Phytohustil® and root extract of Althaea officinalis L. exert anti-inflammatory and anti-oxidative properties and improve the migratory capacity of endothelial cells in vitro.

Introduction: Althaea officinalis L.'s root extract (REA) has been used as a medicinal plant since ancient times to treat a cough. Applying REA leads to a protective film that induces a faster regeneration of the lesioned laryngopharyngeal mucosa caused by dry coughs. The buccopharyngeal mucosa is a highly vascularized tissue. In this regard, anti-inflammatory/-oxidant phytochemicals that improve the repair of the lesion site, e.g., neovascularization in the wound, are critical for promoting healing. For this reason, it is essential to investigate the effects of Phytohustil® and REA on different cellular components of the mucosa under conditions similar to those found in the injured mucosa. Thus, this in vitro study investigated the anti-inflammatory/oxidative and pro-migratory properties of Phytohustil® cough syrup on vascular endothelial cells. Methods: Human umbilical vein endothelial cells (HUVEC) were pretreated (24 h) with Phytohustil®, its excipients, or REA, followed by incubation with hydrogen peroxide (H2O2; 1 h; pro-oxidative) or with lipopolysaccharides (LPS; 3 h; pro-inflammatory). Viability and cytotoxicity were measured by PrestoBlue® assay. Intracellular reactive oxygen species (ROS) were quantified with 20-70-dichlorofluorescein diacetate (DCFDA). The release of interleukin 6 (IL6) was determined by enzyme-linked immunosorbent assay (ELISA). The migratory capacity of HUVEC was measured using a scratch assay. Results: Our results show that Phytohustil®, its excipients and REA were not cytotoxic. Pretreatment of HUVEC (24 h) with Phytohustil® or REA inhibited the LPS-activated IL6 release. Phytohustil® or REA inhibited the H2O2-induced cytotoxicity and intracellular ROS production. Phytohustil® and REA significantly stimulated wound closure compared to the control. Conclusion: Our data show that Phytohustil® and REA have anti-inflammatory/-oxidant properties and improve the migratory capacity of vascular endothelial cells. These properties may contribute to the healing characteristics of Phytohustil® and support the benefit of Phytohustil® in patient's treatment of irritated oral mucosa.

ICP-MS trace element analysis in serum and whole blood.

Trace elements and minerals are compounds that are essential for the support of a variety of biological functions and play an important role in the formation of and the defense against oxidative stress. Here we describe a technique, allowing sequential detection of the trace elements (K, Zn, Se, Cu, Mn, Fe, Mg) in serum and whole blood by an ICP-MS method using single work-up, which is a simple, quick and robust method for the sequential measurement and quantification of the trace elements Sodium (Na), Potassium (K), Calcium (Ca), Zinc (Zn), Selenium (Se), Copper (Cu), Iron (Fe), Manganese (Mn) and Magnesium (Mg) in whole blood as well as Copper (Cu), Selenium (Se), Zinc (Zn), Iron (Fe), Magnesium (Mg), Manganese (Mn), Chromium (Cr), Nickel (Ni), Gold (Au) and Lithium (Li) in human serum. For analysis, only 100 µl of serum or whole blood is sufficient, which make this method suitable for detecting trace element deficiency or excess in newborns and infants. All samples were processed and analyzed by ICP-MS (Agilent Technologies). The accuracy, precision, linearity and the limit of quantification (LOQ), Limit of Blank (LOB) and the limit of detection (LOD) of the method were assessed. Recovery rates were between 80-130% for most of the analyzed elements; repeatabilities (Cv %) calculated were below 15% for most of the measured elements. The validity of the proposed methodology was assessed by analyzing a certified human serum and whole blood material with known concentrations for all elements; the method described is ready for routine use in biomonitoring studies.

Anti-inflammatory and Anti-oxidative Effects of Phytohustil® and Root Extract of Althaea officinalis L. on Macrophages in vitro.

