Effect of Stevioside (Stevia rebaudiana) on Entamoeba histolytica Trophozoites.

Human amoebiasis still represents a major health problem worldwide. Metronidazole has been used as the most common drug to treat the disease; however, it is also known that the drug causes undesirable side effects. This has led to the search for new pharmacological alternatives which include phytochemical compounds with antiamoebic effects. We analyzed the amoebicidal activity of stevioside (STV), a diterpene glycoside present in Stevia rebaudiana, on trophozoites of E. histolytica. Different concentrations of STV were tested, and an inhibitory concentration of 50% of cell viability (IC50) was determined with an exposition of 9.53 mM for 24 h. Trophozoites exposed to STV showed morphological changes evidenced by the decrease in the basic structures related to the movement and adherence to the substrate, as well as ultrastructural features characterized by a loss of regularity on the cell membrane, an increase in cytoplasmic granularity, and an increase in apparent autophagic vacuoles. Also, the decrease in cysteine protease expression and the proteolytic activity of trophozoites to degrade the cell monolayer were analyzed. A histological analysis of hamster livers inoculated with trophozoites and treated with STV showed changes related to the granulomatous reaction of the liver parenchymal tissue. Our results constitute the first report related to the possible use of STV as a therapeutic alternative in amoebiasis.

Naringenin attenuates the progression of liver fibrosis via inactivation of hepatic stellate cells and profibrogenic pathways.

There is no effective treatment for hepatic fibrosis. Previously, we demonstrated that naringenin possesses the ability to prevent experimental chronic liver damage. Therefore, the objective of this work was to investigate whether naringenin could reverse carbon tetrachloride (CCl4)-induced fibrosis in rats and, if so, to search for the mechanisms involved. CCl4 was given to male Wistar rats (400 mg/kg, three times per week, i. p.) for 12 weeks; naringenin (100 mg/kg twice per day, p. o.) was administered from weeks 9-12 of the CCl4 treatment. Liver damage and oxidative stress markers were measured. Masson's trichrome, hematoxylin-eosin staining and immunohistochemistry were performed. Zymography assays for MMP-9 and MMP-2 were carried out. TGF-ß, CTGF, Col-I, MMP-13, NF-κB, IL-1ß, IL-10, Smad7, pSmad3 and pJNK protein levels were determined by western blotting. In addition, α-SMA and Smad3 protein and mRNA levels were studied. Naringenin reversed liver damage, biochemical and oxidative stress marker elevation, and fibrosis and restored normal MMP-9 and MMP-2 activity. The flavonoid also preserved NF-κB, IL-1ß, IL-10, TGF-ß, CTGF, Col-I, MMP-13 and Smad7 protein levels. Moreover, naringenin decreased JNK activation and Smad3 phosphorylation in the linker region. Finally, α-SMA and Smad3 protein and mRNA levels were reduced by naringenin administration. The results of this study demonstrate that naringenin blocks oxidative stress, inflammation and the TGF-ß-Smad3 and JNK-Smad3 pathways, thereby carrying out its antifibrotic effects and making it a good candidate to treat human fibrosis, as previously demonstrated in toxicological and clinical studies.

Rebaudioside A administration prevents experimental liver fibrosis: an in vivo and in vitro study of the mechanisms of action involved.

Rebaudioside A (Reb A) is a diterpenoid isolated from the leaves of Stevia rebaudiana (Bertoni) that has been shown to possess pharmacological activity, including anti-inflammatory and antioxidant properties. However, the ability of Reb A to prevent liver injury has not been evaluated. Therefore, we aimed to study the potential of Reb A (20 mg/kg; two times daily intraperitoneally) to prevent liver injury induced by thioacetamide (TAA) administration (200 mg/kg; three times per week intraperitoneally). In addition, cocultures were incubated with either lipopolysaccharide or ethanol. Antifibrotic, antioxidant and immunological responses were evaluated. Chronic TAA administration produced considerable liver damage and distorted the liver parenchyma with the presence of prominent thick bands of collagen. In addition, TAA upregulated the expression of α-smooth muscle actin, transforming growth factor-ß1, metalloproteinases 9, 2 and 13, and nuclear factor kappaB and downregulated nuclear erythroid factor 2. Reb A administration prevented all of these changes. In cocultured cells, Reb A prevented the upregulation of genes implicated in fibrotic and inflammatory processes when cells were exposed to ethanol and lipopolysaccharide. Altogether, our results suggest that Reb A prevents liver damage by blocking oxidative processes via upregulation of nuclear erythroid factor 2, exerts immunomodulatory effects by downregulating the nuclear factor-κB system and acts as an antifibrotic agent by maintaining collagen content.

Antioxidant and immunomodulatory activity induced by stevioside in liver damage: In vivo, in vitro and in silico assays.

