Hildegard Herman

Differences in the molecular structure of the blood-brain barrier in the cerebral cortex and white matter: an in silico, in vitro, and ex vivo study

The blood-brain barrier (BBB) is the main interface controlling molecular and cellular traffic between the central nervous system (CNS) and the periphery. It consists of cerebral endothelial cells (CECs) interconnected by continuous tight junctions, and closely associated pericytes and astrocytes. Different parts of the CNS have diverse functions and structures and may be subject of different pathologies, in which the BBB is actively involved. It is largely unknown, however, what are the cellular and molecular differences of the BBB in different regions of the brain. Using in silico, in vitro, and ex vivo techniques we compared the expression of BBB-associated genes and proteins (i.e., markers of CECs, brain pericytes, and astrocytes) in the cortical grey matter and white matter. In silico human database analysis (obtained from recalculated data of the Allen Brain Atlas), qPCR, Western blot, and immunofluorescence studies on porcine and mouse brain tissue indicated an increased expression of glial fibrillary acidic protein in astrocytes in the white matter compared with the grey matter. We have also found increased expression of genes of the junctional complex of CECs (occludin, claudin-5, and α-catenin) in the white matter compared with the cerebral cortex. Accordingly, occludin, claudin-5, and α-catenin proteins showed increased expression in CECs of the white matter compared with endothelial cells of the cortical grey matter. In parallel, barrier properties of white matter CECs were superior as well. These differences might be important in the pathogenesis of diseases differently affecting distinct regions of the brain.

Chitosan-Graphene Oxide 3D scaffolds as Promising Tools for Bone Regeneration in Critical-Size Mouse Calvarial Defects

Limited self-regenerating capacity of human skeleton makes the reconstruction of critical size bone defect a significant challenge for clinical practice. Aimed for regenerating bone tissues, this study was designed to investigate osteogenic differentiation, along with bone repair capacity of 3D chitosan (CHT) scaffolds enriched with graphene oxide (GO) in critical-sized mouse calvarial defect. Histopathological/histomorphometry and scanning electron microscopy(SEM) analysis of the implants revealed larger amount of new bone in the CHT/GO-filled defects compared with CHT alone (p < 0.001). When combined with GO, CHT scaffolds synergistically promoted the increase of alkaline phosphatase activity both in vitro and in vivo experiments. This enhanced osteogenesis was corroborated with increased expression of bone morphogenetic protein (BMP) and Runx-2 up to week 4 post-implantation, which showed that GO facilitates the differentiation of osteoprogenitor cells. Meanwhile, osteogenesis was promoted by GO at the late stage as well, as indicated by the up-regulation of osteopontin and osteocalcin at week 8 and overexpressed at week 18, for both markers. Our data suggest that CHT/GO biomaterial could represent a promising tool for the reconstruction of large bone defects, without using exogenous living cells or growth factors.

Protective effects of silymarin on epirubicin-induced mucosal barrier injury of the gastrointestinal tract

Abstract Mucositis is a serious disorder of the gastrointestinal tract that results from cancer chemotherapy. We investigated the protective effects of silymarin on epirubicin-induced mucosal barrier injury in CD-1 mice. Immunohistochemical activity of both pro-apoptotic Bax and anti-apoptotic Bcl-2 markers, together with p53, cyt-P450 expression and DNA damage analysis on stomach, small intestine and colon were evaluated. Our results indicated stronger expression for cyt P450 in all analyzed gastrointestinal tissues of Epi group, which demonstrate intense drug detoxification. Bax immunopositivity was intense in the absorptive enterocytes and lamina connective cells of the small intestine, surface epithelial cells of the stomach and also in the colonic epithelium and lamina concomitant with a decreased Bcl-2 expression in all analyzed tissues. Epirubicin-induced gastrointestinal damage was verified by a goblet cell count and morphology analysis on histopathological sections stained for mucins. In all analyzed tissues, Bax immunopositivity has been withdrawn by highest dose of silymarin concomitant with reversal of Bcl-2 intensity at a level comparable with control. p53 expression was found in all analyzed tissues and decreased by high dose of silymarin. Also, DNA internucleosomal fragmentation was observed in the Epi groups for all analyzed tissues was almost suppressed at 100 mg/kg Sy co-treatment. Histological aspect and goblet cell count were restored at a highest dose of Sy for both small and large intestine. In conclusion, our findings suggest that silymarin may prevent cellular damage of epirubicin-induced toxicity and was effective in reducing the severity indicators of gastrointestinal mucositis in mice.

