Toxicological evaluation of therapeutically active zinc oxide nanoflowers in pre-clinical mouse model.

Our earlier reports established that zinc oxide nanoflowers (ZONF) show significant pro-angiogenic properties, where reactive oxygen species, nitric oxide and MAPK-AKT-eNOS cell signaling axis play an essential task. Considering the significance of angiogenesis in healthcare, our research group has recently demonstrated the in vivo therapeutic application of ZONF (10 mg/kg b.w.) for treating peripheral artery disease. Moreover, based on the angio-neural crosstalk between vascular and neuronal systems, we have further demonstrated the neuritogenic and neuroprotective characteristics of pro-angiogenic nanoflowers (10 mg/kg b.w.) for the treatment of cerebral ischemia. However, it is crucial for a therapeutic material to be non-toxic for its practical clinical applications and therefore assessment of its in vivo toxicity and adverse effect is highly important. Herein, for the first time, we investigate a detailed nanotoxicology of therapeutically active ZONF in Swiss albino mice to evaluate their safety profile and comprehend their aspects for future clinical applications. The maximum tolerated dose (MTD) of ZONF was found to be 512.5 mg/kg b.w. which was employed for acute exposure (2 weeks), showing slight toxicity. However, sub-chronic (4 weeks) and long term chronic (8-12 weeks) studies of nanoflowers exhibited their non-toxic nature particularly at lower therapeutic doses (1-10 mg/kg b.w.). Additionally, in depth genotoxicity study revealed that lower therapeutic dose of ZONF (10 mg/kg b.w.) did not exhibit significant toxicity even in genetic level. Overall, the present nanotoxicology of ZONF suggests their high biocompatible nature at therapeutic dose, offering the basis of their future clinical applications in ischemic and other vascular diseases.

Biochanin-A attenuates DHEA-induced polycystic ovary syndrome via upregulation of GDF9 and BMP15 signaling in vivo.

AIMS: Phytoestrogens can act as natural estrogens owing to their structural similarity to human estrogens. Biochanin-A (BCA) is a well-studied phytoestrogen with a wide variety of pharmacological activities, whereas not reported in the most frequently encountered endocrinopathy called polycystic ovary syndrome (PCOS) in women. PURPOSE: This study aimed to investigate the therapeutic effect of BCA on dehydroepiandrosterone (DHEA) induced PCOS in mice. MAIN METHODS: Thirty-six female C57BL6/J mice were divided into six groups: sesame oil, DHEA-induced PCOS, DHEA + BCA (10 mg/kg/day), DHEA + BCA (20 mg/kg/day), DHEA + BCA (40 mg/kg/day), and metformin (50 mg/kg/day). KEY FINDINGS: The results showed a decrease in obesity, elevated lipid parameters, restoration of hormonal imbalances (testosterone, progesterone, estradiol, adiponectin, insulin, luteinizing hormone, and follicle-stimulating hormone), estrus irregular cyclicity, and pathological changes in the ovary, fat pad, and liver. SIGNIFICANCE: In conclusion, BCA supplementation inhibited the over secretion of inflammatory cytokines (TNF-α, IL-6, and IL-1ß) and upregulated TGFß superfamily markers such as GDF9, BMP15, TGFßR1, and BMPR2 in the ovarian milieu of PCOS mice. Furthermore, BCA reversed insulin resistance by increasing circulating adiponectin levels through a negative correlation with insulin levels. Our results indicate that BCA attenuated DHEA-induced PCOS ovarian derangements, which could be mediated by the TGFß superfamily signaling pathway via GDF9 and BMP15 and associated receptors as first evidenced in this study.

Equine immunoglobulin fragment F(ab')2 displays high neutralizing capability against multiple SARS-CoV-2 variants.

Neutralizing antibody-based passive immunotherapy could be an important therapeutic option against COVID-19. Herein, we demonstrate that equines hyper-immunized with chemically inactivated SARS-CoV-2 elicited high antibody titers with a strong virus-neutralizing potential, and F(ab')2 fragments purified from them displayed strong neutralization potential against five different SARS-CoV-2 variants. F(ab')2 fragments purified from the plasma of hyperimmunized horses showed high antigen-specific affinity. Experiments in rabbits suggested that the F(ab')2 displays a linear pharmacokinetics with approximate plasma half-life of 47 h. In vitro microneutralization assays using the purified F(ab')2 displayed high neutralization titers against five different variants of SARS-CoV-2 including the Delta variant, demonstrating its potential efficacy against the emerging viral variants. In conclusion, this study demonstrates that F(ab')2 generated against SARS-CoV-2 in equines have high neutralization titers and have broad target-range against the evolving variants, making passive immunotherapy a potential regimen against the existing and evolving SARS-CoV-2 variants in combating COVID-19.

