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Macrophages are essential components of the immune system, with significant roles in inflammation modulation. They can be activated into pro-inflammatory M1 or anti-inflammatory M2 phenotypes, depending on their micro-environment. Molecular factors that modulate macrophage polarization are hot targets for therapeutic strategies to counter chronic inflammatory pathological conditions.The current study aimed to elucidate the molecular mechanisms by which Retinoic acid (RA), a potent immunomodulator, suppresses LPS-induced inflammatory response in macrophages.RAW 264.7 macrophages were treated with RA and/or LPS, and analyzed for inflammatory genes and miR-21 by PCR. The roles of miR-21 and NF-ĸB signaling pathway were also assessed by knock-down experiments, immunofluorescence, and ChIP assays.Pretreatment with RA quenched the LPS-induced inflammatory responses, including phagocytosis, ROS generation, and NO production. RA shifted the polarization away from the M1 state by negative regulation of IKKα/β, p65, and miR-21. RA hindered the phosphorylation of IKKα/β, translocation of p65 into the nucleus, and the subsequent upregulation of miR-21. Knock-in and knock-down experiments showed that miR-21 is central for the polarization shift toward the pro-inflammatory M1 state.miR-21 is involved in the LPS-induced pro-inflammatory profile of macrophages and that RA negatively regulates the inflammatory response by targeting NF-ĸB/miR-21 signaling. Our data exposes RA's potential as a pharmacological agent to manipulate miR-21 and counteract hyper-inflammatory response.

Refining nutraceutical conjugated metal nanoparticles (NPs) and understanding their interactions with the cellular micro-environment is necessary for their application in nanomedicine. In the present experiment, we studied the effect of quercetin functionalized gold nanoparticles (AuQurNP) on skin fibroblast and keratinocyte cell migration. Spherical shaped AuQurNPs of 47 nm in size were formed due to the interaction of hydroxyl and carbonyl groups of quercetin with Au atoms as revealed by incremental algorithm-based analysis. AuQurNP containing up to 5 μg l−1 of Au with quercetin (5.2 ± 1.6 ng ml−1) was least toxic to fibroblasts. AuQurNP effectively reduced the generation of intracellular ROS (up to 63%) through free-radical scavenging activity. AuQurNP also enhanced the rate of migration of fibroblasts (24 h) and keratinocytes (20 h) in artificially created wounds. The rate of migration of the cells towards the wound edge was in the order of AuQurNP > control > quercetin > AuNP. AuQurNP also significantly increased the expression of TGFβ1 protein, thereby inducing the downstream SMAD complex (SMAD 2–4). Downregulation of the inhibitory protein SMAD 7 by AuQurNP helped in the nuclear translocation of SMADs 3 and 4. Collectively, the present in vitro study demonstrates the action of AuQurNP on the SMAD family and the interconnected molecular mechanism leading to the cell migration process.

Cardiotoxicity by anthracycline antineoplastic drug doxorubicin is one of the systemic toxicity of the cardiovascular system. The mechanism responsible for doxorubicin cardiotoxicity and lipid metabolism remains elusive. The current study tested the hypotheses that the role of peroxisome proliferator-activated receptor α (PPARα) in the progress of doxorubicin-induced cardiomyopathy and its mechanism behind lipid metabolism. In the present study, male rats were subjected to intraperitoneal injection (5-week period) of doxorubicin with different dosages such as low dosage (1.5 mg/kg body weight) and high dosage (15 mg/kg body weight) to induce doxorubicin cardiomyopathy. Myocardial PPARα was impaired in both low dosage and high dosage of doxorubicin-treated rats in a dose-dependent manner. The attenuated level of PPARα impairs the expression of the genes involved in mitochondrial transporter, fatty acid transportation, lipolysis, lipid metabolism, and fatty acid oxidation. Moreover, it disturbs the reverse triacylglycerol transporter apolipoprotein B-100 (APOB) in the myocardium. Doxorubicin elevates the circulatory lipid profile and glucose. Further aggravated lipid profile in circulation impedes the metabolism of lipid in cardiac tissue, which causes a lipotoxic condition in the heart and subsequently associated disease for the period of doxorubicin treatment. Elevated lipids in the circulation translocate into the heart dysregulates lipid metabolism in the heart, which causes augmented oxidative stress and necro-apoptosis and mediates lipotoxic conditions. This finding determines the mechanistic role of doxorubicin-disturbed lipid metabolism via PPARα, which leads to cardiac dysfunction.

Background More than 140 million people drink arsenic-contaminated groundwater. It is unknown how much arsenic exposure is necessary to cause neurological impairment. Here, we evaluate the relationship between neurological impairments and the arsenic concentration in drinking water (ACDW). Participants and methods A cross-sectional study design was employed. We performed medical examinations of 1867 residents in seven villages in the Thabaung township in Myanmar. Medical examinations consisted of interviews regarding subjective neurological symptoms and objective neurological examinations of sensory disturbances. For subjective neurological symptoms, we ascertained the presence or absence of defects in smell, vision, taste, and hearing; the feeling of weakness; and chronic numbness or pain. For objective sensory disturbances, we examined defects in pain sensation, vibration sensation, and two-point discrimination. We analyzed the relationship between the subjective symptoms, objective sensory disturbances, and ACDW. Results Residents with ACDW ≥ 10 parts per billion (ppb) had experienced a “feeling of weakness” and “chronic numbness or pain” significantly more often than those with ACDW 50 ppb). These data suggest a threshold for the occurrence of peripheral neuropathy due to arsenic exposure, and indicate that the arsenic concentration in drinking water should be less than 10 ppb to ensure human health.

