Liquid plasma promotes angiogenesis through upregulation of endothelial nitric oxide synthase-induced extracellular matrix metabolism: potential applications of liquid plasma for vascular injuries.

BACKGROUND: Applications of nonthermal plasma have expanded beyond the biomedical field to include antibacterial, anti-inflammatory, wound healing, and tissue regeneration. Plasma enhances epithelial cell repair; however, the potential damage to deep tissues and vascular structures remains under investigation. RESULT: This study assessed whether liquid plasma (LP) increased nitric oxide (NO) production in human umbilical vein endothelial cells by modulating endothelial NO synthase (eNOS) phosphorylation and potential signaling pathways. First, we developed a liquid plasma product and confirmed the angiogenic effect of LP using the Matrigel plug assay. We found that the NO content increased in plasma-treated water. NO in plasma-treated water promoted cell migration and angiogenesis in scratch and tube formation assays via vascular endothelial growth factor mRNA expression. In addition to endothelial cell proliferation and migration, LP influenced extracellular matrix metabolism and matrix metalloproteinase activity. These effects were abolished by treatment with NG-L-monomethyl arginine, a specific inhibitor of NO synthase. Furthermore, we investigated the signaling pathways mediating the phosphorylation and activation of eNOS in LP-treated cells and the role of LKB1-adenosine monophosphate-activated protein kinase in signaling. Downregulation of adenosine monophosphate-activated protein kinase by siRNA partially inhibited LP-induced eNOS phosphorylation, angiogenesis, and migration. CONCLUSION: The present study suggests that LP treatment may be a novel strategy for promoting angiogenesis in vascular damage. Video Abstract.

Peptide fraction from B. jararaca snake venom protects against oxidative stress-induced changes in neuronal PC12 cell but not in astrocyte-like C6 cell.

Venom-derived proteins and peptides have prevented neuronal cell loss, damage, and death in the study of neurodegenerative disorders. The cytoprotective effects of the peptide fraction (PF) from Bothrops jararaca snake venom were evaluated against oxidative stress changes in neuronal PC12 cells and astrocyte-like C6 cells. PC12 and C6 cells were pre-treated for 4 h with different concentrations of PF, and then H2O2 was added (0.5 mM in PC12 cells; 0.4 mM in C6 cells) and incubated for 20 h more. In PC12 cells, PF at 0.78 µg mL-1 increased viability (113.6 ± 6.3%) and metabolism (96.3 ± 10.3%) cell against H2O2-induced neurotoxicity (75.6 ± 5.8%; 66.5 ± 3.3%, respectively), reducing oxidative stress markers such as ROS generation, NO production, and arginase indirect activity through urea synthesis. Despite that, PF showed no cytoprotective effects in C6 cells, but potentiated the H2O2-induced damage at a concentration lower than 0.07 µg mL-1. Furthermore, the role of metabolites derived from L-arginine metabolism was verified in PF-mediated neuroprotection in PC12 cells, using specific inhibitors of two of the key enzymes in the L-arginine metabolic pathway: the α-Methyl-DL-aspartic acid (MDLA) to argininosuccinate synthetase (AsS), responsible for the recycling of L-citrulline to L-arginine; and, L-NΩ-Nitroarginine methyl ester (L-Name) to nitric oxide synthase (NOS), which catalyzes the synthesis of NO from L-arginine. The inhibition of AsS and NOS suppressed PF-mediated cytoprotection against oxidative stress, indicating that its mechanism is dependent on the production pathway of L-arginine metabolites such as NO and, more importantly, polyamines from ornithine metabolism, which are involved in the neuroprotection mechanism described in the literature. Overall, this work provides novel opportunities for evaluating whether the neuroprotective properties of PF shown in particular neuronal cells are sustained and for exploring potential drug development pathways for the treatment of neurodegenerative diseases.

Sulfasalazine for the treatment of preeclampsia in a nitric oxide synthase antagonist mouse model.

