Synthase Signaling Pathway Research Articles

Melissa officinalis Regulates Lipopolysaccharide-Induced BV2 Microglial Activation via MAPK and Nrf2 Signaling.

Neuroinflammation and microglial activation play critical roles in neurodegenerative diseases such as Alzheimer's and Parkinson's disease. Modulating microglial activation may help prevent the progression of these disorders. This study aimed to investigate the effects and mechanisms of Melissa officinalis ethanol extract on lipopolysaccharide (LPS)-induced microglial activation in BV2 cells. Cell viability and nitric oxide (NO) production were assessed using MTT assay and Griess reagent, while inflammatory cytokine levels were measured by qPCR. Key inflammatory pathways, including MAPK, TLR4, and antioxidant biomarkers, were analyzed through western blot and immunofluorescence. Rosmarinic acid content in M. officinalis was determined using high-performance liquid chromatography (HPLC). The results demonstrated that M. officinalis ethanol extract significantly inhibited LPS-induced NO production and reduced inflammatory cytokine expression. Additionally, it downregulated inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), TLR4, NF-κB, and MAPK signaling pathways (p38, JNK, ERK), while increasing the expression of antioxidant markers, including Nrf2, HO-1, catalase, and SOD2. In conclusion, M. officinalis ethanol extract exerts neuroprotective effects by modulating inflammation and enhancing antioxidant defenses, suggesting its potential in the prevention and treatment of inflammation-related neurodegenerative diseases.

Porous gelatin microspheres implanted with adipose mesenchymal stromal cells promote angiogenesis via protein kinase B/endothelial nitric oxide synthase signaling pathway in bladder reconstruction

Background aimsCell failure and angiogenesis are the key to bladder wall regeneration. Three-dimensional (3D) culture using porous gelatin microspheres (GMs) as a vehicle promotes stem cell proliferation and improves the paracrine capacity of cells. This study aimed to evaluate the therapeutic potential of GMs constructed from adipose-derived mesenchymal stromal cells (ADSCs) (ADSC-GMs) combined with bladder acellular matrix (BAM) in tissue-engineered bladders. MethodsIsolation of ADSCs, flow cytometry, scanning electron microscopy and cell counting kit-8, β-galactosidase and enzyme-linked immunosorbent assays were performed in vitro to compare two-dimensional (2D) and 3D cultures. In the in vivo study, male Sprague–Dawley rats were randomly divided into three groups: the BAM replacement alone (BAM) group, ADSCs grown on BAM in replacement (ADSC) group and ADSC-GMs combined with BAM followed by replacement (ADSC-GM) group. Bladder function assessed by urodynamics after 12 weeks of bladder replacement, and the rats were sacrificed at 4 and 12 weeks for further experiments. ResultsThe in vitro results showed that GM culture promoted ADSC proliferation, inhibited apoptosis and delayed senescence compared with those in the 2D culture. In addition, ADSC-GMs increased the secretion of the angiogenic factors vascular endothelial growth factor, platelet-derived growth factor-BB, and basal fibroblast growth factor. In vivo experiments revealed that ADSC-GMs adhered to the BAM for longer than ADSCs. Moreover, ADSC-GMs significantly promoted the regeneration of bladder vessels and smooth muscle, thereby facilitating the recovery of bladder function. The expression of phosphorylated protein kinase B (AKT) and phosphorylated endothelial nitric oxide synthase (eNOS) was significantly greater in the ADSC-GMs group compared with the BAM and ADSCs groups. ConclusionsADSC-GMs increased retention of ADSCs on the BAM, thereby promoting the regeneration and functional recovery of the bladder tissue. ADSC-GMs promoted angiogenesis by activating the AKT/eNOS pathway.

Role of hypoxia inducible factor/vascular endothelial growth factor/endothelial nitric oxide synthase signaling pathway in mediating the cardioprotective effect of dapagliflozin in cyclophosphamide-induced cardiotoxicity.