Manufacturing nanoparticles with controlled physicochemical properties using environment-friendly routes have potential to open new prospects for a variety of applications. Accordingly, several approaches have been established for manufacturing metal nanoparticles. Many of these approaches entail the use of hazardous chemicals and could be toxic to the environment, and cannot be used readily for biomedical applications. In the present work, we report a single step bio-friendly approach to formulate gold (Au), silver (Ag), and Au-Ag alloy nanoparticles with desired surface corona and composition using isonicotinylhydrazide (INH) as a reducing agent. INH also functioned as a stabilizing agent by enabling a surface corona around the nanoparticles. Remarkably, within a single step INH could also provide a handle in regulating the composition of Au and Ag in bimetallic systems without any additional chemical modification. The physicochemical and surface properties of the different nanoparticles thus obtained have been examined by analytical, spectroscopic and microscopic techniques. Cell cytotoxicity (release of lactate dehydrogenase), cell viability and intracellular reactive oxygen species (ROS) assays confirmed that the Au, Ag, and Au-Ag bimetallic nanoparticles prepared with INH are biocompatible. Finally, the presence of organic surface corona of INH on the nanoparticles was found to impart nanozyme activity and antimycobacterial sensitivity to the nanoparticles.

Nanomedicine is a new kind of molecular medicine that exploits the ability to control individual atoms and molecules, and associated properties, to generate complex functional drug delivery vehicles, diagnostic and analytical tools for application in medicine. Several types of nanomaterials including liposomes, polymers, metals and metal oxides, and composites are used in nano-drug delivery systems. Further, it has been established that nanomaterials are not benign and that they can affect biological systems at the molecular and cellular levels resulting in detrimental effects. However, over the last decade, considerable efforts have been made to develop suitable in vivo and in vitro toxicity testing assays, and to adapt methods developed originally for bulky materials, to assess nanomaterial-induced toxicity. Thus, the high degree of nanomaterial variables in the context of toxicity testing is a challenge because it is simply impossible to test each nanoparticle’s variant for all its toxicological properties. This leads the chemo-biological interaction upon entry into the biological systems. In addition, nanoparticles may directly interact with proteins, lipids and DNA; resulting in additional biological effects that include protein degradation, membrane damage and mutagenesis. These sources contribute to the production of various oxidants, albeit at different subcellular compartments.

This research aimed to explore the anticancer potential in the protein and hydrolysate protein produced by epiphytic bacteria associated with marine algae on the in vitro model of lung cancer LK-2. Protein was isolated from the bacterium Enterobacter unhas strain SG-A1 in which has an association with brown algae, Sargassum sp. A fractionation method has used in separating the protein. It was used as a dialysis process added by Tris HCl to conduct pre-purified of the fractions, and the pepsin enzyme was used in producing protein hydrolysate (pH 2.0, 37 C) at 3:100 ratio of enzyme-substrate. Pre-screening of toxicity can be conducted using BSLT or Brine Shrimp Lethality Test. The cytotoxic effects of protein and protein hydrolysate on LK-2 cell lines during 16 h were determined by the yellow tetrazolium salt 3-(4, 5- dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The BSLT test revealed that the hydrolysate fractions were very toxic. It showed a higher magnitude of decreasing (significance p < 0.05) in cell viability in LK-2 cells. These findings suggested that the hydrolysate of protein produced by epiphytic bacteria brown algae associated, Sargassum sp. has a high probability of being used as cancer therapy agents.

MicroRNAs can regulate inflammatory responses by modulating macrophage polarization. Although microRNA miR-21 is linked to crucial processes involved in inflammatory responses, its precise role in macrophage polarization is controversial. In this study, we investigated the functional relevance of endogenous miRNA-21 and the role of exosomes. RAW 264.7 macrophages were transfected with miR-21 plasmid, and the inflammatory response was evaluated by flow cytometry, phagocytosis, and real-time PCR analysis of inflammatory cytokines. To understand the signaling pathways' role, the cells were treated with inhibitors specific for PI3K or NFĸB. Exosomes from transfected cells were used to study the paracrine action of miR-21 on naive macrophages. Overexpression of miR-21 resulted in significant upregulation of pro-inflammatory cytokines, pushing the cells towards a pro-inflammatory phenotype, with partial involvement of PI3K and NFĸB signal pathways. The cells also secreted miR-21 rich exosomes, which, on delivery to naive macrophages, caused them to exhibit pro-inflammatory activity. The presence of miR-21 inhibitor quenched the inflammatory response. This study validates the pro-inflammatory property of miR-21 with a tendency to foster an inflammatory milieu. Our findings also reinforce the dual importance of exosomal miR-21 as a biomarker and therapeutic target in inflammatory conditions.

Arsenic is a ubiquitous metalloid compound commonly found in the environment, and it is usually found in combination with sulphur and metals. Arsenic is considered as a therapeutic as well as poisoning agent since ancient times. It causes toxic effects on different organs, mainly the liver. In this review, we focused on the molecular mechanism of arsenic-induced hepatotoxicity. Here we envisaged the bridge between arsenic and hepatotoxicity with particular focus on the level of hepatic enzymes such as ALT, AST, and ALP. Here, we attempted to elucidate the role of arsenic in redox imbalance on increased oxidative stress (elevated level of ROS, MDA and NO) and decreased antioxidant levels such as reduced GSH, catalase, and SOD. Oxidative stress induces mitochondrial dysfunction via apoptosis (AKT-PKB, MAPK, PI3/AKT, PKCδ-JNK, AKT/ERK, p53 pathways), fibrosis (TGF-β/Smad pathway), and necrosis and inflammation (TNF-α, NF-ĸB, IL-1, and IL-6). Along with that, arsenic activates caspases and Bax, decreases Bcl2 through mitochondrial dysfunction, and induces apoptosis regulatory mechanism. We believe the alteration of all these pathways leads to arsenic-induced hepatotoxicity.

Green synthesis of metal-encased nutraceutical nano-hybrids has been a target for research over the last few years. In the present investigation, we have reported temperature dependent facile synthesis of silver nanoparticles using FDA approved c phycocyanin (cPC). The cPC conjugated silver nanoparticles (AgcPCNPs) were characterized by TEM, Zeta Potential, UV-vis, XPS, FTIR, and CD Spectroscopy. The temperature optimization studies suggested the synthesis of stable AgcPCNPs at 40 °C while at higher temperature system shows aggregated appearance. Molecular docking studies predicted the exclusive interaction of C, D, I, and J chains of cPC with the surface of AgNPs. Moreover, AgcPCNPs significantly (p < 0.1 %) counteract the toxic nature of AgNPs on red blood cell by measuring parameters like total RBC count, % hemolysis, % hematocrit, coagulation time, pH, electrolyte concentrations and degree of blood cell lipid peroxidation by the anti-oxidation mechanism. Skin fibroblast in vitro cell migration result suggeststhat AgcPCNPs enhanced the degree of cell movement towards the wound area. Data obtained collectively demonstrate that AgcPCNPs can be a better agent in the dermal wound healing with reduced toxicity with the bi-phasic advantage of cPC as a wound healer and Ag nano-metal as an anti-bacterial agent.

