Amelioration of C-Reactive Protein and Lectin Like Oxidized Low Density Lipoprotein Receptor Complex Induced Endothelial Dysfunction by Oligomeric Proanthocyanidins.

C-reactive protein (CRP) is a well-established biochemical marker for atherosclerosis. Modification of LDL inside the artery wall favors the elevation of this acute phase protein. Hence, this mechanism is considered an important factor to trigger the monocyte to macrophages differentiation which results in the formation of foam cells. Therefore, this key event should be targeted and focused on how this complex (OxLDL + CRP) proceeds to endothelial dysfunction. Oligomeric proanthocyanidins (OPC) is a well-known cardioprotective flavon-3-ols. The present study is challenged between the cardioprotective roles of OPC against the deleterious effect of OxLDL + CRP complex upon endothelial cells. Protein-protein docking was carried out between CRP and LOX-1. This docked protein complex was again docked with OPC to show the inhibitory mechanism of CRP binding with LOX-1. OPC showed a promising inhibitory mechanism against OxLDL + CRP complex. Docking studies showed that in the absence of ligands (OPC), binding of CRP and LOX-1 was greater and vice versa in the presence of ligands. Based on these molecular docking results, in vitro studies have been carried out. The monolayer of endothelial cells was incubated with THP-1 monocytes for 48 h, induced with OxLDL (10 µg/ml) + CRP (15 µg/ml) and cotreated with OPC (100 µg/ml). Morphological changes, cell migration assay, and capillary tube forming assay were carried out. Myeloperoxidase levels were estimated to determine the adhesion of monocytes onto EC monolayer. RT-PCR analysis of L-Selectin was also done. The quantification of NO levels and analysis of mRNA expressions of eNOS was to determine the nitric oxide demand caused due to OxLDL + CRP complex. LOX-1, scavenger receptor levels were analyzed by mRNA expression. Proinflammatory markers such as IL-6, MCP-1, and IL-1ß were studied. Accumulation of ROS levels was measured fluorimetrically using DCF-DA staining. Mitochondrial membrane potential was determined by JC-1 dye and cell cycle analysis was done by FACS analysis. To emphasis the results, the OPC-treated group showed decreased levels of proinflammatory markers, LOX-1 and L-selectin levels. Endothelial nitric oxide levels were increased upon OPC treatment and reduction in the ROS levels was also observed. Endothelial cells apoptosis was prevented by OPC. To conclude, OxLDL + CRP complex inhibitory effects of OPC could maintain the normal homeostasis.

Monocytes treated with ciprofloxacin and oxyLDL express myristate, priming atherosclerosis.

Antibiotics are essential in many life-threatening diseases. On the other hand, improper use of antibiotics can be disastrous. Cell morphological changes were observed in the ciprofloxacin-treated cells starting at 48 hours. Changes in cell morphology were continuously observed up to 14 days, which showed gradual morphological changes from monocyte to plaque-like cells at day 12, and foam cell, which is an intermediate stage in atherosclerosis was observed at day 8, which was confirmed with Oil Red O staining. Flow cytometry data revealed that oxidized LDL (oxyLDL)-induced cells showed 60.16% of CD64 (proinflammatory macrophage markers) and no expression of CD23 (anti-inflammatory macrophage markers), whereas ciprofloxacin-treated cells expressed 67.97% of CD64 and 13.78% of CD23. Chemokine antibody array analysis revealed that ciprofloxacin exposed cells showed a proinflammatory role (ENA78, Eotaxin1, Eotaxin2, IP-10, MIG, MIP-3ß, SDF-1ß, TECK, CXCL16, and Fractalkine). Liquid chromatography with tandem mass spectrometry (LC-MS/MS) revealed that myristic acid was incorporated into a protein with 68 kDa molecular mass in exposing oxyLDL-induced monocytes with ciprofloxacin, which could be a reason for the observed foam cells and in vitro plaque formation. As myristic acid primes atherosclerosis, it is better to limit the intake of antibiotics like ciprofloxacin for common illness, specifically the high-risk patients, which may contribute to atherosclerosis.

In silico approach to study the metabolism and biological activities of oligomeric proanthocyanidin complexes.

OBJECTIVES: Over the past three decades, numerous studies have focused on the biological activities of oligomeric proanthocyanidins (OPCs) in the prevention of many diseases such as neurodegeneration, atherosclerosis, tumorigenesis, and microbial infections. OPC has redox-active metabolites which could modulate the intracellular redox equilibrium to maintain the antioxidant homeostasis. This redox-modulating efficiency of OPC could provide new insights into therapeutic approaches that could reduce the burden of cardiovascular diseases. The main objective of this study was to explore the biological and metabolic activities of OPC using in silico approaches. METHODS: To validate the above objective, chemoinformatic tools were used to predict the metabolism of OPC after ingestion, based on both the ligand and structure of the constituent compounds. RESULTS: OPC showed possible sites for Phase I metabolism by cytochrome P450, and the metabolites obtained thereafter may be responsible for its biological activities. Absorption, distribution, metabolism, elimination, and toxicity properties showed efficient absorption, distribution, and metabolism of OPC, without toxicity. CONCLUSION: Thus, from the results obtained, OPC could be strongly recommended as a cardioprotective drug.