Cyclophosphamide (CP) is a commonly used chemotherapeutic and immunosuppressive alkylating agent. However, cardiac adverse effects of CP interfere with its clinical benefit. Cardio-oncology research is currently an important issue and finding effective cardiopreserving agents is a critical need. For the first time, we aimed to detect if dapagliflozin (DAP) could ameliorate CP-induced cardiac injury and investigated the role of hypoxia inducible factor α (HIF1α)/vascular endothelial growth factor (VEGF)/endothelial nitric oxide synthase (eNOS) pathway. Forty male Wistar albino rats were included in the current model. Studied groups are: control group; CP-induced cardiotoxicity group; CP group treated with DAP; CP group treated with DAP and administered a nitric oxide synthase inhibitor; nitro-ω-L-arginine (L-NNA) before DAP to explore the role of eNOS. Our data revealed that CP could induce cardiac damage as manifested by significant increases in cardiac enzymes, blood pressure, malondialdehyde (MDA), tumor necrosis factor alpha (TNFα), HIF1α, sodium glucose co-transporter 2 (SGLT2) and cleaved caspase-3 levels with toxic histopathological changes. However, there are significant decreases in reduced glutathione (GSH), total antioxidant capacity (TAC), VEGF, and eNOS. On the opposite side, co-administration of DAP showed marked improvement of CP-induced cardiac damage that may be due to its ability to inhibit SGLT2, antioxidant, anti-inflammatory and anti-apoptotic properties. Results showed decreasing the cardioprotective effect of DAP on administration of L-NNA, reflecting the critical effect of eNOS in mediating such protection. DAP could reduce CP cardiotoxicity based upon its ability to modulate SGLT2 and HIF1α/VEGF/eNOS signaling pathway.

TiCPG - a strategy for the simultaneous enrichment of reversibly modified cysteine peptides, phosphopeptides, and sialylated N-Glycopeptides to study cytokines stimulated beta-cells

Diverse post-translational modifications (PTMs) regulate protein function and interaction to fine-tune biological processes. Reversible phosphorylation, cysteines (Cys) modifications, and N-linked glycosylation are all essentially involved in cellular signaling pathways, such as those initiated by the action of pro-inflammatory cytokines, which can induce pancreatic β-cell death and diabetes. Here we have developed a novel strategy for the simultaneous and comprehensive characterization of the proteome and three PTMs including reversibly modified Cysteines (rmCys), phosphorylation, and sialylated N-linked glycosylation from low amount of sample material. This strategy, termed TiCPG, is based on a combination of chemical labeling and titanium dioxide (TiO2) chromatography. We applied the TiCPG strategy to study the proteome and the three PTMs changes in β-cells subject to pro-inflammatory cytokines stimulation. It enabled quantitative analysis of 8346 rmCys sites, 10,321 phosphosites and 962 sialylated N-glycosites from 5496 proteins. Significant regulation was found on 100 proteins at the expression level, while 3020 PTM peptide isoforms from 1468 proteins were significantly regulated. The three PTMs were involved in cytokine mediated β-cell apoptosis, such as the NFκB and the inducible NO synthase signaling pathways. Overall, the TiCPG strategy is a cheap, straightforward, and powerful tool for studies targeting the three PTMs described above. SignificanceThe present study presents a fast and easy method for quantitative assessment of the proteome and three PTMs from minimal amount of sample material. This simple method provides comprehensive and significant knowledge on biological systems and cellular signaling with relatively low analysis time, suitable for younger researchers and researchers that do not have direct access to LC-MSMS in their laboratories. From sub-milligram amount of material, we were able to map known cellular signaling events of proinflammatory cytokine effect on beta-cells and to discover novel PTMs involved in several known signaling pathways.

Effect of the NF-κB/iNOS Signaling Pathway on Spiral Ganglion in Mouse Model of Sensorineural Hearing Loss

