Nitric Oxide Assay Kit Research Articles

Evaluation of Nitric Oxide Activity of Torilis Leptophyllain Indomethacin Induced Gastric Ulcer in Mice

Objective: To evaluate the Nitric Oxide activity of Torilis leptophylla in Indomethacin-induced gastric ulcers in mice. Study Design: Laboratory-based experimental study Place and Duration of Study: Pharmacology Department, University of Health Sciences, Lahore Pakistan, from Jan to Dec2016. Methodology: Thirty-six adult healthy male BALB/C mice with a weight range of 25-35 g were equally divided into six groups and designated as Group-I (Control), Group-II (Positive Control), Groups III to V (Torilis leptophylla extract trial Groups) and Group-VI (Omeprazole). Gastric ulceration was induced by a single dose of Indomethacin (20mg/kg) in Groups II–VI. The plant extract was administered in Groups III-V (100, 200, 300 mg/kg) whereas Omeprazole (3mg/kg) in Group-VI by gavage daily for three days as treatment of gastric ulceration. Nitric Oxide content was measured in gastric juice and serum of all groups by Nitric Oxide assay kit. Results: The positive Control-Group showed little Nitric Oxide in gastric juice and serum. Torilis leptophylla treated Groups (100 mg/kg, 200mg/kg and 300 mg/kg) showed significantly raised Nitric Oxide content in both gastric juice and serum than the Control Group (p<0.001). Conclusion: The study has determined that Torilis leptophylla possesses antiulcer and mucosal protective effects owing to the antioxidant activity of nitric oxide. This supports the use of the plant for the treatment of gastric ulcers.

Arginase-1 promotes lens epithelial-to-mesenchymal transition in different models of anterior subcapsular cataract

BackgroundArginase-1 (ARG1) promotes collagen synthesis and cell proliferation. ARG1 is highly expressed in various tumour cells. The mechanisms of ARG1 in epithelial-to-mesenchymal transition (EMT)-associated cataracts were studied herein.MethodsC57BL/6 mice, a human lens epithelial cell line (HLEC-SRA01/04), and human lens capsule samples were used in this study. The right lens anterior capsule of the mouse eye was punctured through the central cornea with a 26-gauge hypodermic needle. Human lens epithelial cells (HLECs) were transfected with ARG1-targeted (siARG1) or negative control siRNA (siNC). For gene overexpression, HLECs were transfected with a plasmid bearing the ARG1 coding sequence or an empty vector. Medium containing 0.2% serum with or without transforming growth factor beta-2 (TGF-β2) was added for 6 or 24 h to detect mRNA or protein, respectively. The expression of related genes was measured by quantitative real-time polymerase chain reaction (RT–qPCR), western blotting, and immunohistochemical staining. Transwell assays and wound healing assays were used to determine cell migration. Cell proliferation, superoxide levels, nitric oxide (NO) levels, and arginase activity were estimated using Cell Counting Kit-8 assays, a superoxide assay kit, an NO assay kit, and an arginase activity kit.ResultsARG1, alpha-smooth muscle actin (α-SMA), fibronectin, and Ki67 expression increased after lens capsular injury, while zonula occludens-1 (ZO-1) expression decreased.Fibronectin and collagen type I alpha1 chain (collagen 1A1) expression increased, and cell migration increased significantly in ARG1-overexpressing HLECs compared with those transfected with an empty vector after TGF-β2 treatment. These effects were reversed by ARG1 knockdown.The arginase-related pathway plays an important role in EMT. mRNAs of enzymes of the arginase-related pathway were highly expressed after ARG1 overexpression. ARG1 knockdown suppressed these expression changes.Numidargistat (CB-1158) dihydrochloride (CB-1158), an ARG1 inhibitor, suppressed TGF-β2-induced anterior subcapsular cataract (ASC) by reducing the proliferation of lens epithelial cells (LECs) and decreasing fibronectin, α-SMA, collagen 1A1, and vimentin expression.Compared with that in nonanterior subcapsular cataract (non-ASC) patients, the expression of ARG1, collagen 1A1, vimentin, fibronectin, and Ki67 was markedly increased in ASC patients.ConclusionsARG1 can regulate EMT in EMT-associated cataracts. Based on the pathogenesis of ASC, these findings are expected to provide new therapeutic strategies for patients.Graphical abstract

Penfluroidol Attenuates the Imbalance of the Inflammatory Response by Repressing the Activation of the NLRP3 Inflammasome and Reduces Oxidative Stress via the Nrf2/HO-1 Signaling Pathway in LPS-Induced Macrophages.

