Antidiabetic effects of Bisamide Derivative of Dicarboxylic Acid in metabolic disorders

In clinical practice, metabolic syndrome can lead to multiple complications, including diabetes. It remains unclear, which component of the metabolic syndrome (obesity, inflammation, hyperglycemia or insulin resistance) has the strongest inhibitory effect on stem cells involved in beta cell regeneration. This makes it challenging to develop effective treatment options for complications such as diabetes. In our study, experiments were performed on male C57BL/6 mice where metabolic disorders have been introduced experimentally by a combination of streptozotocin-treatment and high-fat diet. We evaluated the biological effects of Bisamide Derivative of Dicarboxylic Acid (BDDA) and its impact on pancreatic stem cells in vivo. To assess the impact of BDDA, we applied a combination of histological and biochemical methods along with a cytometric analysis of stem cell and progenitor cell markers. We show that in mice with metabolic disorders, BDDA has a positive effect on lipid and glucose metabolism. The pancreatic restoration was associated with a decrease of the inhibitory effects of inflammation and obesity factors on pancreatic stem cells. Our data show that BDDA increases the number of pancreatic stem cells. Thus, BDDA could be used as a new compound for treating complications of the metabolic syndrome such as diabetes

Endothelial Progenitor Cells as Pathogenetic and Diagnostic Factors, and Potential Targets for GLP-1 in Combination with Metabolic Syndrome and Chronic Obstructive Pulmonary Disease

In clinical practice, there are patients with a combination of metabolic syndrome (MS) and chronic obstructive pulmonary disease (COPD). The pathological mechanisms linking MS and COPD are largely unknown. It remains unclear whether the effect of MS (possible obesity) has a major impact on the progression of COPD. This complicates the development of effective approaches for the treatment of patients with a diagnosis of MS and COPD. Experiments were performed on female C57BL/6 mice. Introduction of monosodium glutamate and extract of cigarette smoke was modeled to simulate the combined pathology of lipid disorders and emphysema. Biological effects of glucagon-like peptide 1 (GLP-1) and GLP-1 on endothelial progenitor cells (EPC) in vitro and in vivo were evaluated. Histological, immunohistochemical methods, biochemical methods, cytometric analysis of markers identifying EPC were used in the study. The CD31+ endothelial cells in vitro evaluation was produced by Flow Cytometry and Image Processing of each well with a Cytation™ 3. GLP-1 reduces the area of emphysema and increases the number of CD31+ endothelial cells in the lungs of mice in conditions of dyslipidemia and damage to alveolar tissue of cigarette smoke extract. The regenerative effects of GLP-1 are caused by a decrease in inflammation, a positive effect on lipid metabolism and glucose metabolism. EPC are proposed as pathogenetic and diagnostic markers of endothelial disorders in combination of MS with COPD. Based on GLP-1, it is proposed to create a drug to stimulate the regeneration of endothelium damaged in MS and COPD

Modulation of Bleomycin-Induced Lung Fibrosis by Pegylated Hyaluronidase and Dopamine Receptor Antagonist in Mice

