Pirfenidone (Pf), a new broad-spectrum anti-fibrotic agent, is known to offer protection against lung fibrosis in vivo in laboratory animals, and against mitogenesis ...
Molecular and Cellular Biochemistry 204: 119–126, 2000. © 2000 Kluwer Academic Publishers. Printed in the Netherlands.
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Pirfenidone inhibits NADPH-dependent microsomal lipid peroxidation and scavenges hydroxyl radicals* Hara P. Misra and Christine Rabideau Center for Molecular Medicine and Infectious Diseases, Department of Biomedical Sciences and Pathobiology, VirginiaMaryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, Virginia, USA Received 12 April 1999; accepted 17 September 1999
Abstract Pirfenidone (Pf), a new broad-spectrum anti-fibrotic agent, is known to offer protection against lung fibrosis in vivo in laboratory animals, and against mitogenesis and collagen formation by human lung fibroblasts in vitro. Because reactive oxygen species are thought to be involved in these events, we investigated the mechanism(s) by which Pf ameliorates oxidative stress and its effects on NADPH-dependent lipid peroxidation. Pf has been shown to cause inhibit NADPH-dependent lipid peroxidation in sheep liver microsomes in a dose-dependent manner. The concentration of Pf required to cause 50% inhibition of lipid peroxidation was ~ 6 mM. Pf was found to be ineffective as a superoxide radical scavenger. Pf was also ineffective in decomposing H2O2 and chelating iron. In deoxyribose degradation assays, Pf was a potent scavenger of hydroxyl radicals with a rate constant of 5.4 × 109 M–1 sec–1. EPR spectroscopy in combination with spin trapping techniques, using a Fenton type reaction and DMPO as a spin-trapping agent, Pf scavenged hydroxyl radicals in a dose-dependent manner. The concentration of Pf required to inhibit 50% signal height was ~ 2.5 mM. Because iron was used in the Fenton reaction, the ability of Pf in chelating iron was verified in a fluorescent competitive assay using calcein as the fluorescent probe. Pf up to 10 mM concentration was ineffective in chelating either Fe2+ or Fe3+ in this system. We propose that Pf exerts its beneficial effects, at least in part, through its ability to scavenge toxic hydroxyl radicals. (Mol Cell Biochem 204: 119–126, 2000) Key words: pirfenidone, free radicals, lipid peroxidation, EPR, spin trapping, antioxidant, fibrosis
Introduction A new low molecular weight experimental drug, Pirfenidone (5-methyl-L-phenyl-2-(1H)-pyridone) is currently being evaluated clinically as a broad-spectrum antifibrotic agent. Pirfenidone (Pf) has been shown to inhibit the formation of bleomycin- and cyclophosphamide-induced fibrosis in hamsters [1–3], and experimental peritonitis in rats [4].
Hamsters fed Pf in the diet resulted in a marked decrease in fibrosis in an asbestos-induced pulmonary fibrosis animal model [5]. Pf has also been shown to inhibit both TNFinduced and subsequent endotoxin shock in mice [6]. In several in vitro experiments, it has been shown that PF inhibits the proliferation of human lung and skin fibroblasts in culture [7], intracellular adhesion molecule-1 in cultured human synovial fibroblasts [8], fibronectin synthesis by
*Footnote: A part of this paper was presented at the annual meeting of American Society of Biochemistry and Molecular Biology, Washington, D.C., May 16–20, 1998 Address for offprints: H.P. Misra, Center for Molecular Medicine and Infectious Diseases, Department of Biomedical Sciences and Pathobiology, Virginia-Maryland Regional College of Veterinary Medicine, Virginia Polytechnic Institute and State Univesity, Blacksburg, Virginia 240610342, USA
120 culture retinal pigment epithelial cells [9], and leimyoma cell proliferation and collagen production [10]. Because reactive oxygen species are shown to initiate bleomycin-induced fibrosis [11–13], collagen synthesis [14, 15], intracellular adhesion molecule synthesis [16, 17], as well as asbestosinduced pulmonary fibrosis [18], we developed the hypothesis that Pf may be exerting its beneficial effects, at least in part, by an oxyradical scavenging mechanism. A similar involvement of ROS in the expression of TNFα has also been established [19, 20]. Malonaldehyde, an oxyradical mediated end product of lipid peroxidation, has recently been shown to stimulate the synthesis of collagen [21, 22] and has been suggested as a possible mechanism of pulmonary fibrosis [23, 24]. In this study we present evidence that Pf is a potent inhibitor of hydroxyl radicals and is capable of inhibiting microsomal lipid peroxidation.
