Aim
To investigate the effectiveness of antioxidant compounds in modulating mitochondrial oxidative alterations and lipids accumulation in fatty hepatocytes.
Conclusion
Realsil administration effectively contrasts hepatocyte fat deposition, NO derivatives formation, and mitochondrial alterations, allowing the liver to maintain a better glutathione and thioredoxin antioxidant activity.
Methods
Silybin-phospholipid complex containing vitamin E (Realsil(®)) was daily administered by gavage (one pouch diluted in 3 mL of water and containing 15 mg vitamin E and 47 mg silybin complexed with phospholipids) to rats fed a choline-deprived (CD) or a high fat diet [20% fat, containing 71% total calories as fat, 11% as carbohydrate, and 18% as protein, high fat diet (HFD)] for 30 d and 60 d, respectively. The control group was fed a normal semi-purified diet containing adequate levels of choline (35% total calories as fat, 47% as carbohydrate, and 18% as protein). Circulating and hepatic redox active and nitrogen regulating molecules (thioredoxin, glutathione, glutathione peroxidase), NO metabolites (nitrosothiols, nitrotyrosine), lipid peroxides [malondialdehyde-thiobarbituric (MDA-TBA)], and pro-inflammatory keratins (K-18) were measured on days 0, 7, 14, 30, and 60. Mitochondrial respiratory chain proteins and the extent of hepatic fatty infiltration were evaluated.
Results
Both diet regimens produced liver steatosis (50% and 25% of liver slices with CD and HFD, respectively) with no signs of necro-inflammation: fat infiltration ranged from large droplets at day 14 to disseminated and confluent vacuoles resulting in microvesicular steatosis at day 30 (CD) and day 60 (HFD). In plasma, thioredoxin and nitrosothiols were not significantly changed, while MDA-TBA, nitrotyrosine (from 6 ± 1 nmol/L to 14 ± 3 nmol/L day 30 CD, P < 0.001, and 12 ± 2 nmol/L day 60 HFD, P < 0.001), and K-18 (from 198 ± 20 to 289 ± 21 U/L day 30 CD, P < 0.001, and 242 ± 23 U/L day 60 HFD, P < 0.001) levels increased significantly with ongoing steatosis. In the liver, glutathione was decreased (from 34.0 ± 1.3 to 25.3 ± 1.2 nmol/mg prot day 30 CD, P < 0.001, and 22.4 ± 2.4 nmol/mg prot day 60 HFD, P < 0.001), while thioredoxin and glutathione peroxidase were initially increased and then decreased. Nitrosothiols were constantly increased. MDA-TBA levels were five-fold increased from 9.1 ± 1.2 nmol/g to 75.6 ± 5.4 nmol/g on day 30, P < 0.001 (CD) and doubled with HFD on day 60. Realsil administration significantly lowered the extent of fat infiltration, maintained liver glutathione levels during the first half period, and halved its decrease during the second half. Also, Realsil modulated thioredoxin changes and the production of NO derivatives and significantly lowered MDA-TBA levels both in liver (from 73.6 ± 5.4 to 57.2 ± 6.3 nmol/g day 30 CD, P < 0.01 and from 27.3 ± 2.1 nmol/g to 20.5 ± 2.2 nmol/g day 60 HFD, P < 0.01) and in plasma. Changes in mitochondrial respiratory complexes were also attenuated by Realsil in HFD rats with a major protective effect on Complex II subunit CII-30.