The present study aimed to investigate the protective effect of S-propargyl-cysteine (SPRC) on atherosclerosis progression in mice. A mouse model of vulnerable atherosclerotic plaque was created in ApoE^-/- mice by carotid artery tandem stenosis (TS) combined with a Western diet. Macrophotography, lipid profiles, and inflammatory markers were measured to evaluate the antiatherosclerotic effects of SPRC compared to atorvastatin as a control. Histopathological analysis was performed to assess the plaque stability. To explore the protective mechanism of SPRC, human umbilical vein endothelial cells (HUVECs) were cultured in vitro and challenged with oxidized low-density lipoprotein (ox-LDL). Cell viability was determined with a Cell Counting Kit-8 (CCK-8). Endothelial nitric oxide synthase (eNOS) phosphorylation and mRNA expression were detected by Western blot and RT-qPCR respectively. The results showed that the lesion area quantified by en face photographs of the aortic arch and carotid artery was significantly less, plasma total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) were reduced, plaque collagen content was increased and matrix metalloproteinase-9 (MMP-9) was decreased in 80 mg/kg per day SPRC-treated mice compared with model mice. These findings support the role of SPRC in plaque stabilization. In vitro studies revealed that 100 μmol/L SPRC increased the cell viability and the phosphorylation level of eNOS after ox-LDL challenge. These results suggest that SPRC delays the progression of atherosclerosis and enhances plaque stability. The protective effect may be at least partially related to the increased phosphorylation of eNOS in endothelial cells.
Animals ; Humans ; Mice ; Atherosclerosis ; Cholesterol/metabolism* ; Cysteine/pharmacology* ; Human Umbilical Vein Endothelial Cells/metabolism* ; Lipoproteins, LDL/pharmacology* ; Nitric Oxide Synthase Type III/metabolism* ; Phosphorylation ; Plaque, Atherosclerotic/pathology*

In the central nervous system, nitric oxide (NO), a free gas with multitudinous bioactivities, is mainly produced from the oxidation of L-arginine by neuronal nitric oxide synthase (nNOS). In the past 20 years, the studies in our group and other laboratories have suggested a significant involvement of nNOS in a variety of neurological and neuropsychiatric disorders. In particular, the interactions between the PDZ domain of nNOS and its adaptor proteins, including post-synaptic density 95, the carboxy-terminal PDZ ligand of nNOS, and the serotonin transporter, significantly influence the subcellular localization and functions of nNOS in the brain. The nNOS-mediated protein-protein interactions provide new attractive targets and guide the discovery of therapeutic drugs for neurological and neuropsychiatric disorders. Here, we summarize the work on the roles of nNOS and its association with multiple adaptor proteins on neurological and neuropsychiatric disorders.
Humans ; Nitric Oxide Synthase Type I/metabolism* ; Adaptor Proteins, Signal Transducing ; Brain/metabolism* ; Nervous System Diseases

Humans ; Nitric Oxide ; Carcinoma, Non-Small-Cell Lung/drug therapy* ; Lung Neoplasms ; Lung ; Nitric Oxide Synthase ; Macrophages, Alveolar ; Pulmonary Alveoli

Long-chain acyl-coenzyme A (CoA) synthase 4 (ACSL4) is an enzyme that esterifies CoA into specific polyunsaturated fatty acids, such as arachidonic acid and adrenic acid. Based on accumulated evidence, the ACSL4-catalyzed biosynthesis of arachidonoyl-CoA contributes to the execution of ferroptosis by triggering phospholipid peroxidation. Ferroptosis is a type of programmed cell death caused by iron-dependent peroxidation of lipids; ACSL4 and glutathione peroxidase 4 positively and negatively regulate ferroptosis, respectively. In addition, ACSL4 is an essential regulator of fatty acid (FA) metabolism. ACSL4 remodels the phospholipid composition of cell membranes, regulates steroidogenesis, and balances eicosanoid biosynthesis. In addition, ACSL4-mediated metabolic reprogramming and antitumor immunity have attracted much attention in cancer biology. Because it facilitates the cross-talk between ferroptosis and FA metabolism, ACSL4 is also a research hotspot in metabolic diseases and ischemia/reperfusion injuries. In this review, we focus on the structure, biological function, and unique role of ASCL4 in various human diseases. Finally, we propose that ACSL4 might be a potential therapeutic target.
Humans ; Ferroptosis ; Apoptosis ; Phospholipids/metabolism* ; Nitric Oxide Synthase

