eNOS Signaling Pathway Research Articles

Accelerating diabetic wound healing with Ramulus Mori (Sangzhi) alkaloids via NRF2/HO-1/eNOS pathway

Diabetic foot ulcers (DFUs) represent a severe complication of diabetes mellitus. Ramulus Mori (Sangzhi) alkaloids (SZ-A), an approved oral medication for type 2 diabetes, have not been explored for their potential to enhance the processes involved in diabetic wound healing. This study aims to investigate SZ-A's role in diabetic wound healing mechanisms. The in vivo experimentation involves dividing the subjects into NC and SZ-A groups, with SZ-A dosed at 200 and 400 mg/kg, to assess the therapeutic efficacy of SZ-A. The results of the animal studies show that SZ-A intervention accelerates the processes of diabetic angiogenesis and wound healing in a manner dependent on its concentration. Additionally, a pathological model using advanced glycation end products (AGEs) in HUVECs demonstrates SZ-A's cytoprotective effect. In vitro, SZ-A intervention significantly increases cell proliferation, migration and tube formation, protecting HUVECs from oxidative stress injury induced by AGEs. Mechanistically, SZ-A exerts a protective effect on HUVECs from oxidative stress damage through the activation of the NRF2/HO-1/eNOS signaling pathway. The findings suggest that SZ-A exhibits considerable potential as a promising candidate for treating DFUs, which will aid in more effectively integrating plant-based therapies into clinical settings.

Vernodalin Alleviates Cardiotoxicity and Inflammation in Isoproterenol-Mediated Myocardial Infarction through NF-κB/AMPK Signaling Pathways in Rats.

Myocardial infarction (MI) is the foremost cause of mortality in cardiovascular diseases. MI ultimately exacerbates cardiotoxicity due to the release of toxicity biomarkers and inflammatory infiltration. Vernodalin (VN) is a renowned cytotoxic sesquiterpene lactone that possesses antioxidant, anticancer, and anti-inflammatory properties. The cardioprotective mechanism of VN remains concealed. Hence, we explored the cardioprotective efficacy of VN on isoproterenol (ISO)- mediated MI and analyzed its underlying mechanism. Wistar albino rats were injected ISO (85 mg/kg bw) subcutaneously to induce MI to evaluate the cardioprotective potential of VN (10 mg/kg bw) by assessing heart weight/ body weight index, hemodynamic, toxicity enzymes, histopathology, inflammatory mediators, and signaling pathway. ISO enhanced heart weight/body weight index, cardiotoxicity enzymes, biomarkers, inflammation, and histopathological changes while reducing hemodynamic parameters and VEGF-B, AMPK, and eNOS signaling pathways. Treatment with VN could significantly (p<0.05) mitigate the heart weight/body weight index, cardiotoxicity enzymes, biomarkers, inflammatory cytokines, and histopathological changes while enhancing hemodynamic parameters and VEGF-B, AMPK, and eNOS signaling pathways. Collectively, our findings revealed that the VN ameliorated defensive action against MI and averted myocardial injury by reducing the NF-κB-mediated inflammatory pathways in rats. These findings established that VN expressively preserves the myocardium and employs anti-inflammatory actions by regulating NF-κB, VEGF-B, AMPK, and eNOS signaling pathways.

Coronary Artery Disease Risk Variant Dampens the Expression of CALCRL by Reducing HSF Binding to Shear Stress Responsive Enhancer in Endothelial Cells In Vitro.