AIMS: Stevioside is a diterpenoid obtained from the leaves of Stevia rebaudiana (Bertoni) that exhibits antioxidant, antifibrotic, antiglycemic and anticancer properties. Therefore, we aimed to study whether stevioside has beneficial effects in liver injury induced by long-term thioacetamide (TAA) administration and investigated the possible underlying molecular mechanism using in vivo, in vitro and in silico approaches. MAIN METHODS: Liver injury was induced in male Wistar rats by TAA administration (200 mg/kg), intraperitoneally, three times per week. Rats received saline or stevioside (20 mg/kg) twice daily intraperitoneally. In addition, cocultures were incubated with either lipopolysaccharide or ethanol. Liver injury, antioxidant and immunological responses were evaluated. KEY FINDINGS: Chronic TAA administration induced significant liver damage. In addition, TAA upregulated the protein expression of nuclear factor (NF)-κB, thus increasing the expression of proinflammatory cytokines and decreasing the antioxidant capacity of the liver through downregulation of nuclear erythroid factor 2 (Nrf2). Notably, stevioside administration prevented all of these changes. In vitro, stevioside prevented the upregulation of several genes implicated in liver inflammation when cocultured cells were incubated with lipopolysaccharide or ethanol. In silico assays using tumor necrosis factor receptor (TNFR)-1 and Toll-like receptor (TLR)-4-MD2 demonstrated that stevioside docks with TNFR1 and TLR4-MD2, thus promoting an antagonistic action against this proinflammatory mediator. SIGNIFICANCE: Collectively, these data suggest that stevioside prevented liver damage through antioxidant activity by upregulating Nrf2 and immunomodulatory activity by blocking NF-κB signaling.

Stevioside inhibits experimental fibrosis by down-regulating profibrotic Smad pathways and blocking hepatic stellate cell activation.

Liver cirrhosis is associated with increased morbidity and mortality with important health and social consequences; however, an effective treatment has not been found yet. Previous reports have shown some beneficial effects of stevioside (SVT) in different diseases, but the ability of SVT to inhibit liver cirrhosis has not been reported. Therefore, we studied the potential of this diterpenoid to inhibit liver cirrhosis induced by thioacetamide, a model that shares many similarities with the human disease, and investigated the possible underlying molecular mechanism using in vivo and in vitro approaches. Cirrhosis was induced in male Wistar rats by chronic thioacetamide administration (200 mg/kg) intraperitoneally three times per week. Rats received saline or SVT (20 mg/kg) two times daily intraperitoneally. In addition, co-cultures were incubated with either lipopolysaccharide or ethanol. Liver fibrosis, hepatic stellate cells activation, metalloproteinases activity, canonical and non-canonical Smads pathway and expression of several profibrogenic genes were evaluated. Thioacetamide activated hepatic stellate cells and distorted the liver parenchyma with the presence of abundant thick bands of collagen. In addition, thioacetamide up-regulated the protein expression of α-smooth muscle actin, transforming growth factor-ß1, metalloproteinases-9,-2 and -13 and overstimulate the canonical and non-canonical Smad pathways. SVT administration inhibited all of these changes. In vitro, SVT inhibited the up-regulation of several genes implicated in cirrhosis when cells were exposed to lipopolysaccharides or ethanol. We conclude that SVT inhibited liver damage by blocking hepatic stellate cells activation, down-regulating canonical and non-canonical profibrotic Smad pathways.

Stevia Prevents Acute and Chronic Liver Injury Induced by Carbon Tetrachloride by Blocking Oxidative Stress through Nrf2 Upregulation.

The effect of stevia on liver cirrhosis has not been previously investigated. In the present study, the antioxidant and anti-inflammatory properties of stevia leaves were studied in male Wistar rats with carbon tetrachloride- (CCl4-) induced acute and chronic liver damage. Acute and chronic liver damage induced oxidative stress, necrosis, and cholestasis, which were significantly ameliorated by stevia. Chronic CCl4 treatment resulted in liver cirrhosis, as evidenced by nodules of hepatocytes surrounded by thick bands of collagen and distortion of the hepatic architecture, and stevia significantly prevented these alterations. Subsequently, the underlying mechanism of action of the plant was analyzed. Our study for the first time shows that stevia upregulated Nrf2, thereby counteracting oxidative stress, and prevented necrosis and cholestasis through modulation of the main proinflammatory cytokines via NF-κB inhibition. These multitarget mechanisms led to the prevention of experimental cirrhosis. Given the reasonable safety profile of stevia, our results indicated that it may be useful for the clinical treatment of acute and chronic liver diseases.

Quercetin reverses experimental cirrhosis by immunomodulation of the proinflammatory and profibrotic processes.