OBJECTIVE: To investigate the anti-oxidant and anti-inflammatory effects of ethanol extract of Polygala sibirica L. var megalopha Fr. (EEP) on RAW264.7 mouse macrophages. METHODS: RAW264.7 cells were pretreated with 0-200 µg/mL EEP or vehicle for 2 h prior to exposure to 1 µg/mL lipopolysaccharide (LPS) for 24 h. Nitric oxide (NO) and prostaglandin (PGE₂) production were determined by Griess reagent and enzyme-linked immunosorbent assay (ELISA), respectively. The mRNA levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), tumor necrosis factor α (TNF-α), interleukin-1beta (IL-1β), and IL-6 were determined using reverse transcription polymerase chain reaction (RT-PCR). Western blot assay was used to determine the protein expressions of iNOS, COX-2, phosphorylation of extracellular regulated protein kinases (ERK1/2), c-Jun N-terminal kinase (JNK), inhibitory subunit of nuclear factor Kappa B alpha (Iκ B-α) and p38. Immunofluorescence was used to observe the nuclear expression of nuclear factor-κ B p65 (NF-κ B p65). Additionally, the anti-oxidant potential of EEP was evaluated by reactive oxygen species (ROS) production and the activities of catalase (CAT) and superoxide dismutase (SOD). The 2,2-diphenyl-1-picrylhydrazyl (DPPH), hydroxyl (OH), superoxide anion (O₂^-) radical and nitrite scavenging activity were also measured. RESULTS: The total polyphenol and flavonoid contents of EEP were 23.50±2.16 mg gallic acid equivalent/100 g and 43.78±3.81 mg rutin equivalent/100 g. With EEP treatment (100 and 150 µg/mL), there was a notable decrease in NO and PGE₂ production induced by LPS in RAW264.7 cells by downregulation of iNOS and COX-2 mRNA and protein expressions (P<0.01 or P<0.05). Furthermore, with EEP treatment (150 µg/mL), there was a decrease in the mRNA expression levels of TNF-α, IL-1β and IL-6, as well as in the phosphorylation of ERK, JNK and p38 mitogen-activated protein kinase (MAPK, P<0.01 or P<0.05), by blocking the nuclear translocation of NF-κ B p65 in LPS-stimulated cells. In addition, EEP (100 and 150 µg/mL) led to an increase in the anti-oxidant enzymes activity of SOD and CAT, with a concomitant decrease in ROS production (P<0.01 or P<0.05). EEP also indicated the DPPH, OH, O₂^- radical and nitrite scavenging activity. CONCLUSION EEP inhibited inflammatory responses in activated macrophages through blocking MAPK/NF-κ B pathway and protected against oxidative stress.
Animals ; Mice ; Antioxidants/pharmacology* ; Lipopolysaccharides/pharmacology* ; Polygala ; Transcription Factor RelA/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; Ethanol/chemistry* ; Interleukin-6/metabolism* ; Anti-Inflammatory Agents/chemistry* ; Reactive Oxygen Species/metabolism* ; Cyclooxygenase 2/metabolism* ; Nitrites/metabolism* ; NF-kappa B/metabolism* ; Nitric Oxide/metabolism* ; Superoxide Dismutase/metabolism* ; RNA, Messenger ; Nitric Oxide Synthase Type II/metabolism*

