Elk-3 Contributes to the Progression of Liver Fibrosis by Regulating the Epithelial-Mesenchymal Transition

BACKGROUND/AIMS: The role of Elk-3 in the epithelial-mesenchymal transition (EMT) during liver fibrogenesis remains unclear. Here, we determined the expression of Elk-3 in in vitro and in vivo models and in human liver fibrotic tissues. We also investigated the molecular relationships among Elk-3, early growth response-1 (Egr-1), and the mitogen activated protein kinases (MAPK) pathway during EMT in hepatocytes. METHODS: We established anin vitro EMT model in which normal mouse hepatocyte cell lines were treated with transforming growth factor (TGF)-β1 and a CCl4-induced liver fibrosis model. Characteristics of EMT were determined by evaluating the expression levels of related markers. The expression of Elk-3 and its target Egr-1 were analyzed using Western blotting. Gene silencing of Elk-3 was performed using an siRNA knockdown system. RESULTS: The expression levels of mesenchymal markers were increased during TGF-β1-induced EMT of hepatocytes. The expression levels of Elk-3 and Egr-1 were significantly (p<0.05) increased during the EMT of hepatocytes, in CCl₄-induced mouse liver fibrotic tissues, and in human liver cirrhotic tissues. Silencing of Elk-3 and inhibition of the Ras-Elk-3 pathway with an inhibitor suppressed the expression of EMT-related markers. Moreover, Elk-3 expression was regulated by p38 MAPK phosphorylation during EMT. CONCLUSIONS: Elk-3 contributes to the progression of liver fibrosis by modulating the EMT via the regulation of Egr-1 under MAPK signaling.

Oleuropein prevents the progression of steatohepatitis to hepatic fibrosis induced by a high-fat diet in mice

Nonalcoholic steatohepatitis (NASH) is characterized by hepatocyte injury and inflammatory cell infiltration, which has been linked to peripheral insulin resistance and increased levels of triglycerides in the liver. The purposes of this study were to establish a mouse model of NASH by feeding mice a 60% high-fat diet (HFD) and to demonstrate the anti-fibrotic effects of oleuropein, which has been shown to have anti-oxidant and anti-inflammatory properties, in this HFD-induced mouse model of NASH. C57BL/6 mice were divided into three groups: a regular diet group (Chow), a HFD group and an oleuropein-supplemented HFD group (OSD), which was fed a 0.05% OSD for 6 months. The effects of oleuropein in this model were evaluated using biochemical, histological and molecular markers. The expression levels of alpha-smooth muscle actin (alpha-SMA)and collagen type I in the HFD and OSD groups were evaluated using real-time PCR and western blotting. The body weight, biochemical marker levels, nonalcoholic fatty liver disease activity score, homeostasis model of assessment-insulin resistance (HOMA-IR) and leptin levels observed in the HFD group at 9 and 12 months were higher than those observed in the Chow group. The HOMA-IR and leptin levels in the OSD group were decreased compared with the HFD group. In addition, alpha-SMA and collagen type I expression were decreased by oleuropein treatment. We established a NASH model induced by HFD and demonstrated that this model exhibits the histopathological features of NASH progressing to fibrosis. Our results suggest that oleuropein may be pharmacologically useful in preventing the progression of steatohepatitis and fibrosis and may be a promising agent for the treatment of NASH in humans.

Stem cells in musculoskeletal system for clinical application

Mesenchymal stem cells (MSCs) play a crucial role in the proliferation and differentiation of human tissue such as bone, cartilage, muscle, fat, and fibroblasts. Various surgical techniques have been developed for the repair of the musculoskeletal system, but they can be often limited. Thus, the efforts that can be employed in treatment for MSCs population of degenerative musculoskeletal diseases are underway. Patients who have a musculoskeletal disease with low numbers of functional MSCs will be treated using a focus on cell-based therapy. The ideal clinical application is to engineer material/scaffolds that are capable of delivering therapeutic cells that can regenerate and repair damaged tissue. The ability related to MSCs differentiation using biomaterial systems offers a minimally invasive therapeutic option for diseases of the musculoskelecal system and tissue repair. Understanding the natural mechanisms for this delivery is essential to the success of tissue engineering biomaterials that deliver therapeutic cells.

