Nitric Oxide Synthase Expression Research Articles

Absence of PD-L1 signaling hinders macrophage defense against Mycobacterium tuberculosis via upregulating STAT3/IL-6 pathway

The blockade of programmed death-ligand 1 (PD-L1) pathway has been clinically used in cancer immunotherapy, while its effects on infectious diseases remain elusive. Roles of PD-L1 signaling in the macrophage-mediated innate immune defense against M.tb is unclear. In this study, the outcomes of tuberculosis (TB) in wild-type (WT) mice treated with anti-PD-1/PD-L1 therapy and macrophage-specific Pdl1-knockout (Pdl1ΔΜΦ) mice were compared. Treatment with anti-PD-L1 or anti-PD-1 benefited protection against M.tb infection in WT mice, while Pdl1ΔΜΦ mice exhibited the increased susceptibility to M.tb infection. Mechanistically, the absence of PD-L1 signaling impaired M.tb killing by macrophages. Furthermore, elevated STAT3 activation was found in PD-L1-deficient macrophages, leading to increased interleukin (IL)-6 production and reduced inducible nitric oxide synthase (iNOS) expression. Inhibiting STAT3 phosphorylation partially impeded the increase in IL-6 production and restored iNOS expression in these PD-L1-deficient cells. These findings provide valuable insights into the complexity and mechanisms underlying anti-PD-L1 therapy in the context of tuberculosis.

Protective Effects of an Oligo-Fucoidan-Based Formula against Osteoarthritis Development via iNOS and COX-2 Suppression following Monosodium Iodoacetate Injection.

Osteoarthritis (OA) is a debilitating joint disorder characterized by cartilage degradation and chronic inflammation, accompanied by high oxidative stress. In this study, we utilized the monosodium iodoacetate (MIA)-induced OA model to investigate the efficacy of oligo-fucoidan-based formula (FF) intervention in mitigating OA progression. Through its capacity to alleviate joint bearing function and inflammation, improvements in cartilage integrity following oligo-fucoidan-based formula intervention were observed, highlighting its protective effects against cartilage degeneration and structural damage. Furthermore, the oligo-fucoidan-based formula modulated the p38 signaling pathway, along with downregulating cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expression, contributing to its beneficial effects. Our study provides valuable insights into targeted interventions for OA management and calls for further clinical investigations to validate these preclinical findings and to explore the translational potential of an oligo-fucoidan-based formula in human OA patients.

Ion channel Piezo1 activation aggravates the endothelial dysfunction under a high glucose environment

BackgroundVasculopathy is the most common complication of diabetes. Endothelial cells located in the innermost layer of blood vessels are constantly affected by blood flow or vascular components; thus, their mechanosensitivity plays an important role in mediating vascular regulation. Endothelial damage, one of the main causes of hyperglycemic vascular complications, has been extensively studied. However, the role of mechanosensitive signaling in hyperglycemic endothelial damage remains unclear.MethodsVascular endothelial-specific Piezo1 knockout mice were generated to investigate the effects of Piezo1 on Streptozotocin-induced hyperglycemia and vascular endothelial injury. In vitro activation or knockdown of Piezo1 was performed to evaluate the effects on the proliferation, migration, and tubular function of human umbilical vein endothelial cells in high glucose. Reactive oxygen species production, mitochondrial membrane potential alternations, and oxidative stress-related products were used to assess the extent of oxidative stress damage caused by Piezo1 activation.ResultsOur study found that in VECreERT2;Piezo1flox/flox mice with Piezo1 conditional knockout in vascular endothelial cells, Piezo1 deficiency alleviated streptozotocin-induced hyperglycemia with reduced apoptosis and abscission of thoracic aortic endothelial cells, and decreased the inflammatory response of aortic tissue caused by high glucose. Moreover, the knockout of Piezo1 showed a thinner thoracic aortic wall, reduced tunica media damage, and increased endothelial nitric oxide synthase expression in transgenic mice, indicating the relief of endothelial damage caused by hyperglycemia. We also showed that Piezo1 activation aggravated oxidative stress injury and resulted in severe dysfunction through the Ca2+-induced CaMKII-Nrf2 axis in human umbilical vein endothelial cells. In Piezo1 conditional knockout mice, Piezo1 deficiency partially restored superoxide dismutase activity and reduced malondialdehyde content in the thoracic aorta. Mechanistically, Piezo1 deficiency decreased CaMKII phosphorylation and restored the expression of Nrf2 and its downstream molecules HO-1 and NQO1.ConclusionIn summary, our study revealed that Piezo1 is involved in high glucose-induced oxidative stress injury and aggravated endothelial dysfunction, which have great significance for alleviating endothelial damage caused by hyperglycemia.