Excessive inflammatory responses and reactive oxygen species (ROS) formation play pivotal roles in the pathogenesis of sepsis. Penfluroidol (PF), an oral long-acting antipsychotic drug, has been suggested to possess diverse biological properties, including antischizophrenia, antitumour effect, and anti-inflammatory activity. The purpose of this research was to explore the anti-inflammatory and antioxidative effects of penfluroidol on lipopolysaccharide (LPS)-related macrophages. The viability of RAW264.7 and THP-1 cells was measured by Enhanced Cell Counting Kit-8 (CCK-8). The production of nitric oxide was evaluated by the Nitric Oxide Assay Kit. The generation of pro-inflammatory monocytes was detected by qRT-PCR (quantitative real-time PCR) and ELISA (enzyme-linked immunosorbent assay). Oxidative stress was assessed by measuring ROS, malondialdehyde (MDA), and superoxide dismutase (SOD) activity. The protein expression of the Nrf2/HO-1/NLRP3 inflammasome was detected by western blotting. Our results indicated that no cytotoxic effect was observed when RAW264.7 and THP-1 cells were exposed to PF (0-1 μm) and/or LPS (1 μg/ml) for 24 hr. The data showed that LPS, which was repressed by PF, facilitated the generation of the pro-inflammatory molecules TNF-α and IL-6. In addition, LPS contributed to increased production of intracellular ROS compared with the control group, whereas the administration of PF effectively reduced LPS-related levels of ROS. Moreover, LPS induced the generation of MDA and suppressed the activities of SOD. However, PF treatment strongly decreased LPS-induced MDA levels and increased SOD activities in the RAW264.7 and THP-1 cells. Furthermore, our research confirmed that penfluroidol repressed the secretion of pro-inflammatory molecules by limiting the activation of the NLRP3 inflammasome and reducing oxidative effects via the Nrf2/HO-1 signaling pathway. Penfluroidol attenuated the imbalance of the inflammatory response by suppressing the activation of the NLRP3 inflammasome and reduced oxidative stress via the Nrf2/HO-1 signaling pathway in LPS-induced macrophages.

Coelonin protects against PM2 .5 -induced macrophage damage via suppressing TLR4/NF-κB/COX-2 signaling pathway and NLRP3 inflammasome activation in vitro.

One of the important monitoring indicators of the air pollution is atmospheric fine particulate matter (PM2.5 ), which can induce lung inflammation after inhalation. Coelonin can alleviate PM2.5 -induced macrophage damage through anti-inflammation. However, its molecular mechanism remains unclear. We hypothesized that macrophage damage may involve the release of inflammatory cytokines, activation of inflammatory pathways, and pyrosis induced by inflammasome. In this study, we evaluated the anti-inflammation activity of coelonin in PM2.5 -induced macrophage and its mechanism of action. Nitric oxide (NO) and reactive oxygen species (ROS) production were measured by NO Assay kit and dichlorofluorescein-diacetate (DCFH-DA), and apoptosis were measured by Flow cytometry and TUNEL staining. The concentration of inflammatory cytokines production was measured with cytometric bead arrays and ELISA kits. The activation of NF-κB signaling pathway and NLRP3 inflammasome were measured by immunofluorescence, quantitative reverse transcription-polymerase chain reaction and western blot. As expected, coelonin pretreatment reduced NO production significantly as well as alleviated cell damage by decreasing ROS and apoptosis. It decreased generation of interleukin (IL)-6 and tumor necrosis factor (TNF)-α in PM2.5 -induced RAW264.7 and J774A.1 cells. Moreover, coelonin markedly inhibited upregulating the expression of toll-like receptor (TLR)4 and cyclo-oxygenase (COX)-2, blocked activation of p-nuclear factor-kappa B (NF-κB) signaling pathway, and suppressed expression of NLRP3 inflammasome, ASC, GSDMD, IL-18 and IL-1β. In conclusion, the results showed that coelonin could protect against PM2.5 -induced macrophage damage via suppressing TLR4/NF-κB/COX-2 signaling pathway and NLRP3 inflammasome activation in vitro.

Endothelial NOX4 aggravates eNOS uncoupling by decreasing dihydrofolate reductase after subarachnoid hemorrhage.