Ameliorative effect of methanol extract of Rubia cordifolia in N-nitrosodiethylamine-induced hepatocellular carcinoma.

CONTEXT: Rubia cordifolia Linn. (Rubiaceae) is a medicinal plant used in the ayurvedic system of medicine. It is also known as Indian Madder or Manjistha and is traditionally used as an antiinflammatory, antiseptic, and galactopurifier, but its anticancer propertis are yet not known. OBJECTIVE: The ameliorative effect of the Rubia cordifolia methanol extract on N-nitrosodiethylamine-induced experimental hepatocellular carcinogenesis in rats. MATERIALS AND METHODS: Changes in liver weight, serum markers of liver damage, hydroxyl radicals, lipid peroxidation, levels of enzymic and nonenzymic antioxidants; mitochondrial and respiratory chain enzymes were also investigated using various biochemical parameters and histopathological studies. Male albino rats of Wistar strain were divided into four groups for a study period of 3 months. Animals of group I and group IV served as control and drug control, respectively. Hepatocellular carcinoma was induced in animals of groups II and III with 0.02% N-nitrosodiethylamine. RESULTS: Upon Rubia cordifolia methanol extract co-treatment (250, 500, and 750 mg/kg bodyweight) in group III alone levels of serum marker enzymes and antioxidants increased significantly in a dose-dependent manner. The levels of hydroxyl radicals and lipid peroxidation decreased. Mitochondrial enzymes and respiratory chain enzymes, which were decreased in N-nitrosodiethylamine-induced rats, increased significantly in RC treated rats. Further histological analysis of liver confirmed the prevention of pathological changes caused by N-nitrosodiethylamine on Rubia cordifolia supplementation. DISCUSSION AND CONCLUSION: These findings demonstrate that Rubia cordifolia can be a source of potent antioxidants for treatment of diseases such as cancer.

Up-regulation of MUC2 and IL-1ß expression in human colonic epithelial cells by Shigella and its interaction with mucins.

BACKGROUND: The entire gastrointestinal tract is protected by a mucous layer, which contains complex glycoproteins called mucins. MUC2 is one such mucin that protects the colonic mucosa from invading microbes. The initial interaction between microbes and mucins is an important step for microbial pathogenesis. Hence, it was of interest to investigate the relationship between host (mucin) and pathogen interaction, including Shigella induced expression of MUC2 and IL-1ß during shigellosis. METHODS: The mucin-Shigella interaction was revealed by an in vitro mucin-binding assay. Invasion of Shigella dysenteriae into HT-29 cells was analyzed by Transmission electron microscopy. Shigella induced mucin and IL-1ß expression were analyzed by RT-PCR and Immunofluorescence. RESULTS: The clinical isolates of Shigella were found to be virulent by a congo-red binding assay. The in vitro mucin-binding assay revealed both Shigella dysenteriae and Shigella flexneri have binding affinity in the increasing order of: guinea pig small intestinal mucin

Isolation and partial characterisation of a novel lectin from Aegle marmelos fruit and its effect on adherence and invasion of Shigellae to HT29 cells.

Lectins are a class of ubiquitous proteins/glycoproteins that are abundantly found in nature. Lectins have unique carbohydrate binding property and hence have been exploited as drugs against various infectious diseases. We have isolated one such novel lectin from the fruit pulp of Aegle marmelos. The isolated lectin was partially characterised and its effect against Shigella dysenteriae infection was evaluated. The isolated lectin was found to be a dimeric protein with N-acetylgalactosamine, mannose and sialic acid binding specificity. The effect of Aegle marmelos fruit lectin on the adherence of Shigella dysenteriae to human colonic epithelial cells (HT29 cells) was evaluated by Enzyme Linked Immune Sorbent Assay and invasion was analysed. The protective nature of the Aegle marmelos fruit lectin was assessed by analyzing apoptosis through dual staining method. Aegle marmelos fruit lectin significantly inhibited hemagglutination activity of Shigella and its minimum inhibitory concentration is 0.625 µg/well. Further, at this concentration lectin inhibited Shigella dysenteriae adherence and invasion of HT29 cells and protects the HT29 cells from Shigella dysenteriae induced apoptosis. To conclude, isolated lectin dimeric protein with N-acetylgalactosamine, Mannose and sialic acid binding specificity and inhibits adherence and invasion of Shigellae to HT29 cells thus, protects the host.

