Methanolic extract of Phlogacanthus thyrsiflorus Nees leaf induces apoptosis in cancer cells

Phlogacanthus thyrsiflorus Nees is a medicinal herb commonly used in traditional folk medicine, and it is known to possess anticancer activity. Here, we explored the anticancer properties of methanolic extract of P. thyrsiflorus leaves in HeLa and MCF-7 cell lines. We observed nuclear fragmentation as indication of apoptosis in the MPT treated cancer cells using haematoxylin and eosin (H&E) as well as fluorescent dye. DNA from the treated cells showed characteristic laddering of DNA fragments in agarose gel electrophoresis. Cell populations undergoing secondary necrosis following apoptosis could also be detected in FACS by annexin V/propidium iodide (PI) staining. Activated caspase-3 in the treated HeLa cells was detected by polyclonal anti-caspase-3 antibody utilizing immunocytochemistry. Using transmission electron microscopy, sub-cellular changes like rough endoplasmic reticulum, swollen mitochondria, distorted mitochondrial membrane, loss of cristae and matrix were observed in the treated HeLa cells. Extensive plasma membrane blebbing was also observed by scanning electron microscopy. Our findings support that Phlogacanthus thyrsiflorus leaves are natural source of potent anticancer agent

Methanolic extract of Phlogacanthus thyrsiflorus Nees leaf induces apoptosis in cancer cells

153-161Phlogacanthus thyrsiflorus Nees is a medicinal herb commonly used in traditional folk medicine, and it is known to possess anticancer activity. Here, we explored the anticancer properties of methanolic extract of P. thyrsiflorus leaves in HeLa and MCF-7 cell lines. We observed nuclear fragmentation as indication of apoptosis in the MPT treated cancer cells using haematoxylin and eosin (H&E) as well as fluorescent dye. DNA from the treated cells showed characteristic laddering of DNA fragments in agarose gel electrophoresis. Cell populations undergoing secondary necrosis following apoptosis could also be detected in FACS by annexin V/propidium iodide (PI) staining. Activated caspase-3 in the treated HeLa cells was detected by polyclonal anti-caspase-3 antibody utilizing immunocytochemistry. Using transmission electron microscopy, sub-cellular changes like rough endoplasmic reticulum, swollen mitochondria, distorted mitochondrial membrane, loss of cristae and matrix were observed in the treated HeLa cells. Extensive plasma membrane blebbing was also observed by scanning electron microscopy. Our findings support that Phlogacanthus thyrsiflorus leaves are natural source of potent anticancer agent

Effects of vitamin E on mitochondrial dysfunction and asthma features in an experimental allergic murine model

We showed recently that IL-4 causes mitochondrial dysfunction in allergic asthma. IL-4 is also known to induce 12/15-lipoxygenase (12/15-LOX), a potent candidate molecule in asthma. Because vitamin E (Vit-E) reduces IL-4 and inhibits 12/15-LOX in vitro, here we tested the hypothesis that Vit-E may be effective in restoring key mitochondrial dysfunctions, thus alleviating asthma features in an experimental allergic murine model. Ovalbumin (OVA)-sensitized and challenged male BALB/c mice showed the characteristic features of asthma such as airway hyperresponsiveness (AHR), airway inflammation, and airway remodeling. In addition, these mice showed increase in the expression and metabolites of 12/15-LOX, reduction in the activity and expression of the third subunit of mitochondrial cytochrome-c oxidase, and increased cytochrome c in lung cytosol, which indicate that OVA sensitization and challenge causes mitochondrial dysfunction. Vit-E was administered orally to these mice, and 12/15-LOX expression, key mitochondrial functions, ultrastructural changes of mitochondria in bronchial epithelia, and asthmatic parameters were determined. Vit-E treatment reduced AHR, Th2 response including IL-4, IL-5, IL-13, and OVA-specific IgE, eotaxin, transforming growth factor-β1, airway inflammation, expression and metabolites of 12/15-LOX in lung cytosol, lipid peroxidation, and nitric oxide metabolites in the lung, restored the activity and expression of the third subunit of cytochrome-c oxidase in lung mitochondria and bronchial epithelia, respectively, reduced the appearance of cytochrome c in lung cytosol, and also restored mitochondrial ultrastructural changes of bronchial epithelia. In summary, these findings show that Vit-E reduces key mitochondrial dysfunctions and alleviates asthmatic features