Ag2[Fe(CN)5NO]-Fabricated Hydrophobic Cotton as a Potential Wound Healing Dressing: An In Vivo Approach.

There have been reports of different types of wound dressings for various functions and purposes. Cotton being one of the most widely used wound dressing material due to its non-toxic, biodegradable, and other properties is used for fabrication as well as in the form of scaffolds for faster and effective wound closure. Our research team has already demonstrated the role of silver nitroprusside nanoparticles (SNPNPs) for wound healing and antibacterial activity. In the current study, we have developed cotton fabric impregnated with SNPNPs (SNPCFs) which remain photo inert and displayed long-term antimicrobial activity due to the surface modification with the silver nitroprusside complex. These SNPCFs were characterized by various analytical techniques (XRD, FTIR, UV spectroscopy, TGA, TEM, FESEM, EDAX, ICP-OES). The fabricated cotton dressings with nanoparticles showed an improved water contact angle (113-130°) than that of bare cotton gauze (60°) and exhibited more antibacterial property in case of both Gram-negative bacteria (Klebsiella aerogenes and Escherichia coli) and Gram-positive bacteria (Pseudomonas aeruginosa and Bacillus subtilis) even after several washings. The biocompatible nature of SNPCFs was assessed by in vivo chorioallantoic membrane assay that showed no obstruction in the formation of blood vessels. The SNPCFs exhibited better wound healing activity compared to the bare cotton and AgCFs as observed in the C57BL6/J mouse. The histopathological investigation reveals increase in re-epithelialization and deposition of connective tissue. The macrophage (M2) counts in SNPCF-treated skin tissues were supportive of more wound healing activity than mice treated with cotton fabric impregnated with chemically synthesized silver nanoparticles. Based on biodistribution analysis using ICP-OES, the data illustrated that a significant amount of silver is absorbed in the skin tissues of mice as compared to the blood and kidney. Furthermore, the absence of silver from the vital organs (heart, liver, and kidney) corroborates our hypothesis that the SNPCFs can act excellently in treating wounds when topically applied over skin. Thereafter, all these results highlight a strong possibility that SNPCFs exemplify the potential as a new antimicrobial and wound healing agent in future times.

Improved delivery of doxorubicin using rationally designed PEGylated platinum nanoparticles for the treatment of melanoma.

Efficient delivery of chemotherapeutic drugs to tumor cells is one of the crucial issues for modern day cancer therapy. In this article, we report the synthesis of poly ethylene glycol (PEG) assisted colloidal platinum nanoparticles (PtNPs) by borohydride reduction method at room temperature. PtNPs are stable at room temperature for more than 2 years and are stable in serum and phosphate buffer (pH = 7.4) solution for one week. PtNPs show biocompatibility in different normal cell lines (in vitro) and chicken egg embryonic model (ex vivo). Further, we designed and fabricated PtNPs-based drug delivery systems (DDS: PtNPs-DOX) using doxorubicin (DOX), a FDA approved anticancer drug. Various analytical techniques were applied to characterize the nanomaterials (PtNPs) and DDS (PtNPs-DOX). This DDS exhibits inhibition of cancer cell (B16F10 and A549) proliferation, observed by different in vitro assays. PtNPs-DOX induces apoptosis in cancer cells observed by annexin-V staining method. Intraperitoneal (IP) administration of PtNPs-DOX shows substantial reduction of tumor growth in subcutaneous murine melanoma tumor model compared to control group with free drug. Up-regulation of tumor suppressor protein p53 and down regulation of SOX2 and Ki-67 proliferation markers in melanoma tumor tissues (as observed by immunofluorescence and western blot analysis) indicates probable molecular mechanism for the anticancer activity of DDS. Considering the in vitro and pre-clinical (in vivo) results in murine melanoma, it is believed that platinum nanoparticle-based drug delivery formulation could be exploited to develop an alternative therapeutic nanomedicine for cancer therapy in the near future.