The Effects of air-borne particulate matters on the Alveolar Macrophages for the iNOS Expression and Nitric Oxide with Nitrotyrosilated-proteins Formation / 결핵및호흡기질환

BACKGROUND: Particulate matters (PM) when inhaled is known to induce pulmonary diseases including asthma and chronic bronchitis when inhaled. Despite the epidemiological proofevidence, the pathogenesis of PM-related pulmonary diseases is unclearremain poorly understood. METHODS: Primary alveolar macrophages were harvested from the SPF and inflammatory rats by bronchioalveolar lavage (BAL). The cultured primary alveolar macrophages were treated with the medium only, PM only (5~40 microgram/cm2), LPS (5ng/ml) only, and PM with LPS for 24 and 48 hours. The level of secreted nitric oxide (NO) was assayed from the cultured medium by using the Griess reaction. The cultured cells were utilized for the western blotting against the inducible nitric oxide synthase (iNOS) proteins. Immunocyto- chemical staining against the iNOS and NT-proteins were performed in cells that cultured in the Lab-Tek(R) chamber slide after treatments. RESULTS: The PM that utilizein this experiments induced NO formation with iNOS expression in the cultured SPF and inflammatory rats alveolar macrophages, by itself. When the cells were co-treated with PM and LPS, there was a statistically significant synergistic effect on NO formation and iNOS expression over the LPS effect. The cells from the sham control showed minimal immunoreactivity for the NT-proteins. Significantly higher quantities of NT-proteins were detected in the PM and PM with LPS co-treated cells than from the sham control. CONCLUSION: Increased iNOS expression and NO formation with increased NT-proteins formation might be involved in the pathogenesis of PM-induced lung injury.

The Effects of Air-borne Particulate Matters on the Alveolar Macrophages for the TNF-alpha and IL-1beta Secretion / 결핵및호흡기질환

BACKGROUND: PM is known to induce various pulmonary diseases, including asthma, cancer, fibrosis and chronic bronchitis. Despite the epidemiological evidence the pathogenesis of PM-related pulmonary diseases is unclear. METHODS: This study examined the effects of PM exposure on the secretion of TNF-alpha and IL-1beta in the cultured alveolar macrophages. The cultured primary alveolar macrophages were treated with the medium, PM (5~20 microgram/cm2), LPS (5ng/ml), and PM with LPS for 24h and 48h respectively. ELISA was used to assay the secreted TNF-alpha and IL-beta in the culture medium. Western blotting was used to identify and determine the level of proteins isolated from the culture cells. The cells cultured in the Lab-Tek(R) chamber slides were stained with immunocytochemical stains. RESULTS: PM induced TNF-alpha and IL-1beta secretion in the culturing alveolar macrophages, collected from the SPF and inflammatory rats. However, the effects were only dose-dependent in the inflammatory macrophages. When the cells were co-treated with PM and LPS, there was a significant synergistic effect compared with the LPS in the both cell types. CONCLUSION: PM might be play an important role in the induction and/or potentiation of various lung diseases by oversecretion of TNF-alpha and IL-1beta.

HNF1 and/or HNF3 may contribute to the tissue specific expression of glucokinase gene

A possible role of hepatocyte nuclear factor 1 (HNF1) or HNF3, a predominant trans-acting factors of hepatic or pancreatic beta-cells, was examined on the tissue specific interdependent expression of glucokinase (GK) in liver, H4IIE, HepG2, HIT-T15 and MIN6 cell line. The tissues or cell lines known to express GK showed abundant levels of HNF1 and HNF3 mRNA as observed in liver, H4IIE, HepG2, HIT-T15 and MIN6 cells, whereas they were not detected in brain, heart, NIH 3T3, HeLa cells. The promoter of glucokinase contains several HNF3 consensus sequences and are well conserved in human, mouse and rat. Transfection of the glucokinase promotor linked with luciferase reporter to liver or pancreatic beta cell lines showed high interacting activities with HNF1 and HNF3, whereas minimal activities were detected in the cells expressing very low levels of HNFs. The binding of HNF1 or HNF3 to the GK promoter genes was confirmed by electrophoretic mobility shift assay (EMSA). From these data, we propose that the expression of HNF1 and/or HNF3 may, in part, contribute to the tissue specific expression of GK.