Endothelial malfunction is a major contributor to early or delayed vasospasm after subarachnoid hemorrhage (SAH). As a representative form of endothelial dysfunction, endothelial nitric oxide synthase (eNOS) uncoupling leads to a reduction in nitric oxide (NO) generated by endothelial cells. In this study, we investigated how the interaction between endothelial NOX4 (nicotinamide adenine dinucleotide phosphate oxidase 4) and DHFR (dihydrofolate reductase) contributes to eNOS uncoupling after SAH. Setanaxib and the adeno-associated virus (AAV) targeting brain vascular endothelia were injected through the tail vein and the expression and localization of proteins were examined by western blot and immunofluorescence staining. The NO content was measured using the NO assay kit, and laser speckle contrast imaging was used to assess cortical perfusion. ROS (reactive oxygen species) level was detected by DHE (dihydroethidium) staining, DCFH-DA (2',7'-dichlorofluorescin diacetate) staining and H2O2 (hydrogen peroxide) measurement. The Garcia score was employed to examine neurological function. Setanaxib is widely used for its preferential inhibition for NOX1/4 over other NOX isoforms. After endothelial NOX4 was inhibited by Setanaxib in a mouse model of SAH, the endothelial DHFR level was significantly elevated, which attenuated eNOS uncoupling, increased cortical perfusion, and improved the neurological function. The protective role of inhibiting endothelial NOX4, however, disappeared after knocking down endothelial DHFR. Our results suggest that endothelial DHFR decreased significantly because of the elevated level of endothelial NOX4, which aggravated eNOS uncoupling after SAH, leading to decreased cortical perfusion and worse neurological outcome.

Roles of Endothelial Motilin Receptor and Its Signal Transduction Pathway in Motilin-Induced Left Gastric Artery Relaxation in Dogs.

Background: Motilin increases left gastric artery (LGA) blood flow in dogs via the endothelial motilin receptor (MLNR). This article investigates the signaling pathways of endothelial MLNR.Methods: Motilin-induced relaxation of LGA rings was assessed using wire myography. Nitric oxide (NO), and cyclic guanosine monophosphate (cGMP) levels were measured using an NO assay kit and cGMP ELISA kit, respectively.Results: Motilin concentration-dependently (EC50=9.1±1.2×10−8M) relaxed LGA rings precontracted with U46619 (thromboxane A2 receptor agonist). GM-109 (MLNR antagonist) significantly inhibited motilin-induced LGA relaxation and the production of NO and cGMP. N-ethylmaleimide (NEM; G-protein antagonist), U73122 [phospholipase C (PLC) inhibitor], and 2-aminoethyl diphenylborinate [2-APB; inositol trisphosphate (IP3) blocker] partially or completely blocked vasorelaxation. In contrast, chelerythrine [protein kinase C (PKC) inhibitor] and H89 [protein kinase A (PKA) inhibitor] had no such effect. Low-calcium or calcium-free Krebs solutions also reduced vasorelaxation. N-nitro-L-arginine methyl ester [L-NAME; nitric oxide synthase (NOS) inhibitor] and ODQ [soluble guanylyl cyclase (sGC) inhibitor] completely abolished vasodilation and synthesis of NO and cGMP. Indomethacin (cyclooxygenase inhibitor), 18α-glycyrrhetinic acid [18α-GA; myoendothelial gap junction (MEGJ) inhibitor], and K+ channel inhibition through high K+ concentrations or tetraethylammonium (TEA-Cl; KCa channel blocker) partially decreased vasorelaxation, whereas glibenclamide (KATP channel blocker) had no such effect.Conclusion: The current study suggests that motilin-induced LGA relaxation is dependent on endothelial MLNR through the G protein-PLC-IP3 pathway and Ca2+ influx. The NOS-NO-sGC-cGMP pathway, prostacyclin, MEGJ, and K+ channels (especially KCa) are involved in endothelial-dependent relaxation of vascular smooth muscle (VSM) cells.

Nitric oxide delivery using nitric oxide-containing fluid in continuous hemofiltration: an in vitro study.

Administering nitrite has therapeutic effects on ischemic conditions wherein the enzymatic production of nitric oxide depends on oxygen. We developed a supplemental fluid containing nitric oxide (NO) and determined the clearance and supply between the pre- and post-dilution modes of continuous hemofiltration in vitro. Nitric oxide gas, 1000mL or 2000mL, at a concentration of 1000ppm, was injected into 2020mL of conventional supplemental fluid (experimental solution). The same volume of nitrogen gas was injected into the supplemental fluid (control solution). NO concentrations were measured using commercially available NO assay kit. Pre- or post-dilution continuous hemofiltration was performed using a control solution as supplemental fluid to determine the NO clearance. We determined the NO concentration of the outlet blood circuit to confirm the NO supply using the experimental solution as supplemental fluid. Also, using the bovine blood, white blood cell and platelet change rates and the dialysis membrane water flux during continuous hemodiafiltration were evaluated ex vivo as index of the biocompatibilities of a nitric oxide-containing solution. NO was not detected in the control solutions. The experimental solutions significantly increased in nitric oxide concentrations. NO clearance increased as the increase in supplemental and ultrafiltration flow rates using the control solution as supplemental fluid. However, using the experimental solution as supplemental fluid, nitric oxide supply showed a similar trend of NO clearance. Without any changes in biocompatibility using the supplemental fluid containing NO, it could maintain intravascular nitric oxide during continuous renal replacement therapy.