ß-hCG-induced mutant BRCA1 ignites drug resistance in susceptible breast tissue.

ß-hCG expression in breast cancer is highly controversial with reports supporting both protective and tumorigenic effects. It has also been reported that risk of breast cancer at an early age is increased with full-term pregnancies if a woman is a BRCA1 mutation carrier. We have already demonstrated that BRCA1-defective cells express high levels of ß-hCG and that when BRCA1 is restored, ß-hCG level is reduced. Also, BRCA1 can bind to the promoter and reduce the levels of ß-hCG. ß-hCG induces tumorigenicity in BRCA1-defective cells by directly binding to TGFBRII and induces TGFBRII-mediated cell proliferation. In this study, we analyzed the mechanism of action of ß-hCG on BRCA1 expression and its influence on drug sensitivity in breast cancer cells. We demonstrate that ß-hCG induces mutant BRCA1 protein expression in BRCA1 mutant cells; however, in BRCA1 wild-type cells, ß-hCG reduced wild-type BRCA1 protein expression. Transcriptionally, ß-hCG could induce Slug/LSD1-mediated repression of wild-type and mutant BRCA1 messenger RNA levels. However, ß-hCG induces HSP90-mediated stabilization of mutant BRCA1 and hence the overexpression of mutant BRCA1 protein, resulting in partial restoration of homologous recombination repair of damaged DNA. This contributes to drug resistance to HSP90 inhibitor 17AAG in BRCA1-defective cancer cells. A combination of HSP90 inhibitor and TGFBRII inhibitor has shown to sensitize ß-hCG expressing BRCA1-defective breast cancers to cell death. Targeting the ß-hCG-HSP90-TGFBRII axis could prove an effective treatment strategy for BRCA1-mutated breast tumors.

Delivery of BACE1 siRNA mediated by TARBP-BTP fusion protein reduces ß-amyloid deposits in a transgenic mouse model of Alzheimer's disease.

Systemic delivery of nucleic acids to the central nervous system (CNS) is a major challenge for the development of RNA interference-based therapeutics due to lack of stability, target specificity, non-permeability to the blood-brain barrier (BBB), and lack of suitable carriers. Using a designed bi-functional fusion protein TARBP-BTP in a complex with siRNA, we earlier demonstrated knockdown of target genes in the brain of both AßPP-PS1 (Alzheimer's disease, AD) and wild-type C57BL/6 mice. In this report, we further substantiate the approach through an extended use in AßPP-PS1 mice, which upon treatment with seven doses of ß-secretase AßPP cleaving Enzyme 1 (BACE1) TARBP-BTP:siRNA, led to target-specific effect in the mouse brain. Concomitant gene silencing of BACE1, and consequent reduction in plaque load in the cerebral cortex and hippocampus (greater than 60%) in mice treated with TARBP-BTP:siRNA complex, led to improvement in spatial learning and memory. The study validates the efficiency of TARBP-BTP fusion protein as an efficient mediator of RNAi, giving considerable scope for future intervention in neurodegenerative disorders through the use of short nucleic acids as gene specific inhibitors.

Comparative study of toxicological assessment of yttrium oxide nano- and microparticles in Wistar rats after 28 days of repeated oral administration.

