Date Published: February 23, 2017
Publisher: Public Library of Science
Author(s): Marcus Hollenbach, Antje Thonig, Sabine Pohl, Cristina Ripoll, Maurice Michel, Alexander Zipprich, Ratna B. Ray.
High concentrations of methylglyoxal (MGO) cause cytotoxiticy via formation of advanced glycation endproducts (AGEs) and inflammation. MGO is detoxificated enzymatically by glyoxalase-I (Glo-I). The aim of this study was to analyze the role of Glo-I during the development of cirrhosis.
In primary hepatocytes, hepatic stellate cells (pHSC) and sinusoidal endothelial cells (pLSEC) from rats with early (CCl4 8wk) and advanced cirrhosis (CCl4 12wk) expression and activity of Glo-I were determined and compared to control. LPS stimulation (24h; 100ng/ml) of HSC was conducted in absence or presence of the partial Glo-I inhibitor ethyl pyruvate (EP) and the specific Glo-I inhibitor BrBzGSHCp2. MGO, inflammatory and fibrotic markers were measured by ELISA and Western blot. Additional rats were treated with CCl4 ± EP 40mg/kg b.w. i.p. from wk 8–12 and analyzed with sirius red staining and Western blot.
Expression of Glo-I was significantly reduced in cirrhosis in whole liver and primary liver cells accompanied by elevated levels of MGO. Activity of Glo-I was reduced in cirrhotic pHSC and pLSEC. LPS induced increases of TNF-α, Nrf2, collagen-I, α-SMA, NF-kB and pERK of HSC were blunted by EP and BrBzGSHCp2. Treatment with EP during development of cirrhosis significantly decreased the amount of fibrosis (12wk CCl4: 33.3±7.3%; EP wk 8–12: 20.7±6.2%; p<0.001) as well as levels of α-SMA, TGF-β and NF-κB in vivo. Our results show the importance of Glo-I as major detoxifying enzyme for MGO in cirrhosis. The reduced expression of Glo-I in cirrhosis demonstrates a possible explanation for increased inflammatory injury and suggests a “vicious circle” in liver disease. Blunting of the Glo-I activity decrease the amount of fibrosis in established cirrhosis and constitutes a novel target for antifibrotic therapy.
Chronic liver inflammation secondary to different noxious agents can lead to the development of cirrhosis. This inflammation activates hepatic stellate cells (HSC) directly by means of endotoxin (LPS) or indirectly through proinflammatory cytokines. Activated HSC acquire a myofibroblastic phenotype which enhances collagen deposition  and therefore fibrosis. Myofibroblasts lead to activation of factors for cell growth, cell proliferation and cell differentiation, mainly mitogen-activated protein kinases (MAPK), and activation of transcription factors such as nuclear factor-kB (NF-kB) [2,3].
Our study aimed at analyzing the expression and activity of Glo-I in cirrhosis and evaluating the effect of partial Glo-I inhibition in hepatic stellate cells and on progression of cirrhosis. We observed a significant reduction of Glo-I in cirrhosis compared to controls, both on protein and mRNA levels accompanied by elevated levels of MGO in cirrhosis. Furthermore, the reduction in Glo-I expression as well as the increase in MGO concentration were greater with increasing severity of liver disease. This reduction in cirrhosis was found in the whole liver as well in all explored liver cells, namely hepatocytes, hepatic stellate cells and sinusoidal endothelial cells. In addition we observed a decreased activity of Glo-I in hepatic stellate cells. On the other hand, stimulation of non-cirrhotic HSC with LPS as would occur hypothetically in an initial stadium of liver disease, resulted in significant increase of specific Glo-I activity. Finally, we could clearly show that reduction of Glo-I activity with two different inhibitors led to a reduced activation of hepatic stellate cells as shown by a decrease in the secretion of TNF-α, collagen-I and α-SMA. In vivo experiments confirmed the antifibrotic properties of inhibition of Glo-I. Partial inhibition of Glo-I by EP resulted in significantly reduced amount of fibrotic tissue and reduced markers of inflammation and fibrosis. Interestingly, this effect was present starting the administration of EP in already established cirrhosis.