Silibinin induces hepatic stellate cell cycle arrest via enhancing p53/p27 and inhibiting Akt downstream signaling protein expression.

BACKGROUND: Proliferation of hepatic stellate cells (HSCs) plays a pivotal role in the progression of liver fibrosis consequent to chronic liver injury. Silibinin, a flavonoid compound, has been shown to possess anti-fibrogenic effects in animal models of liver fibrosis. This was attributed to an inhibition of cell proliferation of activated HSCs. The present study was to gain insight into the molecular pathways involved in silibinin anti-fibrogenic effect. METHODS: The study was conducted on LX-2 human stellate cells treated with three concentrations of silibinin (10, 50 and 100 µmol/L) for 24 and 96 hours. At the end of the treatment cell viability and proliferation were evaluated. Protein expression of p27, p21, p53, Akt and phosphorylated-Akt was evaluated by Western blotting analysis and Ki-67 protein expression was by immunocytochemistry. Sirtuin activity was evaluated by chemiluminescence based assay. RESULTS: Silibinin inhibits LX-2 cell proliferation in dose- and time-dependent manner; we showed that silibinin upregulated the protein expressions of p27 and p53. Such regulation was correlated to an inhibition of both downstream Akt and phosphorylated-Akt protein signaling and Ki-67 protein expression. Sirtuin activity also was correlated to silibinin-inhibited proliferation of LX-2 cells. CONCLUSION: The anti-proliferative effect of silibinin on LX-2 human stellate cells is via the inhibition of the expressions of various cell cycle targets including p27, Akt and sirtuin signaling.

Effect of isobutyl methacrylate and methacrylic acid eluted from chairside denture hard reliners on enzymatic cellular antioxidants: An in vitro study in human primary buccal mucosal fibroblasts.

AIM: This study was conducted with the objective to evaluate the cytotoxicity of monomers isobutyl methacrylate (IBMA) and methacrylic acid (MA) in human buccal mucosal fibroblast primary cell culture and to study their effect on cellular enzymatic antioxidants-glutathione peroxidase (GPx), superoxide dismutase (SOD), and catalase (CAT). MATERIALS AND METHODS: The tissue for fibroblast cell culture was harvested from oral buccal mucosa of a healthy donor. Fibroblast cells were plated at a density of 1 × 104 cells per well in 96-well tissue culture plates. Cells were exposed to various concentrations of IBMA and MA. The cell viability and various enzyme activities were evaluated 24 h after exposure to the above treatments. All tests were done in triplicate. Cell viability was assessed by trypan blue dye exclusion assay and all enzyme activities were done using assay kits from Cayman Chemicals, Ann Arbor, USA. RESULTS: At all concentrations tested a statistically significant decrease in viability was observed in IBMA- and MA-treated cells. Around 42% cells were viable at the highest test concentration of IBMA (80 µmol/L) and only 20% cells were viable at the highest dose (144 µmol/L) of MA exposure (P < 0.05). Dose-dependent decrease in the GPx and SOD activities was observed in cells treated with IBMA and MA (P < 0.05). CAT activity was not detectable in the controls. However, a fall in CAT activity was detected in cells exposed to IBMA and MA at all concentrations tested (P < 0.05). CONCLUSION: IBMA and MA leaching out from the chairside denture hard reliners are cytotoxic on human buccal fibroblast primary cell cultures. This could be due to the oxidative stress caused by the generation of reactive oxygen species which is evidenced by the fall in activities of antioxidant enzymes (GPx, SOD, and CAT) and cytotoxicity.

Influence of N-acetylcysteine against dimethylnitrosamine induced hepatotoxicity in rats.