Hyperglycemia Induces Remodeling of Nitric Oxide Synthase Activity in Glomerular Podocytes

Background: The regulation of glomerular filtration is influenced by the renin-angiotensin system (RAS) and the release of nitric oxide (NO) by glomerular cells. Several studies suggest that NO levels in glomeruli decrease as renal pathology develops, and restoring NO levels can be beneficial for glomerular function. Despite these findings, the expression of nitric oxide synthase (NOS) subunits in podocytes, particularly under pathological conditions, still requires further investigation. We hypothesized that decreased NO bioavailability in diabetes and hyperglycemia is linked to pathological NOS remodeling, thus influencing redox balance in podocytes and promoting renal damage. Methods: Live-cell confocal imaging was used to investigate NO production and the distribution of NOS in both control and hyperglycemic conditions. First, we used immortalized human podocytes (obtained from M. Saleem lab) loaded with the DAF-FM fluorescent marker in control and hyperglycemic (exposed to high glucose for 12 hours) conditions. Next, freshly isolated glomeruli from Type 2 Diabetic Nephropathy (T2DN) and Wistar male rats (age 26-30 weeks) were used to confirm our data ex vivo. NO production was monitored following Angiotensin II (Ang II) stimulation, and the distribution of NOS was determined using specific NOS1 and NOS2 commercially available inhibitors (Nω-Propyl-L-arginine hydrochloride and L-NIL, respectively). One-way ANOVA (OriginPro) was used for statistical analysis. Results: Upon application of Ang II, rapid NO production was observed in cultured and Wistar rats isolated glomeruli podocytes. The release of NO was blocked by preincubation with corresponding inhibitors targeting NOS. Our findings reveal that NOS1 primarily contributes to signaling in healthy podocytes, accounting for 69±5% of the total NO response, while the remaining NO production mediated by the RAS is attributed to NOS2 (39±5%). In diabetic conditions, the maximal amplitude of NO production mediated by Ang II was decreased by 22±10% compared to the control (one-way ANOVA, n≥24, p<0.001). Furthermore, high glucose conditions significantly altered NOS distribution in podocytes, where NOS2 signaling became predominant (89±7% of the total NO response), and the contribution of NOS1 became less significant. Conclusions: The changes in the distribution of NO sources could be associated with heightened oxidative and nitrosative stress, promoting renal damage. Our research demonstrates that in diabetes, NOS2 activity becomes predominant, potentially playing a direct role in advancing pathological processes in podocytes. Revealing the mechanisms of this fundamental pathway in the glomerulus impacts our progress toward new treatment and preventive measures for diabetic kidney disease development. This research was supported by National Institutes of Health Grants R01 DK126720 (to OP), R01 DK129227 (to AS and OP), and R01 DK115749 (to KS), US Department of Veteran Affairs grants I01CX002391 (to JHL), I01CX001248 (JCO) and I01 BX004024 (to AS). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Physical activity alleviates obesity-associated mesenteric arterial endothelial dysfunction: Role of adiponectin receptor

Introduction: A strong association between cardiovascular disease and obesity has been reported. However, the role of adiponectin in the effect of physical activity on mesenteric circulation and its underlying mechanism has been unknown. We investigated the protective effect of voluntary wheel running (EX) on endothelial function in the mesenteric artery of obese mice and its underlying mechanisms. Methods: C57BL/6J male mice were randomly divided into four groups: (1) control low-fat diet (LF), (2) LF diet with free access to a voluntary running wheel (LF-EX), (3) high-fat diet (HF; 45% of calories from fat), and (4) HF-EX. The protein expressions of total-endothelial nitric oxide synthase (t-eNOS), adiponectin receptors 1 and 2, AMPK, Ob (leptin), inflammatory markers, and proteins regulating apoptosis were quantified by Western blot analysis. Statistical comparisons between groups were performed with two-way ANOVA with Fisher’s LSD test. Results: The protein expressions of t-eNOS, adiponectin receptors 1 and 2, and p-AMPK in HF were lower compared to LF, but EX increased the protein levels ( P < 0.05). Ob in HF was higher compared to LF, but EX decreased it ( P < 0.05). IL-1β, TNF-α, and Caspase-1 in HF were higher compared to LF, but EX decreased the protein levels ( P < 0.05). There was no difference in expressions of AMPK, CHOP, Bcl-2, and GADD153 between groups. Conclusion: Voluntary wheel running alleviates e-NOS-mediated endothelial dysfunction in the mesenteric artery of obese mice possibly via increased adiponectin receptors and decreased inflammation. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Physical activity mitigates serotonin and oxidative stress-associated mesenteric arterial endothelial dysfunction in obesity