The ability of quercetin to reverse an established cirrhosis has not yet been investigated. Therefore, the aim of this study was to examine the efficacy of this flavonoid in reversing experimental cirrhosis. Cirrhosis was induced by intraperitoneal administration of TAA (200 mg/kg of body weight) three times per week for 8 weeks or by intraperitoneal petrolatum-CCl4 (400 mg/kg of body weight) administration three times per week for 8 weeks. To determine the capacity of quercetin to prevent liver fibrosis, the flavonoid (50 mg/kg of body weight, p.o.) was administered daily to rats during the CCl4 or TAA treatment. To evaluate the ability of quercetin to reverse the previously induced cirrhosis, we first treated rats with CCl4 for 8 weeks, as previously described and then the flavonoid was administered for four more weeks. We found that the liver anti-inflammatory and antinecrotic effects of quercetin are associated with its antioxidant properties, to the ability of the flavonoid to block NF-κB activation and in consequence to reduce cytokine IL-1. The ability of quercetin to reverse fibrosis may be associated with the capacity of the flavonoid to decrease TGF-ß levels, hepatic stellate cell activation, and to promote degradation of the ECM by increasing metalloproteinases. The main conclusion is that quercetin, in addition to its liver protective activity against TAA chronic intoxication, is also capable of reversing a well-stablished cirrhosis by blocking the prooxidant processes and by downregulating the inflammatory and profibrotic responses.

Naringenin prevents experimental liver fibrosis by blocking TGFß-Smad3 and JNK-Smad3 pathways.

AIM: To study the molecular mechanisms involved in the hepatoprotective effects of naringenin (NAR) on carbon tetrachloride (CCl4)-induced liver fibrosis. METHODS: Thirty-two male Wistar rats (120-150 g) were randomly divided into four groups: (1) a control group (n = 8) that received 0.7% carboxy methyl-cellulose (NAR vehicle) 1 mL/daily p.o.; (2) a CCl4 group (n = 8) that received 400 mg of CCl4/kg body weight i.p. 3 times a week for 8 wk; (3) a CCl4 + NAR (n = 8) group that received 400 mg of CCl4/kg body weight i.p. 3 times a week for 8 wk and 100 mg of NAR/kg body weight daily for 8 wk p.o.; and (4) an NAR group (n = 8) that received 100 mg of NAR/kg body weight daily for 8 wk p.o. After the experimental period, animals were sacrificed under ketamine and xylazine anesthesia. Liver damage markers such as alanine aminotransferase (ALT), alkaline phosphatase (AP), γ-glutamyl transpeptidase (γ-GTP), reduced glutathione (GSH), glycogen content, lipid peroxidation (LPO) and collagen content were measured. The enzymatic activity of glutathione peroxidase (GPx) was assessed. Liver histopathology was performed utilizing Masson's trichrome and hematoxylin-eosin stains. Zymography assays for MMP-9 and MMP-2 were carried out. Hepatic TGF-ß, α-SMA, CTGF, Col-I, MMP-13, NF-κB, IL-1, IL-10, Smad7, Smad3, pSmad3 and pJNK proteins were detected via western blot. RESULTS: NAR administration prevented increases in ALT, AP, γ-GTP, and GPx enzymatic activity; depletion of GSH and glycogen; and increases in LPO and collagen produced by chronic CCl4 intoxication (P < 0.05). Liver histopathology showed a decrease in collagen deposition when rats received NAR in addition to CCl4. Although zymography assays showed that CCl4 produced an increase in MMP-9 and MMP-2 gelatinase activity; interestingly, NAR administration was associated with normal MMP-9 and MMP-2 activity (P < 0.05). The anti-inflammatory, antinecrotic and antifibrotic effects of NAR may be attributed to its ability to prevent NF-κB activation and the subsequent production of IL-1 and IL-10 (P < 0.05). NAR completely prevented the increase in TGF-ß, α-SMA, CTGF, Col-1, and MMP-13 proteins compared with the CCl4-treated group (P < 0.05). NAR prevented Smad3 phosphorylation in the linker region by JNK since this flavonoid blocked this kinase (P < 0.05). CONCLUSION: NAR prevents CCl4 induced liver inflammation, necrosis and fibrosis, due to its antioxidant capacity as a free radical inhibitor and by inhibiting the NF-κB, TGF-ß-Smad3 and JNK-Smad3 pathways.

Antioxidants in liver health.

Liver diseases are a worldwide medical problem because the liver is the principal detoxifying organ and maintains metabolic homeostasis. The liver metabolizes various compounds that produce free radicals (FR). However, antioxidants scavenge FR and maintain the oxidative/antioxidative balance in the liver. When the liver oxidative/antioxidative balance is disrupted, the state is termed oxidative stress. Oxidative stress leads to deleterious processes in the liver and produces liver diseases. Therefore, restoring antioxidants is essential to maintain homeostasis. One method of restoring antioxidants is to consume natural compounds with antioxidant capacity. The objective of this review is to provide information pertaining to various antioxidants found in food that have demonstrated utility in improving liver diseases.