Despite their enormous advantages, nanoparticles (NPs) have elicited disquiet over their safety. Among the numerous NPs, yttrium oxide (Y2O3) NPs are utilised in many applications. However, knowledge about their toxicity is limited, and it is imperative to investigate their potential adverse effects. Therefore, this study explored the effect of 28 days of repeated oral exposure of Wistar rats to 30, 120 and 480 mg/kg body weight (bw) per day of Y2O3 NPs and microparticles (MPs). Before initiation of the study, characterisation of the particles by transmission electron microscopy, dynamic light scattering, Brunauer-Emmett-Teller and laser Doppler velocimetry was undertaken. Genotoxicity was evaluated using the comet and micronucleus (MN) assays. Biochemical markers aspartate transaminase, alanine transaminase, alkaline phosphatase, malondialdehyde, superoxide dismutase, reduced glutathione, catalase and lactate dehydrogenase in serum, liver and kidney were determined. Bioaccumulation of the particles was analysed by inductively coupled plasma optical emission spectrometry. The results of the comet and MN assays showed significant differences between the control and groups treated with 120 and 480 mg/kg bw/day Y2O3 NPs. Significant biochemical alterations were also observed at 120 and 480 mg/kg bw/day. Haematological and histopathological changes were documented. Yttrium (Y) biodistribution was detected in liver, kidney, blood, intestine, lungs, spleen, heart and brain in a dose- and the organ-dependent manner in both the particles. Further, the highest levels of Y were found in the liver and the lowest in the brain of the treated rats. More of the Y from NPs was excreted in the urine than in the faeces. Furthermore, NP-treated rats exhibited much higher absorption and tissue accumulation. These interpretations furnish rudimentary data of the apparent genotoxicity of NPs and MPs of Y2O3 as well as the biodistribution of Y. A no-observed adverse effect level of 30 mg/kg bw/day was found after oral exposure of rats to Y2O3 NPs.

Novel Cellularly Active Inhibitor Regresses DDAH1 Induced Prostate Tumor Growth by Restraining Tumor Angiogenesis through Targeting DDAH1/ADMA/NOS Pathway.

Dimethylarginine dimethylaminohydrolase1 (DDAH1) inhibitors are important therapeutics by virtue of their ability to control nitric oxide (NO) production by elevating asymmetric dimethylarginine (ADMA) levels. In a screening campaign, we identified that DD1E5 (3-amino-6- tert-butyl-N-(1,3-thiazol-2-yl)-4-(trifluoromethyl)thieno[2,3- b]pyridine-2- carboxamide) inhibits the DDAH1 activity both in vitro and in cultured cells. Mechanistic studies found that DD1E5 is a competitive inhibitor (dissociation constant ( Ki) of 2.05 ± 0.15 µM). Enzyme kinetic assays showed time and concentration dependent inhibition of DDAH1 with DD1E5, which shows tight binding with an inactivation rate constant of 0.2756 ± 0.015 M-1 S-1. Treatment of cancer cells with DDAH1 inhibitors shows inhibition of cell proliferation and a subsequent decrease in NO production with ADMA accumulation. DD1E5 reversed the elevated VEGF, c-Myc, HIF-1α, and iNOS levels induced by exogenous DDAH1 overexpression in PCa cells. Moreover, DD1E5 significantly increased intracellular levels of ADMA and reduced NO production, suggesting its therapeutic potential for cancers in which DDAH1 is upregulated. In in vitro assays, DD1E5 abrogated the secretion of angiogenic factors (bFGF and IL-8) into conditional media, indicating its antiangiogenic potential. DD1E5 inhibited in vivo growth of xenograft tumors derived from PCa cells with DDAH1 overexpression, by reducing tumor endothelial content represented with low CD31 expression. VEGF, HIF-1α, and iNOS expression were reversed in DD1E5 treated tumors compared to respective control tumors. In this work, integrating multiple approaches shows DD1E5 is a promising tool for the study of methylarginine-mediated NO control and a potential therapeutic lead compound against pathological conditions with elevated NO production such as cancers and other diseases.

Acute and subacute oral toxicity of copper oxide nanoparticles in female albino Wistar rats.

The extensive use of copper oxide nanoparticles (CuO-NPs) in various industries and their wide range of applications have led to their accumulation in different ecological niches of the environment. This excess exposure raises the concern about its potential toxic effects on various organisms including humans. However, the hazardous potential of CuO-NPs in the literature is elusive, and it is essential to study its toxicity in different biological models. Hence, we have conducted single acute dose (2000 mg/kg) and multiple dose subacute (30, 300 and 1000 mg/kg daily for 28 days) oral toxicity studies of CuO-NPs in female albino Wistar rats following OECD guidelines 420 and 407 respectively. Blood analysis, tissue aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase and acetylcholinesterase, superoxide dismutase, catalase, lipid malondialdehyde and reduced glutathione assays, and histopathology of the tissues were carried out. The higher dose treatments of the acute and subacute study caused significant alterations in biochemical and antioxidant parameters of the liver, kidney and brain tissues of the rat. In addition, histopathological evaluation of these three organs of treated rats showed significantly high abnormalities in their histoarchitecture when compared to control rats. We infer from the results that the toxicity observed in the liver, kidney and brain of treated rats could be due to the increased generation of reactive oxygen species by CuO-NPs.

