Echinacea purpurea (L.) Moench (EP) is a well-studied plant used for health benefits. Even though there are a lot of data on EP secondary metabolites, its active proteins are not studied well enough. The aim of our experiment was to purify lectin fraction from EP roots and evaluate its biological activity in vitro as well as its effect on kidney morphology in vivo. An EP root glycoprotein fraction was purified by affinity chromatography, identified by LC-MS/MS, and used for biological activity tests in vitro and in vivo. Identified glycoproteins were homologous with the LysM domain containing lectins from the Asteraceae plants Helianthus annuus L., Lactuca sativa L., Cynara cardunculus L. A purified fraction was tested by hemagglutination and hemagglutination inhibition (by carbohydrate reactions) in vitro. We purified the hemagglutinating active ~40 kDa size lactose, D-mannose, and D-galactose specific glycoproteins with two peptidoglycan binding LysM (lysine motif) domains. Purified LysM lectin was tested in vivo. Eight-week old Balb/C male mice (n = 15) were treated with 5 μg of the purified lectin. Injections were repeated four times per week. At the fifth experimental week, animals were sedated with carbon dioxide, then euthanized by cervical dislocation and their kidney samples were collected. Morphological changes were evaluated in hematoxylin and eosin stained kidney samples. The purified LysM lectin induced a statistically significant (p < 0.05) kidney glomerular vacuolization and kidney tubular necrosis (p < 0.001).
A relevant role of osteopontin (OPN) and gremlin 1 (Grem1) in regulating cardiac tissue remodeling and formation of heart failure (HF) are documented, with the changes of OPN and Grem1 levels in blood plasma due to acute ischemia, ischemic heart disease-induced advanced HF or dilatative cardiomyopathy being the primary focus in most of these studies. However, knowledge on the early OPN and Grem1 proteins expression changes within cardiomyocytes during remodeling due to chronic ischemia remains insufficient. The aim of this study was to determine the OPN and Grem1 proteins expression changes in human cardiomyocytes at different stages of ischemic HF. A semi-quantitative immunohistochemical analysis was performed in 105 myocardial tissue samples obtained from the left cardiac ventricles. Increased OPN immunostaining intensity was already detected in the stage A HF group, compared to the control group (p < 0.001), and continued to increase in the stage B HF (p < 0.001), achieving the peak of immunostaining in the stages C/D HF group (p < 0.001). Similar data of Grem1 immunostaining intensity changes in cardiomyocytes were documented. Significantly positive correlations were detected between OPN, Grem1 expression in cardiomyocytes and their diameter as well as the length, in addition to positive correlation between OPN and Grem1 expression changes within cardiomyocytes. These novel findings suggest that OPN and Grem1 contribute significantly to reorganization of cellular geometry from the earliest stage of cardiomyocyte remodeling, providing new insights into the ischemic HF pathogenesis.
Magnesium-sensitive transient receptor potential melastatin (TRPM) ion channels, TRPM6 and TRPM7, are present in several organs, but their roles in the heart remain unclear. Therefore, here, we studied the expression patterns of TRPM6 and TRPM7 in normal and diseased myocardium. Cardiac atrial tissue and cardiomyocytes were obtained from healthy pigs and undiseased human hearts as well as from hearts of patients with ischemic heart disease (IHD) or atrial fibrillation (AF). Immunofluorescence and ELISA were used to detect TRP proteins. TRPM6 and TRPM7 immunofluorescence signals, localized at/near the cell surface or intracellularly, were detected in pig and human atrial tissues. The TRP channel modulators carvacrol (CAR, 100 µM) or 2-aminoethoxydiphenyl borate (2-APB, 500 µM) decreased the TRPM7 signal, but enhanced that of TRPM6. At a higher concentration (2 mM), 2-APB enhanced the signals of both proteins. TRPM6 and TRPM7 immunofluorescence signals and protein concentrations were increased in atrial cells and tissues from IHD or AF patients. TRPM6 and TRPM7 proteins were both detected in cardiac atrial tissue, with relatively similar subcellular localization, but distinctive drug sensitivity profiles. Their upregulated expression in IHD and AF suggests a possible role of the channels in cardiac atrial disease.
Although major pathogenesis mechanisms of heart failure (HF) are well established, the significance of early (mal)adaptive structural changes of cardiomyocytes preceding symptomatic ischemic HF remains ambiguous. The aim of this study is to present the morphological characterization of changes in cardiomyocytes and their reorganization of intermediate filaments during remodeling preceding symptomatic ischemic HF in an adult human heart. A total of 84 myocardial tissue samples from middle-left heart ventricular segments were analyzed histomorphometrically and immunohistochemically, observing the cardiomyocyte’s size, shape, and desmin expression changes in the remodeling process: Stage A of HF, Stage B of HF, and Stages C/D of HF groups (ACC/AHA classification). Values p < 0.05 were considered significant. The cellular length, diameter, and volume of Stage A of HF increased predominantly by the diameter vs. the control group (p < 0.001) and continued to increase in Stage B of HF in a similar pattern (p < 0.001), increasing even more in the C/D Stages of HF predominantly by length (p < 0.001). Desmin expression was increased in Stage A of HF vs. the control group (p < 0.001), whereas it was similar in Stages A and B of HF (p > 0.05), and most intense in Stages C/D of HF (p < 0.001). Significant morphological changes of cardiomyocytes and their cytoskeletal reorganization were observed during the earliest remodeling events preceding symptomatic ischemic HF.
Echinacea purpurea L. (Moench) is used in traditional and conventional medicine. However, there is lack of data on the biological activities of primary plant metabolite lectins. The aim of our experiment was to find out how lectin LysM (lysine motif), which was previously purified, affects the immune response in vivo. Eight-week-old BALB/c male mice (n = 15) received four weekly 250 μg/kg peritonial injections of purified Echinacea purpurea L. (Moench) roots’ LysM lectin. The control animal group (n = 15) received 50 μL peritoneal injections of fresh Echinacea purpurea L. (Moench) root tincture, and the negative control animal group (n = 15) received 50 μL peritoneal injections of physiological solution. At the fifth experimental week, the animals were sedated with carbon dioxide, and later euthanized by cervical dislocation, and then their blood and spleen samples were collected. The leukocytes’ formula and lymphocytes’ count was estimated in blood samples, the T lymphocytes’ density was evaluated in spleen zones. A statistically significant (p < 0.05) difference between each group was observed in the leukocytes’ formula (monocytes’ percentage, also little, medium and giant size lymphocytes). The purple coneflower fresh roots’ tincture significantly decreased (p < 0.05) the T lymphocytes’ quantity in peritoneal lymphoid sheaths (PALS) compared with the physiological solution injection’s group (p < 0.05) and the lectin injection’s group (p < 0.001). Meanwhile, lectin injections caused a significant (p < 0.01) increase in the T lymphocytes in a spleen PALS zone, compared with the physiological solution and tincture injection’s group. Our data suggests that LysM lectin acts as an immunostimulant, while fresh purple coneflower tincture causes immunosuppression.