Anti-Oral Microbial Activity and Anti-Inflammatory Effects of Rosmarinic Acid in Lipopolysaccharide-Stimulated MC3T3-E1 Osteoblastic Cells on a Titanium Surface

Background: The purpose of this study was to investigate the anti-oral microbial activity and anti-inflammatory effects of rosmarinic acid (RA) in lipopolysaccharide (LPS)-stimulated MC3T3-E1 osteoblastic cells on a titanium (Ti) surface during osseointegration, and to confirm the possibility of using RA as a safe natural substance for the control of peri-implantitis (PI) in Ti-based dental implants. Methods: A disk diffusion test was conducted to confirm the antimicrobial activity of RA against oral microorganisms. In order to confirm the anti-inflammatory effects of RA, inflammatory conditions were induced with 100 ng/ml of LPS in MC3T3-E1 osteoblastic cells on the Ti surface treated with or without 14μg/ml of RA. The production of nitric oxide (NO) and prostaglandin E2 (PGE2) in LPS-stimulated MC3T3-E1 osteoblastic cells on the Ti surface was confirmed using an NO assay kit and PGE2 enzyme-linked immunosorbent assay kit. Reverse transcription polymerase chain reaction and western blot analysis were performed to confirm the expression of interleukin (IL)-1β and tumor necrosis factor (TNF)-α in total RNA and protein. Results: RA showed weak antimicrobial effects against Streptococcus mutans and Escherichia coli, but no antimicrobial activity against the bacteria Aggregatibacter actinomycetemcomitans and the fungus Candida albicans. RA reduced the production of pro-inflammatory mediators, NO and PGE2, and proinflammatory cytokines, TNF-α and IL-1β, in LPS-stimulated MC3T3-E1 osteoblastic cells on the Ti surface at the protein and mRNA levels. Conclusion: RA not only has anti-oral microbial activity, but also anti-inflammatory effects in LPS-stimulated MC3T3-E1 osteoblasts on the Ti surface, therefore, it can be used as a safe functional substance derived from plants for the prevention and control of PI for successful Ti-based implants.

P.716 Negr1-deficient mice display anxiolytic effect in the open field but their sensitivity to amphetamine is not altered

MicroRNA-25–5p (miR-25–5p) may be involved in the pathogenesis and processes of vascular diseases. The aim of this study was to investigate the role of miR-25–5p in oxidized low-density lipoprotein (ox-LDL)-treated human brain microvessel endothelial cells (HBMECs) and the underlying mechanisms. RT-qPCR and/or Western blot were used to detect the expression levels of miR-25–5p and neuronal growth regulator 1 (NEGR1). The effect of miR-25–5p overexpression and NEGR1 silencing on cell proliferation, migration, apoptosis and reactive oxygen species (ROS) production of HBMECs were measured by using CCK-8 assay, transwell assay and flow cytometry, respectively. The expression levels of apoptosis-related protein (cleaved caspase-3 and pro-caspase-3) were detected using Western blot, and the nitric oxide (NO) production was measured by a nitric oxide assay kit. The expression level of miR-25–5p was decreased in HBMECs treated with ox-LDL. Compared with the control group, miR-25–5p overexpression significantly promoted the proliferation and migration of HBMECs treated with ox-LDL (p < 0.01). Overexpression of miR-25–5p significantly suppressed cell apoptosis, ROS production and NO reduction of ox-LDL-induced HBMECs (p < 0.01). In addition, the target gene of miR-25–5p was predicted to be NEGR1 through Targetscan online analysis. The effect of NEGR1 silencing on cell proliferation, migration, apoptosis, ROS and NO production of ox-LDL-induced HBMECs was similar to that of miR-25–5p overexpression. Furthermore, miR-25–5p overexpression and NEGR1 silencing significantly downregulated the protein expression levels of JAK2 and STAT3. Thus, miR-25–5p neutralizes the effects of ox-LDL on multiple functions of HBMECs through suppressing the expression of NEGR1 via regulating the JAK/STA signaling pathway.