This study evaluates the hepatoprotective and antioxidant properties of N-acetylcysteine (NAC) on dimethylnitrosamine (DMN) induced hepatotoxicity in male Wistar albino rats. A single intraperitoneal dose of DMN (5 mg/kg b.w.) caused a significant increase in the levels of the serum marker enzymes (aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), glutamyl transpeptidase (γ-GT)) and a subsequent decrease in AST, ALT, ALP and increase in LDH and γ-GT in the liver tissue indicating hepatocellular damage. Elevation in the status of lipid peroxidation, fall in the activities of the enzymic (superoxide dismutase, catalase) and non-enzymic antioxidants (vitamin C, vitamin E) in the liver tissue further confirms oxidative stress and hepatocellular damage induced on DMN administration. Oral administration of NAC (50 mg/kg b.w.) for 7 days significantly prevented the above alterations in the status of the marker enzymes of hepatotoxicity and antioxidant parameters and restored them towards normalcy, which was further substantiated by the histopathological studies of the liver tissue. These results suggest that NAC offers hepatoprotection by ameliorating DMN-induced oxidative stress and hepatotoxicity and this protective effect was attributed to its antioxidant and free radical scavenging properties.

Protective effect of Cassia fistula Linn. on diethylnitrosamine induced hepatocellular damage and oxidative stress in ethanol pretreated rats.

Diethylnitrosamine (DEN), found in many commonly consumed foods, is widely reported to induce cancer in animals and humans. The aim of the present study was to investigate the hepatoprotective and antioxidant activities of the leaf extract of the medicinal plant Cassia fistula Linn. against diethylnitrosamine induced liver injury in ethanol pretreated rats. Albino Wistar rats, pretreated with ethanol for 15 days, were administered a single dose of DEN. Thirty days after DEN administration, hepatotocellular damage was observed histologically, along with elevated levels of serum AST, ALT, ALP, LDH, γ-GT and bilirubin and a simultaneous fall in the levels of the marker enzymes in the liver tissue. Liver oxidative stress was confirmed by elevated levels of lipid peroxidation (LPO) and a decrease in enzymic and non-enzymic antioxidants activities. Oral administration of the ethanolic leaf extract (ELE) of Cassia fistula for 30 days to ethanol + DEN treated rats significantly improved the above alterations in the markers of hepatotoxicity and oxidative stress, resulting in the reversal of most of the parameters studied and were comparable to the standard hepatoprotective drug silymarin.

Antiulcer activity of ethanol leaf extract of Cassia fistula.

The ethanol leaf extract (ELE) of Cassia fistula Linn. (Caesalpinaceae) was evaluated for antiulcer activity against pylorus ligation-induced gastric ulcer. Ranitidine (30 mg/kg b.w.) and ELE at doses of 250, 500, and 750 mg/kg b.w. were administered orally in different groups of rats (n = 6), 1 h prior to pyloric ligation. Four hours after pyloric ligation, the gastric juice was collected for evaluation of various parameters. The antiulcer activity of ELE was evidenced by the significant attenuation of gastric volume, pH, free acidity, and total acidity in the gastric juice of pyloric-ligated rats in a dose-dependent manner, and this protective effect could be due to strengthening of the mucosal defense mechanism. ELE pre-treatment significantly attenuated the fall in status of sialic acid and fucose accompanied by an increase in hexose, hexosamine, total non-amino polysaccharide, total carbohydrate, and C:P ratio in the gastric juice of pylorus-ligated rats, and this effect could be due to protection of the mucosal barrier system. ELE pre-treatment significantly prevented the increase in LPO and SOD accompanied by a fall in CAT, in the gastric juice of pyloric-ligated rats. This protective ability of ELE against pylorus ligation-induced gastric ulcer could be attributed to its free radical scavenging and antioxidant properties. Higher doses of ELE (750 mg/kg b.w.) produced maximum antiulcer activity comparable to ranitidine treatment. In essence, the antiulcer activity of ELE could be attributed to (i) a decrease in gastric acid secretion, (ii) protection of the mucosal barrier and restoration of mucosal secretions, (iii) inhibition of free radical generation or prevention of lipid peroxidation, and (iv) free radical scavenging or antioxidant properties.

Silymarin modulates the oxidant-antioxidant imbalance during diethylnitrosamine induced oxidative stress in rats.