CALCRL (calcitonin receptor-like) protein is an important mediator of the endothelial fluid shear stress response, which is associated with the genetic risk of coronary artery disease. In this study, we functionally characterized the noncoding regulatory elements carrying coronary artery disease that risks single-nucleotide polymorphisms and studied their role in the regulation of CALCRL expression in endothelial cells. To functionally characterize the coronary artery disease single-nucleotide polymorphisms harbored around the gene CALCRL, we applied an integrative approach encompassing statistical, transcriptional (RNA-seq), and epigenetic (ATAC-seq [transposase-accessible chromatin with sequencing], chromatin immunoprecipitation assay-quantitative polymerase chain reaction, and electromobility shift assay) analyses, alongside luciferase reporter assays, and targeted gene and enhancer perturbations (siRNA and clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9) in human aortic endothelial cells. We demonstrate that the regulatory element harboring rs880890 exhibits high enhancer activity and shows significant allelic bias. The A allele was favored over the G allele, particularly under shear stress conditions, mediated through alterations in the HSF1 (heat shock factor 1) motif and binding. CRISPR deletion of rs880890 enhancer resulted in downregulation of CALCRL expression, whereas HSF1 knockdown resulted in a significant decrease in rs880890-enhancer activity and CALCRL expression. A significant decrease in HSF1 binding to the enhancer region in endothelial cells was observed under disturbed flow compared with unidirectional flow. CALCRL knockdown and variant perturbation experiments indicated the role of CALCRL in mediating eNOS (endothelial nitric oxide synthase), APLN (apelin), angiopoietin, prostaglandins, and EDN1 (endothelin-1) signaling pathways leading to a decrease in cell proliferation, tube formation, and NO production. Overall, our results demonstrate the existence of an endothelial-specific HSF (heat shock factor)-regulated transcriptional enhancer that mediates CALCRL expression. A better understanding of CALCRL gene regulation and the role of single-nucleotide polymorphisms in the modulation of CALCRL expression could provide important steps toward understanding the genetic regulation of shear stress signaling responses.

Ginsenoside Rb1 prevents age-related endothelial senescence by modulating SIRT1/caveolin-1/enos signaling pathway

BackgroundAdvancing age is one of the independent risk factors for cardiovascular disorders. The Compendium of Materia Medica, a classic book on traditional Chinese medicine, states that ginseng “harmonizes the five internal organs, calming the spirit and prolonging the years of life." Considered one of the primary bioactive compounds derived from Panax ginseng, ginsenoside Rb1 (g-Rb1) has been scientifically suggested to possess anti-senescence efficacy. More research is needed to explore the vascular pharmacological activity and potential clinical application value of g-Rb1. Aims of the studyOur previous study demonstrated that g-Rb1 could mitigate cellular senescence via the SIRT1/eNOS pathway. This study was performed to explore the exact mechanisms by which g-Rb1 modulates the SIRT1/eNOS pathway. Materials and methodsWe used human primary umbilical vein endothelial cells (HUVECs) to establish a replicative ageing model. Real-time (RT‒PCR), western blotting, small interfering RNA (siRNA), and immunoprecipitation were conducted to detect the effect of g-Rb1 on the SIRT1/caveolin-1/eNOS axis. ResultsG-Rb1 increased NO production and alleviated replicative senescence of HUVECs. The application of g-Rb1 elevated the mRNA and protein abundance of both SIRT1 and eNOS while concomitantly suppressing the expression of caveolin-1. Inhibition of SIRT1 and eNOS by siRNAs suppressed the anti-senescence function of g-Rb1, while caveolin-1 siRNA could enhance it. G-Rb1 decreased the acetylation level of caveolin-1 and increased NO production, which was suppressed by SIRT1 siRNA. Both g-Rb1 and caveolin-1 siRNA could reduce the acetylation level of eNOS and increase NO production. ConclusionG-Rb1 prevents age-related endothelial senescence by modulating the SIRT1/caveolin-1/eNOS signaling pathway.

Periodontal Ligament Stem Cell Exosomes Key to Regulate Periodontal Regeneration by miR-31-5p in Mice Model.