The effect of cinnamon on migraine treatment and blood levels of CGRP and IL-6: A double-blinded randomized controlled clinical trial

Background Migraine is a common disabling disorder characterized by recurrent headache attacks. Evidence showed that increasing in the levels of interleukin-6 (IL-6), Calcitonin-gene-related peptide (CGRP) and Nitric oxide (NO) lead to inflammation and neurogenic pain. Cinnamon has anti-inflammatory and neuroprotective properties, however, to our knowledge, to date, the effect of cinnamon on migraine did not assess. Thus, the aim of this study is to assess the effect of cinnamon on frequency, severity, duration and inflammatory markers of migraine attacks. Methods Fifty eligible migraine patients were randomized into two groups to receive either 3 capsules/day each contained 600 mg of cinnamon powder (intervention group) or receive 3 placebo capsules/day each contained 600 mg of corn starch (control group) for 2 months. At the beginning and the end of the study, blood samples were collected andserumIL-6 and CGRP levels were assessed using ELISA methods and serumNO levels were measured using Nitric Oxide Assay Kit. The frequency, severity and duration of pain attacks were also recorded. Results The serum concentrations of IL6 and NO were significantly reduced in the intervention group compared to the control group (P Conclusion Cinnamon considerably reduced the frequency, severity, and duration of migraine attacks in the migraine patients. This novel finding might be useful in a clinical setting to relief pain and the other complications of migraine.

MicroRNA-25–5p counteracts oxidized LDL-induced pathological changes by targeting neuronal growth regulator 1 (NEGR1) in human brain micro-vessel endothelial cells

MicroRNA-25–5p (miR-25–5p) may be involved in the pathogenesis and processes of vascular diseases. The aim of this study was to investigate the role of miR-25–5p in oxidized low-density lipoprotein (ox-LDL)-treated human brain microvessel endothelial cells (HBMECs) and the underlying mechanisms. RT-qPCR and/or Western blot were used to detect the expression levels of miR-25–5p and neuronal growth regulator 1 (NEGR1). The effect of miR-25–5p overexpression and NEGR1 silencing on cell proliferation, migration, apoptosis and reactive oxygen species (ROS) production of HBMECs were measured by using CCK-8 assay, transwell assay and flow cytometry, respectively. The expression levels of apoptosis-related protein (cleaved caspase-3 and pro-caspase-3) were detected using Western blot, and the nitric oxide (NO) production was measured by a nitric oxide assay kit. The expression level of miR-25–5p was decreased in HBMECs treated with ox-LDL. Compared with the control group, miR-25–5p overexpression significantly promoted the proliferation and migration of HBMECs treated with ox-LDL (p < 0.01). Overexpression of miR-25–5p significantly suppressed cell apoptosis, ROS production and NO reduction of ox-LDL-induced HBMECs (p < 0.01). In addition, the target gene of miR-25–5p was predicted to be NEGR1 through Targetscan online analysis. The effect of NEGR1 silencing on cell proliferation, migration, apoptosis, ROS and NO production of ox-LDL-induced HBMECs was similar to that of miR-25–5p overexpression. Furthermore, miR-25–5p overexpression and NEGR1 silencing significantly downregulated the protein expression levels of JAK2 and STAT3. Thus, miR-25–5p neutralizes the effects of ox-LDL on multiple functions of HBMECs through suppressing the expression of NEGR1 via regulating the JAK/STA signaling pathway.

Ghrelin combined with sodium tanshinone IIA sulfonate pretreatment reduces apoptosis and fractalkine expression induced by high-dose glucose in human umbilical vein endothelial cells.

To explore the regulatory role of ghrelin combined with sodium tanshinone IIA sulfonate (STS) pretreatment in cell apoptosis and fractalkine (FKN) expression of human umbilical vein endothelial cells (HUVECs) induced with high-dose glucose. HUVECs were assigned into control group, high-dose glucose group (HG group), high-dose glucose with ghrelin group (Gr+HG group), and high-dose glucose companied with ghrelin and STS group (Gr+STS+HG group). The apoptosis of HUVECs was determined by Hoechst 33258 straining and flow cytometry (FCM). Nitric oxide (NO) level was measured by total NO assay kit. The mRNA and protein levels of β-catenin, p-GSK-3β and FKN were accessed by Western blot and real-time quantitative polymerase chain reaction (RT-qPCR), respectively. High-dose glucose significantly accelerated apoptosis in HUVECs. The apoptotic rate was lower in Gr+HG group and much lower in Gr+STS+HG group than control group. NO level was significantly reduced in the HG group, which was partly inhibited in Gr+HG group and obviously increased in Gr+STS+HG group than controls. In addition, mRNA levels of GSK-3β and FKN in HUVECs decreased in Gr+HG group, which was more obviously decreased in Gr+STS+HG group. However, ghrelin treatment upregulated β-catenin and p-GSK-3β (Ser9), but downregulated FKN during high-dose glucose treatment, which was more obvious in Gr+STS+HG group. Pretreatment of ghrelin combined with STS reduces the apoptosis rate of HUVECs induced by high glucose environment and inhibits the expression of FKN via β-catenin/Wnt signaling pathway.