To explore the effect of changes in the expression level of necorsis factor (NF)-κB/inducible nitric oxide synthase (iNOS) signaling pathway on hearing loss in a mouse model of sensorineural hearing loss (SNHL) induced by 3-nitropropionic acid (3-NP). The animal model was established by tympanic injection. C57BL/6 male mice were divided into three groups, 3-NP group receiving tympanic injection of 3-NP solution, 3-NP+EVP4593 group receiving tympanic injection of 3-NP solution and intraperitoneal injection of EVP4593 solution, and a control group receiving tympanic injection of phosphate buffered saline (PBS). Auditory brainstem response (ABR) was tested before and after injection. After 4 weeks, the cochlea was harvested and immunohistochemistry and qRT-PCR of NF-κB p65, RelB, iNOS, and Caspase-3 were conducted accordingly. The hearing thresholds of the 3-NP group were higher than those of the control group and the 3-NP+EVP4593 group ( P<0.05), and the hearing thresholds of the 3-NP+EVP4593 group were also higher than those of the control group ( P<0.05). Immunofluorescence staining and qRT-PCR results showed that 3-NP exposure caused an increase in the expressions of NF-κB p65, RelB, and iNOS in the spiral ganglion in comparison with those of the control group ( P<0.05), and their expressions decreased with the administration of EVP4593 ( P<0.05). The expression of Caspase-3 in the spiral ganglion cells in the 3-NP group was higher than that in the control group, while in the 3-NP+EVP4593 group, it was lower than that in the 3-NP group ( P<0.05). This study found that, by activating the NF-κB/iNOS signaling pathway, 3-NP may cause inflammation in the spiral ganglion of the cochlear in the SNHL model mice, which may play an important role in the pathogenesis of SNHL.

Influence of electroacupuncture on ghrelin and the phosphoinositide 3-kinase/protein kinase B/endothelial nitric oxide synthase signaling pathway in spontaneously hypertensive rats

ObjectiveTo 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). MethodsEight 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. ResultsEA 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. ConclusionEA 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.

Citrus maxima and tea regulate AMPK signaling pathway to retard the progress of nonalcoholic fatty liver disease.

BackgroundNonalcoholic fatty liver disease (NAFLD) is a chronic metabolic disease that easily induces hepatitis, cirrhosis, and even liver cancer. The long-term use of NAFLD therapeutic drugs produces toxicity and drug resistance. Therefore, it is necessary to develop high efficiency and low-toxicity active ingredients to alleviate NAFLD.ObjectiveThis study aimed to reveal the role and mechanism of a new functional food CMT in alleviating NAFLD.ResultsIn the ob/ob fatty liver mice models, the CMT extracts significantly inhibited the weight gain of the mice and reduced the accumulation of white fat. The anatomical and pathological results showed that CMT relieved fatty liver in mice and reduced excessive lipid deposition and inflammatory infiltration. Serological and liver biochemical indicators suggest that CMT reduced dyslipidemia and liver damage caused by fatty liver. CMT obviously activated the adenosine 5′-monophosphate-activated protein kinase (AMPK)/acetyl-coA carboxylase (ACC) and AMPK/fatty acid synthase (FAS) signaling pathways, promoted fat oxidation, and inhibited synthesis. Moreover, CMT regulated the expression of inflammatory factors to relieve hepatitis caused by NAFLD.ConclusionThe study explained the role and mechanism of CMT in alleviating NAFLD and suggested that the active ingredients of CMT might be beneficial in NAFLD therapy.

Propionate ameliorates diabetes-induced neurological dysfunction through regulating the PI3K/Akt/eNOS signaling pathway

A large body of research has established diabetes-related cognitive deterioration, sometimes known as “diabetic encephalopathy”. Current evidence supports that oxidative stress, neuronal apoptosis, and cerebral microcirculation weakness are associated with cognition deficits induced by diabetes. The present study explores the effect of propionate on neurological deficits, cerebral blood flow, and oxidative stress in diabetic mice. Propionate in different doses (37.5, 75 and 150 mg/kg) was orally administrated daily. Here, we show that propionate can markedly improve neurological function, which is correlated with its capabilities of stimulating nitrogen monoxide (NO) production, increasing cerebral microcirculation, suppressing oxidative stress, and reducing neuron loss in the hippocampus. In addition, the results of Western Blotting indicated that the brain-protective function of propionate in streptozocin (STZ)-induced type 1 diabetes mellitus (T1DM) mice is related to phosphoinositide 3-kinase (PI3K)/serine-threonine protein kinase (Akt)/endothelial nitrogen monoxide synthase (eNOS) signaling pathway. In a diabetic mouse model, propionate reduces cerebral microcirculation, hippocampus apoptosis, and neurological impairment. Thus, propionate, now employed as a food preservative, may also help slow diabetes-induced cognitive loss.