CB2 receptor activation inhibits melanoma cell transmigration through the blood-brain barrier.

During parenchymal brain metastasis formation tumor cells need to migrate through cerebral endothelial cells, which form the morphological basis of the blood-brain barrier (BBB). The mechanisms of extravasation of tumor cells are highly uncharacterized, but in some aspects recapitulate the diapedesis of leukocytes. Extravasation of leukocytes through the BBB is decreased by the activation of type 2 cannabinoid receptors (CB2); therefore, in the present study we sought to investigate the role of CB2 receptors in the interaction of melanoma cells with the brain endothelium. First, we identified the presence of CB1, CB2(A), GPR18 (transcriptional variant 1) and GPR55 receptors in brain endothelial cells, while melanoma cells expressed CB1, CB2(A), GPR18 (transcriptional variants 1 and 2), GPR55 and GPR119. We observed that activation of CB2 receptors with JWH-133 reduced the adhesion of melanoma cells to the layer of brain endothelial cells. JWH-133 decreased the transendothelial migration rate of melanoma cells as well. Our results suggest that changes induced in endothelial cells are critical in the mediation of the effect of CB2 agonists. Our data identify CB2 as a potential target in reducing the number of brain metastastes originating from melanoma.

Protective effects of silymarin against bisphenol A-induced hepatotoxicity in mouse liver

Bisphenol A (BPA) is an endocrine-disrupting chemical released into the environment, with severe consequences for human health, including metabolic syndrome and associated pathological conditions. Due to limited information on BPA-induced hepatotoxicity, the present study focused on investigating the association between BPA-induced toxicity and inflammatory markers in the liver, and how these injuries may be alleviated using the natural agent silymarin, a flavonoid with antioxidant properties obtained from Silybum marianum. Administration of BPA to male CD-1 mice for 10 days caused a significant increase in the number of cells immunopositive for interleukin 6 and tumor necrosis factor-α, pro-inflammatory cytokines that mediate the hepatic inflammatory response. Treatment with 200 mg/kg of silymarin concurrently with BPA for 10 days resulted in a diminished level of pro-inflammatory cytokines and in significantly reduced ultrastructural injuries. Additionally, silymarin was able to restore the significantly decreased glycogen deposits observed following BPA exposure to normal levels, thus favoring hepatic glycogenesis. This study represents the first report of silymarin ability to reduce hepatic lesions and to counteract inflammation caused by BPA in mice. A dose of 200 mg/kg silymarin was sufficient to induce a protective effect against structural and ultrastructural injuries induced by BPA and to lower the levels of pro-inflammatory cytokines observed in murine liver tissue following exposure to BPA.

Evaluation of Hepatotoxicity with Treatment Doses of Flucytosine and Amphotericin B for Invasive Fungal Infections

Invasive fungal infection is a well-known cause of morbidity and mortality in immunocompromised patients. In this study we aimed to evaluate the hepatotoxicity induced by combined therapy of flucytosine and amphotericin B, at three different doses administered to mice for 14 days: 50 mg/kg flucytosine and 300 μg/kg amphotericin B; 100 mg/kg flucytosine and 600 μg/kg amphotericin B; 150 mg/kg flucytosine and 900 μg/kg amphotericin B. Liver injuries were evaluated by analysis of optic and electron microscopy samples, changes in TNF-α, IL-6, and NF-κB inflammation markers levels of expression, and evaluation of mRNA profiles. Histological and ultrastructural analysis revealed an increase in parenchymal and portal inflammation in mice and Kupffer cells activation. Combined antifungal treatment stimulated activation of an inflammatory pathway, demonstrated by a significant dose-dependent increase of TNF-α and IL-6 immunoreactivity, together with mRNA upregulation. Also, NF-κB was activated, as suggested by the high levels found in hepatic tissue and upregulation of target genes. Our results suggest that antifungal combined therapy exerts a synergistic inflammatory activation in a dose-dependent manner, through NF-κB pathway, which promotes an inflammatory cascade during inflammation. The use of combined antifungal therapy needs to be dose limiting due to the associated risk of liver injury, especially for those patients with hepatic dysfunction.

