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F2-Isoprostanes as markers of oxidative stress and mediators of CCl4-induced liver fibrosis
No full textINTRODUCTION: F2-isoprostanes are considered as the most reliable markers of OS in vivo and mediators of some important biological effects, such as the vasoconstriction of kidney glomerular arterioles. It is known that hepatic fibrosis induced by CCl4 may be linked to oxidative stress (OS). In previous studies, we demonstrated the role of isoprostanes as possible mediators of CCl4-induced hepatic fibrosis. Plasma levels of isoprostanes were found to be elevated in rat chronically treated with CCl4 and correlated with hepatic collagen content. We also demonstrated that in vitro isoprostanes induced a marked increase in DNA and collagen synthesis in cultured hepatic stellate cells (HSC), in the concentration range found in the in vivo studies (1-10 nM). It has been suggested that isoprostanes act through the activation of receptors analogous to those for tromboxane A2 (TxA2r). Thus, the possible occurrence of TxA2r on HSC was investigated. Binding studies with [3H]SQ29548 (a specific antagonist of TxA2r) and competition binding experiments with I-BOP (a specific agonist of TxA2r) demonstrated the existence of TxA2r in HSC and the involvement of TxA2r in Iso-evoked responses. We then examined which signal transduction pathways are set into motion by isoprostanes to exert their fibrogenic effects. METHODS: HSC were isolated from the rat liver and treated with 8-epi-PGE2the most represented isomer). Ins(1,4,5)P3 (IP3) and cAMP levels were determined by commercial kits. Activation of mitogen-activated protein kinases (MAPK) and cyclin 1 was assessed by Western blotting. Cell proliferation and collagen production were assessed by measuring the incorporation of 3H-thymidine and 3H-proline, respectively. RESULTS: 8-epi-PGE2 increased 4 times IP3 and affected cAMP production. The expression of cyclin D1, a molecule involved in cell proliferation, is also increased. Furthermore, 8-epi-PGE2 activated two classes of MAPK: extracellular signal-regulated kinase (ERK) and p38, which have been shown to influence cell proliferation and collagen gene expression. CONCLUSION: On the basis of these data, it is possible to hypothesize that one of the pathways activated by 8-epi-PGF2a is that of Gq/PKC. The binding of isoprostanes to TxA2r could stimulate downstream MAPK activation, via PKC. In particular, p38 is known to increase in HSC collagen production, while ERK is able to increase cyclin D1 expression and then cell proliferation. Thus, fibrogenic effects of isoprostanes in HSC are mediated through TxA2r binding by specific activation of these transduction pathways
Signalling pathways involved in isoprostane-induced fibrogenic effects in hepatic stellate cells
No full textINTRODUCTION: We have previously shown that the levels of plasma F2-Isoprostanes (F2-Iso) are maintained elevated in the model of chronic CCl4 intoxication leading to fibrosis and cirrhosis, and correlated to the increased hepatic collagen. F2-Iso can also potentially mediate some of the adverse effects of oxidant injury. We demonstrated that in hepatic stellate cells (HSC) some fibrogenic events (such as cell proliferation and collagen synthesis) are induced by F2-Iso through the activation of receptors analogous to those for tromboxane A2 (TP receptor), but how isoprostanes act to initiate these events remains unclear. METHODS: HSC were isolated from rat liver and treated with 8-epi-PGF2the most represented isomer). Inositol-3-phosphate (IP3) and cAMP levels were determined by commercial kits. Activation of MAPK and cyclin D1 was assessed by Western blotting. Cell proliferation and collagen production were determined by measuring the incorporation of 3H-thymidine and 3H-proline, respectively. RESULTS: A transient increase of cAMP was observed. 8-epi-PGF2also increased IP3 levels and activated ERK and p38 MAPK which have been shown to influence cell proliferation and collagen gene expression, as well as cyclin D1 expression. Collagen synthesis and cell proliferation induced by 8-epi-PGF2 were completely reversed by PD98059, a specific inhibitor of ERK activation. CONCLUSIONS: The data obtained suggest that one of the pathways activated by 8-epi-PGF2 is that of Gq/PKC. The binding of isoprostanes to TP receptor in HSC could stimulate downstream MAPK activation, leading to an increased collagen production. Furthermore the ability of ERK to increase cyclin D1 expression can stimulate cell proliferation
F2-isoprostanes can mediate bleomycin-induced lung fibrosis
Full text linkF2-isoprostanes (F2-IsoPs) have been considered markers of oxidative stress in various pulmonary diseases, but
little is known about their possible role in pulmonary fibrosis. In this study, we have investigated the potential
key role of F2-IsoPs as markers and mediators of bleomycin (BLM)-induced pulmonary fibrosis in rats. During the
in vivo study, plasma F2-IsoPs showed a peak at 7 days and remained elevated for the entire experimental period.