INTRODUCTION: Development of a therapeutic that targets the pathophysiological elements of preeclampsia would be a major advance for obstetrics, with potential to save the lives of countless mothers and babies. We recently identified anti-inflammatory drug sulfasalazine as a prospective candidate therapeutic for treatment of preeclampsia. In primary human cells and tissues in vitro, sulfasalazine potently decreased secretion of anti-angiogenic sFlt-1 and sENG, increased production of pro-angiogenic PlGF, mitigated endothelial dysfunction, and promoted whole vessel vasodilation. METHODS: Using nitric oxide synthase antagonist Nω-Nitro-l-arginine methyl ester hydrochloride, a preeclampsia-like phenotype was induced in pregnant mice, including high blood pressure, fetal growth restriction, and elevated circulating sFlt-1. Mice were treated with sulfasalazine or vehicle from gestational day (D)13.5, with blood pressure measurements across gestation, fetal measurements at D17.5, and wire myograph assessment of vasoactivity. RESULTS: Sulfasalazine had a modest effect on blood pressure, decreasing diastolic and mean blood pressure on D13.5, but not later in gestation, or systolic blood pressure. Sulfasalazine was not able to rescue fetal growth, in male or female fetuses. There was a suggestion of improved vasoactivity with sulfasalazine, but further clarification is required. DISCUSSION: In this mouse model of preeclampsia, sulfasalazine did not sustain reductions in blood pressure nor affect fetal parameters of size and weight, both desirable attributes of a viable preeclampsia therapeutic. While these data suggest sulfasalazine might improve vasoactivity, murine toxicity considerations limited the dose range of sulfasalazine that could be tested in the current study.

The Dark Side of an Essential Amino Acid: L-Arginine in Spinal Cord Injury.

L-arginine is a semi-essential amino acid involved in a variety of physiological processes in the central nervous system (CNS). It is essential in the survival and functionality of neuronal cells. Nonetheless, L-arginine also has a dark side; it potentiates neuroinflammation and nitric oxide (NO) production, leading to secondary damage. Therefore, modulating the L-arginine metabolism is challenging because both detrimental and beneficial effects are dependent on this semi-essential amino acid. After spinal cord injury (SCI), L-arginine plays a crucial role in trauma-induced neuroinflammation and regenerative processes via the two key enzymes: nitric oxide synthase (NOS) and arginase (ARG). Studies on L-arginine metabolism using ARG and NOS inhibitors highlighted the conflicting role of this semi-essential amino acid. Similarly, L-arginine supplementation resulted in both negative and positive outcomes after SCI. However, new data indicate that arginine depletion substantially improves spinal cord regeneration after injury. Here, we review the challenging characteristics of L-arginine metabolism as a therapeutic target after SCI.

Investigation of the Effects of Opioids on Microglial Nitrite and Nitric Oxide Synthase (iNOS) Production and Phagocytosis during Inflammation.

AIM: This study aimed to investigate how opioids affect phagocytosis and microglial nitrite and nitric oxide synthase (iNOS) production during inflammation. BACKGROUND: Opioids are a group of chemicals that are naturally found in the opium poppy plant and exert a variety of effects on the brain, including pain alleviation in some cases. They are commonly used in surgery and perioperative analgesia. However, research on the impact of opioids on microglial inflammatory factor production and phagocytosis is limited. OBJECTIVES: This study was designed to investigate the effects of opioids on inducible nitric oxide synthase (iNOS) activity and nitric oxide (NO) generation. Moreover, the influence of opioids on the engulfment of C8-B4 microglial cells after stimulation with LPS was also examined. METHODS: C8-B4 mouse microglial cells were exposed to various concentrations of opioids after stimulation with lipopolysaccharide (LPS) and interferon-γ (IFN-γ). Nitrite production was assayed. The iNOS and Cox-2 were determined by Western blotting, and fluorescent immunostaining was performed to assess the percentage of microglia that engulfed fluorescent microspheres in total microglia cultivating with opioids after being activated by LPS. RESULTS: After LPS and IFN-γ stimulation, microglia produced lower amounts of nitric oxide (NO) production with buprenorphine, salvinorin A, and naloxone (P<0.05). When combined with naloxone, no significant differences were found than buprenorphine. It was observed that buprenorphine and salvinorin A could suppress iNOS expression activated by LPS and IFN-γ. Phagocytosis was greatly increased after LPS stimulation, and a significant increase was observed after adding salvinorin A. CONCLUSION: Buprenorphine and salvinorin A were found to reduce NO production and iNOS induction in microglial cells activated by LPS and IFN-γ. Salvinorin A promoted the phagocytosis of microglia cells treated by LPS.