Residues of Culinary-Medicinal Winter Mushroom, Flammulina velutipes (Agaricomycetes), Cultivation as a Potential Source of Functional Skin Substitute with Multiple Bioactivities.

Residues generated during the cultivation of edible mushroom Flammulina velutipes are abundant and utilized with low efficiency. In this study, the composition and bioactivities of a skin substitute named TG05 obtained from residues of the F. velutipes cultivation process were investigated. The main composition of TG05 was considered to be chitin and it inhibited growth of Gram-positive Staphylococcus aureus and Gram-negative Pseudomonas aeruginosa. TG05 also suppressed the inflammatory response through the inducible nitric oxide synthase signaling pathway. Inflammation was attenuated by reducing the expression of tumor necrosis factor-α, interleukin (IL)-1β, IL-6, and prostaglandin E2 at the transcription level. Furthermore, TG05 exhibited antioxidant activities based on hydroxyl, 2,2-diphenyl-1-picryl-hydrazy, 2,2'-azobis-(3-ethylbenzothiazoline-6-sulfonic acid), superoxide anion radical scavenging activity, and reducing power assays. However, the effect of TG05 was independent of hyaluronidase inhibitory activity. Taken together, specific mechanisms related to the notable wound-healing-promoting activity of TG05 were demonstrated, mainly attributable to its antimicrobial, anti-inflammatory, and antioxidant activities. Therefore, TG05 may have potential for use as a functional biomaterial in various applications.

Cancer-Associated Fibroblasts Promote Migration and Invasion of Non-Small Cell Lung Cancer Cells via miR-101-3p Mediated VEGFA Secretion and AKT/eNOS Pathway.

Cancer-associated fibroblasts (CAFs) are major component of tumor microenvironment (TME), which plays crucial roles in tumor growth, invasion and metastasis; however, the underling mechanism is not fully elucidated. Despite many studies are focused on the tumor promoting effect of CAFs-derived cytokines, the upstream regulators of cytokine release in CAFs is largely unknown. Here we found that miR-101-3p was downregulated in primary lung cancer-associated CAFs compared to normal fibroblasts (NFs). Ectopic overexpression of miR-101-3p suppressed CAFs activation, and abrogated the promoting effect of CAFs on migration and invasion of non-small cell lung cancer cells (NSCLC), through attenuating CAFs’ effect on epithelial mesenchymal transition (EMT) process, metastasis-related genes (MMP9, TWIST1) and AKT/endothelial nitric oxide synthase (eNOS) signaling pathway. Further study indicated that vascular endothelial growth factor A (VEGFA) was a novel target of miR-101-3p, and CAFs-derived VEGFA mediated the effect of miR-101-3p on migration and invasion of lung cancer cells, demonstrated by using recombinant VEGFA and VEGFA neutralizing antibody. Interestingly, the analysis of the Cancer Genome Atlas (TCGA) database revealed that lung cancer tissues expressed lower level of miR-101-3p than non-cancerous tissues, and low/medium-expression of miR-101-3p was associated with poor overall survival (OS) rate. Moreover, the mouse xenograft experiment also showed that CAFs accelerated tumor growth whereas miR-101-3p diminished CAFs’ effect. These findings revealed a novel mechanism that CAFs facilitated lung cancer metastasis potential via miR-101-3p/VEGFA/AKT signaling pathway, suggesting miR-101-3p as a potential candidate for metastasis therapy.