Formononetin induces vasorelaxation in rat thoracic aorta via regulation of the PI3K/PTEN/Akt signaling pathway.

BackgroundFormononetin (FMN) is an isoflavone that produces arterial vasodilation. However, the underlying molecular mechanisms are unclear.PurposeThe purpose of this study was to explore the vasorelaxant effect and the potential mechanism of FMN in vascular endothelium in isolated rat aorta.MethodsThe thoracic aortas of Sprague Dawley rats were isolated to test the arterial reactivity in the presence of FMN with or without inhibitors. Bioinformatics analyses, including a Bioinformatics Analysis Tool for Molecular Mechanism of Traditional Chinese Medicine and molecular docking methods, were performed to predict therapeutic targets responsible for the vascular protection produced by FMN. We used rat aortic endothelial cells (RAOECs) as an in vitro model to verify the potential mechanism through molecular biological analyses. The production of nitric oxide (NO) metabolites were evaluated via an NO assay kit according to the manufacturer’s instruction. The mRNA expression of eNOS was analyzed by polymerase chain reaction, and the protein levels of PTEN, phosphorylated Akt, and eNOS were measured by Western blot.ResultsWe found that FMN dilated rat aortic rings in a concentration-dependent manner, which was reduced by endothelium denudation and eNOS inhibition. The bioinformatics analyses indicated that FMN activity was associated with the PI3K/PTEN/Akt signaling pathway. Molecular biological studies demonstrated that FMN significantly elevated the levels of NO and eNOS mRNA and markedly increased the protein expression of phosphorylated Akt and eNOS in RAOECs, and decreased PTEN compared with a dimethyl sulfoxide group.ConclusionFMN performs vasorelaxation of the thoracic aorta through activating the PI3K/PTEN/Akt signaling pathway.

Significance of Serum Nitric Oxide and Superoxide Dismutase in Oral Submucous Fibrosis and Squamous Cell Carcinoma: A Comparative Study.

Introduction:This study aimed at comparative analysis of serum nitric oxide (NO) and superoxide dismutase (SOD) levels as therapeutic and prognostic biomarkers in patients with oral submucous fibrosis (OSMF) and squamous cell carcinoma (SCC).Materials and Methods:Eighty-seven patients were grouped into Group I (n = 29, OSMF), Group II (n = 29, oral SCC), and Group III (n = 29, controls). Two ml of venous blood was collected from patients after overnight fast to avoid any dietary influence on the serum beta-carotene. Standard protocols were followed in transfer, storage, and processing of blood. Modified copper-cadmium reduction method for rapid assay to estimate the serum NO and Enzychrom™ SOD assay kit to determine SOD levels were used.Results:The mean level of NO level in Group I, Group II, and Group III was 42.49, 50.08, and 32.81, respectively, and mean level of SOD in Group I, Group II, and Group III were 207.65, 196.93, and 226.57, respectively. The P values were calculated and were statistically significant (<0.001).Conclusion:An increase in level of oxidant NO in OSMF followed by SCC and decrease in level of antioxidant SOD in OSMF followed by SCC were noted. These levels of NO and antioxidant SOD can be used as prognostic and therapeutic biomarkers.

TWEAK/Fn14 promotes oxidative stress through AMPK/PGC‑1α/MnSOD signaling pathway in endothelial cells.

