Mario G. Moreno

Curcumin prevents and reverses cirrhosis induced by bile duct obstruction or CCl4 in rats: role of TGF-β modulation and oxidative stress

Curcumin is a phytophenolic compound, which is highly efficacious for treating several inflammatory diseases. The aim of this study was to evaluate the efficacy of curcumin in preventing or reversing liver cirrhosis. A 4‐week bile duct ligation (BDL) rat model was used to test the ability of curcumin (100 mg/kg, p.o., daily) to prevent cirrhosis. To reverse cirrhosis, CCl4 was administered chronically for 3 months, and then it was withdrawn and curcumin administered for 2 months. Alanine aminotransferase, γ‐glutamyl transpeptidase, liver histopathology, bilirubin, glycogen, reduced and oxidized glutathione, and TGF‐β (mRNA and protein) levels were assessed. Curcumin preserved normal values of markers of liver damage in BDL rats. Fibrosis, assessed by measuring hydroxyproline levels and histopathology, increased nearly fivefold after BDL and this effect was partially but significantly prevented by curcumin. BDL increased transforming growth factor‐beta (TGF‐β) levels (mRNA and proteins), while curcumin partially suppressed this mediator of fibrosis. Curcumin also partially reversed the fibrosis induced by CCl4. Curcumin was effective in preventing and reversing cirrhosis, probably by its ability of reducing TGF‐β expression. These data suggest that curcumin might be an effective antifibrotic and fibrolitic drug in the treatment of chronic hepatic diseases.

Remission of liver fibrosis by interferon-α2b

Abstract Fibrosis is a dynamic process associated with the continuous deposition and resorption of connective tissue, mainly collagen. Therapeutic strategies are emerging by which this dynamic process can be modulated. Since interferons are known to inhibit collagen production, the aim of this study was to investigate if the administration of interferon-α2b (IFN-α) can restore the normal hepatic content of collagen in rats with established fibrosis. Fibrosis was induced by prolonged bile duct ligation. IFN-α-(100 000 IU/rat/day; s.c.) was administered to fibrotic rats for 15 days. Bile duct ligation increased liver collagen content 6-fold. In addition, serum and liver markers of hepatic injury increased significantly; liver histology showed an increase in collagen deposition, and the normal architecture was lost, with large zones of necrosis being observed frequently. IFN-α-administration reversed to normal the values of all the biochemical markers measured and restored the normal architecture of the liver. Our results demonstrated that IFN-α is useful in reversing fibrosis and liver damage induced by biliary obstruction in the rat. However, further investigations are required to evaluate the therapeutic relevance of interferons on non-viral fibrosis and cholestasis.

N-acetylcysteine prevents carbon tetrachloride-induced liver cirrhosis: role of liver transforming growth factor-beta and oxidative stress

Objectives N-acetylcysteine (NAC) is an antioxidant, a precursor of reduced glutathione, and an inhibitor of the profibrotic cytokine liver transforming growth factor-beta (TGF-&bgr;). Carbon tetrachloride (CCl4) cirrhosis is characterized by oxidative stress and fibrosis. Therefore, the aim of this work was to study the effect of NAC on experimental cirrhosis. Methods CCl4 was chronically administered for 8 weeks along with 300 mg/kg of NAC orally once a day. Alkaline phosphatase, alanine aminotransferase, and &ggr;-glutamyltranspeptidase were measured in plasma. Hydroxyproline, glycogen, lipid peroxidation, glutathione were determined in liver samples by colorimetric methods. TGF-&bgr; was evaluated by western blotting, and a histopathological analysis was performed. Results Serum markers of liver damage increased by CCl4 intoxication (P<0.05), whereas cotreatment with NAC prevented these increases (P<0.05); glycogen was depleted in the cirrhotic group (P<0.05), but preserved by NAC (P<0.05). Lipid peroxidation increased and glutathione decreased by the administration of CCl4 (P<0.05), again NAC prevented both effects (P<0.05). Importantly, collagen increased by about seven-fold in the CCl4 group (P<0.05); administration of NAC preserved the normal levels of collagen (P<0.05). Biochemical determinations were corroborated by hematoxylin and eosin, and trichromic stains. Western blots revealed a four-fold increase in TGF-&bgr; in the group receiving CCl4, NAC cotreatment abolished TGF-&bgr; signal (P<0.05). Conclusion Our results strongly suggest that NAC prevents experimental cirrhosis by two mechanisms: by preventing oxidative stress and by downregulating the profibrogenic cytokine TGF-&bgr;. As NAC is currently used in humans intoxicated with paracetamol, it can be tested in fibrotic or cirrhotic patients under controlled trials.

Antifibrotic and fibrolytic properties of celecoxib in liver damage induced by carbon tetrachloride in the rat

Background: Transforming growth factor‐β (TGF‐β) plays a pivotal role in liver fibrosis, because it activates hepatic stellate cells, stimulating extracellular matrix deposition. Cyclooxygenase‐2 (COX‐2) has been associated with TGF‐β because its inhibition decreases TGF‐β expression and collagen production in some cultured cell types.