Introduction: Serotonin (5-HT) signaling may play a role in modulating vascular endothelial function. Obesity-induced oxidative stress causes the development of cardiovascular disease (CVD). We aimed to investigate the protective effect of voluntary wheel running (EX) on serotonin receptor (5-HT1B) and oxidative stress in the mesenteric artery of obese mice. Methods: Mice were randomly divided into four groups. They were fed a control low-fat diet (LF) or high-fat diet (HF; 45% of calories from fat) and provided with voluntary wheel running (LF-EX or HF-EX) or a sedentary condition. The protein expressions of phosphorylated-endothelial nitric oxide synthase (p-eNOS), 5-HT1B receptor, Akt, and proteins regulating oxidative stress were quantified by Western blot analysis. Statistical comparisons between groups were performed with two-way ANOVA with Fisher’s LSD test. Results: The protein expressions of p-eNOS, 5-HT1B receptor, and p-Akt in HF were lower compared to LF, but EX increased the protein levels ( P < 0.05). NOX2 in HF was higher compared to LF, but EX decreased it ( P < 0.05). SOD1, SOD2, and UCP2 in HF were lower compared to LF, but EX increased the protein levels ( P < 0.05). There was no difference in expressions of Akt, Gαq/11, NOX4, and VDUP1 between groups. Conclusion: Voluntary wheel running improves e-NOS-mediated vascular endothelial function possibly via increased 5-HT1B receptors and reduced oxidative stress, which could mitigate the risk of obesity-induced CVD. This study was supported by grants from the Cardiovascular Research Foundation Asia (#16215 to Y. P.) and the University of Houston (Small Grant Program to Y. P.). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Antimicrobial and anti-inflammatory effects of BenTooth: A natural product blend of burdock root, persimmon leaf extracts, and quercetin on periodontal disease

Periodontal disease represents a condition that exhibits substantial global morbidity, and is characterized by the infection and inflammation of the periodontal tissue effectuated by bacterial pathogens. The present study aimed at evaluating the therapeutic efficacy of BenTooth, an edible natural product mixture comprising burdock root extract, persimmon leaf extract and quercetin, against periodontitis both in vitro and in vivo. BenTooth was examined for antimicrobial properties and its impact on cellular responses related to inflammation and bone resorption. Its effects were also assessed in a rat model of ligature-induced periodontitis. BenTooth demonstrated potent antimicrobial activity against P. gingivalis and S. mutans. In RAW264.7 cells, it notably diminished the expression of inducible nitric oxide synthase and cyclooxygenase-2, as well as reduced interleukin-6 and tumor necrosis factor-α levels triggered by P. gingivalis-derived lipopolysaccharide. Furthermore, BenTooth inhibited osteoclastogenesis mediated by the receptor activator of nuclear factor κB ligand. In the rat model, BenTooth consumption mitigated the ligature-induced expansion in distance between the cementoenamel junction and the alveolar bone crest and bolstered the bone volume fraction. These results present BenTooth as a potential therapeutic candidate for the prevention and remediation of periodontal diseases.

Passive Heat Stress, Like Exercise Training, Leads to Improved Vascular Function

Epidemiological data indicate that repeated heat stress improves cardiovascular health, making passive heat therapy (PHT) a potential alternative for those unable to exercise. Few studies to date have examined the potential exercise mimetic effects in humans, and it is unclear how adaptations compare in magnitude to exercise training. OBJECTIVE: To examine the effects of 6 weeks of localized, muscle-focused PHT on resistance artery vascular function, exercise hemodynamics and performance, molecular regulators of vascular tone, and skeletal muscle capillarization relative to the adaptations observed following high-intensity aerobic exercise training focused on the same muscles. HYPOTHESIS: 6 weeks of PHT, applied to the knee extensor muscles through pulsed shortwave diathermy (2 hr, 3 days/week), would improve both resistance artery function and exercise hemodynamics, as well as increase vascular tone regulators more than a sham treatment, but less than single-leg knee extension (KE) exercise training (EX; 40 min, 3 days/week). Methods: We randomized 34 sedentary but otherwise healthy, young adults (ages 18–36; n = 17 female, 17 male) to receive PHT, EX, or sham heating sessions (SHAM; 2 hr, 3 days/week) over 6 weeks. Vascular function was determined through the blood flow response during both a passive leg movement (PLM) assessment and a knee extension graded exercise test (GXTmax). Muscle biopsies were taken from the vastus lateralis at baseline and after 6 weeks of intervention. Results: Peak blood flow during PLM increased to the same extent in both the EX (~10.5% increase, P=0.009) and PHT groups (~8.5% increase, P=0.044). KE peak flow increased in EX (~19%, P=0.005), but did not change in PHT (P=0.523) and decreased in SHAM (~7%, P=0.020). Peak vascular conductance during KE increased by ~25% in EX (P=0.030) and PHT (P=0.012). KE peak power increased in EX by ~27% (P=0.001) but did not significantly change in PHT (P=0.175) and SHAM groups (P=0.111). EX, but not PHT or SHAM, increased muscle capillary-to-fiber ratio (P = 0.0003), capillary density (P = 0.0428), and the Capillary to Fiber Perimeter Exchange Index (P = 0.0089). Protein expression of endothelial nitric oxide synthase (eNOS) increased significantly in both EX (P=0.0028) and PHT (P=0.0095), but only EX resulted in increased angiogenesis. CONCLUSIONS: 6 weeks of localized PHT, when applied to young healthy individuals, improved resistance artery function at rest and during exercise to the same extent as exercise training. Moreover, our data suggest that these PHT-induced improvements can be primarily attributed to increased expression of the regulators of vascular tone and not neo-angiogenesis. In conclusion, passive heat therapy mimics many, but not all the vascular benefits of exercise training. Funding for this study was provided by the Department of Exercise Sciences at Brigham Young University. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Tectorigenin inhibits inflammatory responses in murineinflammatory bowel disease and LPS-stimulated macrophages via inactivating MAPK signaling pathway.