Evaluation of high-fat high-fructose diet treatment in factor VIII (coagulation factor)-deficient mouse model.

Non-alcoholic fatty liver disease (NAFLD)-like conditions enhance the production and action of clotting factors in humans. However, studies examining the effect of NAFLD due to high-fat high-fructose (HFHF) diet in factor VIII-deficient (haemophilia A) animals or patients have not been reported previously. In this study, we investigated the individual role of factor VIII in the progression of diet-induced NAFLD in the factor 8-/- (F8-/- ) mouse model system and its consequences on the haemophilic status of the mice. The F8-/- mice were fed with HFHF diet for 14 weeks. Physiological, biochemical, haematological, molecular, pathological, and immune histochemical analyses were performed to evaluate the effect of this diet. The F8-/- mice developed hepatic steatosis after 14 weeks HFHF diet and displayed lower energy metabolism, higher myeloid cell infiltration in the liver, decreased platelet count, upregulated de novo fatty acid synthesis, lipid accumulation, and collagen deposition. This study helps to understand the role of factor VIII in NAFLD pathogenesis and to analyse the severity and consequences of steatosis in haemophilic patients as compared to normal population. This study suggests that haemophilic animals (F8-/- mice) are highly prone to hepatic steatosis and thrombocytopenia.

Ferulic acid protects lipopolysaccharide-induced acute kidney injury by suppressing inflammatory events and upregulating antioxidant defenses in Balb/c mice.

Sepsis-induced acute kidney injury (AKI) is responsible for 70-80% mortality in intensive care patients due to elevated levels of endotoxin, Lipopolysaccharide (LPS) caused by gram-negative infections. Ferulic acid (FA), a phenolic phytochemical is known for its renal protection on various induced models of nephrotoxicity. However, the curative effect of FA in LPS-induced AKI is not well studied. This study aimed to investigate the effect of FA on LPS-induced AKI in mice model and to understand the protective mechanisms involved, to provide evidence for FA in the treatment of AKI. Balb/c mice were treated with FA at 50 mg/kg and 100 mg/kg dosages after LPS stimulation (10 mg/kg). At the end of the intervention, we determined the concentrations of serum creatinine and blood urea nitrogen, inflammatory cytokines and histopathological changes in animals. Also, the relative protein expression level of TLR4 mediated NF-κB signaling pathway were studied in kidney tissues. FA treated animals showed upregulation of antioxidant defenses and suppression of inflammatory events by inhibiting TLR-4 mediated NFκB activation. However, LPS alone administered group, resulted in rapid renal damage with increased levels of blood urea nitrogen and modest increase in creatinine; decreased antioxidant defenses and release of inflammatory cytokines. The histopathological analysis also revealed the protective action of the FA against sepsis induced fibrosis and renal damage. Our findings demonstrated that FA exhibits marked protective effects on LPS-induced AKI in mice suggesting its chemopotential role for treating AKI in humans.

Dimethylarginine dimethylaminohydrolase-1 (DDAH1) is frequently upregulated in prostate cancer, and its overexpression conveys tumor growth and angiogenesis by metabolizing asymmetric dimethylarginine (ADMA).

Tissue microarray analysis confirmed higher dimethylarginine dimethylaminohydrolase-1 (DDAH1) expression in prostate cancer (PCa) compared to benign and normal prostate tissues. DDAH1 regulates nitric oxide (NO) production by degrading endogenous nitric oxide synthase (NOS) inhibitor, asymmetric dimethylarginine (ADMA). This study examined whether DDAH1 has any physiological role in PCa progression. Using overexpression of DDAH1 in PCa (PC3 and LNCaP) cell lines, we found that DDAH1 promotes cell proliferation, migration and invasion by lowering ADMA levels, as well as increasing NO production. VEGF, HIF-1α and iNOS were upregulated in DDAH1 expressing cells as result of elevated NO. DDAH1 increased secretion of pro-angiogenic signals bFGF and IL-8, into conditioned media. Treatment of DDAH1-positive PCa cells with NOS inhibitors (L-NAME and 1400 W) attenuated DDAH1 activity to promote cell growth. Xenografts derived from these cells grew significantly faster (> twofold) than those derived from control cells. Proliferation rate of cells stably expressing mutant DDAH1 was same as control cells unlike wild-type DDAH1-positive PCa cells. Xenograft tumors derived from mutant-positive cells did not differ from control tumors. VEGF, HIF-1α and iNOS expression did not differ in DDAH1 mutant-positive tumors compared to control tumors, but was upregulated in wild-type DDAH1 overexpressing tumors. Furthermore, CD31 immunostaining on xenograft tissues demonstrated that DDAH1 tumors had high endothelial content than mutant DDAH1 tumors. These data suggest that DDAH1 is an important mediator of PCa progression and NO/DDAH pathway needs to be considered in developing therapeutic strategies targeted at PCa.