Materials and methods Pirfenidone (Pf) was obtained from Marnac Inc, Dallas, TX, USA. Bovine serum albumin, potassium diphosphate, potassium monophosphate, Trizma base, 2-thiobarbituric acid (TBA), trichloroacetic acid (TCA), NADPH (tetrasodium salt), xanthine oxidase, xanthine, ferricytochrome c, EDTA (trisodium salt), 2-deoxy-D-ribose, ferric chloride, hydrogen peroxide, catalase (bovine liver), epinephrine, superoxide dismutase, calcein and diethylenetrismine-pentaacetic acid (DTPA) were purchased from Sigma Chemical Company. Lascorbic acid, n-butanol, ferrous sulfate (anhydrous), boric acid were products of Fisher Scientific Inc. 5,5-dimethyl-1pyrroline-n-oxide (DMPO) was obtained from Aldrich. All other materials were purchased at the highest available purity. All spectrometric assays were performed at 25°C. A Shimadzu model 160 UV-visible spectrophotometer was used for spectral and kinetic analysis. The fluorescence readings were obtained on a Perkin Elmer LS3 Fluorescence Spectrophotometer. EPR measurements were made on a Bruker D200 X-band spectrometer using a microwave frequency of 100 kHz.
Preparation of sheep microsomes Sheep liver microsomes were prepared by differential centrifugation as described [25]. The washed microsomal pellet was resuspended in 0.05 M Tris-HCl buffer, pH 7.6 to yield a final concentration of 10 mg of microsomal protein per ml. Protein concentration was determined by the Bradford protein assay using bovine serum albumin as the standard [26].
Assay of lipid peroxidation Lipid peroxidation was determined by the thiobarbituric acid assay (TBA) as described [25]. Modifications included the elimination of adenosine diphosphate (ADP) and ferric chloride from the reaction mixture. The reaction mixture containing 4 mg microsomal protein in Tris-HCl buffer, pH 7.6, 200 µM NADPH was incubated at 37°C for 15 min. Lipid peroxidation was initiated by addition of NADPH and terminated by addition of 2 ml of 0.5% (w/v) TBA and 2% trichloroacetic acid. The mixture was heated at 95°C for 60 min and n-butanol was utilized to extract the malondialdehyde (MDA) from the mixture. The samples were centrifuged and MDA concentration was determined by reading at 535 nm against a blank that contained all reagents except NADPH. An extinction coefficient of 1.56 × 105 M–1 cm–1 [27] was used to determine the MDA concentration. Various amounts of pirfenidone (0.1–6.0 mM) were added to the reaction to determine its effect on lipid peroxidation.
Assays for superoxide anion scavenging activity Superoxide scavenging activity of Pf was monitored in terms of its ability to inhibit the superoxide-mediated reduction of ferricytochrome c by the xanthine oxidase system and superoxide-dependent auto-oxidation of epinephrine. These assays were performed as originally described [28–30].
Hydroxyl radical dependent degradation of deoxyribose and determination of rate constant The ability of pirfenidone to act as a hydroxyl scavenger was determined by its role in a Fenton type reaction. The degradation of deoxyribose by hydroxyl radicals and production of a pink chromogen was monitored spectrophotometrically. The reaction mixtures contained, in a final volume of 1 ml, the following reagents: 2.8 mM deoxyribose, 25 µM FeCl3, 30 µM EDTA, 1 mM H2O2, 100 µM ascorbate in 20 mM KH 2 PO 4-KOH buffer, pH 7.4. The reaction mixtures were incubated at 37°C for 1 h and 1 ml of 0.5% TBA (w/v) and one ml of 2% trichloroacetic acid (w/v) was added, and the mixture was heated at 80°C for 30 min. The rate constant of Pf interacting with hydroxyl radical was calculated using the method as originally described [31].