INTRODUCTION: The medicinal plant marshmallow Althaea officinalis L. (A. officinalis), is used for the treatment of cough since centuries. Application of medicinal extracts of marshmallow roots shows immediate effects like a protective film on the inflamed mucosa. Because the soothing layer reduce irritation of the mucous system, a faster regeneration is supported by defense mechanisms required to protect the respiratory tract from environmental injury. Macrophages (MΦ), which belong to a group of multipurpose defensive cells, provide the first line of defense against mucosal invasive pathogens. The present study was performed to investigate, whether the herbal medicinal product has anti-inflammatory or anti-oxidative effects on pro-inflammatorily activated MΦ or after oxidative stress induction. Special attention should be payed to elucidate the effects of A. officinalis on the mechanism of intracellular defense as well as on migratory capacity of the MΦ. RESULTS: Treatment of PMA-differentiated human THP-1 MΦ with Phytohustil® increased their viability without affecting the cell number. Phytohustil® or root extracts of A. officinalis (REAo) - an active component of Phytohustil® - were able to protect human MΦ against H2O2-induced cytotoxicity and H2O2-induced ROS production. Phytohustil®, REAo or diclofenac used as anti-inflammatory reference substance, inhibited the LPS-induced release of tumor necrosis factor-alpha (TNF-α) as well as of IL6 in MΦ. Treatment with Phytohustil®, its excipients or REAo did not impair the mitochondrial membrane potential (MMP). Finally, Phytohustil® and REAo activated the migratory capacity of MΦ. CONCLUSION: The present in vitro investigations indicate protective, i.e., anti-oxidative and anti-inflammatory effects of REAo and Phytohustil®, additionally improving the migratory capacity of MΦ. These antiinflammatory effects were similar or even better than diclofenac. Thus, our data support and may explain the positive effect of Phytohustil® observed in patients during the therapy of inflamed buccal mucosal membranes or treatment of cough.

In Vitro Effects of St. John's Wort Extract Against Inflammatory and Oxidative Stress and in the Phagocytic and Migratory Activity of Mouse SIM-A9 Microglia.

Introduction: Herbal medicinal plants as Hypericum perforatum L., known as St. John's wort (SJW) have been in use for a long time. SJW that is specifically used for the treatment of depressive disorders. Inflammatory cytokines derived from microglia play an important role in the regulation of the synthesis and reuptake of glutamate and influence synaptic function, morphology and neuronal plasticity. The present study was performed to investigate, whether STW3-VI, a special SJW extract has protective effects on mouse SIM-A9 microglia against cytotoxic and proinflammatory effects of ROS, glutamate, NMDA or cortisol. Additionally, we investigated the effects of SJW on migratory and phagocytic properties of microglia. Results: Pre-treatment (48 h) of microglia with STW3-VI (5 or 10 µg/ml)-in contrast to desipramine-inhibited the H2O2-induced TNF-α release by 20-40%. Pre-treatment (48 h) of microglia with STW3-VI (5 or 10 µg/ml) delayed the 3 or 4 mM H2O2-induced intracellular ROS level by 26.9 and 44.4%, respectively. Furthermore, pre-treatment (48 h) of microglia with STW3-VI (5 µg/ml) - in contrast to desipramine - lowered the glutamate-induced cytotoxicity by 13.2%. Besides, pre-treatment (48 h) of microglia with STW3-VI (5 or 10 µg/ml) or desipramine (5 µM) inhibited the NMDA-induced decrease of the viability by 16.5-28.8% or 12%, respectively. Finally, pre-treatment (48 h) of microglia with STW3-VI (5 or 10 µg/ml)-in contrast to desipramine - reduced the cortisol-induced cytotoxicity by 15.5 and 12.9%. Treatment of microglia with STW3-VI (10 or 100 µg/ml) increased the migratory and the phagocytic capacities by 100 and 40%. Conclusion: Our data provide evidence that STW3-VI-in contrast to desipramine - protects microglia from oxidative stress, NMDA- or glutamate-induced cytotoxicity, and has anti-inflammatory properties that are accompanied by improvement of their migratory and phagocytic capacity. These protective (particularly the anti-inflammatory) properties may be beneficial in the treatment of depressive disorders.

Anti-inflammatory effects of Phytodolor® (STW 1) and components (poplar, ash and goldenrod) on human monocytes/macrophages.