Lung F2-IsoP content nearly tripled 7 days following the intratracheal instillation of BLM, and by 28 days, the
value increased about fivefold compared to the controls. Collagen deposition correlated with F2-IsoP content in
the lung. Furthermore, from day 21 onwards, lung sections from BLM-treated animals showed α-smooth muscle
actin (α-SMA) positive cells, which were mostly evident at 28 days. In vitro studies performed in rat lung fibroblasts
(RLF) demonstrated that either BLM or F2-IsoPs stimulated both cell proliferation and collagen
synthesis. Moreover, RLF treated with F2-IsoPs showed a significant increase of α-SMA expression compared to
control, indicating that F2-IsoPs can readily activate fibroblasts to myofibroblasts. Our data demonstrated that
F2-IsoPs can be mediators of key events for the onset and development of lung fibrosis, such as cell proliferation,
collagen synthesis and fibroblast activation. Immunocytochemistry analysis, inhibition and binding studies
demonstrated the presence of the thromboxane A2 receptor (TP receptor) on lung fibroblasts and suggested that
the observed effects may be elicited through the binding to this receptor. Our data added a new perspective on
the role of F2-IsoPs in lung fibrosis by providing evidence of a profibrotic role for these mediators in the pathogenesis
of pulmonary fibrosis
Roflumilast N-oxide, a PDE4 inhibitor, inhibits fibrotic changes induced by isoprostanes in human lung fibroblasts
No full textOxidative stress plays an critical role in the pathogenesis of pulmonary fibrosis. Isoprostanes, the most proximal products of lipid peroxidation, are known mediators of important biological effects and have been found to be increased in fibrosis. Roflumilast, a PDE4 inhibitor, has been demonstrated to mitigate the oxidative stress-induced lung fibrotic response in vivo.
In this study we evaluated in vitro the effect of roflumilast N-oxide (RNO), the active metabolite of roflumilast, on oxidative stress and some markers of fibrotic response induced by isoprostanes. Human lung fibroblasts (HLF) were incubated in the absence or presence of RNO (2nM-1M) for 30 min and then treated with 8-epi-PGE2the most represented isomer of the species).
8-epi-PGE2 (10nM) induced a 1.3-fold increase of radical oxygen species (ROS) generation that was abolished by RNO both at the low (2nM) and at the high (1M) concentration (p<0.05). RNO did not change baseline ROS levels. The time course for total glutathione (GSH) levels showed that 8-epi-PGE2 (10nM) induced an increase (by 19% vs respective control, p<0.05) in total GSH levels after 6 h of incubation. RNO did not add to this effect. On the other hand, RNO (1μM) alone increased total GSH levels by 16% (p<0.05). Based on the latter findings the notion was raised that PDE4 inhibition may enhance total glutathione production possibly by inducing an increased expression of glutamate cysteine ligase (GCLC). In fact, RNO 1μM but also 8-epi-PGE2 (10nM) increased GCLC mRNA expression by 1.3 (p<0.05) and 1.2 fold (p<0.05), respectively. Furthermore, 8-epi-PGE2alpha (10nM) stimulated cell proliferation (+17%; p<0.05) and collagen synthesis (+27%; p<0.05) and RNO (2nM) completely abolished such an increase. Taken together, inhibition of PDE4 (by roflumilast N-oxide) curbs ROS formation, collagen synthesis and proliferation of human lung fibroblasts triggered by the isoprostane 8-epi-PGE2alpha. In addition, the PDE4 inhibitor enhanced total GSH
Roflumilast N-oxide, a PDE4 inhibitor, curbs bleomycin-induced lung fibroblast activation in vitro
No full textPinosilvin administered in monotherapy and in combination with methotrexate reduces oxidative stress in adjuvant arthritis rat model
No full textRheumatoid arthritis (RA) is a common severe joint disease that involves all age groups. This is one of the conditions in which oxidative stress (OS) has been shown to play a role by regulating the progression of the inflammatory response. Adjuvant arthritis (AA) is a rat model of autoimmune erosive arthritis widely used to evaluate etiopathogenetic mechanisms in RA as well as for testing anti-inflammatory drugs. Pinosylvin (PIN), 3,5-dihydroxy-trans-stilbene, is an analogue of resveratrol mainly found in the heartwood of Pinus sylvestris. The aim of the present study was to examine the eventual antioxidant and anti-inflammatory effect of PIN on the progression of AA in rats in monotherapy and in combination with methotrexate (MTX).
AA was induced by a single intradermal injection of heat-inactivated Mycobacterium butyricum in incomplete Freund’s adjuvant. The experiments included healthy animals, arthritic animals not treated, arthritic animals treated with MTX, with PIN, and with a combination of PIN and MTX. The two latter groups received a daily oral dose of 50 mg/kg b.w. of PIN, either alone or with MTX in an oral dose of 0.4 mg/kg b.w. twice a week during 28 experimental days.