Issues: In the area of regenerative medicine paradigm, theranostic nanomedicine is emerging as a promising prototype. It has the advantage of both imaging and therapeutic functions in the platform of personalized medicine. Recently scientists have developed smart and hybrid forms of biological molecules, as carriers for various applications in diagnostics and therapeutics.

Arsenic is a natural metalloid found in abundance, in the environment. Exposure to arsenic can cause health issues due to its carcinogenic nature. The primary source of arsenic contact is drinking water. Exposure to arsenic in drinking water can cause reproductive dysfunction in males through a reduction in testes weight, accessory sex organ weight, viability, and motility of sperm, epididymal sperm count, decreased gonadotrophins level, decreased testosterone, and steroidogenesis disruption. This review focuses on the mechanisms by which arsenic impairs the quality of semen, based on epidemiological observations in humans, and experimental studies in different biological research models. Arsenic-mediated male reproductive toxicity can be induced by various mechanisms such as inhibition of spermatogenesis, testosterone pathway hinderance, oxidative stress, inflammation, genotoxic effects, activation of heat shock proteins, and activation of a signaling pathway in testes (ERK/AKT/NF-kB signaling pathway), among others. The interplay between the principal mechanisms involved needs to be elucidated further in future since an overall examination of arsenic-mediated male reproductive toxicity is still a deficit.

Physicochemical properties of nanoparticles have noteworthy influence on their interaction with biological systems. In the present study, we have developed gold, silver and their bimetallic alloy nanoparticles of different compositions using plant-derived aglycone flavonoid quercetin as a reducing and stabilizing agent. It is interesting that a simple biomolecule can provide a handle to control the composition in a facile manner besides suitable biomolecular corona. Moreover, the presence of quercetin corona in conjugation with specific metal composition exhibited in-vitro radical scavenging capabilities, inherent in-vitro peroxidase like behaviour and antibacterial activities in selective manner. In addition, the cytotoxic effects of these nanoparticles on skin fibroblast cells (M5S) were assessed by membrane leakage of lactate dehydrogenase (LDH), reactive oxygen species (ROS) generation and cellular morphology analysis. All the nanoparticles were found to have composition and surface corona reliant effects on M5S cells. Furthermore, quercetin capping increased cell number and induced the collagen type-1A mRNA expression, confirming collagen synthesis in cells upon treatment. This study reveals that the investigated biological actions of engineered nanoparticles are driven by metal composition of nanoparticles in addition to their surface corona; and fine-tuning of these properties can open new opportunities for a wide range of applications.

Exposure to arsenic in drinking water can stimulate a diverse number of diseases that originate from impaired lipid metabolism in adipose and glucose metabolism, leading to insulin resistance. Arsenic inhibits differentiation of adipocyte and mediates insulin resistance with diminutive information on arsenicosis on lipid storage and lipolysis. This review focused on different mechanisms and pathways involved in adipogenesis and lipolysis in adipose tissue during arsenic-induced diabetes. Though arsenic is known to cause type2 diabetes through different mechanisms, the role of adipose tissue in causing type2 diabetes is still unclear. With the existing literature, this review exhibits the effect of arsenic on adipose tissue and its signalling events such as SIRT3- FOXO3a signalling pathway, Ras -MAP -AP-1 cascade, PI(3)-K-Akt pathway, endoplasmic reticulum stress protein, C/EBP homologous protein (CHOP10) and GPCR pathway with role of adipokines. There is a need to elucidate the different types of adipokines which are involved in arsenic-induced diabetes. The exhibited information brings to light that arsenic has negative effects on a white adipose tissue (WAT) by decreasing adipogenesis and enhancing lipolysis. Some of the epidemiological studies show that arsenic would causes obesity. Few studies indicate that arsenic might induces lipodystrophy condition. Further research is needed to evaluate the mechanistic link between arsenic and adipose tissue dysfunction which leads to insulin resistance.

Anthropogenic sources of arsenic poses and creates unintentional toxico-pathological concerns to humans in many parts of the world. The understanding of toxicity of this metalloid, which shares properties of both metal and non-metal is principally structured on speciation types and holy grail of toxicity prevention. Visible symptoms of arsenic toxicity include nausea, vomiting, diarrhea and abdominal pain. In this review, we focused on the dermal cell stress caused by trivalent arsenic trioxide and pentavalent arsanilic acid. Deciphering the molecular events involved during arsenic toxicity and signaling cascade interaction is key in arsenicosis prevention. FoxO1 and FoxO2 transcription factors, members of the Forkhead/Fox family, play important roles in this aspect. Like Foxo family proteins, ATM/CHK signaling junction also plays important role in DNA nuclear factor guided cellular development. This review will summarize and discuss current knowledge about the interplay of these pathways in arsenic induced dermal pathogenesis.

Besides physicochemical properties, careful design of biomolecular surface corona can dictate therapeutic potential of nanoparticles. Therefore, we demonstrate a new approach to improve the anticancer potential of gold nanoparticles (AuNPs) by developing a strong corona of curcumin and isonicotinic acid hydrazide (INH) around them. Here, curcumin reduced AuNPsCur are surface-functionalized with biologically active INH (AuNPsCur@INH). Our investigations on human lung squamous carcinoma (LK-2) and human lung fibroblast (TIG-120) cells indicate selective toxicity of functionalized nanoparticles. Conjugation of curcumin and INH on the surface of AuNPs induced anticancer action through reactive oxygen species (ROS) generation. The further extension of this study revealed apoptosis and morphological changes in LK-2 and TIG-120 cells, respectively. Moreover, the anticancer potential of these nanoparticles is compared with standard cisplatin. The outcomes of the work determine that the facile functional surfaces on nanoparticles can be important in controlling the biological activities of nanoparticles.