Oxidative stress is a common mechanism contributing to initiation and progression of hepatic damage in a variety of liver disorders. Hence, there is a great demand for the development of agents with potent antioxidant effect. The aim of the present investigation is to evaluate the efficacy of silymarin as a hepatoprotective and an antioxidant against diethylnitrosamine induced hepatocellular damage. Single intraperitoneal administration of diethylnitrosamine (200 mg/kg) to rats resulted in significantly elevated levels of serum aspartate transaminase (AST) and alanine transaminase (ALT), which is indicative of hepatocellular damage. Diethylnitrosamine induced oxidative stress was confirmed by elevated levels of lipid peroxidation and decreased levels of superoxide dismutase (SOD), catalase, glutathione peroxidase, glutathione reductase (GR) and glutathione-S-transferase (GST) in the liver tissue. The status of non-enzymic antioxidants like, vitamin-C, vitamin-E and reduced glutathione (GSH) were also found to be decreased in diethylnitrosamine administered rats. Further, the status of membrane bound ATPases was also altered indicating hepatocellular membrane damage. Posttreatment with the silymarin (50 mg/kg) orally for 30 days significantly reversed the diethylnitrosamine induced alterations in the liver tissue and offered almost complete protection. The results from the present study indicate that silymarin exhibits good hepatoprotective and antioxidant potential against diethylnitrosamine induced hepatocellular damage in rats.

Effect of Cassia fistula Linn. leaf extract on diethylnitrosamine induced hepatic injury in rats.

The hepatoprotective and antioxidant effect of Cassia fistula Linn. leaf extract on liver injury induced by diethylnitrosamine (DEN) was investigated. Wistar rats weighing 200+/-10g were administered a single dose of DEN (200mg/kg b.w., i.p.) and left for 30 days. For hepatoprotective studies, ethanolic leaf extract (ELE) of C. fistula Linn. (500mg/kg b.w., p.o.) was administered daily for 30 days. AST, ALT, ALP, LDH, gamma-GT and bilirubin were estimated in serum and liver tissue. Lipid peroxidation (LPO), SOD and CAT were also estimated in liver tissue as markers of oxidative stress. DEN induced hepatotoxicity in all the treated animals were evident by elevated serum ALT, AST, ALP and bilirubin levels and a simultaneous fall in their levels in the liver tissue after 30 days. Induction of oxidative stress in the liver was evidenced by increased LPO and fall in the activities of SOD and CAT. ELE administration for 30 days prevented the DEN induced hepatic injury and oxidative stress. In conclusion, it was observed that ELE of C. fistula Linn. protects the liver against DEN induced hepatic injury in rats.

Silibinin alleviates N-nitrosodimethylamine-induced glutathione dysregulation and hepatotoxicity in rats.

The present study was designed to evaluate the hepatoprotective and antioxidant potentials of silibinin (SBN) against N-nitrosodimethylamine (DMN)-induced toxic insults in the rat liver. The liver damage was induced in Wistar albino rats by repeated administration of DMN (10 mg·kg(-1) b.w., i.p.) on 3 consecutive days per week for 3 weeks. SBN (100 mg·kg(-1) b.w., p.o.) was given daily to the DMN treated rats for two weeks. The marker enzymes of liver toxicity and second-line enzymic and non-enzymic antioxidants were evaluated in serum and liver tissues before and after SBN treatment. Histopathology of the liver was evaluated by H & E staining. The DMN treatment produced a progressive increase in all the serum marker enzymes (AST, ALT, ALP, LDH, and γ-GT), peaking on Day 21. This treatment produced highly significant decreases in all the second-line antioxidant parameters (GSH, GST, GR, GPx, and vitamins C and E). The SBN treatment significantly reversed the DMN-induced damages, towards normalcy. Histopathological studies confirmed the development of liver toxicity in DMN-treated rats, which was reversed by SBN treatment in corroboration with the aforementioned biochemical results, indicating the hepatoprotective and antioxidant properties of SBN. In conclusion, the DMN-induced degenerative changes in the liver were alleviated by SBN treatment and this protective ability may be attributed to its antioxidant, free radical scavenging, and membrane stabilizing properties.