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) contributes to dysfunction of endothelial cells via its receptor, Fn14. However, its role in the production of reactive oxygen species (ROS), particularly mitochondrial ROS (mtROS) and the subsequent decrease in nitric oxide (NO) in endothelial cells remains unclear. In this study, the effect of TWEAK/Fn14 on generation of ROS, mtROS and NO in endothelial cells and its potential mechanism was investigated. Human umbilical vein endothelial cells (HUVECs) were treated with TWEAK with Fn14 small interfering (si)RNA or negative control RNA. It was demonstrated that TWEAK induced the production of ROS and mtROS in HUVECs, which were detected by fluorescent microscope, and flow cytometry. In addition, TWEAK decreased the generation of NO as indicated using the Nitric Oxide Assay kit. Furthermore, TWEAK aggravated mtDNA damage as measured by quantitative polymerase chain reaction analysis. Inhibition of Fn14 by Fn14 siRNA decreased TWEAK‑induced ROS and mtROS production, as well as mtDNA damage, while it increased the production of NO in endothelial cells. In addition, TWEAK inhibited the expression of active AMP‑activated protein kinase (AMPK) and its downstream protein peroxisome proliferator‑activated receptor‑γ coactivator-1α (PGC‑1α) and manganese superoxide dismutase (MnSOD). Notably, Fn14 siRNA enhanced the expression of the aforementioned proteins. Taken together, TWEAK/Fn14 contributes to endothelial dysfunction through modulation of ROS and mtROS. In addition, the underlying mechanism is implicated in the AMPK/PGC‑1α/MnSOD signaling pathway.

Radix Cyathula officinalis Kuan inhibits arterial remodeling in spontaneously hypertensive rats.

There is still no resolution for arterial remodeling related with hypertension, though hypertension treatment has access to a number of pharmacological agents. The present study aimed at investigating the prevention of Cyathula officinalis Kuan's roots (C. officinalis Kuan) against in arterial remodeling in vitro. Spontaneously hypertensive rats (SHRs) were intragastrically administered 3, 6 or 12 g/kg C. officinalis Kuan or normal saline or enalapril (2.5 mg/kg) once a day for 8 weeks. Hematoxylin and eosin were used to measure blood pressure and stain carotid and arota. The serum concentration of nitric oxide (NO) was measured by NO assay kit (nitrate reductase method). The endothelin-1 transcriptional level, endothelial NO synthase of endothelium as well as angiotensin II receptor type 1 (AT1R) of aorta and carotid was tested by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and the protein level in aorta was also measured by western blotting. The blood pressure in SHR+enalapril, SHR+3 g/kg, SHR+6 g/kg and SHR+12 g/kg C. officinalis Kuan groups was significantly decreased at 4, 6 and 8 weeks post-treatment compared with SHR group. Different doses of C. officinalis Kuan and enalapril treatment showed aortic wall thinness and strengthened NO serum level, but made no impact on the transcriptional level of AT1R in aorta or endothelial NO synthase in carotid. It is suggested by such results that therapy by C. officinalis Kuan is able to fight against arterial remodeling, thus may provide a new means to treat arterial remodeling caused by hypertension.

Glucagon-like peptide-1 affects human umbilical vein endothelial cells in high glucose by the PI3K/Akt/eNOS signaling pathway

Abstract Objectives To investigate the effects of glucagon-like peptide-1 (GLP-1) for the apoptosis of human umbilical vein endothelial cells (HUVECs) in high glucose and the related mechanisms. Material and methods HUVECs were cultured under different conditions for 48 h. The apoptosis rate of cells was detected by flow cytometry, the expression of p-Akt and p-eNOS was measured by Western blot, and the production of nitric oxide (NO) was detected by NO assay kit. Results HUVECs were incubated in high glucose, the apoptosis rate of cells increased, the expression of p-Akt and p-eNOS reduced, and the production of NO decreased. After GLP-1 was added into the high glucose, the apoptosis rate of cells significantly reduced, the expression of p-Akt and p-eNOS and the production of NO obviously increased. After exendin, wortmannine and L-NAME were added into high glucose and GLP-1, respectively, exendin, wortmannine and L-NAME increased the cell apoptosis, down-regulated the expression of p-Akt and p-eNOS and dropped the production of NO, except that L-NAME made no difference on the expression of p-Akt. Conclusion GLP-1 increased the expression of Akt and endothelial nitric oxide synthase (eNOS) in HUVECs via the up-regulation of PI3K/Akt/eNOS pathway and decreased the apoptosis rate of HUVECs in high glucose.

Comparison of Oxidative Stress Intensity in the Seminal Plasma and Serum of Infertile Patients with Different Varicocele Grades and Subjects with No Varicocele: A Cross-Sectional Study