L-Arginine reduces mitochondrial dysfunction and airway injury in murine allergic airway inflammation

Bronchial epithelial injury is the hall mark of asthma which is a chronic airway inflammatory disease. We have shown the mitochondrial ultrastructural changes and dysfunction in bronchial epithelia of OVA induced mice. Reduced L-arginine bioavailability in asthma leads to increased formation of peroxynitrite which could induce mitochondrial dysfunction. We have also shown that L-arginine administration attenuates experimental asthma and reduces peroxynitrite. In this study, we wanted to determine the effect of L-arginine on mitochondrial dysfunction and airway injury in allergic airway inflammation. To determine this, L-arginine was administered to ovalbumin sensitized and challenged mice during allergen challenges. Mitochondrial and cytosolic fractions were purified from the lung to determine key mitochondrial functions, and mitochondrial ultrastructural changes in bronchial epithelia of first generation bronchi were determined. It was found that L-arginine administration increased mitochondrial cytochrome c oxidase activity, reduced cytosolic cytochrome c, increased lung ATP levels, reduced DNA fragmentation in bronchial epithelia and restored the ultrastructural changes of mitochondria of bronchial epithelia. In addition, L-arginine administration reduced the widening of intercellular spaces between adjacent bronchial epithelia. These findings indicated that L-arginine administration reduced airway injury and restored mitochondrial dysfunction in murine allergic airway inflammation

Beneficial effects of high dose of L-arginine on airway hyperresponsiveness and airway inflammation in a murine model of asthma

Background: Disturbance in the delicate balance between L-arginine-metabolizing enzymes such as nitric oxide synthase (NOS) and arginase may lead to decreased L-arginine availability to constitutive forms of NOS (endothelial NOS), thereby increasing the nitro-oxidative stress and airway hyperresponsiveness (AHR). Objective: In this study, we investigated the effects of high doses of L-arginine on L-arginine-metabolizing enzymes and subsequent biological effects such as cyclic guanosine monophosphate production, lipid peroxidation, peroxynitrite, AHR, and airway inflammation in a murine model of asthma. Methods: Different doses of L-arginine were administered to ovalbumin-sensitized and challenged mice. Exhaled nitric oxide, AHR, airway inflammation, TH2 cytokines, goblet cell metaplasia, nitro-oxidative stress, and expressions of arginase 1, endothelial NOS, and inducible NOS in lung were determined. Results: L-arginine significantly reduced AHR and airway inflammation including bronchoalveolar lavage fluid eosinophilia, TH2 cytokines, TGF-β1, goblet cell metaplasia, and subepithelial fibrosis. Further, L-arginine increased ENO levels and cyclic guanosine monophosphate in lung and reduced the markers of nitro-oxidative stress such as nitrotyrosine, 8-isoprostane, and 8-hydroxy-2'-deoxyguanosine. This was associated with reduced activity and expression of arginase 1, increased expression of endothelial NOS, and reduction of inducible NOS in bronchial epithelia. Conclusion: We conclude that L-arginine administration may improve disordered nitric oxide metabolism associated with allergic airway inflammation, and alleviates some features of asthma

Histopathological and immunohistochemical approaches for the diagnosis of Pasteurellosis in swine population of Punjab

Aim: Infectious porcine bronchopneumonia, caused by Pasteurella multocida, is a widespread disease of major economic significance. Thus, the aim of the present study was to diagnose swine Pasteurellosis using gross, histopathological, and immunopathological approaches in the swine population of Punjab and to compare the efficacy of immunohistochemical (IHC) techniques with conventional diagnostic techniques. Materials and Methods: A total of 71 adult swine lung samples showing gross pneumonic changes were collected along with the associated lymph nodes to carry out the study. The collected samples were then processed for histopathological and IHC studies. Results: Out of the total 71 lung samples, 26 samples were found to be suspected for Pasteurellosis as per the microscopic changes observed, and out of these 26 samples, 16 cases were confirmed to be positive for Pasteurellosis by IHC. Varied macroscopic changes noted in lungs were pneumonic patches with consolidation of many lobes, congestion, and focal hemorrhages. Main lesions associated with lymph nodes were its enlargement and hemorrhages. Histologically, the lung showed fibrinous and suppurative bronchopneumonia, multifocal suppuration, thickening of septa with fibrin combined with cellular infiltration and edema. The higher IHC expression of P. multocida was seen in the bronchial epithelium besides in alveolar and bronchial exudate. Moreover, on comparing the histopathological and IHC scores which were calculated on the basis of characteristic microscopic lesions and number of antigen positive cells, respectively, a significant positive correlation (r=0.4234) was found. Conclusion: It was concluded that swine population of Punjab is having P. multocida infection. The gross and histopathological lesions can be helpful in the preliminary diagnosis of Pasteurellosis but needs to be supplemented by other immunodiagnostic tests. Moreover, IHC technique proved to be a specific, reliable, precise, and rapid technique to supplement these conventional methods of diagnosis for Pasteurellosis