Considering the antihepatitis effects of Tectorigenin (TEC), and the same adenosine mitogen-activated protein kinase (MAPK) pathway in both hepatitis and inflammatory bowel disease (IBD) models, exploring the role of TEC in IBD is contributive to develop a new treatment strategy against IBD. The IBD mouse model was constructed by feeding with dextran sodium sulfate (DSS) and injection of TEC. Afterward, the mouse body weight, colon length, and disease activity index (DAI) were tested to assess the enteritis level. Mouse intestine lesions were detected by hematoxylin and eosin staining. Murine macrophages underwent lipopolysaccharide (LPS) induction to establish an inflammation model. Cell viability was determined by cell counting kit-8 assay. Enzyme-linked immunosorbent assay was performed to measure interleukin 6(IL-6) and tumor necrosis factor-α (TNF-α) levels. Cyclooxygenase-2(COX-2) and inducible nitric oxide synthase(iNOS) expressions were quantified via quantitative reverse transcription polymerase chain reaction. Levels of MAPK pathway-related proteins (p-P38, P38, p-Jun N-terminal kinase(JNK), JNK, signal-regulated kinase(ERK), p-ERK), COX-2 and iNOS were quantitated by Western blot. TEC improved the inflammatory response through ameliorating weight loss, shortening colon, and increasing DAI score in IBD mouse. Expressions of intestinal inflammatory factors (IL-6, TNF-α, iNOS and COX-2) and MAPK pathway-related proteins (p-P38, p-JNK, and p-ERK) were increased both in DSS-induced mouse intestinal tissue, but TEC inhibited expressions of inflammatory factors. The same increased trend was identified in LPS-induced macrophages, but TEC improved macrophage inflammation, as evidenced by downregulation of inflammatory factors. TEC mitigates IBD and LPS-induced macrophage inflammation in mice via inhibiting MAPK signaling pathway.

Salvimulticanol from Salvia multicaulis suppresses LPS-induced inflammation in RAW264.7 macrophages: in vitro and in silico studies.

Sustained inflammatory responses can badly affect several vital organs and lead to chronic inflammation-related disorders, such as atherosclerosis, pneumonia, rheumatoid arthritis, obesity, diabetes, Alzheimer's disease, and cancers. Salvia multicaulis is one of the widely distributed plants that contains several biologically active phytochemicals and diterpenoids with anti-inflammatory effects. Therefore, finding alternative and safer natural plant-extracted compounds with good curative anti-inflammatory efficiencies is an urgent need for the clinical treatment of inflammation-related diseases. In the current study, S. multicaulis Vahl was used to extract and isolate two compounds identified as salvimulticanol and candesalvone B methyl ester to examine their effects against inflammation in murine macrophage RAW264.7 cells that were induced by lipopolysaccharide (LPS). Accordingly, after culturing RAW264.7 cells and induction of inflammation by LPS (100ng/ml), cells were exposed to different concentrations (9, 18, 37.5, 75, and 150µM) of each compound. Then, Griess assay for detection of nitric oxide (NO) levels and western blotting for the determination of inducible nitric oxide synthase (iNOS) expression were performed. Molecular docking and molecular dynamics (MD) simulation studies were employed to investigate the anti-inflammatory mechanism. Our obtained results validated that the level of NO was significantly decreased in the macrophage cell suspensions as a response to salvimulticanol treatment in a dose-dependent manner (IC50: 25.1 ± 1.2µM) as compared to the methyl ester of candesalvone B which exerted a weaker inhibition (IC50: 69.2 ± 3.0µM). This decline in NO percentage was comparable with a down-regulation of iNOS expression by western blotting. Salvimulticanol strongly interacted with both the Toll-like receptor 4 (TLR4)/myeloid differentiation factor 2 (MD-2) complex and the inhibitor of nuclear factor kappa-B (NF-κB) kinase subunit beta (IKKβ) to disrupt their inflammatory activation due to the significant hydrogen bonds and effective interactions with amino acid residues present in the target proteins' active sites. S.multicaulis is a rich natural source of the aromatic abietane diterpenoid, salvimulticanol, which exerted a strong anti-inflammatory effect through targeting iNOS and diminishing NO production in LPS-induced RAW264.7 cells in a mechanism that is dependent on the inhibition of TLR4-MD-2 and IKKβ as activators of the classical NF-κB-mediated inflammatory pathway.