Imperatorin a furocoumarin inhibits periplasmic Cu-Zn SOD of Shigella dysenteriae their by modulates its resistance towards phagocytosis during host pathogen interaction.

Shigella dysenteriae continues to be a major health problem, which leads to death, due to diarrhoea and dysentery, predominantly in children below the age of 5. Bacterial invasion of the colonic epithelium leads to severe inflammation together with bacterial dissemination generates abscesses and ulcerations. Periplasmic copper, zinc super oxide dismutase of Shigella protects it from exogenous superoxide produced by host, during its invasion. Hence, in present study an attempt was made to study the effect of aqueous extract of Aegle marmelos on host and pathogen defence. Histology analysis of rat ileal loop showed the loss of virulence in aqueous extract of A. marmelos pre-treated Shigella and their intracellular survival was also decreased, where active component present in aqueous extract of A. marmelos was identified as imperatorin confirmed by UV absorption spectrum and HPLC. Increase in peripheral blood mononuclear cell viability and decreased in intracellular bacterial count along with transmission electron microscope analysis of imperatorin treated S. dysenteriae succumb to host oxidative stress. Loss of virulence is associated with attenuation of copper, zinc super oxide dismutase activity in Shigella, which was confirmed by using activity staining of bacterial cell lysate. Further, by performing docking analysis it has been proved that imperatorin present in aqueous extract of A. marmelos inhibited copper, zinc super oxide dismutase. From the above study, we concluded that Shigella succumb to oxidative stress (host defence) due to inhibition of copper, zinc super oxide dismutase (pathogen's defence) by imperatorin, an active compound aqueous extract of A. marmelos.

Protective effect of grape seed proanthocyanidins against cholesterol cholic acid diet-induced hypercholesterolemia in rats.

BACKGROUND: Dietary cholesterol plays an important role in the development of atherogenesis and cardiovascular diseases. We explored the prospective effect of grape seed proanthocyanidins in controlling hypercholesterolemia induced oxidative injury and apoptosis in atherogenic animals. METHODS: Four groups of male Wistar rats (250-300 g) were used for the study. Group I served as control and received vehicle (saline) alone, Group II served as the induction group fed with a high-cholesterol diet (rat chow supplemented with 4% cholesterol and 1% cholic acid--CC diet) for 30 days, Group III served as drug control and was treated with grape seed proanthocyanidins (100 mg/kg body weight) orally for 30 days, and Group IV animals were fed with CC diet for 30 days along with grape seed proanthocyanidins (100 mg/kg body weight) orally. RESULTS: CC diet induced an abnormal increase in lipid peroxidation, tissue cholesterol, triglyceride, serum low-density lipoprotein, and very low density lipoprotein, and decreased the high-density lipoprotein concentration. Altered activity of cardiac and serum creatine kinase, accompanied by a decreased cardiac enzymatic and nonenzymatic antioxidant defense system and an increase in the expression of cytochrome c and caspases-3, was observed in CC diet-fed rats. These changes were partially restored in the grape seed proanthocyanidin-treated group. CONCLUSION: Grape seed proanthocyanidins have cardioprotective effects against CC diet-induced hypercholesterolemia via their ability to reduce, directly or indirectly, free radicals in the myocardium.

Aberrant expression of epidermal growth factor receptor and its interaction with protein kinase C δ in inflammation associated neoplastic transformation of human esophageal epithelium in high risk populations.

BACKGROUND AND AIM: Esophageal cancer is the second most common cancer among Indian males and is mostly associated with tobacco smoking and alcohol consumption. Epidermal growth factor receptor (EGFR) is a member of Type I tyrosine kinases. Its activation causes the docking of various proteins in its cytosolic tail. In the present study we have analyzed the expression pattern of EGFR, protein kinase C δ (PKCδ), tumor necrosis factor-α (TNF-α), nuclear factor κB (NFκB) and the interactions between EGFR and PKCδ in various pathological conditions. METHODS: Human esophageal biopsies were obtained from 93 patients with a past history of smoking and alcohol consumption: 20 showed normal mucosa, 40 with dysplasia and 33 squamous cell carcinoma (SCC). These pathological conditions were analyzed immunohistochemically for the presence of EGFR expression and then subsequently analyzed using immunoblot and immunoprecipitation. RESULTS: A statistically significant difference of EGFR overexpression was found between low- and high-grade dysplasia and carcinoma (χ² = 3.3, χ² = 3.42: P = 0.07, 0.33). A statistical significance was observed between dysplasia and SCC and in all histopathological types (χ² = 4, χ² = 4.9; P < 0.05, P = 0.18 and χ² = 26.3, 26.6; P < 0.001). EGFR tyrosine phosphorylation and its association with PKCδ was significantly higher in all histopathological types with χ² = 7.965; P < 0.05 and 4.0830; P = 0.2530. CONCLUSION: Altogether, our findings reveal that the activation of EGFR and its subsequent interaction with PKCδ under inflammatory conditions might positively be attributed to the transformation of normal esophageal epithelia to SCC, which could explain ongoing inflammation in normal mucosa in a population prone to smoking and alcoholism.