Tire microplastics exposure in soil induces changes in expression profile of immune-related genes in terrestrial crustacean Porcellio scaber.

Tire particles pose a potential threat to terrestrial organisms because they are deposited in large quantities in the soil by tire wear abrasion, and moreover their chemical complexity poses an additional risk. Microplastics can affect several physiological processes in organisms, including those related to immunity. Therefore, we investigated the expression profile of selected immune-related genes (MnSod, Manganese Superoxide dismutase; Cat, Catalase; CypG, Cyclophilin G; Nos, Nitric oxide synthase; Ppae2a, Prophenoloxidase-activating enzyme 2a; Dscam, Down syndrome cell adhesion molecule; Myd88, Myeloid-differentiation factor 88; Toll4, Toll-like receptor 4; Mas-like, Masquerade-like protein) in haemocytes and the digestive gland hepatopancreas of terrestrial crustacean Porcellio scaber after two different time exposures (4 and 14 days) to tire particles in soil. Our results reveal for the first time the response of P. scaber after microplastic exposure at the transcriptome level. We observed time- and tissue-dependent changes in the expression of the analysed genes, with more pronounced alterations in haemocytes after 14 days of exposure. Some minor changes were also observed in hepatopancreas after 4 days. Changes in the expression profile of the analysed genes are a direct indication of a modulated immune status of the test organism, which, however, does not represent an adverse effect on the test organism under the given conditions. Nevertheless, the question remains whether the observed change in immune status affects the immunocompetence of the test organism.

Nitric oxide synthase inhibition reveals differences in the nitric oxide pathway in previously laminitic ponies.

Previously laminitic (PL) ponies are reported to have higher blood pressure than non laminitic (NL) ponies. This relative hypertension may be related to endothelial cell dysfunction, similar to humans with metabolic syndrome. To investigate the relationship between laminitis predisposition and endothelial dysfunction, the effect of nitric oxide synthase (NOS) inhibition on the change in circulating nitric oxide (NO) concentrations and systemic blood pressure (BP) was determined. An intravenous NO sensor was used to measure changes in blood NO concentration during and after intravenous infusion of the NOS inhibitor L-nitroarginine methyl ester (L-NAME; 4 mg/kg IV) in PL and NL ponies. NO concentrations decreased and BP increased in response to L-NAME infusion, with a significantly (p = 0.02) greater rate of increase in systolic BP in PL (14.62 ± 1.88 mmHg/h) compared to NL (8.54 ± 1.88 mmHg/h) ponies. This greater effect on BP in PL compared to NL ponies is consistent with higher basal NO production in PL ponies, suggesting that the relative hypertension previously seen in PL ponies results in increased basal NO production, rather than being caused by reduced NO production as hypothesised. Up-regulation of the NO system may be a compensatory mechanism stimulated by the higher resting BP in PL ponies. Further investigation of the mechanism underlying the relative hypertension seen in PL ponies is required.

Apigenin attenuates molecular, biochemical, and histopathological changes associated with renal impairments induced by gentamicin exposure in rats.

Gentamicin (GM) is an aminoglycoside antibiotic used to treat bacterial infections. However, its application is accompanied by renal impairments. Apigenin is a flavonoid found in many edible plants with potent therapeutic values. This study was designed to elucidate the therapeutic effects of apigenin on GM-induced nephrotoxicity. Animals were injected orally with three different doses of apigenin (5 mg kg-1 day-1, 10 mg kg-1 day-1, and 20 mg kg-1 day-1). Apigenin administration abolished the alterations in the kidney index and serum levels of kidney-specific functions markers, namely blood urea nitrogen and creatinine, and KIM-1, NGAL, and cystatin C following GM exposure. Additionally, apigenin increased levels of enzymatic (glutathione reductase, glutathione peroxidase, superoxide dismutase, and catalase) and non-enzymatic antioxidant proteins (reduced glutathione) and decreased levels of lipid peroxide, nitric oxide, and downregulated nitric oxide synthase-2 in the kidney tissue following GM administration. At the molecular scope, apigenin administration was found to upregulate the mRNA expression of Nfe2l2 and Hmox1 in the kidney tissue. Moreover, apigenin administration suppressed renal inflammation and apoptosis by decreasing levels of interleukin-1ß, tumor necrosis factor-alpha, nuclear factor kappa-B, Bax, and caspase-3, while increasing B-cell lymphoma-2 compared with those in GM-administered group. The recorded data suggests that apigenin treatment could be used to alleviate renal impairments associated with GM administration.