Nebivolol alleviates liver damage caused by methotrexate via AKT1/Hif1α/eNOS signaling

Despite the wide clinical indications, methotrexate (MTX) use is limited because of serious side effects including liver toxicity. MTX was shown to cause tissue damage by mainly oxidative stress and also inflammation and apoptosis. Thus, Nebivolol (NEB) which has antioxidant and antiapoptotic properties were thought to be effective against MTX-induced injury. This study aimed to evaluate the effects of NEB on MTX-induced liver toxicity via AKT/Hypoxia-Inducible Factor 1 Alpha (HIF1α)/Endothelial Nitric Oxide Synthase (eNOS) signaling pathways. Rats were divided into three groups as control, MTX, and NEB. A single dose of MTX (20 mg/kg intraperitoneally) was given to the rats on the first day of the experiment and NEB (10 mg/kg, daily by oral gavage) was given to the treatment group for a week. At the end of the experiment, bloods were taken for aspartate transaminase (AST), alanine aminotransferase (ALT), and total bilirubin (T-BIL) analyses. Liver tissues were harvested for biochemical (total oxidant status (TOS) and total antioxidant status (TAS), genetic (PCR analyses for AKT1, eNOS, and HIF1a), and histological (Hemotoxylin-Eosin, Masson Trichome, Periodic Acid Schiff-Asien Blue, reticulin for histological, and CD3 for immunohistochemical staining) analyses. MTX increased the levels of TOS values, AST, ALT, T-BIL levels and decreased the expressions of AKT/HIF1α/eNOS. NEB treatment reversed all these changes markedly via decreasing inflammation by nitric oxid (NO) production. In conclusion, NEB treatment significantly preserves the liver by decreasing oxidant levels and inflammatory parameters through HIF1α/eNOS signaling. Due to the antioxidant properties of NEB, it can be used in other liver injury models sharing the same pathway.

Cardioprotective Effects of Glucagon-like Peptide-1 (9-36) Against Oxidative Injury in H9c2 Cardiomyoblasts: Potential Role of the PI3K/Akt/NOS Pathway.

Glucagon-like peptide (GLP)-1(7-36), a major active form of GLP-1 hormone, is rapidly cleaved by dipeptidyl peptidase-4 to generate a truncated metabolite, GLP-1(9-36) which has a low affinity for GLP-1 receptor (GLP-1R). GLP-1(7-36) has been shown to have protective effects on cardiovascular system through GLP-1R-dependent pathway. Nevertheless, the cardioprotective effects of GLP-1(9-36) have not fully understood. The present study investigated the effects of GLP-1(9-36), including its underlying mechanisms against oxidative stress and apoptosis in H9c2 cells. Here, we reported that GLP-1(9-36) protects H9c2 cardiomyoblasts from hydrogen peroxide (H2O2)-induced oxidative stress by promoting the synthesis of antioxidant enzymes, glutathione peroxidase-1, catalase, and heme oxygenase-1. In addition, treatment with GLP-1(9-36) suppressed H2O2-induced apoptosis by attenuating caspase-3 activity and upregulating antiapoptotic proteins, Bcl-2 and Bcl-xL. These protective effects of GLP-1(9-36) are attenuated by blockade of PI3K-mediated Akt phosphorylation and prevention of nitric oxide synthase-induced nitric oxide production. Thus, GLP-1(9-36) represents the potential therapeutic target for prevention of oxidative stress and apoptosis in the heart via PI3K/Akt/nitric oxide synthase signaling pathway.

Phosphoinositide-3-Kinase/Akt–Endothelial Nitric Oxide Synthase Signaling Pathway Mediates the Neuroprotective Effect of Sevoflurane Postconditioning in a Rat Model of Hemorrhagic Shock and Resuscitation

Although extensive reports have demonstrated the neuroprotection of sevoflurane postconditioning in cases of focal and global cerebral ischemia/reperfusion, the underlying mechanisms are not completely elucidated. This study investigated whether this effect is related to endothelial nitric oxide synthase (eNOS) and mediated by the phosphoinositide-3-kinase pathway in a rat model of hemorrhagic shock and resuscitation. Adult male Sprague Dawley rats were subjected to hemorrhagic shock for 60 minutes and then resuscitation for 30 minutes in experimental groups. Sevoflurane postconditioning was performed at the beginning of resuscitation to completion. At 24 hours after resuscitation, the brain infarct volume was evaluated by 2,3,5-triphenyltetrazolium chloride staining. The neuronal morphological changes and apoptosis were determined by hematoxylin and eosin staining and immunohistochemistry analysis, respectively. The activity of phosphorylated Akt and eNOS was evaluated by Western blot analysis. Brain injuries such as the cerebral infarct volume and pathological neuronal changes as well as cell apoptosis were observed in the hippocampus after hemorrhagic shock and resuscitation. Postconditioning with 2.4% sevoflurane significantly attenuated brain injuries. Wortmannin prevented the improvements of neuronal characteristics elicited by sevoflurane postconditioning as well as the hyperactivity of eNOS and phosphorylated Akt. Sevoflurane postconditioning could attenuate brain injury induced by hemorrhagic shock and resuscitation, and this neuroprotective effect may be partly by upregulation of eNOS through the phosphoinositide-3-kinase/Akt signaling pathway.

