Vitamin E and Silymarin Reduce Oxidative Tissue Damage during Gentamycin-Induced Nephrotoxicity.

Aminoglycoside antibiotics and gentamicin (GN), in particular, are still widely used in clinical practice. It is a well-known fact that GN causes nephrotoxicity, and redox disturbances are discussed as a factor in its side effects. Recently, a new type of cell oxidative death, named ferroptosis, was discovered; it is associated with iron accumulation in the cell, glutathione (GSH) depletion and inactivation of glutathione peroxidase-4 (GPX4), reactive oxygen species (ROS) increment with concomitant lipid peroxidation. In this regard, a possible connection between GN-induced renal damage, ferroptosis and the overall antioxidant status of the organism could be investigated. Moreover, due to its beneficial effects, GN is still one of the main choices as a therapeutic agent for several diseases, and the possible reduction of its side effects with the application of certain antioxidants will be of important clinical significance. The study was conducted with adult male white mice divided into several groups (n = 6). GN nephrotoxicity was induced by the administration of GN 100-200 mg/kg i.p. for 10 days. The control group received only saline. The other groups received either Vitamin E (400 mg/kg p.o.) or Silymarin (200 mg/kg p.o.) applied alone or together with GN for the same period. After the end of the study, the animals were sacrificed, and blood and tissue samples were taken for the assessment of biochemical parameters and antioxidant status, as well as routine and specific for GPX4 histochemistry examination. The experimental results indicate that GN-induced nephrotoxicity negatively modulates GPX4 activity and is associated with increased production of ROS and lipid peroxidation. The groups treated with antioxidants demonstrated preserved antioxidant status and better GPX4 activity. In conclusion, the inhibition of ROS production and especially the suppression of ferroptosis, could be of clinical potential and can be applied as a means of reducing the toxic effects of GN application.

Metabolic Markers Associated with Progression of Type 2 Diabetes Induced by High-Fat Diet and Single Low Dose Streptozotocin in Rats.

Science is still searching for readily available, cost-effective biomarkers to assess metabolic disorders occurring before the onset and during the development of type-2 diabetes (T2DM). The aim of the present study was to induce T2DM in rats through a high-fat diet, followed by a single administration of low dose streptozotocin (STZ), and make an assessment of the development of the disease. The rats were divided into two groups-experimental and control-and were monitored for a period of 10 days. Changes in anthropometric parameters, glucose, insulin, lipids, uric acid, advanced oxidation protein products (AOPP), as well as the histological changes in the liver and pancreas, were recorded. To assess insulin resistance, we used the Homeostasis Model Assessment of Insulin Resistance (HOMA-IR) and beta cell function (HOMA-ß) and visceral obesity-adiposity index (AI). The data demonstrate that the increasing values of glucose, HOMA-IR, AI, total cholesterol, triacylglycerols, low- and very-low-density lipoproteins are important markers of the pre-diabetic state. The stable hyperglycemia and increased levels of TC, TG, VLDL, LDL, uric acid and AOPP in experimental rats strongly suggest the development of T2DM. HOMA-IR, HOMA-ß, AI, and uric acid are reliable criteria for T2DM in rats.

The Azadirachta indica (Neem) Seed Oil Reduced Chronic Redox-Homeostasis Imbalance in a Mice Experimental Model on Ochratoxine A-Induced Hepatotoxicity.

