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Oxidative stress has been suggested to play a main role in the pathogenesis of type 2 diabetes mellitus and its complications. As a consequence of this increased oxidative status, a cellular-adaptive response occurs requiring functional chaperones, antioxidant production, and protein degradation. This study was designed to evaluate systemic oxidative stress and cellular stress response in patients suffering from type 2 diabetes-induced nephropathy and in age-matched healthy subjects. Systemic oxidative stress has been evaluated by measuring advanced glycation end-products (pentosidine), protein oxidation (protein carbonyls [DNPH]), and lipid oxidation (4-hydroxy-2-nonenal [HNE] and F2-isoprostanes) in plasma, lymphocytes, and urine, whereas the lymphocyte levels of the heat shock proteins (Hsps) heme oxygenase-1 (HO-1), Hsp70, and Hsp60 as well as thioredoxin reductase-1 (TrxR-1) have been measured to evaluate the systemic cellular stress response. We found increased levels of pentosidine (P Ͻ 0.01), DNPH (P Ͻ 0.05 and P Ͻ 0.01), HNE (P Ͻ 0.05 and P Ͻ 0.01), and F2-isoprostanes (P Ͻ 0.01) in all the samples from type 2 diabetic patients with nephropathy with respect to control group. This was paralleled by a significant induction of cellular HO-1, Hsp60, Hsp70, and TrxR-1 (P Ͻ 0.05 and P Ͻ 0.01). A significant upregulation of both HO-1 and Hsp70 has been detected also in lymphocytes from type 2 diabetic patients without uraemia. Significant positive correlations between DNPH and Hsp60, as well as between the degree of renal failure and HO-1 or Hsp70, also have been found in diabetic uremic subjects. In conclusion, patients affected by type 2 diabetes complicated with nephropathy are under condition of systemic oxidative stress, and the induction of Hsp and TrxR-1 is a maintained response in counteracting the intracellular pro-oxidant status.

Nonalcoholic steatohepatitis (NASH) is associated with increased liver-related mortality. Disturbances in hepatic lipid homeostasis trigger oxidative stress and inflammation (ie, lipotoxicity), leading to the progression of NASH. This study aimed at identifying whether silibinin may influence the molecular events of lipotoxicity in a mouse model of NASH. Eight-week-old db/db mice were fed a methioninecholine deficient (MCD) diet for 4 weeks and treated daily with silibinin (20 mg/kg intraperitoneally) or vehicle. Liver expression and enzyme activity of stearoyl-CoA desaturase-1 and acyl-CoA oxidase, and expression of liver fatty acid-binding protein were assessed. Hepatic levels of reactive oxygen species, thiobarbituric acidreactive substances (TBARS), 3-nitrotyrosine (3-NT), inducible nitric oxide synthase (iNOS), and nuclear factor kappa B (NFkB) activities were also determined. Silibinin administration decreased serum alanine aminotransferase and improved liver steatosis, hepatocyte ballooning, and lobular inflammation in db/db mice fed an MCD diet. Gene expression and activity of stearoyl-CoA desaturase-1 were reduced in db/db mice fed an MCD diet compared with lean controls and were increased by silibinin; moreover, silibinin treatment induced the expression and activity of acylCoA oxidase and the expression of liver fatty acid-binding protein. Vehicle-treated animals displayed increased hepatic levels of reactive oxygen species and TBARS, 3-NT staining, and iNOS expression; silibinin treatment markedly decreased reactive oxygen species and TBARS and restored 3-NT and iNOS to the levels of control mice. db/db mice fed an MCD diet consistently had increased NFkB p65 and p50 binding activity; silibinin administration significantly decreased the activity of both subunits. Silibinin treatment counteracts the progression of liver injury by modulating lipid homeostasis and suppressing oxidative stress-mediated lipotoxicity and NFkB activation in experimental NASH. (Translational Research 2012;159:477-486) Abbreviations: AOX ¼ acyl-CoA oxidase; ALT ¼ alanine aminotransferase; FFA ¼ free fatty acid; iNOS ¼ inducible nitric oxide synthase; L-FABP ¼ liver-fatty acid binding protein;

BackgroundCardiovascular diseases (CVD) in diabetic patients have endothelial dysfunction as a key pathogenetic event. Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase (NOS), plays a pivotal role in endothelial dysfunction. Different natural polyphenols have been shown to preserve endothelial function and prevent CVD. In this study, we assessed the effect of silibinin, a widely used flavonolignan from milk thistle, on ADMA levels and endothelial dysfunction in db/db mice.MethodsEight-week-old db/db mice were administrated a 20 mg/Kg i.p. daily dose of silibinin (n = 6) or vehicle (n = 6) for four weeks. Heterozygous lean db/m mice served as control. Plasma, aorta and liver ADMA levels were determined by ELISA. Vascular reactivity to phenilephrine (PE), acetylcholine (ACh), sodium nitroprusside (SNP) and ADMA was assessed in isolated aortic segments, in wire myograph.ResultsPlasma and aorta ADMA levels were higher in db/db than in control lean mice. Silibinin administration markedly decreased plasma ADMA; consistently, aorta ADMA was reduced in silibinin-treated animals. Plasma and aorta ADMA levels exhibited a positive correlation, whereas liver ADMA was inversely correlated with both plasma and aorta ADMA concentrations. Endothelium-(NO)-dependent vasodilatation to ACh was impaired in db/db mice and was restored in the silibinin group, in accordance with the observed reduction of plasma and vascular levels of ADMA. Endothelium-independent vasodilatation to SNP was not modified by silibinin administration; contractile tone induced in isolated aorta from db/db mice by challenging with exogenous ADMA was not affected by the treatment.ConclusionsSilibinin markedly improves endothelial dysfunction in db/db mice by reducing circulating and vascular ADMA levels. Clinical studies are warranted to assess the efficacy of silibinin for cardiovascular protection.

a b s t r a c tBackground: Nonalcoholic fatty liver disease is a chronic metabolic disorder with significant impact on cardiovascular and liver mortality. Aims: In this study, we examined the effects of silibinin on liver and myocardium injury in an experimental model of nonalcoholic fatty liver disease. Methods: A four-week daily dose of silibinin (20 mg/kg i.p.) was administrated to db/db mice fed a methionine-choline deficient diet. Hepatic and myocardial histology, oxidative stress and inflammatory cytokines were evaluated. Results: Silibinin administration decreased HOMA-IR, serum ALT and markedly improved hepatic and myocardial damage. Silibinin reduced isoprostanes, 8-deoxyguanosine and nitrites/nitrates in the liver and in the heart of db/db fed the methionine-choline deficient diet, whereas glutathione levels were restored to lean mice levels in both tissues. Consistently, liver mitochondrial respiratory chain activity was significantly impaired in untreated mice and was completely restored in silibinin-treated animals. TNF-␣ was increased whereas IL-6 was decreased both in the liver and heart of db/db fed methionine-choline deficient diet. Silibinin reversed heart TNF-␣ and IL-6 expression to control mice levels. Indeed, liver JNK phosphorylation was reduced to control levels in treated animals. Conclusions: This study demonstrates a combined effectiveness of silibinin on improving liver and myocardial injury in experimental nonalcoholic fatty liver disease.