Periodontitis is a chronic inflammatory disease that causes alveolar bone loss. Diabetes is one of the most important factors contributing to periodontitis. Exosomes derived from mesenchymal stem cells (MSCs-Exo) have been reported to promote bone regeneration. This study aimed to examine the function and mechanism of exosomes derived from periodontal ligament stem cells (PDLSCs-Exo) in regulating periodontal regeneration in diabetic periodontitis. Exosomes derived from normal-glucose-cultured PDLSCs (NG-PDLSCs-Exo) and high-glucose-preconditioned PDLSCs (HG-PDLSCs-Exo) were used. Their effects on RAW264.7 cells were investigated by TRAP staining and quantitative real time-polymerase chain reaction (qRT-PCR). The role of exosomal miR-31-5p in osteoclast differentiation was tested using qRT-PCR, double luciferase analysis, and Western blotting. We investigated the effects of these two types of PDLSCs-Exo on alveolar bone loss in vivo in mice with experimental periodontitis. PDLSCs-Exo were transferred to RAW264.7, and HG-PDLSCs-Exo inhibited osteoclast formation to a lesser extent than NG-PDLSCs-Exo. Further studies revealed the effect of PDLSCs-Exo on osteoclastogenesis via the miR-31-5p/eNOS signaling pathway. In mice with experimental periodontitis, PDLSCs-Exo reduced alveolar bone destruction and decreased the number of osteoclasts on the alveolar bone surface. Our results suggest that exosomal miR-31-5p derived from PDLSCs regulates alveolar bone regeneration by targeting eNOS.

A balanced formula of essential amino acids promotes brain mitochondrial biogenesis and protects neurons from ischemic insult.

Mitochondrial dysfunction plays a key role in the aging process, and aging is a strong risk factor for neurodegenerative diseases or brain injury characterized by impairment of mitochondrial function. Among these, ischemic stroke is one of the leading causes of death and permanent disability worldwide. Pharmacological approaches for its prevention and therapy are limited. Although non-pharmacological interventions such as physical exercise, which promotes brain mitochondrial biogenesis, have been shown to exert preventive effects against ischemic stroke, regular feasibility is complex in older people, and nutraceutical strategies could be valuable alternatives. We show here that dietary supplementation with a balanced essential amino acid mixture (BCAAem) increased mitochondrial biogenesis and the endogenous antioxidant response in the hippocampus of middle-aged mice to an extent comparable to those elicited by treadmill exercise training, suggesting BCAAem as an effective exercise mimetic on brain mitochondrial health and disease prevention. In vitro BCAAem treatment directly exerted mitochondrial biogenic effects and induced antioxidant enzyme expression in primary mouse cortical neurons. Further, exposure to BCAAem protected cortical neurons from the ischemic damage induced by an in vitro model of cerebral ischemia (oxygen-glucose deprivation, OGD). BCAAem-mediated protection against OGD was abolished in the presence of rapamycin, Torin-1, or L-NAME, indicating the requirement of both mTOR and eNOS signaling pathways in the BCAAem effects. We propose BCAAem supplementation as an alternative to physical exercise to prevent brain mitochondrial derangements leading to neurodegeneration and as a nutraceutical intervention aiding recovery after cerebral ischemia in conjunction with conventional drugs.

PTTG1 promotes CD34+CD45+ cells to repair the pulmonary vascular barrier via activating the VEGF-bFGF/PI3K/AKT/eNOS signaling pathway in rats with phosgene-induced acute lung injury

Accidental exposure to phosgene can cause acute lung injury (ALI), characterized by uncontrolled inflammation and impaired lung blood-gas barrier. CD34+CD45+ cells with high pituitary tumor transforming gene 1 (PTTG1) expression were identified around rat pulmonary vessels through single-cell RNA sequencing, and have been shown to attenuate P-ALI by promoting lung vascular barrier repair. As a transcription factor closely related to angiogenesis, whether PTTG1 plays a role in CD34+CD45+ cell repairing the pulmonary vascular barrier in rats with P-ALI remains unclear. This study provided compelling evidence that CD34+CD45+ cells possess endothelial differentiation potential. Rats with P-ALI were intratracheally administered with CD34+CD45+ cells transfected with or without PTTG1-overexpressing and sh-PTTG1 lentivirus. It was found that CD34+CD45+ cells reduced the pulmonary vascular permeability and mitigated the lung inflammation, which could be reversed by knocking down PTTG1. Although PTTG1 overexpression enhanced the ability of CD34+CD45+ cells to attenuate P-ALI, no significant difference was found. PTTG1 was found to regulate the endothelial differentiation of CD34+CD45+ cells. In addition, knocking down of PTTG1 significantly reduced the protein levels of VEGF and bFGF, as well as their receptors, which in turn inhibited the activation of the PI3K/AKT/eNOS signaling pathway in CD34+CD45+ cells. Moreover, LY294002 (PI3K inhibitor) treatment inhibited the endothelial differentiation of CD34+CD45+ cells, while SC79 (AKT activator) yielded the opposite effect. These findings suggest that PTTG1 can promote the endothelial differentiation of CD34+CD45+ cells by activating the VEGF-bFGF/PI3K/AKT/eNOS signaling pathway, leading to the repair of the pulmonary vascular barrier in rats with P-ALI.

