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Background and Aims: Inflammation and calcification are major factors responsible for degeneration of bioprosthetic valve and other substitute heart valve implantations. The objective of this study was to evaluate the anti-inflammatory and anti-calcification effects of Entelon150® (consisting of grape-seed extract) in a beagle dog model of intravascular bovine pericardium implantation., Methods: In total, 8 healthy male beagle dogs were implanted with a bovine pericardium bilaterally in the external jugular veins and divided into two groups. Animals in the Entelon150® group (n = 4) were treated with 150 mg of Entelon150® twice daily for six weeks after surgery. The negative control (NC) group (n = 4) was treated with 5 ml of saline using the same method. After six weeks, we measured the calcium content, performed histological examination, and performed molecular analysis., Results: The calcium content of implanted tissue in the Entelon150® group (0.56±0.14 mg/g) was significantly lower than that in the NC group (1.48±0.57 mg/g) (p < 0.05). Histopathological examination showed that infiltration of chronic inflammatory cells, such as fibroblasts and macrophages, occurred around the graft in all groups; however, the inflammation level of the implanted tissue in the Entelon150® group was s lower than that in the NC group. Both immunohistochemical and western blot analyses revealed that bone morphogenetic protein 2 expression was significantly attenuated in the Entelon150® group., Conclusions: Our results indicate that Entelon150® significantly attenuates post-implantation inflammation and degenerative calcification of the bovine pericardium in dogs. Therefore, Entelon150® may increase the longevity of the bovine pericardium after intravascular implantation., Competing Interests: The authors report no conflicts of interest including Sokho Kim and Md. Mahbubur Rahman. It is noted that Sokho Kim and Md. Mahbubur Rahmanis employed by KNOTUS Co., Ltd. This does not alter any adherence to PLOS ONE policies on sharing data and materials.

Diabetes Mellitus (DM)-induced bladder dysfunction is predominantly due to the long-term oxidative stress caused by hyperglycemia. Grape seed proanthocyanidin extract (GSPE) has been reported to possess a broad spectrum of pharmacological and therapeutic properties against oxidative stress. However, its protective effects against diabetic bladder dysfunction have not been clarified. This study focuses on the effects of GSPE on bladder dysfunction in diabetic rats induced by streptozotocin. After 8 weeks of GSPE administration, the bladder function of the diabetic rats was improved significantly, as indicated by both urodynamics analysis and histopathological manifestation. Moreover, the disordered activities of antioxidant enzymes (SOD and GSH-Px) and abnormal oxidative stress levels were partly reversed by treatment with GSPE. Furthermore, the level of apoptosis in the bladder caused by DM was decreased following the administration of GSPE according to the Terminal Deoxynucleotidyl Transferase (TdT)-mediated dUTP Nick-End Labeling (TUNEL) assay. Additionally, GSPE affected the expression of apoptosis-related proteins such as Bax, Bcl-2 and cleaved caspase-3. Furthermore, GSPE showed neuroprotective effects on the bladder of diabetic rats, as shown by the increased expression of nerve growth factor (NGF) and decreased expression of the precursor of nerve growth factor (proNGF). GSPE also activated nuclear erythroid2-related factor2 (Nrf2), which is a key antioxidative transcription factor, with the concomitant elevation of downstream hemeoxygenase-1 (HO-1). These findings suggested that GSPE could ameliorate diabetic bladder dysfunction and decrease the apoptosis of the bladder in diabetic rats, a finding that may be associated with its antioxidant activity and ability to activate the Nrf2 defense pathway.

Vibrio cholerae produces cholera toxin (CT), an AB5 protein toxin that is primarily responsible for the profuse watery diarrhea of cholera. CT is secreted into the extracellular milieu, but the toxin attacks its Gsα target within the cytosol of a host cell. Thus, CT must cross a cellular membrane barrier in order to function. This event only occurs after the toxin travels by retrograde vesicular transport from the cell surface to the endoplasmic reticulum (ER). The catalytic A1 polypeptide then dissociates from the rest of the toxin and assumes an unfolded conformation that facilitates its transfer to the cytosol by a process involving the quality control system of ER-associated degradation. Productive intoxication is blocked by alterations to the vesicular transport of CT and/or the ER-to-cytosol translocation of CTA1. Various plant compounds have been reported to inhibit the cytopathic activity of CT, so in this work we evaluated the potential anti-CT properties of grape extract. Two grape extracts currently sold as nutritional supplements inhibited CT and Escherichia coli heat-labile toxin activity against cultured cells and intestinal loops. CT intoxication was blocked even when the extracts were added an hour after the initial toxin exposure. A specific subset of host-toxin interactions involving both the catalytic CTA1 subunit and the cell-binding CTB pentamer were affected. The extracts blocked toxin binding to the cell surface, prevented unfolding of the isolated CTA1 subunit, inhibited CTA1 translocation to the cytosol, and disrupted the catalytic activity of CTA1. Grape extract could thus potentially serve as a novel therapeutic to prevent or possibly treat cholera.