Polydatin alleviates alcohol-induced acute liver injury in mice: Relevance of matrix metalloproteinases (MMPs) and hepatic antioxidants.

BACKGROUND: Alcohol, a most commonly consumed beverage, is the foremost cause of liver injury throughout the world. Polydatin, a stilbenoid glucoside, was known to possess antioxidant and anti-inflammatory properties and is being investigated for use in various disorders. PURPOSE: The present study was intended at investigating the hepatoprotective efficacy of polydatin against acute-alcohol induced liver injury model in mice. STUDY DESIGN: C57BL/6 mice were fed with five doses of 50% ethyl alcohol (10ml/kg body weight) to induce acute liver injury. Effect of polydatin against alcohol induced hepatic injury was investigated by giving 50 or 100mg/kg polydatin, orally, for 8 days. METHODS: Serum markers of liver injury, morphology, histology and fibrosis of liver tissue, levels of enzymatic and non-enzymatic antioxidants and the mitochondrial respiratory enzyme activities in liver tissue were investigated. The activities and the protein expression of matrix metalloproteinases (MMP-2 and -9), the expression of NF-κB in the liver tissue were also studied. RESULTS: Polydatin pre-treatment significantly alleviated the alcohol induced hepatic injury by reducing the serum liver injury markers, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), attenuating oxidative stress and restoring antioxidant balance in the hepatic tissue. Simultaneously, polydatin pre-treatment also prevented alcohol induced mitochondrial damage and refurbished the matrix metalloproteinases levels of the hepatic tissue. CONCLUSION: The findings of the present study suggest that polydatin may have a potential benefit in preventing alcohol-induced acute hepatic injury.

Increased sensitivity of BRCA defective triple negative breast tumors to plumbagin through induction of DNA Double Strand Breaks (DSB).

We have earlier shown that Plumbagin (PB) can induce selective cytotoxicity to BRCA1 defective ovarian cancer cells; however, the effect of this molecule in BRCA1 mutated breast cancers has not been analyzed yet. Here, we report that reactive oxygen species (ROS) induced by PB resulted in DNA DSB and activates downstream signaling by ATR/ATM kinases and subsequent apoptosis. PB reduces DNA- dependent protein kinase (DNA-PK) expression and inhibits NHEJ (Non Homologous End Joining) activity in BRCA1 defective breast cancer cells. Also, PB induces apoptosis in two different BRCA1 conditional knock out murine models: MMTV-Cre; BRCA1(Co/Co) and WAP-Cre; BRCA1(Co/Co), at 2 mg/kg body weight, but 32 mg/kg of carboplatin (CN) was needed to induce apoptosis in them. This is the first study where two different tissue specific promoter driven transgenic mice models with BRCA1 exon 11 deletions are used for preclinical drug testing. The apoptosis induced by PB in HR (Homologous Recombination) defective triple negative BRCA1 mutant cell lines and in mouse models occur by inducing ROS mediated DNA DSB. The toxicity profile as compared with CN in transgenic mice provides evidence for PB's safer disposition as a therapeutic lead in breast cancer drug development.

Resveratrol attenuates monocyte-to-macrophage differentiation and associated inflammation via modulation of intracellular GSH homeostasis: Relevance in atherosclerosis.