Estrogen, Exercise Training, and Nitric-oxide Mediated Sympatholysis

Our laboratory has previously reported that nitric oxide (NO)-mediated inhibition of sympathetic vasoconstriction in contracting muscle (sympatholysis) was enhanced in female, compared with male rats. Estrogen has been shown to modulate NO synthase (NOS) expression and NO-mediated sympatholysis. Therefore, this study investigated the hypothesis that NO-mediated inhibition of sympathetic vasoconstriction in resting and contracting skeletal muscle of female rats would be a function of estrogen bioavailability. Further, exercise training has been shown to enhance sympatholysis. Thus, we also investigated the hypothesis that exercise training would restore sympatholysis in estrogen-deficient rats. Female Sprague-Dawley rats were randomized to sedentary ovary-intact (SOI, n=10), sedentary ovariectomized (SOVX, n=9), exercise-trained (10 weeks, 5 days/week, 600m, 40m·min−1, 5% grade) ovary-intact (TOI, n=13), or exercise-trained ovariectomized (TOVX, n=10) groups. OVX rats had both ovaries surgically removed. Following sedentary behavior or exercise training, rats were anesthetized and instrumented for measurement of blood pressure, femoral vascular conductance (FVC), stimulation of the lumbar sympathetic chain, and contraction of the triceps surae muscle group. The percentage change of FVC (%FVC) in response to sympathetic chain stimulation delivered at 2 and 5 Hz was determined at rest and during muscle contraction (60% maximal contractile force) in control, selective neuronal NOS (nNOS) inhibition (SMTC; 0.6 mg·kg−1 IV), and non-selective NOS inhibition (L-NAME; 10 mg·kg−1 IV) conditions. In the Control condition, sympathetic vasoconstrictor responsiveness (%FVC) in resting and contracting skeletal muscle was not different between groups. Under resting conditions, sympathetic vasoconstrictor responsiveness was not different following selective nNOS inhibition (p>0.05), but was increased (p<0.05, main effect of drug) following non-selective NOS inhibition compared with control and selective nNOS inhibition conditions at both 2 Hz and 5 Hz. During muscle contraction, sympathetic vasoconstrictor responsiveness to 2 Hz sympathetic stimulation was unchanged (p>0.05) in the presence of selective nNOS inhibition, but was increased (p<0.05, main effect of drug) following non-selective NOS inhibition compared with control and selective nNOS inhibition conditions. At 5 Hz, sympathetic vasoconstrictor responsiveness was increased (p<0.05, main effect of drug) by selective nNOS inhibition, and increased further (p<0.05, main effect of drug) by non-selective NOS inhibition. The magnitude of sympatholysis at 2 Hz was not different (p>0.05) between groups or drug conditions. At 5 Hz, sympatholysis was blunted (p<0.05, main effect of drug) in the selective nNOS inhibition condition, and further reduced (p<0.05, main effect of drug condition) following non-selective NOS inhibition in all groups. Sympatholysis was not different (p>0.05) between sedentary and exercise trained groups. In conclusion: 1) sympathetic vasoconstrictor responsiveness in resting and contracting skeletal muscle was not dependent on estrogen bioavailability; 2) NO blunted vasoconstriction in all groups, however, NO-mediated sympatholysis was not altered by estrogen status, and; 3) exercise training did not alter sympathetic vasoconstrictor responsiveness or sympatholysis regardless of estrogen bioavailability. NSERC, Canada, Canadian Foundation for Innovation. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Vascular endothelial growth factor receptor 2 contributes to shear stress-induced endothelial nitric oxide synthase expression in human lung microvascular endothelial cells