Salidroside inhibits endothelial‑mesenchymal transition via the KLF4/eNOS signaling pathway.

Homocysteine (Hcy) was discovered to be an independent risk factor for the development of atherosclerosis (AS). Moreover, endothelial-mesenchymal transition (EndMT) was found to be one of main mechanisms contributing to the pathogenesis of AS. Salidroside (SAL) has diverse pharmacological activities, including anti-inflammatory, anti-cancer, anti-oxidative and anti-fibrosis properties. However, whether SAL serves a beneficial role in Hcy-induced EndMT remains unknown. The present study aimed to investigate whether SAL exerted its effects on Hcy-induced EndMT via the Kruppel-like factor 4 (KLF4)/endothelial nitric oxide (NO) synthase (eNOS) signaling pathway. HUVECs were pretreated with high and low doses (10 or 50 µmol/l) of SAL for 2 h, followed by 1 mmol/l Hcy for 48 h to induce EndMT. Western blotting was used to analyze the protein expression levels of the endothelial marker, VE-cadherin, the mesenchymal cell marker, α-smooth muscle actin (SMA), and the nuclear transcription factors, KLF4 and eNOS. Wound healing assays were used to determine the cell migratory ability, and the levels of NO in the cell culture supernatants were measured using a nitrate reductase assay. Cellular immunofluorescence was used to analyze the expression and localization of KLF4. Small interfering (si)RNA targeting KLF4 (siKLF4) was used to knock down KLF4 expression in HUVECs. The results of the present study revealed that treatment with SAL upregulated the expression levels of VE-cadherin, downregulated the expression levels of α-SMA, reduced cell migration and activated the eNOS/NO signaling axis, as well as downregulated KLF4 expression and translocation to the nucleus. Compared with the SAL + siKLF4 co-administration group, no significant differences were observed in the expression levels of the phenotypic markers in the SAL or siKLF4 groups. In conclusion, the findings of the present study revealed that SAL may inhibit Hcy-induced EndMT via regulation of the KLF4/eNOS signaling pathway.

Angiotensin (1-7) Alleviates Postresuscitation Myocardial Dysfunction by Suppressing Oxidative Stress Through the Phosphoinositide 3-Kinase, Protein Kinase B, and Endothelial Nitric Oxide Synthase Signaling Pathway.

There is increasing evidence that angiotensin (1-7) [Ang (1-7)] is an endogenous biologically active component of the renin-angiotensin system. However, the role of the Ang (1-7)-MasR axis in postresuscitation myocardial dysfunction (PRMD) and its associated mechanism are still unclear. In this study, we investigated the effect of the Ang (1-7)-MasR axis on myocardial injury after cardiac arrest-cardiopulmonary resuscitation-restoration of spontaneous circulation. We established a model of oxygen/glucose deprivation-reperfusion in myocardial cells in vitro and a rat model of cardiac arrest-cardiopulmonary resuscitation-restoration of spontaneous circulation in vivo. The cell apoptosis rate and the expression of the superoxide anion 3-nitrotyrosine were decreased in the Ang (1-7) group in vitro and in vivo. The mean arterial pressure was decreased, whereas +LVdp/dtmax and -LVdp/dtmax were increased in rats in the Ang (1-7) group. The mRNA and protein levels of Ang II type 1 receptor, MasR, phosphoinositide 3-kinase, protein kinase B, and endothelial nitric oxide synthase were increased in the Ang (1-7) group in vivo. These results indicate that the Ang (1-7)-MasR axis can alleviate PRMD by reducing myocardial tissue damage and oxidative stress through activation of the phosphoinositide 3-kinase-protein kinase B-endothelial nitric oxide synthase signaling pathway and provide a new direction for the clinical treatment of PRMD.