Osteoporosis is a bone pathology leading to increased fracture risk and challenging the quality of life. The aim of this study was to evaluate the effect of an anthraquinone glycoside, aloin, on osteogenic induction of MC3T3-E1 cells. Aloin increased alkaline phosphatase (ALP) activity, an early differentiation marker of osteoblasts. Aloin also increased the ALP activity in adult human adipose-derived stem cells (hADSC), indicating that the action of aloin was not cell-type specific. Alizarin red S staining revealed a significant amount of calcium deposition in cells treated with aloin. Aloin enhanced the expression of osteoblast differentiation genes, Bmp-2, Runx2 and collagen 1a, in a dose-dependent manner. Western blot analysis revealed that noggin and inhibitors of p38 MAPK and SAPK/JNK signals attenuated aloin-promoted expressions of Bmp-2 and Runx2 proteins. siRNA mediated blocking of Wnt-5a signaling pathway also annulled the influence of aloin, indicating Wnt-5a dependent activity. Inhibition of the different signal pathways abrogated the influence of aloin on ALP activity, confirming that aloin induced MC3T3-E1 cells into osteoblasts through MAPK mediated Wnt and Bmp signaling pathway.

Arsenic exposure through drinking water is a major public health problem. It causes a number of toxic effects on skin. Arsenic has been reported to inhibit cell proliferation in in vitro conditions. However, reports about the molecular mechanisms are limited. Here, we investigated the mechanism involved in arsenic acid-mediated inhibition of cell proliferation using mouse skin fibroblast cell line. The present study found that 10 ppm arsenic acid inhibited cell proliferation, without any effect on cell death. Arsenic acid induced the generation of reactive oxygen species (ROS), resulting in oxidative stress to DNA. It also activated the mammalian Ste20-like protein kinase 1 (MST1); however the serine/threonine kinase Akt was downregulated. Forkhead box O (FOXO) transcription factors are activated through phosphorylation by MST1 under stress conditions. They are inhibited by phosphorylation by Akt through external and internal stimuli. Activation of FOXOs results in their nuclear localization, followed by an increase in transcriptional activity. Our results showed that arsenic induced the nuclear translocation of FOXO1 and FOXO3a, and altered the cell cycle, with cells accumulating at the G2/M phase. These effects caused cellular senescence. Taken together, our results indicate that arsenic acid inhibited cell proliferation through cellular senescence process regulated by MST1-FOXO signaling pathway.

Plastic and polythene as hydrophobic materials become a grave concern due to their non-biodegradable nature, cumbersome recycling and waste management. Cuticular wax derived from Calotropis procera is explored as an eco-friendly and safe hydrophobic material. The effects of duration of exposure to solvent, solvent type, size and side of the leaf on cuticular wax yield have been studied. Leaf with the smallest area (10 cm2–25 cm2) was found to be the most suitable to isolate the wax. GC–MS analysis of the wax revealed that the wax consists of mainly esters, alkane and alkene. Mitochondrial reductase (MTT) and lactate dehydrogenase (LDH) assay have been carried out on M5S cell line at various concentrations and the results indicate that up to 1 μg/ml (acetone as solvent) and 3 μg/ml (chloroform as solvent) use of wax has no toxic effect. To evaluate the hydrophobic potential of the wax in developing hydrophobic paper water regains and contact angle has been measured. The gain in hydrophobicity of the paper is evident from the rise in contact angle (≥90˚) of paper coated with wax. Scanning electron micrograph and FTIR spectra generated physical and chemical evidence of coating of wax on paper.

Osteopenic disorders such as osteoporosis and rheumatoid arthritis are characterized by excessive bone resorption by osteoclasts relative to bone formation by osteoblasts. MicroRNAs are emerging as key players in bone remodeling, modulating the functions of both osteoblasts and osteoclasts. Among them, miR-21 is highly expressed in osteoclast precursors and is known to regulate genesis, differentiation, and apoptosis of osteoclasts. The pro-osteoclastogenic nature of miR-21 makes it a potential candidate as a therapeutic target to treat bone disorders. We had previously demonstrated that anthroglycoside aloin derived from Aloe vera was effective in promoting osteoblastogenesis and inhibiting osteoclastogenesis. The present study investigated the role of miR-21 in aloin’s inhibitory effect on osteoclast differentiation. Aloin effectively suppressed receptor activator of nuclear factor kappa-B (NFĸB) ligand (RankL)-induced miR-21 expression via repression of NFĸB activation. MiR-21 suppression resulted in upregulation of osteoclast suppressor programmed cell death protein 4 (PDCD4), and downregulation of osteoclast marker cathepsin K. Knockdown or gain-of-function studies revealed that miR-21 was pivotal to aloin’s inhibitory effect on osteoclastogenesis. This study also highlights the dynamic potential of aloin as a therapeutic agent to treat osteopenic disorders.

The appearance of Mycobacterium tuberculosis strains leading to drug resistance has caused new problems in TB treatment in various parts of the world and forces WHO to declare TB as a global emergency. With the increase of TB drug resistance, it is convinced that a more effective vaccine development will stop the epidemic of TB. Some M. tuberculosis antigens, one of which is MPT83, have been examined as TB vaccine candidate. MPT83 antigen, which is very immunogenic in lipoprotein micro bacteria, is identified as surface cell interrelated to antigen with cytometry circulation. Having TB resistance from BCG vaccine, MPT83 is considered TB vaccine candidate that can protect people against TB at adult age. The purpose of this research is to conduct amplification of MPT83 antigen cloning, and expression of its antigen on E. coli bacteria. From the result of the research, it is expected that raw material to produce TB vaccine as well as a high-quality antigen can be obtained. The band of DNA in PCR product is 660 bp, while the one in pGEMT-Easy-Mpt83 recombinant plasmid is 3678 bp. This is expressed in E. coli BL21 strain and produces 48 kDa protein as well as GST-MPT83 fusion protein.