Enhancement of silymarin anti-fibrotic effects by complexation with hydroxypropyl (HPBCD) and randomly methylated (RAMEB) β-cyclodextrins in a mouse model of liver fibrosis

Silymarin (Sy) shows limited water solubility and poor oral bioavailability. Water-soluble hydroxypropyl (HPBCD) and randomly methylated (RAMEB) β-cyclodextrins were designed to enhance anti-fibrotic efficiency of silymarin in CCl4-induced liver fibrosis in mice. Experimental fibrosis was induced by intraperitoneal injection with 2 ml/kg CCl4 (20% v/v) twice a week, for 7 weeks. Mice were orally treated with 50 mg/kg of Sy-HPBCD, Sy-RAMEB and free silymarin. For assessment of the spontaneous reversion of fibrosis, CCl4 treated animals were investigated after 2 weeks of recovery time. The CCl4 administration increased hepatic oxidative stress, augmented the expression of transforming growth factor-β1 (TGF-β1) and Smad 2/3, and decreased Smad 7 expression. Furthermore, increased α-smooth muscle actin (α-SMA) expression indicated activation of hepatic stellate cells (HSCs), while up-regulation of collagen I (Col I) and matrix metalloproteinases (MMPs) expression led to an altered extracellular matrix enriched in collagen, confirmed as well by trichrome staining and electron microscopy analysis. Treatment with Sy-HPBCD and Sy-RAMEB significantly reduced liver injury, attenuating oxidative stress, restoring antioxidant balance in the hepatic tissue, and significantly decreasing collagen deposits in the liver. The levels of pro-fibrogenic markers’ expression were also significantly down-regulated, whereas in the group for spontaneous regression of fibrosis, they remained significantly higher, even at 2 weeks after CCl4 administration was discontinued. The recovery was significantly lower for free silymarin group compared to silymarin/β cyclodextrins co-treatments. Sy-HPBCD was found to be the most potent anti-fibrotic complex. We demonstrated that Sy-HPBCD and Sy-RAMEB complexes decreased extracellular matrix accumulation by inhibiting HSC activation and diminished the oxidative damage. This might occur via the inhibition of TGF-β1/Smad signal transduction and MMP/tissue inhibitor of MMPs (TIMP) rebalance, by blocking the synthesis of Col I and decreasing collagen deposition. These results suggest that complexation of silymarin with HPBCD or RAMEB represent viable options for the its oral delivery, of the flavonoid as a potential therapeutic entity candidate, with applications in the treatment of liver fibrosis.

Dose-Dependent Antifibrotic Effect of Chrysin on Regression of Liver Fibrosis: The Role in Extracellular Matrix Remodeling

Liver fibrosis represents an overaccumulation of extracellular matrix (ECM). This study was designed to investigate the effect of chrysin on established ECM overproduction in carbon tetrachloride (CCl4) mouse liver fibrosis. Experimental fibrosis was induced by intraperitoneal injection of 2 mL/kg CCl4 twice a week, for 7 weeks. Mice were orally treated with 3 doses of chrysin (5,7-dihydroxyflavone). For the assessment of the spontaneous reversion of fibrosis, CCl4-treated mice were investigated after 2 weeks of recovery time. Silymarin was used as a standard of liver protection. In fibrotic livers, the results showed the upregulation of collagen I (Col I) and tissue inhibitors of metalloproteinase 1 (TIMP-1) and modulation of matrix metalloproteinases (MMPs), which led to an altered ECM enriched in Col, confirmed as well by electron microscopy investigations. Treatment with chrysin significantly reduced ultrastructural changes, downregulated Col I, and restored TIMP-1/MMP balance, whereas in the group observed for the spontaneous regression of fibrosis, they remained in the same pattern with fibrotic livers. In this study, we have shown chrysin efficacy to attenuate dose-dependent CCl4-stimulated liver ECM accumulation by regulation of MMP/TIMP imbalance and inhibition of Col production. We have shown the dose-dependent chrysin efficiency in attenuation of CCl4-induced liver ECM accumulation by regulation of MMP/TIMP imbalance and inhibition of Col production. Our findings suggest that chrysin oral administration may introduce a new strategy for treating liver fibrosis in humans.