Protective role of nebivolol via AKT1/Hif-1α/eNOS signaling pathway: nephrotoxicity caused by methotrexate in a rat model.

Methotrexate (MTX) is an antineoplastic and anti-inflammatory agent, which is used in severe diseases. Its use should be limited due to side effects such as nephrotoxicity, myelotoxicity, and hepatotoxicity. Nebivolol (NBV), which is a beta-blocker used in the treatment of hypertension, also contributes to vasodilation in tissues by activating the endothelial nitric oxide synthase (eNOS) enzyme. The purpose of this study is to research the effect of NBV on MTX-induced nephrotoxicity through the AKT1/hypoxia-inducible factor 1-alpha (Hif-1α)/eNOS signaling pathway. The rats were randomly divided into three groups of eight each. The groups were control, MTX, and MTX + NBV. A single dose of 20 mg/kg MTX was given intraperitoneally to the rats on the first day of the study and 10 mg/kg NBV was given orally to the treatment group for 7 days. At the end of the study, rats' blood and kidney tissues were taken for histopathological, immunohistochemical, and biochemical examinations. MTX administration significantly decreased the expression levels of AKT1, eNOS, and Hif-1αcompared with the control group (p<0.001 for all), and NBV treatment increased these values compared with the MTX group (p<0.001 for all). In conclusion, NBV treatment ameliorated the MTX-induced nephrotoxicity via AKT1/Hif-1α/eNOS signaling pathway.

Vitexin Regulates Angiogenesis and Osteogenesis in Ovariectomy-Induced Osteoporosis of Rats via the VDR/PI3K/AKT/eNOS Signaling Pathway

It is extremely important to promote angiogenesis-dependent osteogenesis and ameliorate bone loss for the prevention and treatment of osteoporosis (OP) development. Vitexin, as one of the major active components in pigeonpea leave, promoted the proliferation of osteoblast and HUVECs in hypoxia. The present study aimed to investigate the effect of vitexin on alleviating osteoporosis in ovariectomized (OVX) rats and further explore its underlying mechanisms. Herein, the OVX rat model was established and treated with vitexin (10 mg kg-1) for 3 months. After being sacrificed, we performed hematoxylin-eosin (H&E) staining and micro-computed tomography (micro-CT) to assess bone mass, which found that trabecular bone was damaged in the OVX rat model. Vitexin could repair bone injury and promote osteoblast biochemical indicators and angiogenesis indicators. Furthermore, EAhy926 cells were used to further explore the effect of vitexin on improving hypoxia-induced endothelial injury in vitro. Vitexin had a protective effect on hypoxia-treated EAhy926 cells and up-regulated vitamin D receptor (VDR) signaling and promoted phosphorylation of phosphatidylinositol-3-kinase (PI3K), protein kinase B (AKT), and endothelial NO synthase (eNOS), which enhanced endothelial cell migration and tube formation. VDR small-interfering RNA (siRNA) transfection significantly decreased both VDR and p-eNOS proteins, and VDR siRNA transfection + vitexin could not further increase VDR and downstream proteins. Overall, this study presented that vitexin regulates angiogenesis and osteogenesis in ovariectomy-induced osteoporosis of rats via the VDR/eNOS signaling pathway.