Background ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) proteins play an important pathological role in matrix degeneration. Aggrecan degradation is a significant and critical event in early-stage osteoarthritis. To determine the effect of hemoglobin (Hb) on the ability of synovial tissues to produce ADAMTS family members, we examined the influence of Hb by synovial cells in an in vitro experimental system. Methods Synovial tissues were obtained from five young patients with meniscal injury under arthroscopic surgery. Primary cultures of human knee synovial cells were treated with different doses of human Hb (0, 25, 50, 100 μg/ml). The culture media were collected 24 h after Hb-treatment. In the time-course studies, cells were treated with and without 100 μg/ml Hb, and culture media were taken at 6, 12, and 24 h. To identify the proteins responsible for aggrecanase activity, Western blot analysis using antibodies against human ADAMTS-5, −8, −9, and −10; enzyme-linked immunosorbent assay (ELISA); and gene expression for ADAMTS-5 and -9 were examined. Statistical comparisons between each group were performed using paired t-tests. Results Western blot analysis revealed that Hb-treatment resulted in the expression of ADAMTS-5 and -9. Neither control group nor Hb-treated medium showed immunoreactivity against ADAMTS-8 or −10. In a dose-dependency study, the Hb-treated group showed significantly higher levels of ADAMTS-5 and -9 compared with the control (p

Transforming growth factor beta1 (TGFβ1) is a pleiotropic growth factor with a very broad spectrum of effects on wound healing. Chronic non-healing wounds such as diabetic foot ulcers express reduced levels of TGFβ1. On the other hand, our previous studies have shown that the microRNA miR-21 is differentially regulated in diabetic wounds and that it promotes migration of fibroblast cells. Although interplay between TGFβ1 and miR-21 are studied in relation to cancer, their interaction in the context of chronic wounds has not yet been investigated. In this study, we examined if TGFβ1 could stimulate miR-21 in fibroblasts that are subjected to high glucose environment. MiR-21 was, in fact, induced by TGFβ1 in high glucose conditions. The induction by TGFβ1 was dependent on NFκB activation and subsequent ROS generation. TGFβ1 was instrumental in degrading the NFκB inhibitor IκBα and facilitating the nuclear translocation of NFκB p65 subunit. EMSA studies showed enhanced DNA binding activity of NFκB in the presence of TGFβ1. ChIP assay revealed binding of p65 to miR-21 promoter. NFκB activation was also required for the nuclear translocation of Smad 4 protein and subsequent direct interaction of Smad proteins with primary miR-21 as revealed by RNA-IP studies. Our results show that manipulation of TGFβ1-NFκB-miR-21 pathway could serve as an innovative approach towards therapeutics to heal diabetic ulcers.

Hypertension and oxidant stress predispose to the onset and progression of arteriosclerotic diseases. In this study, the components of two kinds of porcine liver hydrolysates (LH-I and LH-II) were analyzed, and the antioxidant effects and angiotensin converting enzyme (ACE)-inhibiting effects of LH-I and LH-II were examined in vitro. Furthermore, the effects of LH-I and LH-II on the blood pressure were examined in spontaneously hypertensive rats (SHR). The results showed that peptides and amino acids accounted for 70% or more of the constituents of both LH-I and LH-II. The results of gel filtration HPLC showed that most of the nitrogen-containing components were peptides or amino acids with molecular weights of 6000 or less. The DPPH radical scavenging activities of LH-I and LH-II were 55.6 and 38.1 μM Trolox Equivalent/g, respectively. The IC(50) values for the ACE-inhibiting activity of LH-I and LH-II were 0.18 and 0.31 mg/mL, respectively. Oral administration of 1 g/rat of LH-I or LH-II to SHR resulted in significant lowering of the blood pressure. These findings indicate that both LH-I and LH-II have antioxidant activity and ACE-inhibiting activity. Moreover, both exerted a blood pressure-lowering effect in SHR. The antioxidant activity and ACE-inhibiting activity of LH-I and LH-II are presumed to be based on the actions of the component peptides.

Arsenic (As) toxicity is a global health problem, affecting millions of people. Exposure to arsenic, mostly via drinking water, has been associated with cancer of skin, lungs, and blood, in addition to several kinds of skin lesions. The present study focused on the effect of arsenic trioxide (As2O3) on normal skin fibroblast cells. Specifically, the effect of As2O3 on ROS generation and oxidative stress was investigated. Proteins involved in the DNA damage signaling pathway and cell cycle were also studied. As2O3 induced the generation of intracellular ROS. Immunohistochemistry analysis revealed a dose-dependent increase in the number of 8-OHdG-positive cells, an indication of oxidative stress. Cell cycle analysis by flow cytometry demonstrated that As2O3 caused a significant percentage of cells to accumulate in the G0/G1 phase with a concomitant reduction in the S phase. Increases in the activated forms of DNA damage signaling proteins, ATM and ATR, and their effector molecules, Chk2 and p53, were also observed. In addition, expression of oncogene p21 was also increased. The study shows that exposure of normal skin fibroblast cells to As2O3 could lead to cell cycle arrest through ATM/ATR and DNA damage signaling pathways. In conclusion, we report here that arsenic trioxide increases cellular oxidative stress leading to shift in cell cycle and leads to DNA damage through ATM/ATR and the CHK-dependent signaling pathway.

1. The present study examined the role of C-phycocyanin (C-pc) in relation to growth factors and cell migration during wound healing. 2. Histological and biochemical studies showed that C-pc scaffold significantly (P < 0.01) increased hydroxyl proline, total hexamine and protein content, and decreased uronic acid content in the wound tissues during a time course study in newly formed skin. 3. Reverse transcription polymerase chain reaction array of mouse growth factors in wound tissue showed overexpression (up to 10-fold) of growth factors, such as Cxcl12, Fgf18, Lefty 1, Lefty 2, Rabep 1 and Zip91, and downregulation (up to -10-fold) of Amh, Bmp 7 and Nodal genes in a 6-day period in C-pc treated groups. Also, Csf 3, Fgf 22, Mdk, Igf 2, transforming growth factor (TGF)-α 1 and interleukin (IL)-1β showed an upregulation of more than 30-fold than the control groups. TGF-β subfamily cytokine growth factors, such as Bmp 2, 4 and 8b, and other growth factors, such as Cxcl 1, showed the highest activity on day 3, showing a transient type of regulation. Western blot analysis showed a positive correlation between gene activity and protein expressions of Bmp 8b, Bmp4, Bmp2 and Cxcl 1. Day 6 in the C-pc group showed the highest csf3 and IL-1β expression. 4. C-pc had no direct effect on keratinocyte migration. However, keratinocytes that were co-cultured with fibroblasts showed a significantly higher rate of migration in the presence of C-pc, showing an indirect effect of C-pc on keratinocyte migration. 5. In conclusion, biodegradable C-pc scaffold might help to serve as an alternate scaffold material for wound healing.