EPR spectroscopic analysis Effects of Pf on scavenging hydroxyl radicals were studied using EPR in combination with spin trapping techniques. Hydroxyl radicals were generated in a Fenton-type reaction
121 and were detected as DMPO-OH adduct, exhibiting a wellcharacterized 1:2:2:1 signal pattern with AN = AHB = 14.92 G. The scan conditions were as follows: microwave frequency, 100 kHz, power 10 mw; modulation amplitude of 1 G, time constant 0.64 sec, scan rate 200 sec; receiver gain of 5 × 105 and center field setting 3483 G. The reaction mixture contained the following reagents at the final concentration: 1.67 mM hydrogen peroxide, 1.67 mM ferrous sulfate, 33 mM DMPO and 0.2 M boric acid/borate buffer, pH 7.8. The reaction was initiated by the addition of ferrous sulfate. Various concentrations of Pf (2.5–10 mM) were used in the above system.
Fluorescence iron chelation assay A fluorescent iron chelation assay was performed using calcein as a fluorescent probe [32] with slight modification. The fluorescence wavelengths used for excitation was 487 nm and for emission was 517 nm. The reaction mixture contained 50 µM calcein in 0.25 M Tris-HCl buffer, pH 7.6. Iron was added (2 µM ferrous ammonium sulfate or 2 µM ferric chloride) to the reaction mixture and fluorometric readings were taken every minute for three minutes, the sample was removed and either Pf (1.0–6.0 mM) or DTPA (250 µM) was added. Fluorescent readings were recorded every min for 3 consecutive min.
Fig. 1. Effect of pirfenidone on NADPH-dependent lipid peroxidation. The reaction mixture contained 4 mg/ml microsomes and 200 µM NADPH in Tris-HCl buffer, pH 7.6. Indicated concentrations of Pf (0.1–6 mM) were added. The reaction mixtures were incubated for 15 min at 37°C. Lipid peroxidation was initiated by addition of NADPH and was terminated by addition of 2 ml 0.5% TBA in 2% TCA. The reaction mixtures were heated at 95°C for 60 min and MDA concentrations were determined as described in ‘Materials and methods’. The inset is the reciprocal plot suggesting the inhibition of lipid peroxidation by Pf is kinetically simple.
Results Effect of Pf on lipid peroxidation Lipid peroxidation of microsomes was induced by incubating the liver microsomes with 200 µM NADPH, without the addition of extraneous iron to the medium, and the production of TBA-reactive products was measured as an index of lipid peroxidation. The production of TBA-reactive product was dependent on the concentrations of NADPH (up to 200 µM) as well as microsomal proteins (up to 4 mg/ml). Preincubation with Pf inhibited the NADPH-dependent lipid peroxidation in a dose dependent manner (Fig. 1). As shown in Fig. 1, lipid peroxidation was strongly inhibited by Pf and the concentration of Pf required to cause 50% inhibition was found to be ~ 6 mM. When these data were presented on reciprocal coordinates (Fig. 1, inset), the inhibition appeared to be kinetically simple. Effect of Pf on reactive oxygen species In an attempt to identify the reactive species directly involved in lipid peroxidation and which Pf can scavenge,
we investigated the role of Pf in various known oxyradicalgenerating systems. Superoxide anions are known to be produced when xanthine oxidase acts on xanthine in the presence of molecular oxygen. The superoxide radicals so generated can reduce ferricytochrome c. Superoxide dismutase inhibits this reaction by effectively competing with ferricytochrome c for the flux of superoxide anion (O2–). This reaction has been used as a convenient assay for superoxide dismutase [33]. We tested the effects of Pf in this system as a possible superoxide dismutase mimic. Pf at 1–15 mM concentrations did not inhibit the rate of ferricytochrome c reduction (data not shown). Although Pf had no effect on ferricytochrome c reduction, there is reason to believe that Pf could be interfering in xanthine oxidase reaction and/or reacting with superoxide anion to form itself a reactive species, thus maintaining a steady-state level of electron flux for the reduction of ferricytochrome c. This was not the case as illustrated by the following experiments. Xanthine oxidase is known to react on xanthine under aerobic conditions to yield uric acid and the production of uric acid can be conveniently monitored spectrometrically at