Effects of electroconvulsive therapy on nitrosative stress and oxidative DNA damage parameters in patients with a depressive episode.

BACKGROUND: Few studies have investigated the relationship between electroconvulsive therapy (ECT) and markers of nitrosative stress and oxidative DNA damage. OBJECTIVE: The aim of this study is to examine changes in nitrosative stress and oxidative DNA damage in patients with a depressive episode treated with ECT. METHODS: The current study included 48 patients with a depressive episode treated with ECT and 30 healthy control participants. First, the serum nitrosative stress markers of nitric oxide (NO•), nitric oxide synthase (NOS), and peroxynitrite (ONOO-) and the oxidative DNA damage marker 8-hydroxy-2'-deoxyguanosine (8-OHdG) were compared between the study and control groups. These parameters were also compared pre- and post-treatment for the study group. RESULTS: NO•, NOS, and ONOO- levels were significantly higher in patients with depressive disorder (DD) than in the control group. NO• and NOS levels significantly decreased in the ECT group after treatment while 8-OHdG levels significantly increased. CONCLUSIONS: The study findings suggest that ECT may have reduced nitrosative stress levels while increasing oxidative DNA damage. More research is now needed to better understand the issue.KEY POINTSNitrosative stress levels can increase in patients with depressive disorder.Electroconvulsive therapy may reduce nitrosative stress while increasing oxidative DNA damage.These results suggest that nitrosative stress plays an important role in the mechanism of action of electroconvulsive therapy.

Dietary L-citrulline influences body temperature and inflammatory responses during nitric oxide synthase inhibition and endotoxin challenge in chickens.

Recent studies have revealed the role of L-citrulline (L-CIT) in thermoregulation, but very little is known about the mechanisms involved. In this study, nitric oxide synthase inhibition and endotoxin-induced fever were used to investigate the effects of L-CIT on body temperature and inflammatory responses. In experiment 1, NW-nitro-L-arginine methyl ester (L-NAME, 150 mg/kg BW), was i. p. injected into chicks fed with basal (CON) or L-CIT diets for 14 days. In experiment 2, Lipopolysaccharide (LPS, 2 mg/kg BW) was i. p. injected following 21d feeding with CON or L-CIT diets. In experiment 3, chickens were injected with either L-NAME, LPS, or L-NAME + LPS following 26 days feeding with CON or L-CIT diets. The rectal (RT), ear (ET), and core body temperature (CBT) of chickens were examined. Results showed that L-NAME effectively decreased the RT, ET, CBT, and plasma NO concentration. In contrast, LPS increased NO levels and initiated hyperthermia by increasing RT, ET, CBT, and PGE2 levels. L-CIT diet reduced the mean CBT in experiment 1 and diminished the NO level, PGE2 level, and mean RT in experiment 3. Co-administration of L-CIT + LPS upregulated IL-6 expression, whereas, LPS injection alone induced IL-10, IL-1ß, and TLR4 gene expressions. Therefore, this study reveals that L-CIT-induced hypothermia was related to NO inhibition and a decrease in PGE2 concentration. Conversely, LPS induced hyperthermia was associated with an increase in both NO and PGE2 concentrations.

The effect of long term nicotine exposure on endothelial function in rats.