Neutralization of radical toxicity by temperature-dependent modulation of extracellular SOD activity in coral bleaching pathogen Vibrio shiloi and its role as a virulence factor.

Vibrio shiloi is the first and well-documented bacterium which causes coral bleaching, particularly, during summer, when seawater temperature is between 26 and 31 degrees C. Coral bleaching is the disruption of the symbiotic association between coral hosts and their photosynthetic microalgae zooxanthellae. This is either due to lowered resistance in corals to infection or increased virulence of the bacterium at the higher sea surface temperature. The concentration of the oxygen and resulting oxygen radicals produced by the zooxanthellae during photosynthesis are highly toxic to bacteria, which also assist corals in resisting the infection. Hence, in this study we examined the effect of different temperatures on the activity of a novel extracellular SOD in V. shiloi. We also partially characterized the SOD and clearly confirmed that the extracellular SOD produced by V. shiloi is Mn-SOD type, as it was not inhibited by H2O2 or KCN. Performing chemical susceptibility killing assay, we confirmed that extracellular SOD may act as first line of defense for the bacteria against the reactive oxygen species. Since, increased activity of novel Mn-SOD at higher temperature, leads to the neutralization of radical toxicity and facilitates the survival of V. shiloi. Hence, the extracellular Mn-SOD may be considered as a virulence factor.

Morin fosters apoptosis in experimental hepatocellular carcinogenesis model.

Here we investigated the in vivo effect of morin (500ppm in diet) in fostering apoptosis in diethylnitrosamine (DEN) (200mg/kg bodyweight) mediated experimental hepatocellular carcinogenesis model. We analyzed the expression of cytosolic protein Akt and their important apoptotic downstream targets like caspase-9, Bcl-2, Bax, GSK-3betain vivo, by immunoblot analysis. In silico docking studies indicated that morin could serve as a better inhibitor than the classical PI3K inhibitor LY294002. The results obtained from in vivo studies confirm this. We also demonstrate here that morin's interaction with a defined set of amino acids of PI3K p110gamma catalytic subunit resulted in the down-regulation of p-Akt(Ser473), p-Akt(Thr308) and total Akt causing the attenuation of its downstream targets in DEN-induced hepatocellular carcinoma. Further, morin caused the up-regulation of tumor suppressor PTEN, an important negative regulator of Akt, thus initiating apoptosis. Supplementation of morin to experimental animals modulated Bcl-2/Bax ratio causing the release of cyt C and up-regulation of caspase-3 and -9. Morin was also found to prevent the Akt-mediated suppression of GSK-3beta possibly causing cell cycle arrest at the G1/S phase. These observations were supported by the DNA fragmentation and transmission electron microscopy results, which showed the occurrence of apoptosis. In conclusion, our findings demonstrate that morin begets apoptosis in DEN-induced hepatocellular carcinoma.

Differential expression of ompC and ompF in multidrug-resistant Shigella dysenteriae and Shigella flexneri by aqueous extract of Aegle marmelos, altering its susceptibility toward beta-lactam antibiotics.

Steadily increasing resistance among Shigella to beta-lactams, aminoglycosides, and tetracycline has compromised the utility of these commonly used antimicrobial agents. Also, undesirable side effects of certain antibiotics have triggered immense interest in search of alternative therapies using medicinal plants. One such medicinal plant used since ancient times to cure diarrhea is Aegle marmelos. The present study exemplifies the susceptibility of beta-lactam-resistant Shigella dysenteriae and Shigella flexneri toward beta-lactam antibiotics, when grown in the presence of aqueous extract of A. marmelos (AEAM), by altering porin channels. This was demonstrated by antibiotic sensitivity test using disc diffusion method and MIC test. Susceptibility toward beta-lactam antibiotic is associated with changes in outer membrane porins OmpC (approximately 42 kDa) and OmpF (approximately 38 kDa) and cytosolic proteins of approximately 26 kDa, OmpR, a transcriptional regulator. Expression of ompF is increased in S. dysenteriae and S. flexneri grown in the presence of AEAM due to down-regulation of ompR, which is conformed by reverse transcriptase polymerase chain reaction. In conclusion, AEAM influences susceptibility of beta-lactam-resistant Shigella toward beta-lactam antibiotics by altering porin channels. Hence, AEAM along with beta-lactam can be used for treatment of multidrug-resistant Shigella.