Numerous evidences suggest that plant polyphenols may have therapeutic benefits in regulating oxidative stress and providing neuroprotection in many neurodegenerative diseases, including multiple sclerosis (MS). However, these mechanisms are not yet completely understood. In this study, we investigated the effect of olive leaf polyphenols on oxidative stress through oxidation marker level and activity (TBARS, SOD, and GPX) and their protein expression (SOD1, SOD2, and GPX1), as well as the protein expression of Sirtuin 1 (SIRT1) and microglia markers (Iba-1, CD206, and iNOS) and myelin integrity (proteolipid protein expression) in the brain of rats with induced experimental autoimmune encephalomyelitis (EAE) and subjected to olive leaf therapy. Experiments were performed in male EAE DA rats, which were randomly divided into 2 main groups: EAE groups treated with the therapy of olive leaf (EAE+TOL) and untreated EAE control groups. The EAE treated groups consumed olive leaf tea instead of drinking water (ad libitum) from the beginning to the end of the experiment. In addition, olive leaf extract was injected intraperitoneally (i.p.) for the 10 continuous days and started on the 8th day after EAE induction. The clinical course was monitored in both groups until the 30th day after EAE induction. Our results demonstrated that TOL attenuated the clinical course of EAE; reduced the oxidative stress (by decreasing the concentration of MDA); upregulated antioxidant enzymes (SOD1, SOD2, and GPX1), SIRT1 (overall and microglial), and anti-inflammatory M2 microglia; downregulated proinflammatory M1 type; and preserved myelin integrity. These data support the idea that TOL may be an effective therapeutic approach for treating MS and other neurodegenerative diseases.

Metallothioneins (MTs) are small, cysteine-rich proteins which have been implicated in various forms of stress providing cytoprotective action against oxidative injury, DNA damage and apoptosis. Owing to their high affinity for physiological metals, such as zinc and copper MTs are also critical components of regulatory proteins involved in cell growth and multiplication, as well as in the maintenance of immune homeostasis. To elucidate the role of MTs in the pathomechanisms of autoimmune CNS disorders we estimated the expression of MT I+II proteins and the content of free Zn ions in the brain, spinal cord and in the liver early in the course of chronic relapsing experimental autoimmune encephalomyelitis (CR-EAE) pathogenesis, i.e. before the onset of any clinical symptoms. Disease was induced in the genetically susceptible Dark Agouti (DA) rats by subcutaneous injection of bovine brain homogenate in CFA. Control animals were treated with CFA alone. The data, obtained by immuno-histochemistry and in situ fluorescent labeling of free zinc ions, have shown that in the presymptomatic phase of CR-EAE (on the seventh postimmunization day) MTs I+II were markedly upregulated in the cells that form blood-brain and blood-cerebrospinal fluid barriers, as well as in the cerebellar parenchyma and hippocampal dentate gyri. Furthermore, we found that the liver also becomes a site of extensive MTs I+II synthesis shortly after immunization. Simultaneously, tissue content of free zinc ions increased at the sites of MTs induction, reflecting their antioxidative activity. The data, described in this paper point to regulatory and neuroprotective role of MTs in the pathogenesis of CR-EAE.

Skeletal muscles are high-insulin tissues responsible for disposing of glucose via the highly regulated process of facilitated glucose transporter 4 (GLUT4). Impaired insulin action in diabetes, as well as disorders of GLUT4 vesicle trafficking in the muscle, are involved in defects in insulin-stimulated GLUT4 translocation. Since the Rab GTPases are the main regulators of vesicular membrane transport in exo- and endo-cytosis, in the present work, we studied the effect of olive leaf polyphenols (OLPs) on Rab8A, Rab13, and Rab14 proteins of the rat soleus muscle in a model of streptozotocin (SZT)-induced diabetes (DM) in a dose-dependent manner. Glucose, cholesterol, and triglyceride levels were determined in the blood, morphological changes of the muscle tissue were captured by hematoxylin and eosin histological staining, and expression of GLUT4, Rab8A, Rab13, and Rab14 proteins were analyzed in the rat soleus muscle by the immunofluorescence staining and immunoblotting. OLPs significantly reduced blood glucose level in all treated groups. Furthermore, significantly reduced blood triglycerides were found in the groups with the lowest and highest OLPs treatment. The dynamics of activation of Rab8A, Rab13, and Rab14 was OLPs dose-dependent and more effective at higher OLP doses. Thus, these results indicate a beneficial role of phenolic compounds from the olive leaf in the regulation of glucose homeostasis in the skeletal muscle.

Objectives: Compared to the Dark Agouti (DA), the Albino Oxford (AO) rat strain exhibits lower susceptibility to the induction of experimental autoimmune encephalomyelitis (EAE). Here, we investigated the potential contribution of the heavy metal-binding proteins metallothioneins (MTs) I/II to these effects. Methods: Rats were immunized with bovine brain homogenate emulsified in complete Freund's adjuvant or only with complete Freund's adjuvant. The expression patterns of MTs mRNA and proteins and tissue concentrations of Zn²⁺ and Cu²⁺ were estimated in the brain and in the liver on days 7 and 12 after immunization, by real-time PCR, immunohistochemistry and inductively coupled plasma spectrometry, respectively. Additionally, the hepatic transforming growth factor beta and nuclear factor kappa B immunoreactivities were tested. Results: Clinical signs of EAE were not induced in AO rats, but they upregulated the expression of MT I/II proteins in the brain (hippocampus and cerebellum) and in the liver, similarly as DA rats. The transcriptional activation of MT-I occurred, however, only in DA rats, which accumulated also more zinc in the brain and in the liver. In contrast, intact AO rats had greater hepatic MT-I mRNA immunoreactivity and more Cu²⁺ in the hippocampus. Besides, in immunized AO rats a high upregulation of transforming growth factor beta and nuclear factor kappa B immunoreactivities was found in several hepatic structures (vascular endothelium, Kupffer cells and hepatocytes). Conclusions: Our data show that AO and DA rats differ in constitutive and inductive MT-I gene expression in the brain and in the liver, as well as in the hepatic cytokine profile, suggesting that these mechanisms may contribute to the discrepancy in the susceptibility to EAE.

In a search of peripheral factors that could be responsible for the discrepancy in susceptibility to EAE in Albino Oxford (AO) and Dark Agouti (DA) rats, we estimated the expression of metallothioneins I/II (MT), heat shock protein-gp96, interleukin (IL)-6, and transforming growth factor (TGF)-β in the livers of these animals. Rats were immunized with bovine brain homogenate (BBH) emulsified in complete Freund adjuvant (CFA) or only with CFA. Western blot and immunohistochemical analyses were done on day 12 after the immunization, as well as in intact rats. The data have shown that during the first attack of EAE only the EAE prone-DA rats markedly upregulated the hepatic MTs, gp96, IL-6, and TGF-β. In contrast, AO rats had a significantly higher expression of MT I/II, IL-6, and TGF-β in intact liver (P < 0,001), suggesting that the greater constitutive expression of these proteins contributed to the resistance of EAE. Besides, since previously we found that AO rats reacted on immunization by an early upregulation of TGF-β on several hepatic structures (vascular endothelium, Kupffer cells, and hepatocytes), the data suggest that the specific hepatic microenvironment might contribute also to the faster recovery of these rats from EAE.