Monocyte-to-macrophage differentiation promotes an inflammatory environment within the arterial vessel wall that causes a mal-adaptive immune response, which contributes to the progression of atheromatous plaque formation. In the current study, we show that resveratrol, a well-known antioxidant, dose-dependently attenuated phorbol myristate acetate (PMA)-induced monocyte-to-macrophage differentiation, as measured by cell adhesion, increase in cell size, and scavenger receptor expression in THP-1 monocytes. Also, resveratrol significantly inhibited PMA-induced pro-inflammatory cytokine/chemokine and matrix metalloprotease (MMP-9) production. This inhibitory effect of resveratrol on monocyte differentiation results from its ability to restore intracellular glutathione (GSH) status, as resveratrol in the presence of buthionine sulfoximine (BSO) failed to affect monocyte differentiation. Furthermore, PMA-induced monocyte differentiation and inflammation was greatly inhibited when cells were co-treated with N-Acetyl-l-cysteine (NAC), a GSH precursor, while the presence of BSO aggravated these processes. These results also show that resveratrol mediated up-regulation of GSH is due to AMP-activated protein kinase (AMPK)-α activation, as compound C (AMPK inhibitor) treatment drastically depleted intracellular GSH and exacerbated PMA-induced monocyte differentiation and pro-inflammatory cytokine production. More importantly, chronic administration of resveratrol efficiently prevented monocyte infiltration and markedly diminished angiotensin (Ang)-II-induced atheromatous plaque formation in apolipoprotein-E knockout (ApoE(-/-)) mice. We conclude that, intracellular GSH status plays a critical role in regulating monocyte-to-macrophage differentiation and inflammation and resveratrol, by restoring GSH levels, inhibits these processes. Taken together, these results suggest that resveratrol can attenuate atherosclerosis, at least, in part, by inhibiting monocyte differentiation and pro-inflammatory cytokines production.

An Atypical System for Studying Epithelial-Mesenchymal Transition in Hepatocellular Carcinoma.

Intrahepatic and extrahepatic metastases are frequently detected in hepatocellular carcinoma (HCC). Epithelial-mesenchymal transition (EMT) is believed to drive metastasis. There are not many well-established model systems to study EMT in HCC. Here we identified an atypical EMT while characterizing a population of mesenchymal cells in Huh7.5 hepatoma cell cultures. Cells with distinct morphology appeared during geneticin treatment of Huh7.5 cultures. Molecular characterization of geneticin resistant Huh7.5M cells confirmed EMT. Huh7.5M cells expressed cancer stem cell markers. p38MAPK and ERK1/2 were substantially activated in Huh7.5M cells. Their Inhibition elevated E-Cadherin expression with concerted suppression of Vimentin and anchorage independent growth in Huh7.5M cells. TGFß could not induce EMT in Huh7.5 cultures, but enriched mesenchymal populations, similar to geneticin. Huh7.5M cells formed more aggressive solid tumors, primarily comprising cells with epithelial morphology, in nude mice. Canonical EMT-TFs did not participate in this atypical EMT, indicating that the established canonical EMT-TFs do not drive every EMT and there is a dire need to identify additional factors. The system that we characterized is a unique model to study EMT, MET and biphasic TGFß signaling in HCC and offers considerable potential to facilitate more insightful studies on deeper questions in tumor metastasis.

Mitochondria-targeted esculetin alleviates mitochondrial dysfunction by AMPK-mediated nitric oxide and SIRT3 regulation in endothelial cells: potential implications in atherosclerosis.

Mitochondria-targeted compounds are emerging as a new class of drugs that can potentially alter the pathophysiology of those diseases where mitochondrial dysfunction plays a critical role. We have synthesized a novel mitochondria-targeted esculetin (Mito-Esc) with an aim to investigate its effect during oxidative stress-induced endothelial cell death and angiotensin (Ang)-II-induced atherosclerosis in ApoE(-/-) mice. Mito-Esc but not natural esculetin treatment significantly inhibited H2O2- and Ang-II-induced cell death in human aortic endothelial cells by enhancing NO production via AMPK-mediated eNOS phosphorylation. While L-NAME (NOS inhibitor) significantly abrogated Mito-Esc-mediated protective effects, Compound c (inhibitor of AMPK) significantly decreased Mito-Esc-mediated increase in NO production. Notably, Mito-Esc promoted mitochondrial biogenesis by enhancing SIRT3 expression through AMPK activation; and restored H2O2-induced inhibition of mitochondrial respiration. siSIRT3 treatment not only completely reversed Mito-Esc-mediated mitochondrial biogenetic marker expressions but also caused endothelial cell death. Furthermore, Mito-Esc administration to ApoE(-/-) mice greatly alleviated Ang-II-induced atheromatous plaque formation, monocyte infiltration and serum pro-inflammatory cytokines levels. We conclude that Mito-Esc is preferentially taken up by the mitochondria and preserves endothelial cell survival during oxidative stress by modulating NO generation via AMPK. Also, Mito-Esc-induced SIRT3 plays a pivotal role in mediating mitochondrial biogenesis and perhaps contributes to its anti-atherogenic effects.