Melatonin receptor agonist ramelteon alleviates experimental acute ocular inflammation via HIF-1Α/VEGF/E-NOS signaling.

Ramelteon (RML) is a potent, selective agonist of the high-affinity melatonin receptor 1 and 2 receptors. In addition, RML is known to have antioxidant and anti-inflammatory effects. In this study, we aimed to investigate the effects of RML on HIF-1α, VEGF and e-NOS signaling pathway in a rat model of endotoxin-induced uveitis (EIU). Twenty-eight Wistar albino rats were divided into 4 groups as controls, lypopolysaccharide (LPS) group (5 mg/kg i.p.), LPS + RML group (5 mg/kg i.p and 8 mg/kg orally, respectively) and RML group (8 mg/kg orally). EIU was induced by a single intraperitoneal LPS injection. Histopathological and genetical analyses were performed. In histopathological analysis, LPS caused mild anterior uveitis characterized by increased surface area of iris leaflets and ciliary body due to edema, mild to moderate congestion, and inflammatory infiltrate 6 h following the injection. The pathological findings were reduced by RML. Higher inflammation levels seen in LPS group were significantly reduced in LPS + RML group. Also, HIF-1 α, eNOS and VEGF expressions increased in LPS and decreased in LPS + RML group. RML treatment reversed the changes in the HIF-1α /eNOS/ VEGF signaling pathway in LPS-induced uveitis in rats, preventing the progression of the damage and showed positive effects.

Protective effects of trehalose preconditioning on cardiac and coronary endothelial function through eNOS signaling pathway in a rat model of ischemia-reperfusion injury.

Coronary endothelial dysfunction is a major cause of ischemia-reperfusion (I/R) injury. Trehalose, a natural disaccharide, has been reported to ameliorate endothelial dysfunction during aging by activating endothelial nitric oxide synthase (eNOS); however, its role in I/R injury is unknown. This study evaluated the effects of trehalose preconditioning on cardiac and coronary endothelial function after I/R. Langendorff-perfused rat hearts underwent 30min of global ischemia followed by 80min of reperfusion with or without trehalose preconditioning. Rate pressure product (RPP) and coronary flow (CF) were measured during reperfusion. Perivascular edema was assessed by hematoxylin and eosin staining. Myocardial oxidative stress and apoptosis were evaluated by immunohistochemistry and TUNEL staining, respectively. eNOS dimerization was determined by western blotting. An eNOS inhibitor was used to examine the role of eNOS. Trehalose preconditioning showed a higher recovery rate after I/R as indicated by high RPP (control vs. trehalose, 28 ± 6% vs. 46 ± 9%; P = 0.017, Cohen's d = 2.3) and CF values (35 ± 10% vs. 55 ± 9%; P = 0.025, d = 1.7). Furthermore, trehalose preconditioning reduced perivascular edema, myocardial oxidative stress, and apoptosis. The eNOS dimerization ratio was increased by trehalose (1.2 ± 0.2 vs. 1.6 ± 0.2; P = 0.023, d = 2.1), which was associated with the recovery of RPP and CF. These effects of trehalose were abolished by the eNOS inhibitor. Trehalose preconditioning showed protective effects on cardiac and coronary endothelial function after I/R through the eNOS signaling pathway.

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.

Tiaojing Cuyun Recipe Enhances Pregnancy Outcome via the VEGF/PI3K/AKT/eNOS Signaling Pathway in EID Mice.