BACKGROUND: Populus tremula L. (Poplar), Fraxinus excelsior L. (ash) and Solidago virgaurea L. (goldenrod) have been used for medicinal purposes through centuries, to treat pain, fever and inflammation, but their mechanisms of action are still not fully understood. The present study was performed to investigate, whether the herbal medicinal product Phytodolor® (STW 1) and its components have anti-inflammatory effects on activated human monocytes and differentiated human macrophages to elucidate their modes of action in comparison with well-known analgesic, non-steroidal anti-inflammatory drug (NSAIDs) as diclofenac. METHODS: Adherent human monocytes obtained from peripheral blood mononuclear cells (PBMCs) were cultured in serum-free medium and pre-treated with 50-100 µg/ml of diclofenac, STW 1, their components, poplar, ash or goldenrod or its combination (0.05% to 2%). Thereafter, monocytes were activated with 0.1 or 1 µg/ml LPS for 24 h. The intracellular expressions of TNF-α or PTGS2 were determined by cell-based ELISA. Apoptotic cells were identified by YO-PRO-1 staining. Protein or total RNA were isolated to perform SDS-PAGE/Western blot and qRT-PCR analyses. PMA-differentiated human THP-1 macrophages were pre-treated with diclofenac (50 µg/ml) or STW1 (0.1%) and afterwards with LPS (1 µg/ml) and the translocation of the intracellular p62 NF-κB subunit was detected by immunofluorescence. RESULTS: STW 1 inhibited the intracellular content of TNF-α and PTGS2 protein, as well as of TNF-α and PTGS2 gene expression and induced apoptosis in LPS-activated human monocytes under serum free conditions. Furthermore, STW 1 inhibited the translocation of the p65 subunit of the redox-regulated NF-κB into the nucleus in LPS-activated human macrophages. CONCLUSION: The present in vitro investigations suggest a significant anti-inflammatory activity of STW 1 and its components by inhibiting pro-inflammatory cytokine as TNF-α and the key enzyme PTGS2 in LPS-activated human monocytes, which is, at least partly mediated through the suppression of NF-κB activation. Our results provide evidence for distinctive anti-inflammatory effects of STW 1 and its components on LPS-activated human monocytes/macrophages and, thus, for the therapeutic use of STW 1 in inflammation and pain related disorders.

Krill Oil-In-Water Emulsion Protects against Lipopolysaccharide-Induced Proinflammatory Activation of Macrophages In Vitro.

BACKGROUND: Parenteral nutrition is often a mandatory therapeutic strategy for cases of septicemia. Likewise, therapeutic application of anti-oxidants, anti-inflammatory therapy, and endotoxin lowering, by removal or inactivation, might be beneficial to ameliorate the systemic inflammatory response during the acute phases of critical illness. Concerning anti-inflammatory properties in this setting, omega-3 fatty acids of marine origin have been frequently described. This study investigated the anti-inflammatory and LPS-inactivating properties of krill oil (KO)-in-water emulsion in human macrophages in vitro. MATERIALS AND METHODS: Differentiated THP-1 macrophages were activated using specific ultrapure-LPS that binds only on the toll-like receptor 4 (TLR4) in order to determine the inhibitory properties of the KO emulsion on the LPS-binding capacity, and the subsequent release of TNF-α. RESULTS: KO emulsion inhibited the macrophage binding of LPS to the TLR4 by 50% (at 12.5 µg/mL) and 75% (at 25 µg/mL), whereas, at 50 µg/mL, completely abolished the LPS binding. Moreover, KO (12.5 µg/mL, 25 µg/mL, or 50 µg/mL) also inhibited (30%, 40%, or 75%, respectively) the TNF-α release after activation with 0.01 µg/mL LPS in comparison with LPS treatment alone. CONCLUSION: KO emulsion influences the LPS-induced pro-inflammatory activation of macrophages, possibly due to inactivation of the LPS binding capacity.

Intraperitoneal oxidative stress in rabbits with papillomavirus-associated head and neck cancer induces tumoricidal immune response that is adoptively transferable.