Se-Methylselenocysteine Alleviates Liver Injury in Diethylnitrosamine (DEN)-Induced Hepatocellular Carcinoma Rat Model by Reducing Liver Enzymes, Inhibiting Angiogenesis, and Suppressing Nitric Oxide (NO)/Nitric Oxide Synthase (NOS) Signaling Pathway.

BackgroundHepatocellular carcinoma is the third leading cause of cancer-associated mortality. This study aimed to investigate the effects of se-methylselenocysteine (MSC) on oncogenesis of diethylnitrosamine (DEN)-induced hepatocellular carcinoma.Material/MethodsA hepatocellular carcinoma rat model was established by administering DEN. Rat models were divided into Model (0.1 mg/kg MSC), Model+0.3 mg/kg MSC, Model+1 mg/kg MSC, and Model+3 mg/kg MSC groups. A Normal control group consisted of mice not administered MSC. Hematoxylin and eosin staining was used to determine liver injury. Immunohistochemical analysis was conducted to identify CD34 and vascular endothelial growth factor (VEGF) expression. VEGF gene transcription was detected with RT-PCR. Biochemical analyses were performed to determine alanine aminotransferase, aspartate aminotransferase, total bilirubin, γ-glutamyl transpeptidase, alkaline phosphatase, and albumin levels in serum, and nitric oxide (NO)/nitric oxide synthase (NOS) levels in liver tissues. Transmission electron microscopy was used to observe the ultra-microstructures of hepatocytes.ResultsMSC treatment markedly alleviated liver injury and nuclear lesions in the treatment groups compared to the Model group. MSC treatment significantly improved liver functions in the treatment groups compared to the Model group (P<0.05). MSC treatment significantly decreased CD34 expression and NO and NOS levels in liver tissues and suppressed VEGF expression compared to the Model group (all P<0.05).ConclusionsMSC administration alleviated liver injury in a DEN-induced hepatocellular carcinoma rat model through reducing liver enzymes, inhibiting angiogenesis, and suppressing the NO/NOS signaling pathway.

CX3CR1-microglia mediates neuroinflammation and blood pressure regulation in the nucleus tractus solitarii of fructose-induced hypertensive rats

BackgroundInflammation is a common pathophysiological trait found in both hypertension and cardiac vascular disease. Recent evidence indicates that fractalkine (FKN) and its receptor CX3CR1 have been linked to inflammatory response in the brain of hypertensive animal models. Here, we investigated the role of CX3CR1-microglia in nitric oxide (NO) generation during chronic inflammation and systemic blood pressure recovery in the nucleus tractus solitarii (NTS).MethodsThe hypertensive rat model was used to study the role of CX3CR1-microglia in NTS inflammation following hypertension induction by oral administration of 10% fructose water. The systolic blood pressure was measured by tail-cuff method of non-invasive blood pressure. The CX3CR1 inhibitor AZD8797 was administered intracerebroventricularly (ICV) in the fructose-induced hypertensive rat. Using immunoblotting, we studied the nitric oxide synthase signaling pathway, NO concentration, and the levels of FKN and CX3CR1, and pro-inflammatory cytokines were analyzed by immunohistochemistry staining.ResultsThe level of pro-inflammatory cytokines IL-1β, IL-6, TNF-α, FKN, and CX3CR1 were elevated two weeks after fructose feeding. AZD8797 inhibited CX3CR1-microglia, which improved the regulation of systemic blood pressure and NO generation in the NTS. We also found that IL-1β, IL-6, and TNF-α levels were recovered by AZD8797 addition.ConclusionWe conclude that CX3CR1-microglia represses the nNOS signaling pathway and promotes chronic inflammation in fructose-induced hypertension. Collectively, our results reveal the role of chemokines such as IL-1β, IL-6, and TNF-α in NTS neuroinflammation with the involvement of FKN and CX3CR1.