Fisetin, a dietary flavonoid, ameliorates experimental colitis in mice: Relevance of NF-κB signaling.

Fisetin, a dietary flavonoid, is commonly found in many fruits and vegetables. Although studies indicate that fisetin has an anti-inflammatory property, little is known about its effects on intestinal inflammation. The present study investigated the effects of the fisetin on dextran sulphate sodium (DSS)-induced murine colitis, an animal model that resembles human inflammatory bowel disease. Fisetin treatment to DSS-exposed mice significantly reduced the severity of colitis and alleviated the macroscopic and microscopic signs of the disease. Moreover, fisetin reduced the levels of myeloperoxidase activity, the production of proinflammatory cytokines, tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1ß) and interleukin-6 (IL-6) and the expressions of COX-2 and iNOS in the colon tissues. Further studies revealed that fisetin suppressed the activation of NF-κB (p65) by inhibiting IκBα phosphorylation and NF-κB (p65)-DNA binding activity and attenuated the phosphorylation of Akt and the p38, but not ERK and JNK MAPKs in the colon tissues of DSS-exposed mice. In addition, DSS-induced decline in reduced glutathione (GSH) and the increase in malondialdehyde (MDA) levels were significantly restored by oral fisetin. Furthermore, the results from in vitro studies showed that fisetin significantly reduced the pro-inflammatory cytokine and mediator release and suppressed the degradation and phosphorylation of IκBα with subsequent nuclear translocation of NF-κB (p65) in lipopolysaccharide (LPS)-stimulated mouse primary peritoneal macrophages. These results suggest that fisetin exerts anti-inflammatory activity via inhibition of Akt, p38 MAPK and NF-κB signaling in the colon tissues of DSS-exposed mice. Thus, fisetin may be a promising candidate as pharmaceuticals or nutraceuticals in the treatment of inflammatory bowel disease.

Baicalein alleviates doxorubicin-induced cardiotoxicity via suppression of myocardial oxidative stress and apoptosis in mice.

AIMS: Doxorubicin is a widely used anthracycline derivative anticancer drug. Unfortunately, the clinical use of doxorubicin has the serious drawback of cardiotoxicity. In this study, we investigated whether baicalein, a bioflavonoid, can prevent doxorubicin-induced cardiotoxicity in vivo and we delineated the possible underlying mechanisms. MAIN METHODS: Male BALB/c mice were treated with either intraperitoneal doxorubicin (15 mg/kg divided into three equal doses for 15 days) and/or oral baicalein (25 and 50 mg/kg for 15 days). Serum markers of cardiac injury, histology of heart, parameters related to myocardial oxidative stress, apoptosis and inflammation were investigated. KEY FINDINGS: Treatment with baicalein reduced doxorubicin-induced elevation of serum creatine kinase-MB isoenzyme (CK-MB), lactate dehydrogenase (LDH), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels and ameliorated the histopathological damage. Baicalein restored the doxorubicin-induced decrease in both enzymatic and non-enzymatic myocardial antioxidants and increased the myocardial expression of nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1). Further studies showed that baicalein could inverse the Bax/Bcl-2 ratio, suppress doxorubicin-induced p53, cleaved caspase-3 and PARP expression and prevented doxorubicin-induced DNA damage. Baicalein treatment also interferes with doxorubicin-induced myocardial NF-κB signaling through inhibition of IκBα phosphorylation and nuclear translocation of p65 subunit. Doxorubicin elevated iNOS and nitrites levels were also significantly decreased in baicalein treated mice. However, we did not find any significant change (p>0.05) in the myocardial TNF-α and IL-6 levels in control and treated animals. SIGNIFICANCE: Our finding suggests that baicalein might be a promising molecule for the prevention of doxorubicin-induced cardiotoxicity.