Purpose In this study, we evaluated the effect of Tiaojing Cuyun Recipe (TJCYR) on embryo implantation dysfunction- (EID-) induced damage of endometrial receptivity in mice and investigated the mechanisms underlying the effect. Methods The main compounds of TJCYR were identified by high-performance liquid chromatography (HPLC). One hundred and twenty pregnant mice were randomly divided into six groups: control, EID only, progesterone (Prog)+EID, TJCYR-low-dose+EID, TJCYR-medium-dose+EID, and TJCYR-high-dose+EID. Mifepristone was injected to make the EID model. On the fourth day of pregnancy, serum was obtained to analyze hormone level by radioimmunoassay, the uterus was collected to analyze morphology by hematoxylin and eosin (H&E) and scanning electron microscopy (SEM), and a combination of immunofluorescence and Western blot was used to identify the related proteins. On the eighth day of pregnancy, the mice were sacrificed and the number of uterus-implanted blastocysts was counted. Results Treatment with TJCYR significantly improved the number of implanted sites, the number of well-developed pinopodes, and microvascular formation in the mice. Moreover, TJCYR significantly activated PI3K/Akt/eNOS signaling pathways to promote angiogenesis, resulting in significantly improved endometrial receptivity and fertility outcomes when compared to the model group. Conclusion These findings demonstrate that TJCYR was able to protect embryo implantation of EID mice due to TJCYR-mediated improvement in endometrial receptivity by promoting endometrial angiogenesis.

Baicalin Targets HSP70/90 to Regulate PKR/PI3K/AKT/eNOS Signaling Pathways.

Baicalin is a major active ingredient of traditional Chinese medicine Scutellaria baicalensis, and has been shown to have antiviral, anti-inflammatory, and antitumor activities. However, the protein targets of baicalin have remained unclear. Herein, a chemical proteomics strategy was developed by combining baicalin-functionalized magnetic nanoparticles (BCL-N3@MNPs) and quantitative mass spectrometry to identify the target proteins of baicalin. Bioinformatics analysis with the use of Gene Ontology, STRING and Ingenuity Pathway Analysis, was performed to annotate the biological functions and the associated signaling pathways of the baicalin targeting proteins. Fourteen proteins in human embryonic kidney cells were identified to interact with baicalin with various binding affinities. Bioinformatics analysis revealed these proteins are mainly ATP-binding and/or ATPase activity proteins, such as CKB, HSP86, HSP70-1, HSP90, ATPSF1β and ACTG1, and highly associated with the regulation of the role of PKR in interferon induction and the antiviral response signaling pathway (P = 10−6), PI3K/AKT signaling pathway (P = 10−5) and eNOS signaling pathway (P = 10−4). The results show that baicalin exerts multiply pharmacological functions, such as antiviral, anti-inflammatory, antitumor, and antioxidant functions, through regulating the PKR and PI3K/AKT/eNOS signaling pathways by targeting ATP-binding and ATPase activity proteins. These findings provide a fundamental insight into further studies on the mechanism of action of baicalin.

Type-2 diabetic rat heart: The effect of kolaviron on mTOR-1, P70S60K, PKC-α, NF-kB, SOD-2, NRF-2, eNOS, AKT-1, ACE, and P38 MAPK gene expression profile

It has been established that genetic factors partially contribute to type-2 diabetes and vascular disease development. This study determined the effect of kolaviron on the expression profile of genes associated with the insulin signaling pathway and involved in regulating glucose and lipid metabolism, oxidative stress, inflammation, vascular functions, pro-survival and the apoptosis pathway in the heart of type-2 diabetic rats. After induction and confirmation of type-2 diabetes seven days after, the rats were treated with kolaviron for twenty-eight days before being euthanized. Organs were harvested and stored at − 80 °C in a biofreezer. Total RNA was extracted from the ventricle, reverse transcribed to cDNA followed by a real-time quantitative polymerase chain reaction (RT-qPCR) analysis of the expression of mTOR-1, P70S60K, PKC-α, NF-kB, SOD-2, NRF-2, eNOS, AKT-1, ACE, p38 MAPK and the reference gene (GAPDH), after which they were normalized/standardized. The results show an increase in the relative mRNA expression of mTOR/P70S60K/PKCα /P38MAPK/NF-KB/ACE and a decrease in the relative mRNA expression of NRF2/SOD/AKT/eNOS in the heart of the diabetic rats. Nevertheless, kolaviron modulated the expression profile of these genes, which suggest a therapeutic effect and target for vascular dysfunction and complications in type-2 diabetes through the activation of the NRF-2/AKT-1/eNOS signaling pathway and suppression of the NF-kB/PKC signaling pathway.