Introduction: Physiological shear stress (pSS) helps to maintain endothelial homeostasis via numerous intracellular signaling pathways. We previously showed that pSS upregulates mRNA and protein levels of endothelial nitric oxide synthase (eNOS) in primary human lung microvascular endothelial cells (hLMVECs), and that phosphorylation of Akt (p-Akt), a phosphatidylinositol 3-kinase (PI3K) target, is increased. Upstream regulatory mechanisms for these responses have not been fully studied. Since vascular endothelial growth factor receptor 2 (VEGFR2) is an integral component of the mechanosensory complex that transduces shear stress-mediated signaling pathways in endothelial cells, we hypothesized that VEGFR2 is required for shear stress-induced eNOS expression and PI3K activation in hLMVECs. Methods: We cultured early passage (p≤7) primary hLMVECs from 4 unique donor lots in standard 6-well culture plates using a fixed volume (depth) and viscosity of tissue culture media. We exposed cells to shear stress for 24 hours using an orbital shaker and calculated the magnitude of shear stress exerted on hLMVECs using the extended solution to Stokes’ second problem. We defined the magnitude of pSS as 12 dyn/cm2. In some experiments, we exposed cells to pSS in the presence of chemical inhibitors of VEGFR2 (SU5416; 5 or 10 μM) or PI3K (LY294002; 10 μM). We collected whole cell lysates and performed immunoblotting to probe for phosphorylated eNOS (p-eNOS; Serine 1177), total eNOS, p-Akt (Serine 473), total Akt and housekeeping protein β-tubulin. Potential interactions between chemical inhibitors and pSS on protein expression and phosphorylation were analyzed by two-way ANOVA. Results: After 24 hours of pSS exposure, total eNOS levels were significantly (p<0.05) increased in hLMVECs, although the p-eNOS/eNOS ratio was not changed (p=0.85). Conversely, 24 hours of pSS exposure increased the p-Akt/Akt ratio (p<0.05) but did not increase total Akt levels (p=0.88). Inhibition of VEGFR2 activity with SU5416 attenuated pSS-induced changes in total eNOS levels (p<0.05 for interaction) but did not attenuate pSS-induced increases in the p-Akt/Akt ratio (p=0.91 for interaction). Inhibition of PI3K activity using LY294002 did not modify the effects of pSS on total eNOS levels (p=0.25 for interaction) or p-Akt/Akt ratio (p=0.47 for interaction). Conclusions: We conclude that VEGFR2 activity is necessary for pSS-mediated increases in eNOS protein levels, but not PI3K activity, in hLMVECs. Furthermore, neither VEGFR2 nor PI3K activity appear to be required for pSS-mediated eNOS phosphorylation in these cells. Collectively, these data suggest that shear stress regulates hLMVEC eNOS signaling via multiple signaling pathways. This complex regulation may have important implications for local nitric oxide bioavailability under pathological conditions that modify shear stress (e.g., pulmonary embolism, pulmonary hypertension). This work is supported by NIH grants HL126514 and HL159906. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Stress during pubertal development affects female sociosexual behavior in mice

Puberty is a crucial phase for the development of female sexual behavior. Growing evidence suggests that stress during this period may interfere with the development of sexual behavior. However, the neural circuits involved in this alteration remain elusive. Here, we demonstrated in mice that pubertal stress permanently disrupted sexual performance without affecting sexual preference. This was associated with a reduced expression and activation of neuronal nitric oxide synthase (nNOS) in the ventrolateral part of the ventromedial hypothalamus (VMHvl). Fiber photometry revealed that VMHvl nNOS neurons are strongly responsive to male olfactory cues with this activation being substantially reduced in pubertally stressed females. Finally, treatment with a NO donor partially restored sexual performance in pubertally stressed females. This study provides insights into the involvement of VMHvl nNOS in the processing of olfactory cues important for the expression of female sexual behavior. In addition, exposure to stress during puberty disrupts the integration of male olfactory cues leading to reduced sexual behavior.

Scutellarin Attenuates Microglia Activation in LPS-Induced BV-2 Microglia via miRNA-7036a/MAPT/PRKCG/ERK Axis.