Objective: To investigate the changes of intestinal wall barrier function and its correlation with infection occurrence in patients with cirrhotic portal hypertension. Methods: 263 patients with cirrhotic portal hypertension were split into: the clinically evident portal hypertension (CEPH) combined with infection group (n = 74); CEPH group (n = 104); and Non-CEPH group (n = 85). Among them, 20 CEPH patients and 12 non-CEPH patients in non-infection status were subjected to sigmoidoscopy. Immunohistochemical staining was used to detect the expression of trigger receptor-1 (TREM-1), CD68, CD14, the inducible nitric oxide synthase molecule, and Escherichia coli (E.coli) in the medullary cells of the colon mucosa. An enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of soluble myeloid cell trigger receptor-1 (sTREM-1), soluble leukocyte differentiation antigen-14 subtype (sCD14-ST) and intestinal wall permeability index enteric fatty acid binding protein (I-FABP). Fisher's exact probability method, one-way ANOVA, Kruskal-Wallis-H test, Bonferroni method, and Spearman correlation analysis were used for statistical analysis. Results: The serum sTREM-1 and I-FABP levels were higher in CEPH patients than those of non-CEPH patients in the non-infectious state (P < 0.05), but the difference in blood sCD14-ST levels was not statistically significant (P > 0.05). Serum levels of sTREM-1, sCD14-ST, and I-FABP in infected patients were higher than those in patients without a concurrent infection (P < 0.05). Serum sCD14-ST levels were positively correlated with serum sTREM-1, C-reactive protein (CRP), and procalcitonin (PCT), and sTREM-1 levels were also positively correlated with CRP and PCT (r > 0.5, P < 0.001). The rates of CD68, inducible nitric oxide synthase, CD14-positive cells, and E.coli-positive glands were higher in the intestinal mucosa of the CEPH group than those of the control group (P < 0.05). Spearman's correlation analysis showed that the rate of E.coli-positive glands in CEPH patients was positively correlated with the expression of molecular markers CD68 and CD14 in the lamina propria macrophages. Conclusion: Patients with cirrhotic portal hypertension have increased intestinal permeability and inflammatory cells, accompanied by bacterial translocation. Serum sCD14-ST and sTREM-1 can be used as indicators to predict and evaluate the occurrence of infection in patients with cirrhotic portal hypertension.
Humans ; Nitric Oxide Synthase Type II ; Lipopolysaccharide Receptors ; Prospective Studies ; Biomarkers ; C-Reactive Protein/analysis* ; Liver Cirrhosis/complications* ; Hypertension, Portal

OBJECTIVES: Endothelium-dependent vasodilation dysfunction is the pathological basis of diabetic macroangiopathy. The utilization and adaptation of endothelial cells to high glucose determine the functional status of endothelial cells. Glycolysis pathway is the major energy source for endothelial cells. Abnormal glycolysis plays an important role in endothelium-dependent vasodilation dysfunction induced by high glucose. Pyruvate kinase isozyme type M2 (PKM2) is one of key enzymes in glycolysis pathway, phosphorylation of PKM2 can reduce the activity of pyruvate kinase and affect the glycolysis process of glucose. TEPP-46 can stabilize PKM2 in its tetramer form, reducing its dimer formation and phosphorylation. Using TEPP-46 as a tool drug to inhibit PKM2 phosphorylation, this study aims to explore the impact and potential mechanism of phosphorylated PKM2 (p-PKM2) on endothelial dependent vasodilation function in high glucose, and to provide a theoretical basis for finding new intervention targets for diabetic macroangiopathy. METHODS: The mice were divided into 3 groups: a wild-type (WT) group (a control group, C57BL/6 mice) and a db/db group (a diabetic group, db/db mice), which were treated with the sodium carboxymethyl cellulose solution (solvent) by gavage once a day, and a TEPP-46 group (a treatment group, db/db mice+TEPP-46), which was gavaged with TEPP-46 (30 mg/kg) and sodium carboxymethyl cellulose solution once a day. After 12 weeks of treatment, the levels of p-PKM2 and PKM2 protein in thoracic aortas, plasma nitric oxide (NO) level and endothelium-dependent vasodilation function of thoracic aortas were detected. High glucose (30 mmol/L) with or without TEPP-46 (10 μmol/L), mannitol incubating human umbilical vein endothelial cells (HUVECs) for 72 hours, respectively. The level of NO in supernatant, the content of NO in cells, and the levels of p-PKM2 and PKM2 protein were detected. Finally, the effect of TEPP-46 on endothelial nitric oxide synthase (eNOS) phosphorylation was detected at the cellular and animal levels. RESULTS: Compared with the control group, the levels of p-PKM2 in thoracic aortas of the diabetic group increased (P<0.05). The responsiveness of thoracic aortas in the diabetic group to acetylcholine (ACh) was 47% lower than that in the control group (P<0.05), and that in TEPP-46 treatment group was 28% higher than that in the diabetic group (P<0.05), while there was no statistically significant difference in the responsiveness of thoracic aortas to sodium nitroprusside (SNP). Compared with the control group, the plasma NO level of mice decreased in the diabetic group, while compared with the diabetic group, the phosphorylation of PKM2 in thoracic aortas decreased and the plasma NO level increased in the TEPP-46 group (both P<0.05). High glucose instead of mannitol induced the increase of PKM2 phosphorylation in HUVECs and reduced the level of NO in supernatant (both P<0.05). HUVECs incubated with TEPP-46 and high glucose reversed the reduction of NO production and secretion induced by high glucose while inhibiting PKM2 phosphorylation (both P<0.05). At the cellular and animal levels, TEPP-46 reversed the decrease of eNOS (ser1177) phosphorylation induced by high glucose (both P<0.05). CONCLUSIONS p-PKM2 may be involved in the process of endothelium-dependent vasodilation dysfunction in Type 2 diabetes by inhibiting p-eNOS (ser1177)/NO pathway.
Animals ; Humans ; Mice ; Carboxymethylcellulose Sodium/pharmacology* ; Diabetes Mellitus, Type 2/metabolism* ; Endothelium, Vascular/metabolism* ; Glucose/metabolism* ; Human Umbilical Vein Endothelial Cells ; Mice, Inbred C57BL ; Nitric Oxide/metabolism* ; Nitric Oxide Synthase Type III/metabolism* ; Phosphorylation ; Pyruvate Kinase/metabolism* ; Vasodilation