Agmatine mitigates palmitate (PA)-induced mitochondrial and metabolic dysfunction in microvascular endothelial cells.

Agmatine is an arginine metabolite that has neuroprotective capacity. Recently, it has been found to ameliorate atherosclerosis progression in rabbits. However, further molecular mechanisms of its anti-atherosclerotic properties remain unclear. High plasma levels of free fatty acids (FFAs) are an important risk factor for atherosclerosis due to their detrimental effects on vascular endothelial cells (ECs). Here, we used palmitate (PA), a kind of FFA, to induce endothelial dysfunction in human microvascular endothelial cells (HMECs) to determine the possible biological functions of agmatine. We found that PA caused ECs dysfunction in HMEC-1 cells, decreased cell viability, and elevated lactate dehydrogenase (LDH) release which could be reversed by agmatine treatment. Agmatine also improved the nitric oxide (NO) production and endothelial nitric oxide synthase (eNOS) activity in PA-induced HMEC-1 cells. The PA-caused mitochondrial dysfunction of HMEC-1 cells was diminished after agmatine treatment, as proven by the increased intracellular Adenosine Triphosphate (ATP) level, decreased mitochondrial reactive oxygen species (ROS) level, and increased mitochondrial oxygen consumption rate (OCR). Further, agmatine could alleviate PA-caused lipid accumulation with increased levels of Triglyceride (TG) and total cholesterol (TC) in HMEC-1 cells. Furthermore, Western blot analysis revealed that agmatine administration markedly decreased the expression levels of phosphorylated-AMP-activated protein kinase α (p-AMPKα), p-protein kinase B (p-AKT), and p-eNOS in PA-induced HMEC-1 cells. Inhibition of AMPK by compound C reversed the protective effects of agmatine on PA-induced HMEC-1 cells. Taken together, we hypothesize that agmatine mitigated PA-induced HMEC-1 cell dysfunction by alleviating mitochondrial and metabolic dysfunction via the AMPK/PI3K/Akt/eNOS signaling pathway.

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.

Pravastatin attenuates sepsis-induced acute lung injury through decreasing pulmonary microvascular permeability via inhibition of Cav-1/eNOS pathway

BackgroundDisruption of alveolar endothelial barrier caused by inflammation drives the progression of septic acute lung injury (ALI). Pravastatin, an inhibitor of HMG Co-A reductase, has potent anti-inflammatory effects. In the present study, we aim to explore the beneficial role of pravastatin in sepsis‐induced ALI and its related mechanisms. MethodsA septic ALI model was established by cecal ligation and puncture (CLP) in mice. The pulmonary microvascular endothelial cells (PMVECs) were challenged with lipopolysaccharide (LPS). The pathological changes in lung tissues were examined by HE staining. The pulmonary microvascular permeability was determined by lung wet-to-dry (W/D) weight ratio and Evans blue staining. The total protein concentration in bronchoalveolar lavage fluid (BALF) was detected by BCA assay. The levels of TNF-α, IL-1β, and IL-6 were assessed by qRT-PCR and ELISA. Apoptosis was determined by flow cytometry and TUNEL. Western blotting was performed for detection of target protein levels. The expression of VE-Cadherin in lung tissues was evaluated by immunohistochemical staining. ResultsPravastatin improved survival rate, attenuated lung pathological changes and reduced pulmonary microvascular permeability in septic mice. In addition, pravastatin restrained sepsis-induced inflammatory response and apoptosis in the lung tissues and PMVECs. Moreover, pravastatin up-regulated the levels of junction proteins ZO-1, JAM-C, and VE-Cadherin. Finally, pravastatin suppressed inflammation, apoptosis and enhanced the expression of junction proteins via regulating Cav-1/eNOS signaling pathway in LPS-exposed PMVECs. ConclusionPravastatin ameliorates sepsis-induced ALI through improving alveolar endothelial barrier disruption via modulating Cav-1/eNOS pathway, which may be an effective candidate for treating septic ALI.

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.