This study aimed to identify a novel angiotensin I-converting enzyme (ACE) inhibitory peptide from porcine skeletal myosin B. Proteins were hydrolyzed with pepsin and the hydrolysates were then subjected to various types of chromatography to isolate the active peptides. The 50% inhibitory concentrations of Lys–Arg–Val–Ile–Gln–Try (M6 a novel peptide) and Val–Lys–Ala–Gly–Phe (A5, a peptide discovered by Ukeda et al. (1991)) were 6.1 and 20.3 μM, respectively. As a result of a homology search, it was determined that the M6 peptide originated from myosin and peptide A5 was of actin origin. M6 is a novel ACE inhibitory peptide, whose activity was shown to be the strongest amongst the previously published myosin-originated peptides. Kinetic evaluations showed that both peptides are competitive inhibitors of ACE. Based on their activity against ACE, M6 was classified as a pro-drug conformer and A5 was classified as a substrate conformer. When both peptides were administered orally to spontaneously hypertensive rats at doses of 10 mg/kg, temporal hypertension was observed after 6 h. This study suggests that M6 and A5 are peptides that may serve several purposes. Based on their remarkable antihypertensive activity, we suggest that M6 and A5 may have potential applications as functional food, which could be used as nutraceutical compounds.

Angiogenesis plays an important role in tumor metastasis and progression, and thus inhibiting angiogenesis is a promising strategy for treatment of cancer. However, tumor-associated angiogenesis is influenced by various angiogenic factors in the tumor microenvironment. Thymidine phosphorylase (TP, EC 2. 4. 2. 4), an enzyme involved in the reversible conversion of thymidine to thymine, is an important mediator of angiogenesis, tumorigenicity, metastasis and invasion. The angiogenic effect of TP requires the enzymatic activity of TP. TP activity is expressed at higher levels in a wide variety of solid tumors than in adjacent non-neoplastic tissue. The tumor microenvironment (hypoxia, acidosis) regulates the expression of TP, and TP expression in tumor tissue shows significant correlation with microvessel density and poor prognosis. 2-Deoxy-D-ribose (D-dRib), one of the degradation products of thymidine generated by TP activity, promotes angiogenesis and the chemotactic activity of endothelial cells and also confers resistance to hypoxia-induced apoptosis in some cancer cell lines. These findings suggest that D-dRib is a downstream mediator of TP function. 2-Deoxy-L-ribose, a stereoisomer of D-dRib, can inhibit D-dRibs anti-apoptotic effects and suppress metastasis and invasion of TP-expressing tumors in mice. Although the mechanism of action of D-dRib is still unknown, the physiological activities of D-dRib have recently been reported by several groups. We review the role of D-dRib in tumor progression and discuss inhibition of D-dRib as a promising approach for chemotherapy of various tumors.

A novel angiotensin-converting enzyme (ACE) inhibitory peptide was isolated and purified from chicken bone extract by enzymatic digestion. The peptide was defined as an ACE inhibitor, and it demonstrated antihypertensive activity following oral administration to spontaneously hypertensive rats (SHRs). The results of this study suggest that peptides derived from an extract of chicken bones, administered orally, have the ability to reduce the blood pressure of SHRs significantly over a short period of time (3 h). Moreover, the blood pressure then remains low for 3 h. This peptide derived from chicken bones may therefore have great value as a short-term remedy for chronic conditions such as high blood pressure. The amino acid sequence of the peptide was YYRA (Tyr-Tyr-Arg-Ala), which was the origin of the Ig heavy chain V region (27-30 position). The IC₅₀ value of its synthetic peptide was 33.9 μg/mL. We suggest that the ACE inhibitory and antihypertensive peptides derived from chicken bone extract may contribute to develop physiologically functional foods or improve food functionality.

Urokinase plasminogen activator (uPA) system is important for several biological processes that call for extracellular proteolysis, fibrinolysis, cell migration, proliferation and angiogenesis. The current study highlights the fibrinolytic and wound healing potential of emodin, an anthraquinone, with relevance to the uPA system. Emodin increased the fibrinolytic activity of fibroblast cells in a dose-dependent manner. Zymography linked the activity to increased uPA activity. Subsequent RT-PCR and western analyses demonstrated uPA gene upregulation. Interestingly, PAI-1, the inhibitor of uPA was also upregulated. EMSA showed the upregulation occurred independent of emodin's effect on nuclear factor kappa B (NFkappaB). The effect on uPA system is supposedly via generation of reactive oxygen species (ROS) since cotreatment with ascorbic acid, an anti-oxidant, attenuated the activity. In addition to profibrinolytic potential, emodin also demonstrated wound healing activity in in vitro wound models. Presence of emodin in the medium enhanced the rate of migration of fibroblasts into the wounded region. These in vitro experiments reveal that emodin is a potent profibrinolytic and wound healing agent.

Wound repair requires both recruitment and well co-ordinated actions of many cell types including inflammatory cells, endothelial cells, epithelial cells and importantly fibroblast cells. Urokinase-type plasminogen activator (uPA) system plays a vital role in wound healing phenomenon. We have previously demonstrated that C-phycocyanin (C-pc), a biliprotein from blue-green algae, transcriptionally regulates uPA through cAMP-dependent protein kinase A (PKA) pathway. To date, a role for C-pc in wound-healing scenario is not elucidated. This study was designed to examine the wound-healing property of C-pc in relation to fibroblast proliferation and migration. C-pc increased fibroblast proliferation in a dose-dependent manner. It also enhanced G1 phase of cell cycle and increased the expressions of cyclin-dependent kinases 1 and 2, which facilitate cell cycle progression, in a uPA-independent manner. In vitro wound healing and migration assays revealed the pro-migratory properties of C-pc. Short-interference RNA studies demonstrated that uPA was necessary for C-pc-induced fibroblast migration. C-pc also significantly elevated the expressions of chemokines (MDC, RANTES, Eotaxin, GRO alpha, ENA78 and TARC) and Rho-GTPases (Cdc 42 and Rac 1) in a uPA-dependent manner. Pre-treatment of C-pc-stimulated cells with pharmacological inhibitor of PI-3K (LY294002) annulled the expression of GTPases implying that Rac 1 and Cdc 42 were induced through PI-3K pathway. C-pc-induced cellular migration towards wounded area was also negatively affected by PI-3K inhibition. In vivo wound-healing experiments in mice validated our finding that C-pc accelerates wound healing. Our data provides conclusive evidence of a novel therapeutic usage for C-pc as a wound-healing agent. C-pc is a food and drug administration (FDA)-approved health supplement. We believe this compound can also be beneficial in healing of internal wounds, such as ulcers.