Zidovudine and isoniazid induced liver toxicity and oxidative stress: Evaluation of mitigating properties of silibinin.

HIV/AIDS patients are more prone for opportunistic TB infections and they are administered the combined regimen of anti-retroviral drug zidovudine (AZT) and isoniazid (INH) for therapy. However, AZT+INH treatment has been documented to induce injury and remedial measures to prevent this adversity are not clearly defined. Silibinin (SBN) is a natural hepatoprotective principle isolated from medicinal plant Silybum marianum and is currently used for therapy of various liver diseases. This study investigate the hepatotoxic potentials of AZT alone, INH alone and AZT+INH treatments and the mitigating potentials of SBN against these drugs induced toxic insults of liver in rats. Separate groups of rats (n=6 in each group) were administered AZT alone (50mg/kg b.w.), INH alone (25mg/kg, b.w.), AZT+INH (50mg/kg, b.w. and 25mg/kg, b.w.), SBN alone (100mg/kg, b.w.) and SBN+AZT+INH daily for sub-chronic period of 45days orally. The control rats received saline/propylene glycol. INH alone and AZT+INH-induced parenchymal cell injury and cholestasis of liver was evidenced by highly significant increase in the activities of marker enzymes (aspartate and alanine transaminase, alkaline phosphatase, argino succinic acid lyase), bilirubin, protein, oxidative stress parameters (lipid peroxidation, superoxide dismutase, catalase, reduced glutathione, vitamins C and E) and membrane bound ATPases were evaluated in serum/liver tissue homogenates. Histopathological studies show ballooning degradation, inflammatory lesions, lipid deposition and hydropic changes in the liver tissue. All the above biochemical and pathological changes induced by AZT+INH treatments were mitigated in rats receiving SBN simultaneously with these hepatotoxins, indicating its hepatoprotective and antioxidant potentials against AZT+INH-induced hepatotoxicity. The moderate hepatoprotective and oxidant potentials of SBN could be due to its low bioavailability and this deficiency could be prevented by supplementation of phosphatidylcholines and studies are warranted on these lines to improve the therapeutic efficiency of SBN.

Silibinin Inhibits Proliferation and Migration of Human Hepatic Stellate LX-2 Cells.

BACKGROUND: Proliferation of hepatic stellate cells (HSCs) play pivotal role in the progression of hepatic fibrosis consequent to chronic liver injury. Silibinin (SBN), a flavonoid compound, has shown to possess cell cycle arresting potential against many actively proliferating cancers cell lines. The objective of this study was to evaluate the anti-proliferative and cell cycle arresting properties of SBN in rapidly proliferating human hepatic stellate LX-2 cell line. METHODS: LX-2 cells were fed with culture medium supplemented with different concentrations of SBN (10, 50 and 100 µM). After 24 and 96 h of treatment, total cell number was determined by counting. Cytotoxicity was evaluated by trypan blue dye exclusion test. The expression profile of cMyc and peroxisome proliferator-activated receptor-γ (PPAR-γ) protein expressions was evaluated by Western blotting. Oxidative stress marker genes profile was quantified using qPCR. The migratory response of HSCs was observed by scrape wound healing assay. RESULTS: SBN treatments significantly inhibit the LX-2 cell proliferation (without affecting its viability) in dose dependent manner. This treatment also retards the migration of LX-2 cells toward injured area. In Western blotting studies SBN treatment up regulated the protein expressions of PPAR-γ and inhibited cMyc. CONCLUSION: The present study shows that SBN retards the proliferation, activation and migration of LX-2 cells without inducing cytotoxicity and oxidative stress. The profound effects could be due to cell cycle arresting potential of SBN.

Influence of silymarin administration on hepatic glutathione-conjugating enzyme system in rats treated with antitubercular drugs.