<div><p>Hyaluronidases are groups of enzymes that degrade hyaluronic acid (HA). To stop enzymatic hydrolysis we modified testicular hyaluronidase (HYAL) by activated polyethylene oxide with the help of electron-beam synthesis. As a result we received pegylated hyaluronidase (pegHYAL). Spiperone is a selective D₂ dopamine receptor antagonist. It was demonstrated on the model of a single bleomycin damage of alveolar epithelium that during the inflammatory phase monotherapy by pegHYAL or spiperone reduced the populations of hematopoietic stem /progenitor cells in the lung parenchyma. PegHYAL also reduced the levels of transforming growth factor (TGF)-β, interleukin (IL)-1β, tumor necrosis factor (TNF)-α in the serum and lungs, while spiperone reduced the level of the serum IL-1β. Polytherapy by spiperone and pegHYAL caused the increase of the quantity of hematopoietic stem/ progenitor cells in the lungs. Such an influx of blood cell precursors was observed on the background of considerable fall level of TGF-β and the increase level of TNF-α in the serum and lungs. These results show pegHYAL reduced the bleomycin-induced fibrosis reaction (production and accumulation of collagen) in the lung parenchyma. This effect was observed at a single and repetitive bleomycin damage of alveolar epithelium, the antifibrotic activity of pegHYAL surpassing the activity of testicular HYAL. The antifibrotic effect of pegHYAL is enhanced by an additional instillation of spiperone. Therapy by pegHYAL causes the flow of CD31^‒CD34^‒CD45^‒CD44⁺CD73⁺CD90⁺CD106⁺-cells into the fibrous lungs. These cells are incapable of differentiating into fibroblast cells. Spiperone instillation separately or together with pegHYAL reduced the MSC-like cells considerably. These data enable us to assume, that pegHYAL is a new and promising instrument both for preventive and therapy of toxic pneumofibrosis. The blockage of D₂ dopamine receptors with the following change of hyaluronan matrix can be considered as a new strategy in treatment of pneumofibrosis.</p></div

Analysis of murine pan-hematopoietic stem cells (CD45⁺-cells) and CD45^- -cells derived from lung (21^st day of the experiment).

<p>Cells were analyzed by FACS with antibody to identify mouse CD45, CD90.1, CD34, CD73, CD106 (VCAM-1), CD44 and CD31 (PECAM-1) (BD Biosciences, USA). Cells for analysis were derived from lungs of C57BL/6 mice of control and experimental groups. The CD45 negative cells population and CD45 positive cells population were sorted from one sample. The CD45 negative cells population was defined as CD34 and CD31 negative and positive for CD106, CD73, CD44, and CD90.1 at the same time in the two specimens (1 specimen—subpopulation of cells with phenotype CD45^-, CD73⁺, CD90⁺, CD106⁺, CD44⁺ and 2 specimen—subpopulation of cells with phenotype CD45^-, CD73⁺, CD90⁺, CD31^-, CD34^-). Dot plots are representative figures of three independent experiments with the mean from three independent experiments.</p

Effects of pegylated hyaluronidase (pegHYAL) and spiperone treatment on HSCs and hematopoietic progenitor cells.

<p>The content of HSCs and hematopoietic progenitor cells in the lung of C57BL/6 mice on the 7th day of the experiment after single dose intratracheal BLM instillation. Membrane receptor's expression of murine HSCs derived from lung were assayed according to the protocol for BD Mouse Hematopoietic Stem and Progenitor Cell Isolation Kit (BD Biosciences, USA). The HSC population taken through a Lin^- selection, is shown gated Sca1⁺ and c-kit⁺, then displayed for CD34^- and CD34⁺. The Lin^- Sca1⁺c-kit⁺CD34^- -cells are LT-HSCs and the Lin^-Sca1⁺c-kit⁺CD34⁺-cells are considered ST-HSCs. Thus, all two of these populations can be readily sorted from one sample. Dot plots are representative figures of three independent experiments with the mean from three independent experiments.</p><p>Results are presented as mean and SEM.</p><p>*—significance of the difference with the mice, that received intratracheal 0.9% NaCl (P <0.05),</p><p>&—significance of the difference with the mice, that received intratracheal BLM and treated with 0.9% NaCl (P<0.05), t test was used.</p><p>Effects of pegylated hyaluronidase (pegHYAL) and spiperone treatment on HSCs and hematopoietic progenitor cells.</p

Characterization of hematopoietic stem cells isolated from lung of C57BL/6 mice (7th day of the experiment).