Our data demonstrated that PIN potentiated both the anti-arthritic (decrease of hind paw volume) and the antioxidant effect of MTX (TBARS in plasma). The level of inflammatory protein CRP in plasma and activity of GGT in spleen were not improved by addition of PIN to MTX due to the prominent effect of MTX alone on these parameters. Arthritic animals showed increased OS, also evaluated as plasma levels of isoprostanes. PIN alone or in combination with MTX strongly reduced isoprostane levels (about 50%). Anti-inflammatory and antioxidant functions have been attributed to heme oxygenase (HO-1). Our data show a significant decline in HO-1 (about 40%) in the lung from AA rats. In these animals, PIN alone increased the levels of HO-1 by about 30% more than MTX. Moreover, the combination therapy was the most effective in increasing the levels of HO-1 (3-fold in respect to AA values). Finally, activated NF-B plays an important role in the expression of pro-inflammatory genes. In our model, we observed a marked increase in NF-B in the lung and liver from AA animals. This increase was strongly reduced by PIN alone as well as in combination with MTX. These results suggest that the anti-inflammatory activity of PIN can be mediated by suppression of NF-B activation.
In conclusion, PIN is able to reduce OS in AA rat model. In fact, the combined administration of PIN and MTX suppressed arthritic progression more effectively than did MTX alone. This natural compound may then be useful in the treatment of rheumatoid arthritis.
Acknowledgement: VEGA 2/0045/11, APVV-052-10, CNR/SAV bilateral project 2010-2012: In vitro and in vivo models of arthritic processes for studying the mechanisms of inflammation and oxidative stress link-up. New perspectives for arthritis therapy. Pinosylvin used in this study was prepared by Prof. Jan Šmidrkal (Institute of Chemical Technology, Prague, Czech Republic) and Ing. Juraj Harmatha, PhD (Institute of Organic Chemistry and Biochemistry, Academy of Sciences of Czech Republic)
Activity of pinosylvin administered in monotherapy and in combination with methotrexate on the development of rat adjuvant arthritis
No full textOxidative stress (OS) has been implicated in various pathological conditions involving several diseases and aging. Rheumatoid arthritis (RA) is a common severe joint disease that involves all age groups. The pathogenesis of RA is associated predominantly with the formation of free radicals at the site of inflammation. However, knowledge on the role of OS in the progression of RA is scarce and the link between OS and inflammation status in arthritis should be more precisely investigated. Pinosylvin (PIN), 3,5-dihydroxy-trans-stilbene, is mainly found in the heartwood of Pinus sylvestris. PIN used in this study was synthesized in the Institute of Organic Chemistry and Biochemistry, Academy of Sciences, Prague, Czech Republic by Ing. Juraj Harmatha, PhD. The aim of the present study was to examine the effect of PIN on the progression of adjuvant-induced arthritis (AA) in rats in monotherapy and in combination with methotrexate (MTX), which is a classical immunosuppressant drug.
AA was induced by a single intradermal injection of heat-inactivated Mycobacterium butyricum in incomplete Freund’s adjuvant. The experiments included healthy animals, arthritic animals not treated, arthritic animals treated with MTX, with PIN, and with a combination of PIN and MTX. The two latter groups received a daily oral dose of 50 mg/kg b.w. of PIN, either alone or with MTX in an oral dose of 0.4 mg/kg b.w. twice a week during 28 experimental days.
We found that PIN potentiated both the antiarthritic (decrease of hind paw volume) and the antioxidant effect of MTX (reduction of plasmatic levels of TBARS). Activity of GGT in spleen, level of MCP-1 and CRP in plasma were not improved by addition of PIN to MTX due to the prominent effect of MTX alone on these parameters. Arthritic animals showed increased OS, evaluated as plasma levels of isoprostanes. PIN alone or in combination with MTX strongly reduced isoprostane levels (about 50%). On the contrary, a significant decline in Nrf2-regulated antioxidant defences, such as hemeoxygenase-1 (HO-1), was observed in the lung (about 40%) but not in the liver from AA rats. In the AA lung, PIN alone increased the levels of HO-1 by about 30% more than MTX. Moreover, the combination therapy was the most effective in increasing the levels of HO-1 (3-fold in respect to AA values). OS can also activate NF-B, which plays a critical role in the transcription of proinflammatory genes. Our data showed a marked increase in NF-B in the lung and liver from AA animals. This increase was strongly reduced by PIN alone as well as in combination with MTX. Our results suggest that the anti-inflammatory activity of PIN is mediated by suppression of NF-kB activation in the liver and lung of arthritic animals.
In summary, combined administration of PIN and MTX suppressed arthritic progression in rats more effectively than did MTX alone. This natural compound is able to reduce OS in vivo and may help improve the treatment of rheumatoid arthritis.
Acknowledgement: VEGA 2/0045/11, APVV-0315-07, CNR/SAV bilateral project 2010-2012: In vitro and in vivo models of arthritic processes for studying the mechanisms of inflammation and oxidative stress link-up. New perspectives for arthritis therapy
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
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