PURPOSE: How tumors evade or suppress immune surveillance is a key question in cancer research, and overcoming immune escape is a major goal for lengthening remission after cancer treatment. Here, we used the papillomavirus-associated rabbit auricular VX2 carcinoma, a model for studying human head and neck cancer, to reveal the mechanisms underlying the antitumorigenic effects of intraperitoneal oxidative stress following O3/O2-pneumoperitoneum (O3/O2-PP) treatment. EXPERIMENTAL DESIGN: Solid auricular VX2 tumors were induced in immune-competent adult New Zealand White Rabbits. Animals were O3/O2-PP- or sham-treated, after which they underwent tumor ablation upon reaching no-go criteria. CD3(+) tumor-infiltrating lymphocytes (TIL) were evaluated by immunohistochemistry, and expression levels of 84 immune response genes were measured by quantitative real-time PCR. Adoptive transfer of peripheral blood leukocytes (PBL)-derived from animals with tumor regression-into control animals with progressing tumors was implemented to assess acquired tumor resistance functionally. RESULTS: Auricular VX2 tumors regressing after O3/O2-PP treatment exhibited increased levels of CD3(+) TILs; they also exhibited enhanced expression of genes that encode receptors involved in pattern recognition, molecules that are required for antigen presentation and T cell activation, and inflammatory mediators. Adoptive cell transfer of PBLs from donor rabbits with regressing tumors to recipient rabbits with newly implanted VX2 carcinoma resulted in acquired tumor resistance of the host and tumor regression. CONCLUSION: Intraperitoneal oxidative stress effectively converts the immune response against the papillomavirus-associated rabbit VX2 carcinoma from tumor permissive to tumoricidal and leads to a sustainable, adoptively transferable oncolytic immune response.

Cytoprotection by omega-3 fatty acids as a therapeutic drug vehicle when combined with nephrotoxic drugs in an intravenous emulsion: Effects on intraglomerular mesangial cells.

During therapeutic interventions, blood concentrations of intravenously applied drugs are higher, and their onset of pharmacological action is faster than with other routes of drug administration. However, acute drug therapy often produces nephrotoxic side effects, as commonly seen after treatment with Ketorolac or Gentamicin leading to questions about their use, especially for patients at risk for acute renal failure. Omega-6(n-6) and omega-3(n-3) polyunsaturated fatty acids (PUFA) affect eicosanoid metabolism, which plays a role in the regulation of inflammation. Eicosanoids derived from n-6 FA have proinflammatory and immunoactive functions, whereas eicosanoids derived from n-3 PUFA have anti-inflammatory and cytoprotective properties. We hypothesized that providing such injectable drugs with nephrotoxic potential in combination with n3-PUFAs from the outset, might afford rapid cytoprotection of renal cells, given the recent evidence that intravenously administered n3-PUFAs are rapidly incorporated into cell membranes. We used intraglomerular mesangial cells (MES13) that are sensitive to treatment with Ketorolac or Gentamicin instead of proximal tubular cells which do not respond to Ketorolac. We found a significant inhibition of Ketorolac (0.25, 0.5, 1 mM) or Gentamicin (2.5, 5 mM) induced cytotoxicity after pretreatment of MES13 cells with 0.01% of 20%w/v LipOmega-3 Emulsion 9/1, containing 90:10 wt/wt mixture of fish oil derived triglycerides to medium chain triglycerides.

In vitro anti-proliferative effects of the willow bark extract STW 33-I.

The well-known anti-inflammatory and analgesic effects of the phytopharmacon willow bark extract have been attributed to the content of salicin; however, pharmacological studies have shown that salicin alone, despite being involved in its therapeutic action, cannot fully explain its clinical efficacy. In addition to reducing inflammation and pain, acetylsalicylic acid (ASA, CAS 50-78-2), like other synthetic non-steroidal anti-inflammatory drugs (NSAIDs), has been shown to exert anti-proliferative effects and to induce apoptosis in a variety of cell lines, e.g., colon, stomach, and prostate cancer cells. To investigate the mechanism of action and possible anti-proliferative and proapoptotic effects of willow bark, a water extract (STW 33-I) and a polyphenol rich fraction (fraction E) have been tested by using the colon-carcinoma cell line HT-29. Both, STW 33-I and its fraction E showed significant anti-proliferative and (1) Introduction The most well-known component of willow bark extract is salicin, which is metabolized in vivo to salicylic acid. The standardized aqueous willow bark extract STW 33-I, which is an effective analgesic and anti-inflammatory drug, contains 23-26% total salicin derivatives and additionally flavonoids, condensed tannins and polyphenols. Typical representatives of the flavonoids are glycosides of naringenin, isosalipurpuroside or eriodictyol. In vitro experiments have demonstrated for pro-apoptotic effects on HT-29 cancer cells. Related to the salicin content of the willow bark extract, a higher dosage of ASA was needed. Furthermore, compared to ASA and to diclofenac (Diclo, CAS 15307-79-6), the COX-1 and COX-2 mRNA expressions were influenced differently by STW 33-I and fraction E. ASA and Diclo inhibited both the COX-1 and COX-2 mRNA expressions, whereas STW 33-I and its fraction E increased the COX-1 mRNA expression. In addition to the already well-known anti-inflammatory and analgesic effects, willow bark extract has been found to possess anti-proliferative and pro-apoptotic effects similar to NSAIDs. The different influence of willow bark on the COX-1 and COX-2 mRNA expressions in comparison to NSAIDs might be relevant, e.g., for prevention of undesirable side effects such as gastric erosions.