Protective effects of allopurinol against acute liver damage and cirrhosis induced by carbon tetrachloride: Modulation of NF-κB, cytokine production and oxidative stress

BACKGROUND The aim of this work was to evaluate the hepatoprotective ability of allopurinol to prevent the liver injury induced by carbon tetrachloride (CCl(4)). METHODS Acute liver damage was induced with CCl(4) (4g/kg, by gavage); allopurinol (50mg/kg, by gavage) was given 1h before and 1h after CCl(4) intoxication and two daily doses for the previous three days. Cirrhosis was established by CCl(4) administration (0.4g/kg, i. p. three times a week, eight weeks); allopurinol was administered (100mg/kg, by gavage, daily) during the long-term of CCl(4) treatment. Alanine aminotransferase (ALT), γ-glutamyl transpeptidase (γ-GTP), xanthine oxidase (XO), lipid peroxidation, reduced and oxidized glutathione (GSH, GSSG, respectively), hydroxyproline and histopathologycal analysis were performed. Nuclear factor-κB (NF-κB), pro-inflammatory and anti-inflammatory cytokines, transforming growth factor-β (TGF-β) and metalloproteinase-13 (MMP-13) were analyzed by Western blots. RESULTS Acute injury increased ALT and γ-GTP activities, additionally enhanced NF-κB nuclear translocation and cytokines production such as tumor necrosis factor-α, interleukine-1β, and interleukine-6. Allopurinol partially prevented these effects, while increased interleukine-10. Acute and chronic CCl(4) treatments altered the levels of XO activity, lipid peroxidation, and GSH/GSSG ratio, while these remained within normal range with allopurinol administration. Necrosis, fibrosis and TGF-β production induced in chronic injury were partially prevented by allopurinol, interestingly, this drug induced MMP-13 activity. CONCLUSIONS Allopurinol possesses antioxidant, anti-inflammatory and antifibrotic properties, probably by its capacity to reduce NF-κB nuclear translocation and TGF-β expression, as well as to induce MMP-13. General significance Allopurinol might be effective treatment of liver diseases.

Effects of acetyl salycilic acid and ibuprofen in chronic liver damage induced by CCl4

Non‐steroidal anti‐inflammatory drugs (NSAIDs) are drugs used primarily to treat inflammation, pain and fever. Their main mechanism of action is cyclooxygenase (COX) inhibition, and this enzyme has been linked to hepatotoxicity. The association of COX and liver injury has been, in part, due to the presence of COX‐2 isoform in damaged liver and the possible induction of this enzyme by profibrotic molecules like Transforming Growth Factor‐β (TGF‐β). The aim of this work was to evaluate the effects of two of the most used NSAIDs, acetyl salicylic acid (ASA) and ibuprofen (IBP), on experimental liver fibrosis. We formed experimental groups of rats including vehicle and drug controls, damage induced by chronic CCl4 (0.4 g kg−1, i.p., three times per week, for 8 weeks) administration, and CCl4 plus ASA (100 mg kg−1, p.o., daily) or IBP (30 mg kg−1, p.o., daily). Both drugs showed important antifibrotic properties. They inhibited COX‐2 activity, prevented oxidative stress measured as lipid peroxidation and glutathione content, and ASA inhibited partially and IBP totally increased TGF‐β expression and collagen content. ASA and IBP prevented translocation of NFκB to the nucleus and, interestingly, ASA induced MMP‐2 and MMP‐13 whereas IBP induced MMP‐2, MMP‐9 and MMP‐13. As a whole, these effects explain the beneficial effects of ASA and IBP on experimental liver fibrosis. Copyright

Methyl palmitate prevents CCl4‐induced liver fibrosis

Liver fibrosis is characterized by an excess of collagen fiber deposition, and it is known that Kupffer cells play an important role by immunomodulation of the toxic response. Methyl palmitate (MP) is an effective Kupffer cell inhibitor. The aim of this work was to evaluate the effect of MP on experimental liver fibrosis. Four groups were formed: the control group, which received the vehicles only; CCl4 group (0.4 g kg−1, i.p., three times a week, for eight weeks); CCl4 plus MP (300 mg kg−1, i.p., daily); and MP alone. Alanine aminotransferase was increased by CCl4, and MP did not prevent this increase. Lipid peroxidation was increased markedly by CCl4; again, MP was not able to prevent this effect. Fibrosis increased nearly 6‐fold (measured as liver hydroxyproline content) in the CCl4 group; MP preserved the normal content of collagen. These results were corroborated by histopathology. To elucidate the antifibrogenic mechanism of MP, we measured the production of TGF‐β; CCl4 increased this cytokine several‐fold, and MP abolished this increase. Collectively the present results indicate that MP possesses a strong antifibrogenic effect at least in the CCl4 model of fibrosis. The antifibrotic effect of MP is probably associated with its ability to reduce TGF‐β content, maybe by immunomodulation of Kupffer cells functioning. Copyright

Sulfasalazine prevents the increase in TGF-β, COX-2, nuclear NFκB translocation and fibrosis in CCl4-induced liver cirrhosis in the rat.