Nicotine is one of the main chemicals in the cigarettes responsible for addiction formation. Many researches investigating the effects of nicotine on coronary heart disease and atherosclerosis have been published. The robustness of endothelial cells is very important in the development of atherosclerosis. The aim of this study is to evaluate the effect of nicotine exposure on the indicators of endothelial function either by examining the vascular reactivity of aorta taken from rats exposed to nicotine during prenatal (starting by the mating period) and postnatal periods (6 weeks after delivery), or by determining the protein expression of nitric oxide synthase (NOS) enzymes, NADPH oxidase (Nox) and nitrotyrosine. Chronic nicotine exposure at 6 mg/L in drinking water produced a significant decrease in phenylephrine contractility of thoracic aortic rings compared to control and low dose exposure group (0.4 mg/L, p < 0.001). Endothelium-dependent relaxations to acetylcholine increased dose-dependently while no changes were observed in endothelium-independent relaxations to sodium nitroprusside and protein expressions in rat thoracic aorta. It has been concluded that long term nicotine exposure does not have serious effects on endothelial vasodilator response directly and does not change protein expression of NOS or Nox enzymes. However, more studies should be done for the exact mechanisms responsible for the effect of nicotine on endothelial function.

Angiotensin converting enzyme inhibitor potentiates the hypoglycaemic effect of NG-nitro-L-arginine methyl ester (L-NAME) in rats.

The inhibition of renin angiotensin system pathway has been largely documented to be effective in the control of cardiovascular events. The present study investigated the effect of angiotensin converting enzyme (ACE) inhibitor on fasting blood glucose level in hypertension induced by the inhibition of nitric oxide synthase (NOS) in male Wistar rats. Hypertension was induced by the inhibition of NOS using a non-selective NOS inhibitor, NG-nitro-L-arginine methyl ester (L-NAME). The blockade of NOS resulted in an increase in blood pressure, ACE, angiotensin II and endothelin-1 levels, and a decrease in fasting blood glucose and nitric oxide (NO) levels. The hypertensive rats treated with ACE inhibitor (ramipril) recorded a decrease in blood pressure, ACE, angiotensin II, endothelin-1, NO and fasting blood glucose levels, and an increase in prostacyclin level. In conclusion, ACE inhibitor potentiated the hypoglycaemic effect of NOS inhibitor and this effect is independent of NO and pancreatic insulin release.

Nω-nitro-L-arginine, a nitric oxide synthase inhibitor, attenuates nickel-induced neurotoxicity.

The various mediums of exposure to nickel (Ni) compounds have raised enormous public health concerns, as it has been illustrated to exert toxic effects in biological organs, including the brain. We have previously implicated the involvement of elevated nitric oxide (NO) in Ni-induced oxidative stress in the brain. Hence, the present study investigated the ameliorative potential of Nω-nitro-L-arginine (L-NA), a NO synthase inhibitor, following Ni-induced neurotoxicity. Adult male rats were divided into four groups; control (normal saline), 10 mg/kg Ni chloride (NiCl2) only, 1 mg/kg L-NA, or 2 mg/kg L-NA co-administered with NiCl2. The administration was via daily intraperitoneal injections for three weeks. Neurobehavioural assessments performed thereafter ascertained short-term spatial memory and anxiety. Furthermore, histological evaluations of the cortex, hippocampus, and striatum were carried out using routine hematoxylin and eosin technique, while the phosphotungstic acid hematoxylin method was used to express the degree of astrogliosis. Biochemical analysis of NO levels was examined along with other oxidative stress markers (superoxide dismutase, catalase, glutathione, glutathione S transferase, glutathione peroxidase, myeloperoxidase, and lipid peroxidation). The results illustrated altered behavioral responses, a higher population of degenerating neurons, and astrocytes in the NiCl2 group. There was also an elevation in the NO level and a corresponding reduction in antioxidant activities. However, these debilitating changes were ameliorated in the L-NA treated groups. These results demonstrate an association between alterations in NO synthesis pathway and Ni neurotoxicity, which may render neuronal cells susceptible to damage by oxidative stress. This may yet be another mechanism and useful therapeutic marker in deciphering Ni-induced neurotoxicity.

Nitric Oxide Synthase Potentiates the Resistance of Cancer Cell Lines to Anticancer Chemotherapeutics.