Background: Varicocele is the leading cause of male infertility throughout the world. Objectives: This study aimed to identify the effects of varicocele on oxidative stress, total antioxidant capacity (TAC), and semen parameters. Methods: In this cross-sectional study, serum and semen samples were collected from subjects with no varicocele (n, 35) and patients with varicocele (n, 86), who were referred to infertility and in vitro fertilization (IVF) centers of Babol Clinic Hospital, Babol, Iran during 2015 - 2016. The semen samples were analyzed according to the world health organization (WHO) guidelines. After the ultrasound and physical examinations, varicoceles were classified into three grades: G1, G2, and G3. TAC and malondialdehyde (MDA) levels were measured in the serum and seminal plasma of subjects, using the ferric reducing ability of plasma (FRAP) and thiobarbituric acid reaction (TBAR) methods, respectively. Nitric oxide (NO) was also measured using Nitric Oxide assay kit. Results: The mean total sperm count in the G1, G2, and G3 groups were significantly lower than the control group (P = 0.037; P = 0.003, and P = 0.044, respectively). A trend of lower semen volume was observed in the G3 group, compared with the control group (P = 0.06). A significant positive correlation was observed between elevated serum MDA level and varicocele degree (P G2 > G1 > controls. There was also a significant negative correlation between the serum and seminal plasma TAC levels (P < 0.05). Varicocele patients with G3 degree had a significantly lower mean TAC level in the serum and seminal plasma, compared with the control group (P = 0.001 and P = 0.008, respectively). No significant difference was found in the mean Nitric Oxide level between the groups. However, an increasing trend for the mean Nitric Oxide level was observed in the serum and seminal plasma of varicocele patients. Conclusions: The reduced level of seminal plasma antioxidants, which is associated with increased lipid peroxidation, is one of the main reasons for low sperm quality in patients with varicocele. Antioxidant therapy may be useful in decreasing oxidative stress intensity and improving the condition of varicocele patients.

Pudilan xiaoyan oral liquid alleviates LPS-induced respiratory injury through decreasing nitroxidative stress and blocking TLR4 activation along with NF-ΚB phosphorylation in mice

Ethnopharmacological relevancePudilan xiaoyan oral liquid (PDL), collected in Chinese Pharmacopoeia, has been used clinically for treating inflammatory diseases such as upper respiratory tract infection diseases. However, its potential anti-inflammation and the mechanism are still unclear. Materials and methodslipopolysaccharide (LPS) was used to induce respiratory inflammation of mice by intratracheal administration. UPLC/MS was performed for components analysis of PDL. Enzyme-linked immune sorbent assay (ELISA) was conducted for determining interleukin-6(IL-6), interleukin-1β(IL-1β) and tumor necrosis factor-α(TNF-α) in serum and supernatant of tracheal tissue while Nitric oxide assay kit for nitric oxide (NO) content. Hematoxylin-Eosin (HE) staining was applied to evaluate pathological lesions. Western blotting analysis (WB) and Immunohistochemistry(IHC) were employed for the determination of Toll-like receptors 4(TLR4), TNF-α, IL-6, inducible nitric oxide synthase(iNOS) and nuclear factor-kappa B p65 (NF-κB p65) protein expressions. ResultsSeven major compounds of PDL were analyzed simultaneously. The treatment of PDL could attenuate LPS-induced histopathological damage of tracheal tissues, followed by reducing pro-inflammation mediators including TNF-α and IL-6 in serum and supernatant of tracheal tissue. LPS-induced nitroxidative stress including NO content and iNOS expression was inhibited significantly by PDL. Furthermore, PDL also down-regulated NF-kB p65 phosphorylation and TLR4 expressions. ConclusionThe results indicated that the PDL had a protective effect on LPS-induced respiratory inflammation injury in mice. Our findings for the first time provide experimental evidence for the application of PDL on respiratory inflammation injury in clinical practice.

Thromboxane A2 receptor antagonist SQ29548 suppresses the LPS‑induced release of inflammatory cytokines in BV2 microglia cells via suppressing MAPK and NF‑κB signaling pathways.

Inflammation in the brain, characterized by the activation of microglia, is hypothesized to participate in the pathogenesis of neuronal disorders. It is proposed that thromboxane A2 receptor (TXA2R) activation is involved in thrombosis/hemostasis and inflammation responses. In the present study, the anti-inflammatory effects of SQ29548 on lipopolysaccharide (LPS)-stimulated BV2 microglial cells and its molecular mechanisms were investigated. In the BV2 cell line, LPS-stimulated nitric oxide (NO) and inflammatory cytokine release, and the phosphorylation of mitogen-activated protein kinases (MAPKs) and the nuclear factor (NF)-κB were assessed using an NO assay kit, reverse transcription-quantitative polymerase chain reaction and western blotting, respectively. In vitro studies demonstrated that SQ29548 inhibited LPS-stimulated BV2 activation and reduced the mRNA expression levels of interleukin (IL)-1β, IL-6, tumor necrosis factor-α and inducible NO synthase via inhibition of MAPKs and the NF-κB signaling pathway. SQ29548 inhibited the LPS-induced inflammatory response by blocking MAPKs and NF-κB activation in BV2 microglial cells.