Scutellarin is an herbal agent which can exert anti-neuroinflammatory effects in activated microglia. However, it remains uncertain if it can inhibit microglia-mediated neuroinflammation by regulating miRNAs. This study sought to elucidate the upstream regulatory mechanisms by endogenous microRNAs and its target gene in activated microglia in lipopolysaccharide (LPS)-induced BV-2 microglia. Results show that scutellarin suppressed the expression of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and inducible nitric oxide synthase (iNOS) significantly in LPS-stimulated BV-2 microglia. As with the results of miRNAs function classification in vitro, the expression levels of mir-7036a-5p are upregulated in LPS-activated BV-2 microglia, but are downregulated by scutellarin. Rescue experiments indicated that mir-7036a-5p is a pro-inflammatory factor in activated BV-2 microglia. mir-7036a-5p agomir promoted the expression of phosphorylated tau proteins (p-tau), protein kinase C gamma type (PRKCG), extracellular regulated protein kinases (ERK1/2), but the is reversed by mir-7036a-5p antagomir in vitro. It is shown here that mir-7036a-5p is involved in microglia-mediated inflammation in LPS-induced BV-2 microglia. More important is the novel finding that scutellarin mitigated microglia inflammation by down-regulating the mir-7036a-5p/MAPT/PRKCG/ERK signaling pathway.

Morphofunctional changes in ventral and dorsal hippocampus in adult rats after chronic mild stress: a preclinical experimental study

Background. Stressful influences, depending on their severity and duration, can cause the development of pathological conditions. Repeated episodes of stress cause functional and structural changes in the central nervous system and can cause the development of depressive conditions. Depression is one of the leading mental illnesses. One of the most stress-sensitive brain structures is the hippocampus. Objective. To study is to evaluate structural changes in the hippocampus, which is considered as a heterogeneous structure with separate dorsal and ventral regions, to evaluate the expression of inducible nitric oxide synthase, endothelial nitric oxide synthase, serine racemase, synaptophysin in a mild stress model.Methods. A study of the effects of mild stress was carried out on 16 adult male Wistar rats (age 12 months, body weight 350–400 g). After acclimatization, the rats were divided into two equal groups (n = 8): intact (control) and stressed. When keeping animals, modeling and removing them from the experiment, we were guided by the Regulations for Carrying Out Work Using Laboratory Animals and the Declaration of Helsinki. Experimental modeling of depression in animals was induced by mild stress exposure for 7 days (30 minutes daily). Euthanasia was performed in a CO2 incubator. The brain was fixed in neutral buffered 10% formalin. Paraffin sections were made in the frontal plane, stained with hematoxylin and eosin, thionin using the Nissl method and examined at a level from –2.40 to –3.96 mm relative to bregma using an Axio Lab A1 microscope (Carl Zeiss Microscopy GmbH, Germany). Photo documentation was carried out with an AxioCam 105 color camera (Carl Zeiss Microscopy GmbH, Germany). Using the Image Analysis module of the ZEN 1.1.2.0 program (Carl Zeiss Microscopy GmbH, Germany) in the pyramidal layer of the hippocampus. Statistical analysis was performed with Microsoft Office Excel 2016 (Microsoft, USA) and Prism 6 (GraphPad Software Inc., USA). Comparisons of two conditions were made by nonparametric Mann-Whitney-U test to avoid a statistical bias of unequal data distribution. The level of significance was set at p < 0.05. The summarized data were presented as a as mean ± standard error of mean.Results. Functional research methods and assessment of pathological changes in hippocampal neurons are presented. An increase in the relative number of wrinkled hyperchromatic pyramidal neurons in the dorsal cornu ammonis field 3 in stressed rats was noted by 23.6% (p < 0.05) compared to the control. There was an increase in the relative number of inducible nitric oxide synthase-immunopositive neurons in the dorsal cornu ammonis field 3 by 40% ( p < 0.05) and the relative area of inducible nitric oxide synthase-immunoreactive material by 35% (p < 0.05) in the pyramidal layer of cornu ammonis field 3 in stressed rats. A decrease in the relative area of synaptophysin-immunopositive material in stressed rats was found in the ventral cornu ammonis field 3 compared to the control group by 16.8% (p < 0.05); decrease in the relative area of serine racemase-immunopositive material in dorsal cornu ammonis field 3 by 4.3% (p < 0.05) and ventral cornu ammonis field 3 by 7.8% (p < 0.05).Conclusion. The results of the study demonstrate that mild stress is an adequate model of depression in rats. In animals exposed to mild stress, pronounced morphological signs of damage to hippocampal neurons were revealed; motor and indicative exploratory activity decreases. Differences were found in morphofunctional changes in the dorsal and ventral parts of the hippocampus under the influence of mild stress. In cornu ammonis field 3 of the dorsal hippocampus, in contrast to the ventral section, more pronounced signs of damage to pyramidal layer neurons were observed. The increase in the relative number of inducible nitric oxide synthase-immunopositive neurons and the relative area of inducible nitric oxide synthase-immunoreactive material in the cornu ammonis field 3 pyramidal layer in stressed rats indicates an increase in nitric oxide production and the participation of nitrooxide-dependent free radical mechanisms of damage to hippocampal neurons. The decrease in the relative area of synaptophysin-immunoreactive material in stressed rats may contribute to changes in synaptic plasticity. A decrease in the relative area of serine racemase-immunoreactive material in the dorsal and ventral parts of cornu ammonis field 3 is considered to be a sign of a possible decrease in N-methyl-D-aspartate-dependent neurotransmission in the hippocampus under stress.

