proteoglycan. Among which, collage is the most important components of ECM.Plasminogen activators include tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA). Both of them belong to serine protease family. They activate proenzyme plasminogen (Plg) to biologically active plasmin. tPA, as a FDA permitted drug is wildly used in clinical as a thrombolytic agent. The main biological function of tPA is to cleave Plg into plasmin, which degrades fibrin of thrombus. However, uPA, combines its receptor uPAR, is found to play an important role in tumor invasion and metastasis. Recently, it is understood that plasmin not only directly degrades fibrin, but also activate the family of metalloproteinases (MMPs), which are the main enzyme family to degrade ECM. So, we hypothesize that plasminogen activators might play important roles in liver fibrosis.In order to study the function of tPA in the process of liver fibrosis, we used tPA gene knock out C57/bl mice. Firstly, we took injection intraperitoneally 25% carbon tetrachloride (CCM) (dissolved in olive oil) to mice twice per week for 4 weeks and set up liver fibrosis animal model in both wild-type and tPA gene knock out mice. Mice treated with intraperitoneal injection the same dosage of olive oil as the comparable controls. After 4 weeks, the mice were sacrificed. By means of zymography, we found that tPA activity was unregulated markedly in CCM induced wild-type mice liver compared to olive oil administrated mice liver. As expected, there was no tPA activity found in tPA knock out mice. It seems that tPA might plays an important role in liver fibrosis.HE (Hematoxylin and Eosin) staining found that mice lacking tPA developed more severe morphological injury with lots of inflammatory cells existing. VG (Van Gieson) staining and hydroxyproline analysis showed that the deposition of collagen protein was increased in tPA knock out mice liver after after CCl4 administration compared with wild-type counterparts. It was showed that deficiency of tPA gene accelerated mice liver fibrosis.Liver fibrosis is usually to be caused by excess ECM deposition, we analysis both ECM generation and ECM degradation in wild-type mice and tPA knock out mice.α-smooth muscle actin (α-SMA) is the molecular marker for hepatocyte stellate cells (HSCs) transforming to myofibroblasts (MFs), which is the main cell sources of ECM production. On the other hand, MMPs is the main enzymes to degrade ECM. Tissue inhibitor of metalloproteinase-1 (TIMP-1) is the main inhibitor of MMPs. We examined the protein expression of a-SMA, matrix metalloproteinase-13 (MMP-13), TIMP-1 and the protein activity of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9).α-SMA expression in CC14 administrated tPA knock out mice livers compared with wild-type mice was increased and MMP-2, MMP-9 activities, MMP-13, TIMP-1 expression were found decreased in the liver of CCl4 administrated tPA knock out mice compared with wild-type counterparts. All the results illustrated that deficiency of tPA aggravated liver fibrosis through promoting HSCs activation and inhibiting ECM degradation by decreasing MMP-2, MMP-9 activities and disrupting the balance between MMP-13 and TIMP-1.Recently, many studies showed that overexpression of uPA in the liver may initiate upstream of matrix proteolysis cascade to degrade the deposition of ECM and reverse hepatic fibrogenesis. We used uPA knock out mice model to study the effect of uPA in liver fibrosis. The method used in setting up liver fibrosis mice model is the same as mentioned above. Through observing the mice liver, we found that after CCl4 administration, the surface of uPA knock out mice liver was granulated; the color was much whiter and was harder compared with wild-type counterparts. HE staining showed that mice lacking uPA developed more severe morphological injury and lots of hepatocytes showed vacuole liking changes. VG staining and hydroxyproline analysis displayed an increased deposition of collagen in the uPA knock out mice liver, moreover, the mRNA analysis of collagen I, which is the main ECM gradient in liver fibrosis, showed that collagen mRNA expression increased in uPA knock out mice liver after CCl4 administration compared with wild-type counterparts. The results above illuminated that disruption of uPA accelerated liver fibrosis. Then, we analysisα-SMA mRNA and protein expression, the results showed that deficiency of uPA increasedα-smooth muscle actin both mRNA and protein expression in the mice livers. And deletion of uPA increased TIMP-1 mRNA and protein expression in the mice liver. However, although, uPA null mice showed decreased MMP-13 protein expression, the mRNA expression was higher compared with wild type mice. It needs to be studied further. These results illustrated that deficiency of uPA aggravated liver fibrosis through promoting HSCs activation and inhibiting ECM degradation by promoting TIMP-1 and decreasing MMP-13 expression.