BACKGROUND: Despite the advancement in the fields of medical science and molecular biology, cancer is still the leading cause of death worldwide. Chemotherapy is a choice for treatment; however, the acquisition of chemoresistance is a major impediment for cancer management. Many mechanisms have been postulated regarding the acquisition of chemo-resistance in breast cancer and the impact on cellular signalling and the induction of apoptosis in tumour cells. The mechanism of the apoptotic mutation ofp53 and bcl-2 proteins is commonly associated with increased resistance to apoptosis and, therein, to chemotherapy. OBJECTIVES: The current study was aimed to investigate A172 and MDA-MB-231 cancer cells'sensitivity against chemotherapeutic drugs, including cisplatin, doxorubicin, and paclitaxel with different doses. Moreover, it estimates resistance of cancer cells by evaluating Nitric Oxide Synthase (NOS) expression and evaluate its correlation with the expression profile proteins of the apoptosis regulating Bcl-2 family. METHODS: Dose-dependent sensitivity to cisplatin, doxorubicin or paclitaxel was evaluated on spheroid cultured A172 and MDA-MB-231 cells lines, was measured by time-lapse microscopy over a 72h period. Expressions of two Nitric Oxide (NO) synthases isoforms (iNOS, eNOS), anti-apoptotic (Bcl-2, phospho-Bcl-2, Mcl-1, and Bcl-xL) and proapoptotic (BID, Bim, Bok, Bad, Puma, and Bax) were evaluated by Western blot. The effect of NO modulation on antiand pro-apoptotic molecule expression was also studied using Western blot. RESULTS: A172 cells show more resistance to chemotherapy drugs than MDA-MB-231 cancer cells, therefore, they need higher doses for apoptosis. Resistance of gliomas might be returned to higher significant expression of endothelial eNOS expression. It was clear that there is not a significant effect of NO modulation on the expression of pro- andantiapoptotic proteins on both cell lines. CONCLUSION: The present work provides a putative mechanism for the acquisition of drug resistance in breast cancer and glioma, which might be significant for clinical outcomes.

A Cannabinoid Type 2 (CB2) Receptor Agonist Augments NOS-Dependent Responses of Cerebral Arterioles During Type 1 Diabetes.

INTRODUCTION: While activation of cannabinoid (CB2) receptors has been shown to be neuroprotective, no studies have examined whether this neuroprotection is directed at cerebral arterioles and no studies have examined whether activation of CB2 receptors can rescue cerebrovascular dysfunction during a chronic disease state such as type 1 diabetes (T1D). The goal was to test the hypothesis that administration of a CB2 agonist (JWH-133) would improve impaired endothelial (eNOS)- and neuronal (nNOS)- dependent dilation of cerebral arterioles during T1D. METHODS: In vivo diameter of cerebral arterioles in nondiabetic and diabetic rats was measured in response to an eNOS-dependent agonist (adenosine 5'-diphosphate; ADP), an nNOS-dependent agonist (N-methyl-Daspartate; NMDA), and an NOS-independent agonist (nitroglycerin) before and 1 hour following JWH-133 (1 mg/kg IP). In a second series of experiments, to determine the role of CB2 receptors, rats were injected with AM-630 (3 mg/kg IP). AM-630 has been shown to be a specific antagonist to CB2 receptors. After 30 minutes, the nondiabetic and T1D rats were treated with JWH-133 (1 mg/kg IP). One hour after the injection of JWH-133, responses of arterioles to the agonists were again examined. In a third series of experiments, a potential time-dependency in reactivity of cerebral arterioles to the agonists was examined. Initially responses of arterioles to ADP, NMDA and nitroglycerin were examined. Then, one hour after injection of vehicle (ethanol) for JWH-133 and AM-630 responses of arterioles to the agonists were again examined. RESULTS: Baseline diameter of cerebral arterioles was similar in nondiabetic and T1D rats across all groups of rats. In addition, treatment of the rats with JWH-133, JWH-133 and AM-630 or vehicle (ethanol) did not produce a change in baseline diameter in either nondiabetic or T1D rats. Dilation of cerebral arterioles to ADP and NMDA was greater in nondiabetic than in diabetic rats. Treatment with JWH-133 increased responses of cerebral arterioles to ADP and NMDA in both nondiabetic and diabetic rats. Responses of cerebral arterioles to nitroglycerin were similar between nondiabetic and diabetic rats, and JWH-133 did not influence responses to nitroglycerin in either group. The restoration in responses to the agonists by JWH-133 could be inhibited by treatment with a specific inhibitor of CB2 receptors. CONCLUSIONS: This study showed that acute treatment with a specific activator of CB2 receptors could potentiate dilation of cerebral resistance arterioles to eNOS- and nNOS-dependent agonists in both nondiabetic and T1D rats. In addition, the influence of activation of CB2 receptors on cerebral vascular function could be attenuated by treatment with a specific antagonist of CB2 receptors (AM-630). Based on these findings, one could speculate that treatment with CB2 receptor agonists may have potential therapeutic benefits for the treatment of cerebral vascular disease that can contribute to the pathogenesis of stroke.