<p>The phenotype of cells from lung was studied according to the protocol for hematopoietic stem cells (BD Biosciences). The HSC population taken through a Lin^- selection (not shown) and then Sca1⁺ and c-kit⁺ (not shown), is shown gated displayed for CD34^- (P7) and CD34⁺ (P8). The Lin^-Sca1⁺c-kit⁺CD34^- cells are LT-HSCs and the Lin^-Sca1⁺c-kit⁺CD34⁺ cells are considered ST-HSCs. Thus, all two of these populations can be readily sorted from one sample. (<b>A</b>) HSCs isolated from mice after intratracheal 0.9% NaCl administration; (<b>B</b>) HSCs isolated from mice after intratracheal BLM administration; (<b>C</b>) HSCs isolated from mice after intratracheal BLM administration and treated spiperone; (<b>D</b>) HSCs isolated from mice after intratracheal BLM administration and treated pegHYAL; (<b>E</b>) HSCs isolated from mice after intratracheal BLM administration and treated spiperone and pegHYAL; (<b>F</b>) HSCs isolated from mice after intratracheal BLM administration and treated HYAL. Dot plots are representative figures of three independent experiments with the mean from three independent experiments.</p

Effects of hyaluronidase (HYAL) and Spiperone treatment on collagen type I, hydroxyproline, total soluble collagen, hyaluronic acid levels after the single dose intratracheal bleomycin instillation.

<p>Hydroxyproline, collagen type I, total soluble collagen and hyaluronic acid levels were measured in homogenate of right lung lobes from C57BL/6 mice on 21^st day after BLM treatment. Hydroxyproline, collagen type I and HA were assayed by ELISA according to manufacturer instructions (Cusabio Biotech CO., Ltd, China). The right lung lobes were excised and snap frozen after having measured the wet weight. Sensitivities were >1.95 ng/mL for hydroxyproline, >0.039 ng/mL for collagen type I and >15.6 pg/mL for HA. The total soluble collagen was determined using a standard curve for the Sircol^TM assay (Biocolor Ltd, UK) according to manufacturer’s instructions. Results were expressed as μg collagen per right lung.</p><p>Data represent mean of 3 independent experiments, n = 5/group. Results are presented as mean and SEM.</p><p>*—compared to the mice received intratracheal 0.9% NaCl (P <0.05),</p><p>●—compared to the mice received intratracheal BLM and 0.9% NaCl treated (P<0.05), t test was used.</p><p>Effects of hyaluronidase (HYAL) and Spiperone treatment on collagen type I, hydroxyproline, total soluble collagen, hyaluronic acid levels after the single dose intratracheal bleomycin instillation.</p

Photomicrographs of representative lung sections obtained from C57BL/6 mice after a single bleomycin instillation (stained by hematoxylin-eosin).

<p>Tissues were stained with hematoxylin-eosin to investigate inflammatory cells accumulation (21st day of the experiment). (<b>A</b>) Mice receiving intratracheal 0.9% NaCl, (<b>B</b>) Mice receiving intratracheal BLM, (<b>C</b>) Mice with fibrosis spiperone treated, (<b>D</b>) Mice with fibrosis HYAL treated, (<b>E</b>) Mice with fibrosis pegHYAL treated, (<b>F</b>) Mice with fibrosis pegHYAL and spiperone treated. The photomicrographs were taken using an Axio Lab.A1 (Carl Zeiss MicroImaging GmbH; Göttingen, Germany) microscope and AxioCam ERc5s digital camera. All photomicrographs were at 100 × magnification.</p

Effect of bleomycin on the lung architecture in C57Bl/6 mice.

<p>Comparison of the lung architecture in C57Bl/6 mice after BLM instillation (21st day of the experiment—partially reversible pulmonary fibrosis or 60th day of the experiment—irreversible pulmonary fibrosis) or 0.9% NaCl (sham treatment), as shown by hematoxylin-eosin (<b>A</b>) and picrofuchsin (<b>B</b>) staining of representative tissue sections. The photomicrographs were taken using an Axio Lab.A1 (Carl Zeiss MicroImaging GmbH; Göttingen, Germany) microscope and AxioCam ERc5s digital camera. All photomicrographs were at 100 × magnification.</p