Liver damage severity depends on both the dose and the exposure duration. Oxidative stress may increase the Ochratoxine-A (OTA) hepatotoxicity and many antioxidants may counteract toxic liver function. The present study aims to investigate the hepatoprotective potential of Azadirachta indicaA (A. indica; neem oil) seed oil to reduce acute oxidative disorders and residual OTA toxicity in a 28-day experimental model. The activity of antioxidant and hepatic enzymes, cytokines and the levels of oxidative stress biomarkers -MDA, GSPx, Hydroxiproline, GST, PCC, AGEs, PGC-1, and STIR-1 were analyzed by ELISA. The free radicals ROS and RNS levels were measured by EPR. The protective effects were studied in BALB/C mice treated with A. indica seed oil (170 mg/kg), alone and in combination with OTA (1.25 mg/kg), by gavage daily for 28 days. At the end of the experiment, mice treated with OTA showed changes in liver and antioxidant enzymes, and oxidative stress parameters in the liver and blood. A. indica oil significantly reduced oxidative stress and lipid peroxidation compared to the OTA group. In addition, the hepatic histological evaluation showed significant adipose tissue accumulation in OTA-treated tissues, while treatment with 170 mg/kg A. indica oil showed moderate adipose tissue accumulation.

Effects of heat and freeze on isolated erythrocyte submembrane skeletons.

In this study we heated insoluble residues, obtained after Triton-X-100 (0.1 v/v%) extraction of erythrocyte ghost membranes (EGMs). Specific heat capacity, electric capacitance and resistance, and optical transmittance (280 nm) sustained sharp changes at 49°C (TA) and 66°C (TC), the known denaturation temperatures of spectrin and band 3, respectively. The change at TA was selectively inhibited by diamide (1 mM) and taurine mustard (1 mM) while its inducing temperature was selectively decreased by formamide in full concert with the assumed involvement of spectrin denaturation. In the residues of EGMs, pretreated with 4,4'-diiso-thiocyanato stilbene-2,2'-disulfonic acid (DIDS), the change at TC was shifted from 66 to 78°C which indicated the involvement of band 3 denaturation. The freeze and rapid thaw of EGM residues resulted in a strong reduction of cooperativity of band 3 denaturation while the slow thaw completely eliminated the peak of this denaturation. These effects of freeze-thaw were prevented in residues obtained from DIDS-treated EGMs. The freeze-thaw of residues slightly affected spectrin denaturation at 49°C although an additional denaturation appeared at 55°C. The results indicate preserved molecular structure and dynamics of the membrane skeleton in Triton-X-100 extracts of EGMs. The freeze-thaw inflicted strong damage on band 3 and spectrin-actin skeleton of EGM extracts which is relevant to cryobiology, cryosurgery and cryopreservation of cells.

In vitro studies on radioprotective efficacy of silymarin against γ-irradiation.

UNLABELLED: Abstract Purpose: Silymarin has been widely exploited for its hepatoprotective activities. This study aimed to evaluate the protective efficacy of silymarin against γ-radiation. MATERIALS AND METHODS: The radioprotective properties of silymarin were studied using different assays. Cytotoxicity of silymarin on Human embryonic kidney (HEK) cells was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. Protective efficacy against γ-radiation was assessed by studying reduction in micronuclei frequency and free radical generation using 2',7'-dichlorodihydroflurescin diacetate (H2DCFDA). Radiation-induced apoptosis was estimated by Annexin V-PI (propidium iodide) analysis and cell cycle analysis. γ-radiation induced changes in mitochondrial membrane potential (MMP) and DNA damage was estimated employing flow-cytometry and comet assay respectively. RESULTS: MTT assay and Annexin V-PI studies showed that pre-incubation of HEK cells with silymarin protected them from γ-irradiation. Significant reduction in apoptosis (76.36%) was observed. Silymarin also decreased the percentage of radiation-induced micronuclei (> 69%) (p < 0.05 ). Measurement of intracellular reactive oxygen species (ROS) by H2DCFDA revealed a reduction in ROS (21%) at 0.5 h. Cell cycle analysis revealed G1 block in the unirradiated control, which declined in the silymarin pretreated irradiated group (0.5 h). Silymarin treatment resulted in a significant increase in MMP (2 h) against the radiation control. Moreover, the presence of silymarin during irradiation significantly decreased the DNA damage (as measured by comet assay). CONCLUSIONS: Protection against radiation-induced cell-death and DNA damage by silymarin could be attributed to a reduction in ROS induced by γ-radiation. In vitro experiments on HEK cells explicitly prove that silymarin is a promising, effective and safe radiation countermeasure agent.