Soybean isoflavones of genistein and biochanin A, its analogue, promote the activity for generating tissue-plasminogen activator (tPA) from human cervical cancer cells (HeLa S3) and human umbilical vein endothelial cells (HUVEC). At a concentration of 50 µM, each of 14 types of isoflavones were added to HeLa culture solution and incubated. After 24 h, the culture solution was replaced, and then incubated for another 24 h. When fibrinolytic activity was checked in the resulting culture solution using the fibrin plate method, substantial fibrinolytic activity was confirmed for two types of isoflavones. Genistein showed the highest level of fibrinolytic activity at 12.4 times the control, and for biochanin A, an analogue of genistein, the level was 3.5 times the control. Checking fibrinolytic activity and molecular weight of the protein bands separated by zymography, a rise in the protein band concentration in proportion to the concentration of the reagents added was confirmed for the protein band with activity in the same position as the standard reference tPA, which has a molecular weight of about 68 kDa. ELISA also demonstrated that the concentration of tPA in the culture solution was higher than that of plasminogen activator-1. Fibrinolytic activity of HUVEC incubated with 25 µM of biochanin A was much higher than that of the control, which suggests that these soybean isoflavones could have beneficial effects on blood circulation in vivo.

Background Osteoporosis is a bone pathology leading to increased fracture risk and challenging the quality of life. As current treatments can exhibit deleterious side effects, the use of phyto-compounds with therapeutic and preventive activities against orthopaedic related problems represents a promising alternative. Purpose We investigated the effect of aloin, an anthrocyclic compound, on inhibition of osteoclastogenesis using receptor of the nuclear factor κB (NF-κB) ligand (RANKL)-induced RAW264.7 macrophage cells. Study design/methods The inhibitory effect of aloin on in vitro osteoclastogenesis was evaluated by reduction in tartrate-resistant acid phosphatase (TRAP) content and expression levels of osteoclast-specific gene, cathepsin K. Multinuclear formation of osteoclast was assessed with haematoxylin and eosin staining. F4/80 content the marker of the murine monocyte/macrophage cells, was evaluated by immunocytochemistry. The underlining mechanisms were assessed by Western blots and EMSA. Effect of aloin on generation of intracellular reactive oxygen species (ROS) was estimated by dichlorofluorescein diacetate (DCFH-DA). Bone degradation effect was evaluated by bone pit assay. The bone pit culture supernatant was studied by Fluorescein assay. Results We demonstrated that aloin reduced TRAP content and levels of osteoclast-specific gene and protein, cathepsin K. Treatment with aloin (0.75 µM) prevented multinuclear formation (haematoxylin and eosin staining), reduced intracellular TRAP content (TRAP Staining) and increased F4/80 content (F4/80 immunohistochemistry) in RANKL (20 ng/ml) treated RAW cells. Treatment of the RAW cells with aloin suppressed RANKL-induced NF-κB pathway components like IKKα, IKKβ, Phospho.IKK α/β, NF-κB-p65, Phospho NF-κB-p65 and IκBα. EMSA studies showed aloin dose dependently reduced DNA binding activity of NF-κB. Additionally, in vitro bone pit assay revealed that aloin prevented bone degradation and also decreased the fluorescence content in cells, thus confirming the role of aloin in inhibition of osteoclastogenesis . Conclusion Collectively, this study identifies aloin as a potent inhibitor of osteoclastogenesis and bone resorption. The action of aloin was in par with alendronate sodium trihydrate and may provide evidence for its therapeutic potential to treat diseases involving abnormal bone lysis.

c-Phycocyanin (c-pc), a blue coloured, fluorescent protein was purified from blue-green alga, Spirulina fusiformis and its effect on fibrinolytic system in vascular endothelial cells was investigated. The c-pc consisted of two subunits, alpha and beta, whose molecular masses were 16 and 17 kDa, respectively. N-terminal sequences of both subunits were well conserved compared with other blue green algal phycobiliproteins. Fibrinolytic activity in the medium conditioned by calf pulmonary arterial endothelial cells was measured by the fibrin plate method. The c-pc increased the fibrinolytic activity in dose- and time-dependent manners. Fibrin zymographic studies indicated that c-pc-induced urokinase-type plasminogen activator in the cells. These in vitro results suggest that c-pc from S. fusiformis is a potent profibrinolytic protein in the vascular endothelial system.

Intra-articular bleeding causes degradation of articular cartilage leading to joint disorders, but the mechanisms is not well understood. The present study examined the effect of hemoglobin on the ability of synovial tissues to produce plasminogen activators and matrix metalloproteinases that play important roles in the degradation of articular cartilage. Human Hb added to primary cultures of human knee synovial cells markedly increased fibrinolytic activity and gelatinolytic activity. The fibrinolytic activity was due to an increase in uPA activity. Western blot analysis and gelatin zymography indicated that the increased gelatinolytic activity was due to increased MMP-2 and -9. In order to know whether the effect of Hb on cultured synovial tissue is also true in in vivo system or not, rabbit hemoglobin was injected into rabbit knee joints. Coinciding with in vitro study, hemoglobin elicited considerable increase in fibrinolytic and gelatinolytic activity. The level of proteoglycan fragments in the hemoglobin-treated joint fluid was significantly elevated, indicating cartilage matrix degradation. Cartilage damage after hemoglobin treatment was also confirmed by histological study. These findings suggest that hemoglobin stimulates the secretion of uPA, MMP-2 and MMP-9 by synovial tissues, and raise a possible role of hemoglobin in joint damage after intra-articular bleeding.