Aberrant insulin receptor signaling and amino acid homeostasis as a major cause of oxidative stress in aging.

The mechanisms leading to the increase in free radical-derived oxidative stress in "normal aging" remains obscure. Here we present our perspective on studies from different fields that reveal a previously unnoticed vicious cycle of oxidative stress. The plasma cysteine concentrations during starvation in the night and early morning hours (the postabsorptive state) decreases with age. This decrease is associated with a decrease in tissue concentrations of the cysteine derivative and quantitatively important antioxidant glutathione. The decrease in cysteine reflects changes in the autophagic protein catabolism that normally ensures free amino acid homeostasis during starvation. Autophagy is negatively regulated by the insulin receptor signaling cascade that is enhanced by oxidative stress in the absence of insulin. This synopsis of seemingly unrelated processes reveals a novel mechanism of progressive oxidative stress in which decreasing antioxidant concentrations and increasing basal (postabsorptive) insulin receptor signaling activity compromise not only the autophagic protein catabolism but also the activity of FOXO transcription factors (i.e., two functions that were found to have an impact on lifespan in several animal models of aging). In addition, the aging-related decrease in glutathione levels is likely to facilitate certain "secondary" disease-related mechanisms of oxidative stress. Studies on cysteine supplementation show therapeutic promise.

Anti-atherosclerotic properties of telmisartan in advanced atherosclerotic lesions in apolipoprotein E deficient mice.

Clinical studies have demonstrated that the inhibition of the renin-angiotensin system (RAS) by either an angiotensin-converting enzyme (ACE)-inhibitor or an angiotensin II receptor type 1 (AT(1))-antagonist reduces cardiovascular disease. The objective of this study was to evaluate underlying mechanisms of the AT(1)-antagonist telmisartan in comparison to the ACE-inhibitor ramipril on advanced atherosclerotic lesions. Thirty-two-week-old apolipoprotein E deficient mice (n=60) exhibiting advanced atherosclerotic lesions were fed a chow diet supplemented with ramipril or telmisartan for 16 weeks. Twenty mice received a standard diet. Mice receiving telmisartan had a 38% and mice receiving ramipril had a 18% reduction in progression of atherosclerotic lesion size within the innominate artery. Signs of plaque instability such as frequency of intra-plaque hemorrhage and size of the necrotic cores were reduced in mice receiving telmisartan. Furthermore, telmisartan-treated mice had fewer macrophages and reduced expression of early growth response gene-1 (Egr-1) within the lesions. Electrophoretic mobility shift assays revealed reduced DNA-binding activity of nuclear factor kappaB (NFkappaB) in the aorta of telmisartan-treated mice. In vitro studies in mouse macrophages demonstrated enhanced promoter activation of the nuclear transcription factor peroxisome proliferators-activated receptor gamma (PPARgamma). Target genes of PPARgamma, such as inducible nitric oxide synthase, NFkappaB and Egr-1, showed reduced activity after telmisartan pretreatment. These data suggest that chronic inhibition of the RAS by telmisartan prevails in reducing advanced atherosclerosis and promoting plaque stability over ramipril, possibly through the reduced activity of the pro-inflammatory transcription factors NFkappaB and Egr-1 and through the activation of PPARgamma.

The deficit in low molecular weight thiols as a target for antiageing therapy.