122 295 nm. We have investigated the effects of Pf on the rate of accumulation of uric acid at 295 nm when xanthine oxidase was allowed to react with xanthine at pH 7.8. In this assay, up to 10 mM Pf was found to have little effect on the rate of conversion of xanthine to uric acid indicating that Pf does not interfere in xanthine oxidase activity. Further, we have investigated the effects of Pf on superoxide radicals in an assay in which O2– acted as an oxidant, not as reductant, as in ferricytochrome c assay. We tested the O2– scavenging ability of Pf in O2–-dependent epinephrine auto-oxidation assay [29]. In this assay, the rate of adrenochrome formation as measured at 480 nm was not effected by the presence of 1, 6, and 15 mM Pf (data not shown). These results indicate that Pf is not an effective O2– scavenger. The hydroxyl radicals generated in a Fenton type system (Fe2+ + H2O2 → .OH + OH– + Fe3+) yield spin adducts with DMPO [34]. Thus, as presented in Fig. 2, a well characterized 1:2:2:1 splitting pattern of DMPO-OH with AN = AHB = 14.92 G, consistent with the reported values [35, 36] was obtained when 1.67 mM FeSO4 was added to 1.67 mM H2O2 in the presence of 33 mM DMPO in 0.2 M borate buffer, pH 7.8. The EPR signal of DMPO-OH adducts was stable for several minutes. As shown in Fig. 2, Pf inhibited the DMPOOH adduct formation in a dose-dependent manner. When signal height of the second peak was used to calculate the percent inhibition, the molar concentration of Pf required to cause 50% inhibition was found to be ~ 2.5 mM. If inhibition of DMPO-OH adduct formation by Pf, as shown above, is truly a reflection of interaction of the drug
Fig. 2. Inhibition of DMPO-OH adduct formation by Pf. The ability of Pf to compete with DMPO for the available .OH radicals, generated in a Fenton type reaction, was studied by EPR spectroscopic analysis. The reaction mixture contained 1.67 mM H2O2, 1.67 mM FeSO4 and 33 mM DMPO in 0.2 M boric acid/borate buffer, pH 7.8. Reaction was initiated up on addition of ferrous sulfate. (A): Control (no Pf); (B): 2.5 mM Pf; (C): 10 mM Pf; D: no Fe 2+ or Pf.
with the .OH, then similar results should be obtained with a different .OH-dependent reaction. That this was the case is illustrated in Fig. 3 when a different assay, deoxyribose colorimetric assay [31], was adopted. In this assay, reacting FeCl3-EDTA and ascorbate generated .OH radicals with H2O2 at pH 7.4. The .OH radicals so generated can be conveniently measured by their ability to degrade deoxyribose into fragments that generate a pink chromogen upon heating with TBA at low pH. In this assay, Pf was able to compete with deoxyribose effectively in preventing the TBA-reactive color formation. As shown in Fig. 3, the amount of Pf required to inhibit 50% of this reaction was ~ 5 mM. The second order rate constants for the reaction of Pf with .OH was calculated [31] and was found to be 5.4 × 109 M–1 sec–1 (Fig. 3, inset). Control experiments showed that Pf does not interfere with the measurement of deoxyribose degradation nor itself react with .OH to give TBA-reactive products. Thus, when 6 mM Pf was added to the reaction mixture at the end of the incubation time, before the addition of TBA, little protection of deoxyribose degradation was observed. Further, when Pf at 6 mM was allowed to react with the .OH-generating system in the absence of deoxyribose, no TBA-reactive products were observed at 535 nm.
Fig. 3. Inhibition of hydroxyl radical-dependent degradation of deoxyribose by Pf. The reaction mixture contained 2.8 mM deoxyribose, 25 µM FeCl3, 30 µM EDTA, 1 mM H2O2, 100 µM ascorbate in 0.2 M phosphate buffer, pH 7.4. The reaction was initiated by adding H2O2. Indicated concentrations of Pf were added before the initiation of the reaction. The reaction mixtures were incubated for 60 min at 37°C, then one ml of 0.2% TBA in 2.0% TCA was added to stop the reaction. The reaction mixtures were then heated for 30 min at 80°C. The inset yields the data necessary to calculate the rate constant as described in ‘Material and methods’.