The proteinase inhibitor, type-1 plasminogen activator inhibitor (PAI-1), is a major regulator of the plasminogen activator system involved in plasmin formation and fibrinolysis. The present study explores the effects of intracellular iron on the expression of PAI-1 and associated cell-surface plasmin activity in human lung fibroblasts; and reports the presence of a novel iron-responsive protein. ELISA revealed a dose-dependent increase in PAI-1 antigen levels expressed in the conditioned medium of cells treated with deferoxamine, in the three cell lines studied. A concomitant increase in mRNA levels was also observed by Northern analyses. Presaturation with ferric citrate quenched the effect of deferoxamine. Experiments with transcription and translation inhibitors on TIG 3-20 cells demonstrated that intracellular iron modulated PAI-1 expression at the post-transcriptional level with the requirement of de-novo protein synthesis. Electrophoretic mobility shift assay and UV crosslinking assays revealed the presence of an approximately 81-kDa nuclear protein that interacted with the 3'-UTR of PAI-1 mRNA in an iron-sensitive manner. Finally, we demonstrated that the increased PAI-1 is functional in suppressing cell-surface plasmin activity, a process that can affect wound healing and tissue remodeling.

This study reports the stability of mRNA of type-1 plasminogen activator inhibitor (PAI-1), the major physiologic inhibitor of plasminogen activation, by deferoxamine-aided iron deprivation, in PC3 adenocarcinoma cells. ELISA and Northern analyses studies revealed dose-dependent increase in PAI-1 expression by deferoxamine-treated cells. Co-treatment with ferric citrate quenched the effect of deferoxamine, confirming the role of iron in PAI-1 regulation. DRB-based RNA chase experiments suggested that post-transcriptional mechanism was involved in PAI-1 regulation. De-novo protein synthesis was necessary for this regulation. Electrophoretic mobility shift assay revealed the presence of a nuclear protein, binding to the 3'-UTR of PAI-1 mRNA in an iron-mediated manner. This is the first report of iron-mediated mRNA-protein interaction in PAI-1, involved in mRNA stability.

In order to obtain basic data on the effect of broad-spectrum protease inhibitor against local symptoms of Viperidae snake envenomation, inhibitory capacity of rat murinoglobulin on local hemorrhagic and edematogenic activities of venoms from Crotalus atrox, Bothrops jararaca, Lachesis muta muta, Trimeresurus flavoviridis and Echis carinatus sochureki were examined. Murinoglobulin, pre-incubated with the crude venoms at 37 °C for 15 min, inhibited hemorrhagic activity of all five venoms to various extents. The activity of C. atrox was almost completely inhibited at the murinoglobulin/venom ratio (w/w) of 20. The activity of B. jararaca, Lachesis muta muta and T. flavoviridis venoms was considerably inhibited at the ratio of 20 (77.2, 80.0 and 86.2% inhibition, respectively), however some of the activity still remained even at the ratio of 40 (84.2, 79.8 and 86.2% inhibition, respectively). Among the five venoms, E. c. sochureki venom is quite resistant to murinoglobulin treatment and statistically significant inhibition was only found at the ratio of 40 (64.1% inhibition). Fibrinolytic and gelatinase activities were more susceptible to murinoglobulin inhibition. The treatment at the ratios of 10 and 20 almost completely inhibited respectively the fibrinolytic and the gelatinase activities of all the venoms. Murinoglobulin treatment also significantly inhibited the edematogenic activity of L. muta muta, T. flavoviridis and Echis carinatus sochureki. The treatment of murinoglobulin at the ratio of 40 considerably suppressed the swelling up to 60 min after subcutaneous injection of L. muta muta and E. c. sochureki venoms, and up to 30 min after T. flavoviridis venom injection. Murinoglobulin is a potent inhibitor against local effects of multiple snake venoms in Viperidae family.

Coagulopathy is one of the major complications following envenomations by crotalid and viperid snakes. The present study was undertaken to examine the effect of a hemorrhagic metalloproteinase in Bothrops jararaca venom, jararafibrase I (JF I), on the development of coagulopathy using rat snakebite model. Coagulation parameters were monitored after subcutaneous injection of B. jararaca crude venom, JF I-neutralized venom and purified JF I in rats. Crude venom induced unclottable blood and fibrinogen consumption, while JF I-neutralized venom and purified JF I did not induce coagulopathy. Plasma venom antigen level of rats given JF I-neutralized venom was lower than that of rats given crude venom. We conclude that venom hemorrhagic metalloproteinases play an important role in the development of coagulopathy through rapid spreading of venom coagulation components from the injected area into systemic circulation.

Background. Although extensive studies on the detailed mechanisms of ischemia-reperfusion injury have been conducted, the implication of the fibrinolytic system has not been known. To determine the role of the fibrinolytic system in ischemia-reperfusion injury, we used tranexamic acid, a synthetic specific plasmin and tissue-type plasminogen activator inhibitor, to suppress fibrinolytic activity in a rabbit lung ischemia-reperfusion model. Methods. New Zealand White rabbits were randomly divided into two groups: a simple ischemia group and a group injected with tranexamic acid before left hilar occlusion. After 2 hours of warm ischemia, plasma was collected from pulmonary vessels. Fibrin zymography was used to ascertain fibrinolytic activity, and enzyme-linked immunosorbent assay was used to determine soluble thrombomodulin levels as a marker for endothelial cells damage. Changes in left pulmonary function including arterial oxygen tension, peak airway pressure, and pulmonary vascular resistance were recorded during reperfusion after the 2 hours of warm ischemia. Results. Fibrinolytic activity and soluble thrombomodulin levels increased in the vessels of the ischemic lung, indicating endothelial cell injury. The increased fibrinolytic activity and the rise in soluble thrombomodulin were suppressed by the preadministration of tranexamic acid, resulting in remarkably improved pulmonary function during reperfusion. After 2 hours of reperfusion, the wet-to-dry weight ratios and histological studies showed reduced pulmonary edema in the group that had received tranexamic acid. Conclusion. These findings suggest that the fibrinolytic system is involved in the onset mechanism of ischemia-reperfusion injury through induced endothelial cell damage and increased vascular permeability.