The popular use of antioxidative vitamins illustrates the growing awareness of oxidative stress as an important hazard to our health and as an important factor in the ageing process. Superoxide radicals and superoxide-derived reactive oxygen species (ROS) are constantly formed in most cells and tissues. To ensure that ROS can function as biological signaling molecules without excessive tissue damage, ROS are typically scavenged by antioxidants such as glutathione and the vitamins A, C, and E. "Oxidative stress" occurs if the production of ROS is abnormally increased or antioxidant concentrations are decreased. Genetic studies in mice, Drosophila, and C.elegans suggested that ageing may be mechanistically linked to oxidative stress. Several manifestations of oxidative stress were shown to increase with age, whereas tissue levels of vitamin E, plasma concentrations of vitamin C, and intracellular glutathione concentrations decrease with age. In at least two independent studies, cysteine supplementation on top of the normal protein diet has shown significant beneficial effects on each of several different parameters relevant to ageing, including skeletal muscle functions. As the quality of life in old age is severely compromised by the loss of skeletal muscle function, and as muscle function can be measured with satisfactory precision, loss of muscle function is one of the most attractive surrogate parameters of ageing. The mechanisms by which a deficit in glutathione and its precursor cysteine contributes to various ageing-related degenerative processes appears to be related largely but not exclusively to the dysregulation of redox-regulated biological signaling cascades.

Gallium-68-DOTA-albumin as a PET blood-pool marker: experimental evaluation in vivo.

Investigations into tumor angiogenesis and antiangiogenic treatment have renewed interest in tumor perfusion. To image tumor blood-pool by PET, suitable tracers are not generally available. In this experimental study, we characterized a 68Ga-labeled 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) conjugate of rat serum albumin (68Ga-DOTA-RSA) in vivo using a generator-produced isotope. Biodistribution was determined in ACI rats after intravenous administration of 3-6 MBq of 68Ga-DOTA-RSA. Three ACI rats were imaged over 1 h by dynamic PET after intravenous administration of 15-25 MBq of 68Ga-DOTA-RSA while the blood-pool activity was recorded simultaneously in a closed extracorporeal loop (ECL) between the carotid artery and the jugular vein. Time-activity curves (TACs) were obtained from volume of interest (VOI) analysis and from the ECL data. Stability and metabolites in plasma and urine were analyzed by size exclusion HPLC (SE-HPLC) 1 h after intravenous injection of 67Ga-DOTA-RSA. Blood radioactivity decreased by 10% and 18% from 10 to 60 min p.i. by biodistribution and PET or ECL, respectively. Tissue sampling between 10 and 60 min p.i. showed slight increases in the uptake of spleen, myocardium, kidney and skeletal muscle while hepatic accretion remained unchanged. Total urinary excretion after 60 min amounted to 9% of the injected dose. HPLC demonstrated a single urinary metabolite corresponding in size to gallium-labeled DOTA. 68Ga-DOTA-RSA is a blood-pool tracer whose physical and biological half-life is well suited for PET. Our findings support clinical imaging using 68Ga-DOTA-labeled human serum albumin (HSA). The generator-produced label makes 68Ga-DOTA-labeled albumin continuously available even to centers lacking an in-house cyclotron.

Effect of thiol antioxidant on body fat and insulin reactivity.

Insulin signaling is enhanced by moderate concentrations of reactive oxygen species (ROS) and suppressed by persistent exposure to ROS. Diabetic patients show abnormally high ROS levels and a decrease in insulin reactivity which is ameliorated by antioxidants, such as N-acetylcysteine (NAC). A similar effect of NAC has not been reported for non-diabetic subjects. We now show that the insulin receptor (IR) kinase is inhibited in cell culture by physiologic concentrations of cysteine. In two double-blind trials involving a total of 140 non-diabetic subjects we found furthermore that NAC increased the HOMA-R index (derived from the fasting insulin and glucose concentrations) in smokers and obese patients, but not in nonobese non-smokers. In obese patients NAC also caused a decrease in glucose tolerance and body fat mass. Simultaneous treatment with creatine, a metabolite utilized by skeletal muscle and brain for the interconversion of ADP and ATP, reversed the NAC-mediated increase in HOMA-R index and the decrease in glucose tolerance without preventing the decrease in body fat. As the obese and hyperlipidemic patients had lower plasma thiol concentrations than the normolipidemic subjects, our results suggest that low thiol levels facilitate the development of obesity. Supplementation of thiols plus creatine may reduce body fat without compromising glucose tolerance.