It has been demonstrated that this sulfasalazine (SF) inhibits the nuclear factor κB (NFκB) pathway, which regulates important genes during inflammation and immune answer. The aim of this work was to evaluate the effects of SF on carbon tetrachloride (CCl4)-induced liver fibrosis. We formed the following experimental groups of rats: controls, damage induced by chronic CCl4 (0.4 g/kg, intraperitoneally, three times a week for 8 weeks) administration and CCl4 + SF (100 mg/kg/day, postoperatively for 8 weeks) administration. We determined the activities of alanine aminotransferase (ALT), γ-glutamyl transpeptidase (γ-GTP), cyclooxygenase (COX)-1 and COX-2, lipid peroxidation, glutathione levels, collagen content, expression of transforming growth factor-β (TGF-β) and nuclear translocation of NFκB. SF was capable to inhibit the ALT and γ-GTP elevated levels induced with the CCl4 administration. SF had antioxidant properties, prevented the lipid peroxidation and the imbalance of reduced and oxidized glutathione produced by CCl4. Importantly, SF blocked the accumulation of collagen in the liver, the expression of TGF-β, the nuclear translocation of NFκB and the activity of COX-2, all induced with the administration of CCl4 in the rat. These results show that SF has strong antifibrotic properties because of its antioxidant properties and its ability to prevent nuclear translocation of NFκB and consequently the expression of TGF-β and the activity of COX-2.

D1 and D2 antagonists reverse the effects of appetite suppressants on weight loss, food intake, locomotion, and rebalance spiking inhibition in the rat NAc shell

Obesity is a worldwide health problem that has reached epidemic proportions. To ameliorate this problem, one approach is the use of appetite suppressants. These compounds are frequently amphetamine congeners such as diethylpropion (DEP), phentermine (PHEN), and bupropion (BUP), whose effects are mediated through serotonin, norepinephrine, and dopaminergic pathways. The nucleus accumbens (NAc) shell receives dopaminergic inputs and is involved in feeding and motor activity. However, little is known about how appetite suppressants modulate its activity. Therefore, we characterized behavioral and neuronal NAc shell responses to short-term treatments of DEP, PHEN, and BUP. These compounds caused a transient decrease in weight and food intake while increasing locomotion, stereotypy, and insomnia. They evoked a large inhibitory imbalance in NAc shell spiking activity that correlated with the onset of locomotion and stereotypy. Analysis of the local field potentials (LFPs) showed that all three drugs modulated beta, theta, and delta oscillations. These oscillations do not reflect an aversive-malaise brain state, as ascertained from taste aversion experiments, but tracked both the initial decrease in weight and food intake and the subsequent tolerance to these drugs. Importantly, the appetite suppressant-induced weight loss and locomotion were markedly reduced by intragastric (and intra-NAc shell) infusions of dopamine antagonists SCH-23390 (D1 receptor) or raclopride (D2 receptor). Furthermore, both antagonists attenuated appetite suppressant-induced LFP oscillations and partially restored the imbalance in NAc shell activity. These data reveal that appetite suppressant-induced behavioral and neuronal activity recorded in the NAc shell depend, to various extents, on dopaminergic activation and thus point to an important role for D1/D2-like receptors (in the NAc shell) in the mechanism of action for these anorexic compounds.

Trolox Down‐Regulates Transforming Growth Factor‐β and Prevents Experimental Cirrhosis

Cirrhosis is a very common disease and its treatment is limited due to lack of effective drugs. Some studies indicate that this disease is associated with oxidative stress. Therefore, we decided to study the effect of trolox, an effective antioxidant, on experimental cirrhosis. Cirrhosis was induced by CCl4 administration (0.4 g/kg, intraperitoneally, three times per week, for 8 weeks) to Wistar male rats. Trolox was administered daily (50 mg/kg, orally). Fibrosis was assessed histologically and by measuring liver hydroxyproline content. Glutathione, lipid peroxidation and glycogen were measured in liver; serum markers of liver damage were also quantified. Transforming growth factor-beta (TGF-beta) was determined by Western blot and quantified densitometrically. Alkaline phosphatase, gamma-glutamyl transpeptidase and alanine aminotransferase increased in the group receiving CCl4; trolox completely or partially prevented these alterations. Glycogen was almost depleted by CCl4 but was partially preserved by trolox. Lipid peroxidation increased while glutathione decreased by CCl4 administration; trolox corrected both effects. Histology showed thick bands of collagen, necrosis and distortion of the hepatic parenchyma in the CCl4 group, such effects were prevented by trolox. Hydroxyproline content increased 5-fold by CCl4, while the group receiving both CCl4 and trolox showed no significant difference compared to the control group. CCl4 increased 3-fold TGF-beta, while trolox completely prevented this increase. We found that trolox effectively prevented cirrhosis induced with CCl4 in the rat. Our results suggest that the beneficial effects of trolox may be associated to its antioxidant properties and to its ability to reduce the profibrogenic cytokine TGF-beta expression.