Mathematical Model Predicts Effective Strategies to Inhibit VEGF-eNOS Signaling.

The endothelial nitric oxide synthase (eNOS) signaling pathway in endothelial cells has multiple physiological significances. It produces nitric oxide (NO), an important vasodilator, and enables a long-term proliferative response, contributing to angiogenesis. This signaling pathway is mediated by vascular endothelial growth factor (VEGF), a pro-angiogenic species that is often targeted to inhibit tumor angiogenesis. However, inhibiting VEGF-mediated eNOS signaling can lead to complications such as hypertension. Therefore, it is important to understand the dynamics of eNOS signaling in the context of angiogenesis inhibitors. Thrombospondin-1 (TSP1) is an important angiogenic inhibitor that, through interaction with its receptor CD47, has been shown to redundantly inhibit eNOS signaling. However, the exact mechanisms of TSP1′s inhibitory effects on this pathway remain unclear. To address this knowledge gap, we established a molecular-detailed mechanistic model to describe VEGF-mediated eNOS signaling, and we used the model to identify the potential intracellular targets of TSP1. In addition, we applied the predictive model to investigate the effects of several approaches to selectively target eNOS signaling in cells experiencing high VEGF levels present in the tumor microenvironment. This work generates insights for pharmacologic targets and therapeutic strategies to inhibit tumor angiogenesis signaling while avoiding potential side effects in normal vasoregulation.

M1-like macrophage-derived exosomes suppress angiogenesis and exacerbate cardiac dysfunction in a myocardial infarction microenvironment.

The roles and the underlying mechanisms of M1-type macrophages in angiogenesis and postmyocardial infarction (MI) cardiac repair have remained unclear. In this study, we investigated the role of M1-like macrophage-derived exosomes in a MI microenvironment. We found that the proinflammatory M1-like-type macrophages released an extensive array of proinflammatory exosomes (M1-Exos) after MI. M1-Exos exerted an anti-angiogenic effect and accelerated MI injury. They also exhibited highly expressed proinflammatory miRNAs, such as miR-155. miR-155 was transferred to endothelial cells (ECs), leading to the inhibition of angiogenesis and cardiac dysfunction by downregulating its novel target genes, including Rac family small GTPase 1 (RAC1), p21 (RAC1)-activated kinase 2 (PAK2), Sirtuin 1 (Sirt1), and protein kinase AMP-activated catalytic subunit alpha 2 (AMPKα2). M1-Exos depressed Sirt1/AMPKα2-endothelial nitric oxide synthase and RAC1-PAK2 signaling pathways by simultaneously targeting the five molecule nodes (genes), reduced the angiogenic ability of ECs, aggravated myocardial injury, and restrained cardiac healing. The elucidation of this mechanism provides novel insights into the functional significance of M1 macrophages and their derived exosomes on angiogenesis and cardiac repair. This mechanism can be used as a novel potential therapeutic approach for the prevention and treatment of MI.

Protective effects of phenolic acid extract from ginseng on vascular endothelial cell injury induced by palmitate via activation of PI3K/Akt/eNOS pathway.

Elevated free fatty acids may impair insulin-mediated signaling to eNOS that contributes to the pathophysiology of endothelial dysfunction. Previous studies have indicated the protective effect of ginseng and the regulatory potential of phenolic acid components from other plants on endothelial function. Therefore, this study investigated the protective effects of phenolic acid extract from ginseng (PG2) on endothelial cells against palmitate-induced damage. We found that PG2 increases cell viability, inhibits the palmitate-induced intracellular accumulation of lipids, and the overexpression of endothelin-1 (ET-1) through enhancing the phosphorylation of the phosphatidylinositol 3-kinase/Akt/endothelial nitric oxide synthase (PI3K/Akt/eNOS) signaling pathway. The results of this study may be valuable for the development of PG2 to combat the endothelial cell damage caused by hyperlipidemia. PRACTICAL APPLICATION: We proved that phenolic acid extract from ginseng has a protective effect on free fatty acid-induced endothelial dysfunction in vitro. This study provides experimental data for the application of ginseng-derived phenolic acids in treating cardiovascular disease.