A novel antimicrobial peptide derived from modified N-terminal domain of bovine lactoferrin: Design, synthesis, activity against multidrug-resistant bacteria and Candida

AbstractLactoferrin (LF) is believed to contribute to the host's defense against microbial infections. This work focuses on the antibacterial and antifungal activities of a designed peptide, L10 (WFRKQLKW) by modifying the first eight N-terminal residues of bovine LF by selective homologous substitution of amino acids on the basis of hydrophobicity, L10 has shown potent antibacterial and antifungal properties against clinically isolated extended spectrum beta lactamases (ESBL), producing gram-negative bacteria as well as Candida strains with minimal inhibitory concentrations (MIC) ranging from 1 to 8μg/mL and 6.5μg/mL, respectively. The peptide was found to be least hemolytic at a concentration of 800μg/mL. Interaction with lipopolysaccharide (LPS) and lipid A (LA) suggests that the peptide targets the membrane of gram-negative bacteria. The membrane interactive nature of the peptide, both antibacterial and antifungal, was further confirmed by visual observations employing electron microscopy. Further analyses, by means of propidium iodide based flow cytometry, also supported the membrane permeabilization of Candida cells. The peptide was also found to possess anti-inflammatory properties, by virtue of its ability to inhibit cyclooxygenase-2 (COX-2). L10 therefore emerges as a potential therapeutic remedial solution for infections caused by ESBL positive, gram-negative bacteria and multidrug-resistant (MDR) fungal strains, on account of its multifunctional activities. This study may open up new approach to develop and design novel antimicrobials

Baicalein reduces IL-13 induced mitochondrial dysfunction and bronchial epithelial injury.

<p>A) Mitochondria and cytosols were isolated from fresh lungs. 13-S-HODE levels in lung cytosol (A), activities of complex I (B) and cytochrome c oxidase (C), cytochrome c (D) and caspase 3 (E) were estimated. F) TUNEL apoptosis assay in lung tissue sections. Brown color indicates the TUNEL positive apoptotic cells. Data were mean ± SEM of three independent experiments. *P<0.05 vs. BSA/PBS group, and ^†P<0.05 vs. rIL-13/PBS group; n = 5–6 mice in each group. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062916#pone-0062916-t001" target="_blank">Table 1</a>. Effects of Baicalein on IL-4, IL-13, IFN-γ, TLR-2, TLR-4, OVA specific IgE, IgG2a, eotaxin levels and airway inflammation score.</p

Effect of Baicalein (BAIC) pretreatment on airway hyperresponsiveness (AHR) to Methacholine.

<p>A) Male Balb/c mice were sensitized and challenged as shown. AHRs were determined on Day 33. B) In dose titration experiments, single chamber plethysmography results were expressed as MCh PC200 [the partial concentration of methacholine which is required to double the baseline enhanced pause (Penh)]. C) In preventive model, invasive airway mechanics results were expressed as airway resistance. D) Representative photomicrographs of Haematoxylin and Eosin staining were shown. Br, bronchus; V, vessel; a, alveolus; Image are shown at 20X magnifications. E) Absolute cell count in BAL fluid. Absolute number of eosinophils (Eosino), macrophages (Macro), mononuclear agranulocytes (Mono), and neutrophils (Neutro) in BAL fluid was determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062916#s4" target="_blank">Materials and Methods</a>. Data were mean ± SEM. *P<0.05 vs. SHAM/PBS/VEH and ^†P<0.05 vs. OVA/OVA/VEH; n = 5–6 mice in each group.</p