123 Although Pf was found to scavenge .OH in both the EPR studies and deoxyribose assay, both these assays used H2O2 and Fe2+ in a Fenton type system (Fe2+ + H2O2 → .OH + OH– + Fe3+). Therefore, there is reason to believe that Pf could directly interact with H2O2 and/or Fe2+ and could lower their concentration in the system, thus reducing the rate of generation of .OH and appear to inhibit the .OH-dependent reaction. In order to lessen the likelihood of this subtle artifact, we have investigated the effect of Pf on both H2O2 and Fe2+. On the basis of the absorption curves of peroxide solutions [37], the activity of catalase can be determined by direct measurements of the decrease of light absorption at 240 nm caused by the decomposition of H 2O 2 by catalase. By replacing catalase with Pf, no such change in absorption at 240 nm was observed when 2 mM H2O2 was allowed to act on 1 or 6 mM Pf in 0.01 M phosphate buffer, pH 7.0 for 10 min at 25°C (data not shown). These data indicate that Pf does not decompose or interact directly with H2O2. Free iron was expected to be present in all the reaction systems described above. A fluorescent iron chelation assay was performed using calcein as a fluorescent probe [32]. The relative fluorescence of calcein was decreased with time when monitored at excitation wavelength of 487 nm and emission at 517 nm (Fig. 4). As shown in Fig. 4, addition of iron (2 µM ferrous ammonium sulfate) to this calcein solution accelerated the fluorescence loss with time. In a similar reaction mixture addition of 250 µM DTPA (a known iron chelator) inhibited the rate of decrease of fluorescence caused by iron. Pf (1 or 6 mM) was found have little effect on irondependent calcein fluorescence. Further, when 2 µM ferric chloride was added in place of ferrous ammonium sulfate, Pf at 1 or 6 mM had no detectable effects on the rate of calcein fluorescence change (data not shown). These data indicate that Pf does not interact with either Fe2+ or Fe3+ under our experimental conditions.
Discussion The experimental drug Pf has been found to be markedly reduce the lung fibrosis induced by bleomycin, asbestos or cyclophosphamide in experimental animals [1–3, 5]. However, the mechanism of such cytoprotection has not been elucidated. Oxidative damage appears to play a relevant role in the pathogenesis of fibrotic diseases. Besides its tumorkilling properties, the chemotherapeutic drug bleomycin enhances lipid peroxidation [11, 48] and causes inflammation, alveolar cell damage, and eventually fibrosis [46, 47]. Asbestos and the antitumor agent cyclophosphamide have also been considered to induce pulmonary fibrosis and oxidative stress seems to play a secondary role in the pathogenesis of lung fibrosis [18, 49, 50]. The results reported
Fig. 4. Calcein fluorescence assay. The protocol was as described in ‘Materials and methods.’ The reaction mixture contained: 0.05 mM calcein, 2 mM ferrous ammonium in 0.25 M Tris-HCl, pH 7.6. Indicated concentrations of Pf or 250 µM DTPA was added at the indicated time intervals.
here clearly demonstrate that Pf is a potent scavenger of .OH and inhibitor of membrane lipid peroxidation, suggesting that the protective effects of Pf against the action of the fibrotic agents may at least in part be related to its antioxidant properties. In the present study, we have demonstrated that the experimental antifibrotic drug Pf inhibits NADPH-dependent lipid peroxidation in a dose-dependent manner (Fig. 1). A link between lipid peroxidation and fibrogenesis was demonstrated in vivo by the evidence that antioxidant supplementation was able to depress procollagen α1 mRNA over expression [38] and collagen deposition in fibrotic liver as a consequence of chronic CCl4 administration [39, 40]. Recent studies have also demonstrated the role of MDA, an aldehydeic end product of lipid peroxidation, on the synthesis of collagen [10, 21]. Because lipid peroxide products are also known to stimulate the synthesis of collagen [21, 22] and have been suggested as a possible mechanism of pulmonary fibrosis [23, 24], it is reasonable to suggest that the antifibrotic effects of Pf observed in several experimental models [1–5, 7] may, in part, be attributed to the reduction of MDA production. Because reactive oxygen species are known to be involved in lipid peroxidation [25, 43, 52, 53] and are suggested to be culprits in developing pulmonary fibrosis in experimental models [14, 15, 18], attempts were made to identify the