Berberis vulgaris extract/β-cyclodextrin complex increases protection of hepatic cells via suppression of apoptosis and lipogenesis pathways

Berberis vulgaris (Bv) is well known worldwide for its healing properties. However, limited information is available concerning its mechanism of action and the increased hepatoprotective activity of formulated extracts. This study evaluated the protective effect of Bv bark extract against CCl4-induced cytotoxicity in Huh7 cells, as well whether β-cyclodextrin complexation of the extract resulted in increased hepatoprotective effects. Huh7 cells were incubated for 48 h with 5, 7.5 and 10 µg/ml of unformulated or formulated Bv extract alone and in co-treatment with CCl4. The effects on Huh7 cell growth and apoptosis were evaluated by MTT assay, caspase-3/7 activity and caspase-3 expression, whereas fatty acid changes were investigated by Oil red O staining and the detection of peroxisome proliferator-activated receptor-γ (PPARγ) expression using immunofluorescence. Ultrastructural alterations were observed by electron microscopy. The MTT assay showed that co-exposure to CCl4 and 7.5 µg/ml formulated extract led to a 1.25-fold increase in cell viability compared with the non-formulated extract. Caspase-3/7 activity decreased by 50% and 70% following co-treatment with unformulated or formulated extract, compared with that in cells treated with CCl4 alone. Furthermore, hepatocyte ultrastructure was protected from CCl4-induced injury in the two co-treated groups, intracytoplasmic lipid accumulation decreased significantly and PPARγ expression was restored, in comparison with CCl4-treated cells alone. Formulated and unformulated extracts were efficient against the anti-proliferative and pro-apoptotic actions of CCl4 through suppression of CCl4-induced caspase-3 activation and lipid accumulation. The protective effect of the formulated extract was more pronounced than that of the unformulated one, which may be due to its increased solubility.

Flucytosine and Amphotericin B Coadministration Induces Dose-Related Renal Injury

Invasive fungal infections remain an important clinical problem, and despite recent approaches, they bring high morbidity and mortality. Combination therapies are the most effective; however, adverse effects need to be considered. In this study, we aimed to evaluate the nephrotoxicity induced by combined therapy of flucytosine (FL) and amphotericin B (AMF) at 3 different doses administered to mice for 14 days: 300 μg/kg AMF+50 mg/kg FL; 600 μg/kg AMF+100 mg/kg FL; 900 μg/kg AMF+150 mg/kg FL. Antifungal coadministration triggered nuclear translocation of NF-κB and upregulated nuclear factor kappa-light-chain-enhancer of activated B cells subunit p65 (NF-κB p65) messenger RNA mRNA level in dose-dependent manner. The immunopositivity of tumor necrosis factor-α and interleukin-6 (IL-6), together with IL-6 gene expression, increased both in tubular and glomerular cells. Amphotericin B–flucytosine cotreatment increased significantly the number of terminal deoxy-nucleotidyl transferase (TdT)-mediated dUTP nick end-labeling positive nuclei. Apoptotic cells in renal tubuli were confirmed by electron microscopy. Histopathological analysis revealed collagen accumulation at the glomerular level. Collagen was also evidenced in the glomeruli at the dose of 900 μg/kg AMF+150mg/kg FL by Masson-Goldner trichrome staining and electron microscopy. Moreover, antifungal cotherapy induced upregulation of transforming growth factor beta 1 (TGF-β1) gene expression in a dose-dependent manner. Inflammation and epithelial tubular apoptosis are associated with TGF-β1 activation and initiation of the early stage of glomerular fibrosis at higher doses, leading to tubule–interstitial fibrosis.