OBJECTIVE: This study evaluated the influence of simultaneous administration of silymarin (SIL), a hepatoprotective and antioxidant agent, on the status of glutathione (GSH) and its metabolising enzymes in the liver tissue of rats treated with antitubercular drugs, i.e. isoniazid (INH), rifampicin (RIF) and pyrazinamide (PYR). METHODS: Male Wistar albino rats (n = 24) were randomly divided into four groups. Group I received saline as they served as controls. Group II rats were administered antitubercular drugs (INH 25 mg/kg + RIF 50 mg/kg + PYR 140 mg/kg orally) daily for 45 days. Group III animals were treated with SIL (50 mg/kg orally) simultaneously with the antitubercular drugs for the same period. Group IV animals were treated with SIL alone. The status of GSH, glutathione peroxidase (GPx), glutathione reductase (GR) and glutathione-s-transferase (GST) in liver tissue was evaluated at the end of the study. RESULTS: Administration of antitubercular drugs caused a significant decrease (p < 0.001) in the status of GPx, GST and GR and of non-enzymic (GSH) antioxidants in liver tissue when compared with saline-treated control rats. Simultaneous treatment of SIL with antitubercular drugs completely prevented decreases in the levels of all the above parameters. Treatment with SIL alone enhanced the activities of GST (p < 0.001) and GPx (p < 0.05) and did not alter glutathione levels compared with control. CONCLUSION: A fall in the status of glutathione and its conjugating enzymes upon administration of antitubercular drugs denotes an impairment of the antioxidant defence mechanism. Simultaneous administration of SIL afforded complete protection of the liver against this abnormality, an effect that could have been due to the strong antioxidant properties of SIL.

Ameliorative effect of silibinin against N-nitrosodimethylamine-induced hepatic fibrosis in rats.

The protective effect of silibinin (SBN) against hepatic fibrosis induced by repeated intermittent administration of N-nitrosodimethylamine (DMN) was investigated in rats. Oral administration of SBN recovered body and liver weight loss and reversed the elevation of serum AST, ALT and ALP accompanied by their fall in the liver tissue in DMN-induced fibrotic rats. Severe oxidative stress induced in fibrotic rats was evidenced by two to three fold elevation in MDA and protein carbonyl levels associated with a fall in the activities of SOD and CAT in repeated DMN treatment and this adversity was protected by SBN post-treatment. Further, the fall in the activities of ATPases and increase in the levels of hydroxyproline and collagen observed in the liver tissue of DMN treated rats was prevented and reversed back toward normalcy by SBN post-treatment. Recovery of rat liver tissue against DMN-induced hepatocellular necrosis, inflammatory changes and hepatic fibrosis by SBN treatment is also confirmed by both H & E and Masson's trichrome stained histopathological evaluation of liver tissue. In conclusion, SBN exhibit hepatoprotective, antioxidant, free radical scavenging, membrane stabilizing and anti-fibrotic activity against DMN-induced hepatic fibrosis suggesting that it may be useful as a therapeutic agent toward amelioration of hepatic fibrosis.

Protective effect of Cassia fistula Linn. on diethylnitrosamine induced hepatocellular damage and oxidative stress in ethanol pretreated rats

Diethylnitrosamine (DEN), found in many commonly consumed foods, is widely reported to induce cancer in animals and humans. The aim of the present study was to investigate the hepatoprotective and antioxidant activities of the leaf extract of the medicinal plant Cassia fistula Linn. against diethylnitrosamine induced liver injury in ethanol pretreated rats. Albino Wistar rats, pretreated with ethanol for 15 days, were administered a single dose of DEN. Thirty days after DEN administration, hepatotocellular damage was observed histologically, along with elevated levels of serum AST, ALT, ALP, LDH, γ-GT and bilirubin and a simultaneous fall in the levels of the marker enzymes in the liver tissue. Liver oxidative stress was confirmed by elevated levels of lipid peroxidation (LPO) and a decrease in enzymic and non-enzymic antioxidants activities. Oral administration of the ethanolic leaf extract (ELE) of Cassia fistula for 30 days to ethanol + DEN treated rats significantly improved the above alterations in the markers of hepatotoxicity and oxidative stress, resulting in the reversal of most of the parameters studied and were comparable to the standard hepatoprotective drug silymarin.