Nanosensor Chemical Cytometry for Characterizing the Efflux Heterogeneity of Nitric Oxide from Macrophages.

Macrophages are a critical part of the human immune response, and their collective heterogeneity is implicated in disease progression and prevention. A nondestructive, label-free tool does not currently exist for profiling the dynamic, antigenic responses of single macrophages in a collection to correlate with specific molecular expression and correlated biophysical properties at the cellular level, despite the potential for diagnosis and therapeutics. Herein, we develop a nanosensor chemical cytometry (NCC) that can profile the heterogeneity of inducible nitric oxide synthase (iNOS) responses from macrophage populations. By integrating a near-infrared (nIR) fluorescent nanosensor array and collagen layer with microfluidics, the cellular lensing effect of the macrophage was utilized to characterize both nitric oxide (NO) efflux and refractive index (RI) changes at a single-cell level. Using a parallel, multichannel approach, distinct iNOS heterogeneities of macrophages can be monitored at an attomolar (10-18 mol) sensitivity in a nondestructive and real-time manner with a throughput of exceeding the 200 cells/frame. We demonstrate that estimated mean NO efflux rates of macrophage populations are elevated from 342 (σ = 199) to 464 (σ = 206) attomol/cell·hr with a 3% larger increase in the heterogeneity, and estimated RI of macrophage decrease from 1.366 (σ = 0.015) to 1.359 (σ = 0.009) with trimodal subpopulations under lipopolysaccharide (LPS) activation. These measured values are also in good agreement with Griess assay results and previously reported measurements. This work provides an efficient strategy for single-cell analysis of macrophage populations for cellular manufacturing and biopharmaceutical engineering.

Modulation of Nitric Oxide Bioavailability Attenuates Ischemia-Reperfusion Injury in Type II Diabetes.

BACKGROUND: Diabetes mellitus increases the susceptibility of free tissue transplantations to ischemia-reperfusion injury. The aim of this study was to enhance nitric oxide (NO) bioavailability through exogenous NO synthase and the substrate L-arginine to attenuate ischemia reperfusion-induced alterations in a type 2 diabetes rodent model. MATERIAL AND METHODS: Sixty-four Wistar rats were divided into 8 experimental groups. Type 2 diabetes was established over 3 months with a combination of a high-fat diet and streptozotocin. A vascular pedicle isolated rat skin flap model that underwent 3 h of ischemia was used. At 30 min before ischemia, normal saline, endothelial NOSs (eNOSs), inducible NOSs, neuronal NOSs (1 and 2 IU), and L-arginine (50 mg/kg body weight) were administered by intravenous infusion alone or in combination. Ischemia-reperfusion-induced alterations were measured 5 days after the operation. RESULTS: The three isoforms of NOS significantly increased the flap vitality rate (VR) between 20% and 28% as compared to the control group (3%). Sole L-arginine administration increased the VR to 33%. The combination of L-arginine with NOS resulted in a further increase in flap VRs (39%-50%). Best results were achieved with the combination of eNOS and L-arginine (50%). An increase in enzyme dosage led to decreased VRs in all NOS isoforms alone and even in combination with L-arginine. CONCLUSION: Modulation of NO bioavailability through the exogenous application of NOSs and L-arginine significantly attenuated ischemia-reperfusion-induced alterations in a type 2 diabetic skin flap rat model. The combination of enzyme and substrate result in the highest VRs. Higher enzyme dosage seems to be less effective. This pharmacological preconditioning could be an easy and effective interventional strategy to support the conversion of L-arginine to NO in ischemic and in type 2 diabetic conditions.

Mitochondria-targeted magnolol inhibits OXPHOS, proliferation, and tumor growth via modulation of energetics and autophagy in melanoma cells.