Diabetic nephropathy is a microvascular complication of diabetes. Its etiology involves metabolic disorder-induced endothelial dysfunction. Endothelium-derived nitric oxide (NO) plays an important role in a number of physiological processes, including glomerular filtration and endothelial protection. NO dysregulation is an important pathogenic basis of diabetic nephropathy. Hyperglycemia and dyslipidemia can lead to oxidative stress, chronic inflammation and insulin resistance, thus affecting NO homeostasis regulated by endothelial nitric oxide synthase (eNOS) and a conglomerate of related proteins and factors. The reaction of NO and superoxide (O₂^.-) to form peroxynitrite (ONOO^-) is the most important pathological NO pathway in diabetic nephropathy. ONOO^- is a hyper-reactive oxidant and nitrating agent in vivo which can cause the uncoupling of eNOS. The uncoupled eNOS does not produce NO but produces superoxide. Thus, eNOS uncoupling is a critical contributor of NO dysregulation. Understanding the regulatory mechanism of NO and the effects of various pathological conditions on it could reveal the pathophysiology of diabetic nephropathy, potential drug targets and mechanisms of action. We believe that increasing the stability and activity of eNOS dimers, promoting NO synthesis and increasing NO/ONOO^- ratio could guide the development of drugs to treat diabetic nephropathy. We will illustrate these actions with some clinically used drugs as examples in the present review.
Diabetes Mellitus ; Diabetic Nephropathies/drug therapy* ; Endothelium, Vascular ; Humans ; Nitric Oxide/metabolism* ; Nitric Oxide Synthase Type III/therapeutic use* ; Oxidative Stress ; Peroxynitrous Acid/therapeutic use*

OBJECTIVE: To investigate the influence of electroacupuncture (EA) on ghrelin and the phosphoinositide 3-kinase/protein kinase B/endothelial nitric oxide synthase (PI3K/Akt/eNOS) signaling pathway in spontaneously hypertensive rats (SHRs). METHODS: Eight Wistar-Kyoto rats were used as the healthy blood pressure (BP) control (normal group), and 32 SHRs were randomized into model group, EA group, EA plus ghrelin group (EA + G group), and EA plus PF04628935 group (a potent ghrelin receptor blocker; EA + P group) using a random number table. Rats in the normal group and model group did not receive treatment, but were immobilized for 20 min per day, 5 times a week, for 4 continuous weeks. SHRs in the EA group, EA + G group and EA + P group were immobilized and given EA treatment in 20 min sessions, 5 times per week, for 4 weeks. Additionally, 1 h before EA, SHRs in the EA + G group and EA + P group were intraperitoneally injected with ghrelin or PF04628935, respectively, for 4 weeks. The tail-cuff method was used to measure BP. After the 4-week intervention, the rats were sacrificed by cervical dislocation, and pathological morphology of the abdominal aorta was observed using hematoxylin-eosin (HE) staining. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of ghrelin, nitric oxide (NO), endothelin-1 (ET-1) and thromboxane A2 (TXA2) in the serum. Isolated thoracic aortic ring experiment was performed to evaluate vasorelaxation. Western blot was used to measure the expression of PI3K, Akt, phosphorylated Akt (p-Akt) and eNOS proteins in the abdominal aorta. Further, quantitative real-time polymerase chain reaction (qRT-PCR) was conducted to measure the relative levels of mRNA expression for PI3K, Akt and eNOS in the abdominal aorta. RESULTS: EA significantly reduced the systolic BP (SBP) and diastolic BP (DBP) (P < 0.05). HE staining showed that EA improved the morphology of the vascular endothelium to some extent. Results of ELISA indicated that higher concentrations of ghrelin and NO, and lower concentrations of ET-1 and TXA2 were presented in the EA group (P < 0.05). The isolated thoracic aortic ring experiment demonstrated that the vasodilation capacity of the thoracic aorta increased in the EA group. Results of Western blot and qRT-PCR showed that EA increased the abundance of PI3K, p-Akt/Akt and eNOS proteins, as well as expression levels of PI3K, Akt and eNOS mRNAs (P < 0.05). In the EA + G group, SBP and DBP decreased (P < 0.05), ghrelin concentrations increased (P < 0.05), and the concentrations of ET-1 and TXA2 decreased (P < 0.05), relative to the EA group. In addition, the levels of PI3K and eNOS proteins, the p-Akt/Akt ratio, and the expression of PI3K, Akt and eNOS mRNAs increased significantly in the EA + G group (P < 0.05), while PF04628935 reversed these effects. CONCLUSION EA effectively reduced BP and protected the vascular endothelium, and these effects may be linked to promoting the release of ghrelin and activation of the PI3K/Akt/eNOS signaling pathway.
Animals ; Electroacupuncture ; Ghrelin/pharmacology* ; Nitric Oxide/metabolism* ; Nitric Oxide Synthase Type III/pharmacology* ; Phosphatidylinositol 3-Kinase/pharmacology* ; Phosphatidylinositol 3-Kinases/metabolism* ; Proto-Oncogene Proteins c-akt/pharmacology* ; Rats ; Rats, Inbred SHR ; Rats, Inbred WKY ; Signal Transduction