124 reactive species that may be scavenged by Pf. Utilizing EPR spectroscopy in combination with spin trapping techniques as well as colorimetric techniques, we found that Pf is not an effective superoxide scavenger. This was confirmed by using two different assays. In both cytochrome c assay, where O2– acts as a reductant, and in epinephrine assay, where O2– acts as an oxidant, Pf up to 15 mM concentration had little effect. However, Pf was found to be a potent scavenger of hydroxyl radicals. This was demonstrated both in the EPR spintrapping studies as well as in the deoxyribose degradation assays (Figs 2 and 3). Although it appears that high concentrations (mM) of Pf is required to scavenge the hydroxyl radicals, it should be noted that Pf was allowed to compete with reactants (in vitro) that were reacting at or near diffusion limiting rates. Thus, when .OH radicals were generated in a Fenton-type reaction and detected as DMPO-OH adducts by EPR spectroscopic analysis, Pf inhibited the DMPO-OH adduct signals in a dose-dependent manner. In this assay the rate of reaction of DMPO with .OH is known to be extremely fast, 3.4 × 109 M–1 sec–1 [41]. As little as 2.5 mM Pf was found to be able to compete with 33 mM DMPO for the available .OH to cause a 50% reduction of DMPO-OH adduct formation (Fig. 3). The .OH scavenging property of Pf was confirmed in a colorimetric assay where .OH radicals react with deoxyribose to generate TBA reactive product [31]. In this assay Pf inhibited the TBA reactive product formation in a dosedependent manner. The rate at which Pf scavenged .OH was calculated to be 5.4 × 109 M–1 sec–1. Compared to the rate constants reported for some of the known .OH scavengers [31], Pf was found to be approximately twenty times more sensitive than citrate, two times more sensitive than allopurinol, ethanol, histidine and mannitol, and ~ 50% as effective as dimethyl sulfoxide. Reactive oxygen species, such as .OH, are proposed to be the direct initiators of lipid peroxidation by concerted addition-abstraction reactions with the diene bonds of unsaturated lipids [42–45]. It was proposed that .OH are also involved in the propagation of lipid peroxidation [42], which in turn can produce membrane damage and cell dysfunction [43]. Pf was found to be a powerful antioxidant, which can scavenge .OH and prevent membrane lipid peroxidation. Therefore, the protective effects of Pf against membrane lipid peroxide formation may, at least in part, be attributed to the reactive oxygen scavenging properties of this drug. Although the percent inhibition of lipid peroxidation by Pf was dose dependent, the observed effects were not a linear function of the concentration of Pf (Fig. 1). This may be explained by the fact that lipid peroxidation is not a simple reaction. The hydroxyl radicals could initiate the chain reaction, but the propagation of the reaction could be carried out by at least three other reactive species, such as L., LO. and LOO..
Initiation LH + .OH → L. + H2O Propagation L. + O2 → LOO. LOO. + LH → LOOH + L. LOOH + Fe(II) → LO. + Fe(III) Degradation Malondialdehyde cleavage Termination LOO. + L. → LOOH + LH or L. + Pf → LH + Pf. Pf. + L. → LH + Pf(ox) Thus, lipid peroxidation can be initiated by a free radical compound, such as the hydroxyl radical, which abstracts hydrogen from the polyunsaturated lipid (LH), resulting in the formation of a lipid radical (L.). The free radical chain reaction is propagated by the addition of O2, which forms the lipid peroxy radical (LOO.) and lipid peroxide (LOOH). Rearrangement of the single electron results in degradation of the lipid and release of 3C units as malondialdehyde. The chain reaction can be terminated by antioxidants, such as Pf, which could donate single electrons in two subsequent steps to form a stable oxidized compound. Although the rate at which Pf scavenges .OH was found to be 5.4 × 109 M–1 sec–1, some .OH species would escape the PF action and react with LH or LO., generating malondialdehydes. Some of these reactions are known to operate close to the diffusion controlled rates [54, 55]. It is possible that Pf could also interact with lipid radical (L.) as shown under termination reaction and may not be able to react with other reactive species, such as peroxy radicals (LOO.) or alkoxy radicals (LO.), thus letting in the accumulation of malondialdehyde at a slower rate. This may, in part, be the reason for the abrupt change in the slope of the curve in Fig. 1, where relatively higher dose of Pf was required for a modest increase in the inhibition of lipid peroxidation. Cytokines, such as TGFβ, PDGF and TNFα, are recognized to play a role in modulating inflammatory processes [51] and dietary supplementation with vitamin E afforded a significant down modulation of these cytokines and collagen I formation [19, 20, 38]. Because reactive oxygen species are known to be involved in the expression of some of the cytokines [19, 20], the observed protective effects of Pf on TNF-induced and subsequent endotoxin shock in mice [6] may also be attributed to its antioxidant properties. Taken together, the reported findings suggest that Pf exerts the antifibrotic effects by scavenging highly reactive oxygen species generated during exposure to some of the pro-oxidant
125 fibrotic agents. The cytoprotective effect of Pf may now be attributed to its antioxidant properties.
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