Effects of castration-induced visceral obesity and antioxidant treatment on lipid profile and insulin sensitivity in New Zealand white rabbits.

Molecular mechanisms, responsible for the impaired insulin-sensitivity state due to the obesity are not fully understood in both humans and animals. The purpose of this study was to investigate the effects of castration-induced visceral obesity and the influence of two antioxidants on constituents of blood lipid profile and insulin sensitivity in New Zealand white rabbits. Twenty-six clinically healthy male New Zealand white rabbits were used in the experiment and were divided into 3 groups: first group (CI, n=7) - castrated-obese and treated with antioxidants "Immunoprotect" for 2months; second group (CO, n=7) - castrated-obese; third group (NC, n=12) - control group (non-castrated, non-obese). At the end of the follow-up period of 2months after castration an intravenous glucose tolerance test (IVGTT) was performed after a 12-h fasting period as the blood samples for determination of glucose and insulin and their kinetic parameters were obtained at 5 and 0min before and at 5, 10, 30, 60 and 120min after the infusion of the glucose. The constituents of lipid profile, triglycerides (TG), total cholesterol (TC) and HDL-cholesterol (HDL-C) were also assessed in the overnight fasting blood samples. The body weight (BW), body mass index (BMI), amount of the visceral fat (VF) and VF/BW ratio were both measured and calculated before the IVGTT and at the end of the experimental period. All measured markers of obesity (BW, BMI, VF, VF/BW) were significantly higher in both groups of castrated rabbits than in the control group. Apart HDL-C, the plasma concentrations of all constituents of lipid profile (TG, TC, HDL-C) were the highest in CO group. There were generally no differences between CI and NC groups for the same traits. After glucose injection blood glucose concentrations and glucose and insulin kinetic parameters were considerably higher (except of glucose elimination rate) in CO rabbits than in NC ones. Castrated rabbits treated with "Immunoprotect" showed lower fasting plasma insulin and improved glucose kinetics dynamics than CO rabbits, but commensurable values of glucose and insulin kinetics parameters than NC group. The results of the current study clearly indicated that castration-induced visceral obesity affected negatively the lipid profile and insulin sensitivity and/or responsiveness. Treatment with antioxidant supplementation, consisted of d-limonene and vitamin E, improved blood lipid profile, fatty liver, glucose homeostasis and insulin sensitivity in obese rabbits. In addition, based on our results we may suggest that castrated male New Zealand white rabbits might be considered as an appropriate animal model to study various metabolic abnormalities related to visceral obesity, such as dyslipidemia and impaired insulin sensitivity.

Evaluation of silymarin as a promising radioprotector.

Silymarin, a purified extract of seeds of Silybum marianum L. and well known for its hepatoprotective abilities, has been evaluated for inherent utility as a radioprotective agent. A fraction (INM-7035) was authenticated by characterizing the percentage composition of silybin A and B (39.9% and 57.4%). Free radical scavenging activities of INM-7035 against superoxide radicals (>68%), hydroxyl radicals (>33.75%), DPPH (67.2%), and ABTS (32.4%) were also evaluated. The fraction chelated (>30%) ferrous ions, thereby able to restrict amplification. INM-7035 exhibited >50% peroxyl radical scavenging activity in the lipid phase along with dose-dependent (R2 = 0.990) reducing power in the aqueous phase. Radiation-induced free radical flux can lead to disruption of biomolecules like membrane lipids. INM-7035 completely inhibited lipid peroxidative stress in case of membranes against supralethal radiation stress in the liposomal system. The ability of INM-7035 to modulate the levels of NF-kappaB, indicated its inherent potential as a radioprotective bioactive constituent.