OBJECTIVE: To investigate the effect of biflavonoid 4'-O-methylochnaflavone (MF) on palmitic acid-induced endothelial dysfunction in rat cavernous endothelial cells (RCECs). METHODS: The isolated RCECs were commercially available and randomly divided into four groups: normal+BSA group (NC group), palmitic acid (PA) group, MF group, and icariside Ⅱ (ICA Ⅱ) group. The protein expression levels of protein kinase B (PKB/AKT) and endothelial nitric oxide synthase (eNOS) in each group were evaluated via Western blotting. The differences in the intracellular nitric oxide of RCECs treated by MF or ICA Ⅱ were detected by DAF-FM DA that served as a nitric oxide fluorescent probe. Effects of MF and ICA Ⅱ on cell proliferation of PA-stimulated RCECs were determined via CCK-8 assay. RESULTS: The content of nitric oxide in RCECs was significantly increased after the treatment of MF and ICA Ⅱ in comparison with the NC group (P < 0.05). Moreover, compared with ICA Ⅱ group, MF demonstrated a more obvious effect in promoting nitric oxide production (P < 0.05). Compared with the NC group, the expression levels of eNOS and AKT in the PA group were significantly decreased, indicating that a model for simulating the high-fat environment in vitro was successfully constructed (P < 0.05). Meanwhile, the intervention of MF and ICA Ⅱ could effectively increase the expression of eNOS and AKT, suggesting that MF and ICA Ⅱ could promote the recovery of endothelial dysfunction caused by high levels of free fatty acids (P < 0.05). The results of CCK-8 assays showed that PA could significantly reduce the proli-feration ability of RCECs (P < 0.05). Furthermore, the decreased cell viability induced by PA was significantly elevated by treatment with ICA Ⅱ and MF (P < 0.05). CONCLUSION In RCECs, MF and ICA Ⅱ could effectively increase the content of nitric oxide. The down-regulation of the expression of proteins associated with the AKT/eNOS pathway after PA treatment revealed that this pathway was involved in the development of endothelial dysfunction, which could be effectively reversed by MF and ICA Ⅱ. In addition, the cell proliferation ability was significantly decreased following PA treatment, but MF and ICA Ⅱ could restore the above changes. Overall, biflavonoid MF has an obvious repairing effect on PA-stimulated endothelial dysfunction.
Animals ; Biflavonoids/pharmacology* ; Cells, Cultured ; Endothelial Cells/metabolism* ; Nitric Oxide/pharmacology* ; Nitric Oxide Synthase Type III/pharmacology* ; Palmitic Acid/pharmacology* ; Phosphorylation ; Proto-Oncogene Proteins c-akt/metabolism* ; Rats ; Signal Transduction ; Sincalide/pharmacology*