Background: Atherosclerosis is one of the major complications of type 2 diabetic patients (T2DM), leading to morbidity and mortality. Grape seed procyanidin B2 (GSPB2) has demonstrated protective effect against atherosclerosis, which is believed to be, at least in part, a result of its antioxidative effects. The aim of this study is to identify the target protein of GSPB2 responsible for the protective effect against atherosclerosis in patients with DM., Methods and Results: GSPB2 (30 mg/kg body weight/day) were administrated to db/db mice for 10 weeks. Proteomics of the aorta extracts by iTRAQ analysis was obtained from db/db mice. The results showed that expression of 557 proteins were either up- or down-regulated in the aorta of diabetic mice. Among those proteins, 139 proteins were normalized by GSPB2 to the levels comparable to those in control mice. Among the proteins regulated by GSPB2, the milk fat globule epidermal growth factor-8 (MFG-E8) was found to be increased in serum level in T2DM patients; the serum level of MFG-E8 was positively correlated with carotid-femoral pulse wave velocity (CF-PWV). Inhibition of MFG-E8 by RNA interference significantly suppressed whereas exogenous recombinant MFG-E8 administration exacerbated atherogenesis the db/db mice. To gain more insights into the mechanism of action of MFG-E8, we investigated the effects of MFG-E8 on the signal pathway involving the extracellular signal-regulated kinase (ERK) and monocyte chemoattractant protein-1 (MCP-1). Treatment with recombinant MFG-E8 led to increased whereas inhibition of MFG-E8 to decreased expression of MCP-1 and phosphorylation of ERK1/2., Conclusion: Our data suggests that MFG-E8 plays an important role in atherogenesis in diabetes through both ERK and MCP-1 signaling pathways. GSPB2, a well-studied antioxidant, significantly inhibited the arterial wall changes favoring atherogenesis in db/db mice by down-regulating MFG-E8 expression in aorta and its serum level. Measuring MFG-E8 serum level could be a useful clinical surrogate prognosticating atherogenesis in DM patients.

Lung cancer remains the leading cause of cancer-related deaths worldwide, and non-small cell lung cancer (NSCLC) represents approximately 80% of total lung cancer cases. The use of non-toxic dietary phytochemicals can be considered as a chemotherapeutic strategy for the management of the NSCLC. Here, we report that grape seed proanthocyanidins (GSPs) induce apoptosis of NSCLC cells, A549 and H1299, in vitro which is mediated through increased expression of pro-apoptotic protein Bax, decreased expression of anti-apoptotic proteins Bcl2 and Bcl-xl, disruption of mitochondrial membrane potential, and activation of caspases 9, 3 and poly (ADP-ribose) polymerase (PARP). Pre-treatment of A549 and H1299 cells with the caspase-3 inhibitor (z-DEVD-fmk) significantly blocked the GSPs-induced apoptosis of these cells confirmed that GSPs-induced apoptosis is mediated through activation of caspases-3. Treatments of A549 and H1299 cells with GSPs resulted in an increase in G1 arrest. G0/G1 phase of the cell cycle is known to be controlled by cyclin dependent kinases (Cdk), cyclin-dependent kinase inhibitors (Cdki) and cyclins. Our western blot analyses showed that GSPs-induced G1 cell cycle arrest was mediated through the increased expression of Cdki proteins (Cip1/p21 and Kip1/p27), and a simultaneous decrease in the levels of Cdk2, Cdk4, Cdk6 and cyclins. Further, administration of 50, 100 or 200 mg GSPs/kg body weight of mice by oral gavage (5 d/week) markedly inhibited the growth of s.c. A549 and H1299 lung tumor xenografts in athymic nude mice, which was associated with the induction of apoptotic cell death, increased expression of Bax, reduced expression of anti-apoptotic proteins and activation of caspase-3 in tumor xenograft cells. Based on the data obtained in animal study, human equivalent dose of GSPs was calculated, which seems affordable and attainable. Together, these results suggest that GSPs may represent a potential therapeutic agent for the non-small cell lung cancer.