INTRODUCTION: Melanoma is an aggressive form of skin cancer for which there are no effective drugs for prolonged treatment. The existing kinase inhibitor antiglycolytic drugs (B-Raf serine/threonine kinase or BRAF inhibitors) are effective for a short time followed by a rapid onset of drug resistance. PRESENTATION OF CASE: Here, we show that a mitochondria-targeted analog of magnolol, Mito-magnolol (Mito-MGN), inhibits oxidative phosphorylation (OXPHOS) and proliferation of melanoma cells more potently than untargeted magnolol. Mito-MGN also inhibited tumor growth in murine melanoma xenografts. Mito-MGN decreased mitochondrial membrane potential and modulated energetic and mitophagy signaling proteins. DISCUSSION: Results indicate that Mito-MGN is significantly more potent than the FDA-approved OXPHOS inhibitor in inhibiting proliferation of melanoma cells. CONCLUSION: These findings have implications in the treatment of melanomas with enhanced OXPHOS status due to metabolic reprogramming or drug resistance.

Magnolol Ameliorates Behavioral Impairments and Neuropathology in a Transgenic Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is a common neurodegenerative disease characterized by progressive memory loss. Magnolol (MN), the main active ingredient of Magnolia officinalis, possesses anti-AD effects in several experimental models of AD. In this study, we aimed to explore whether MN could ameliorate the cognitive deficits in TgCRND8 transgenic mice and to elucidate its molecular mechanisms. Male TgCRND8 mice were orally administered with MN (20 and 40 mg/kg) daily for 4 consecutive months, followed by assessing the spatial learning and memory functions using the open-field, radial arm maze, and novel object recognition tests. The results demonstrated that MN (20 and 40 mg/kg) could markedly ameliorate the cognitive deficits in TgCRND8 mice. In addition, MN significantly increased the expression of postsynaptic density protein 93 (PSD93), PSD-95, synapsin-1, synaptotagmin-1, synaptophysin (SYN), and interleukin-10 (IL-10), while markedly reduced the protein levels of tumor necrosis factor alpha (TNF-α), IL-6, IL-1ß, Aß 40, and Aß 42, and modulated the amyloid precursor protein (APP) processing and phosphorylation. Immunofluorescence showed that MN significantly suppressed the activation of microglia (Iba-1) and astrocytes (GFAP) in the hippocampus and cerebral cortex of TgCRND8 mice. Mechanistic studies revealed that MN could significantly increase the ratios of p-GSK-3ß (Ser9)/GSK-3ß, p-Akt (Ser473)/Akt, and p-NF-κB p65/NF-κB p65. These findings indicate that MN exerted cognitive deficits improving effects via suppressing neuroinflammation, amyloid pathology, and synaptic dysfunction through regulating the PI3K/Akt/GSK-3ß and NF-κB pathways, suggesting that MN is a promising naturally occurring polyphenol worthy of further developing into a therapeutic agent for AD treatment.

Renal protective effects of vicenin-2 and scolymoside in a mouse model of sepsis

This study was initiated to determine whether 2 structurally related flavonoids found in Cyclopia subternata-vicenin-2 (VCN) and scolymoside (SCL)-could modulate renal functional damage in a mouse model of sepsis, and to elucidate the relevant underlying mechanisms. The potential of VCN and SCL treatment to reduce renal damage induced by cecal ligation and puncture (CLP) surgery in mice was measured via assessment of serum creatinine, blood urea nitrogen (BUN), lipid peroxidation, total glutathione, glutathione peroxidase activity, catalase activity, and superoxide dismutase activity. Treatment with either VCN or SCL resulted in elevated plasma levels of BUN and creatinine, and of protein in the urine of mice with CLP-induced renal damage. Moreover, both VCN and SCL inhibited nuclear factor κB activation and reduced the induction of nitric oxide synthase and excessive production of nitric acid. VCN and SCL treatment also reduced the plasma levels of interleukin-6, and tumor necrosis factor-α, reduced lethality due to CLP-induced sepsis, increased lipid peroxidation, and markedly enhanced the antioxidant defense system by restoring the levels of superoxide dismutase, glutathione peroxidase, and catalase in kidney tissues. The present results suggest that VCN and SCL protect mice from sepsis-triggered renal injury