The cGAS/STING signaling pathway is involved in sevoflurane induced neuronal necroptosis via regulating microglia M1 polarization

ObjectiveThe specific mechanisms of sevoflurane-induced neurotoxicity are still undetermined. The aim of the current study was to investigate the role of the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signaling pathway in sevoflurane-induced neuronal necroptosis. MethodsBV2 microglial cells were divided into a control group and a 4% sevoflurane exposure group. Western blotting was used to detect expression of the M1 polarization marker inducible nitric oxide synthase (iNOS). RNA was collected for RNA sequencing analysis. After STING knockdown in microglia, western blotting was performed to examine expression of the pro-inflammatory markers CD16 and CD32. The tumor necrosis factor-α (TNF-α) level in media was detected using an enzyme-linked immunosorbent assay. BV2 microglia conditioned media was collected to incubate HT22 neuronal cells, and their cell activity was measured using a CCK8 assay. Calcium was observed by fluorescence. Western blotting was performed to evaluate receptor-interacting protein kinase 1 (RIPK1), RIPK3, and mixed lineage kinase domain-like (MLKL) expression. Neuronal necroptosis rate were detected using flow cytometry. ResultsSevoflurane exposure promoted microglial M1 polarization. The cGAS/STING pathway was screened and identified by RNA sequencing analysis of sevoflurane-exposed microglia and the control group. Compared with the control group, STING knockdown in microglia rescued the amoeboid morphology, inhibited TNF-α release, and significantly decreased iNOS, CD16, and CD32 expression. Moreover, calcium ions and necroptosis within neurons were decreased, and RIPK1, RIPK3, and p-MLKL expression was markedly decreased in microglia media culture with STING knockdown. ConclusionThese results suggest that sevoflurane can regulate microglial M1 polarization by activating the cGAS/STING signaling pathway and increasing immune factor release, thus accelerating the neuronal necroptosis induced by calcium overload.

Expression of inducible nitric oxide synthase, nitrotyrosine, eosinophilic peroxidase, eotaxin-3, and galectin-3 in patients with gastroesophageal reflux disease, eosinophilic esophagitis, and in healthy controls: a semiquantitative image analysis of 3,3'-diaminobenzidine-stained esophageal biopsies.

Eosinophilic esophagitis (EoE) and gastroesophageal reflux disease (GERD) share many histopathological features; therefore, markers for differentiation are of diagnostic interest and may add to the understanding of the underlying mechanisms. The nitrergic system is upregulated in GERD and probably also in EoE. Esophageal biopsies of patients with EoE (n = 20), GERD (n = 20), and healthy volunteers (HVs) (n = 15) were exposed to antibodies against inducible nitric oxide synthase (iNOS), nitrotyrosine, eosinophilic peroxidase, eotaxin-3, and galectin-3. The stained object glasses were randomized, digitized, and blindly analyzed regarding the expression of DAB (3,3'-diaminobenzidine) by a protocol developed in QuPath software. A statistically significant overexpression of iNOS was observed in patients with any of the two inflammatory diseases compared with that in HVs. Eotaxin-3 could differentiate HVs versus inflammatory states. Gastroesophageal reflux patients displayed the highest levels of nitrotyrosine. Neither iNOS nor nitrotyrosine alone were able to differentiate between the two diseases. For that purpose, eosinophil peroxidase was a better candidate, as the mean levels increased stepwise from HVs via GERD to EoE. iNOS and nitrotyrosine are significantly overexpressed in patients with EoE and GERD compared with healthy controls, but only eosinophil peroxidase could differentiate the two types of esophagitis. The implications of the finding of the highest levels of nitrotyrosine among gastroesophageal reflux patients are discussed.

Design, synthesis and molecular modeling of novel D-ring substituted steroidal 4,5-dihydropyrazole thiazolinone derivatives as anti-inflammatory agents by inhibition of COX-2/iNOS production and down-regulation of NF-κB/MAPKs in LPS-induced RAW264.7 macrophage cells

It has been reported that 4,5-dihydropyrazole and thiazole derivatives have many biological functions, especially in the aspect of anti-inflammation. According to the strategy of pharmacophore combination, we introduced thiazolinone and dihydropyrazole moiety into steroid skeleton to design and synthesize a novel series of D-ring substituted steroidal 4,5-dihydropyrazole thiazolinone derivatives, and assessed their in vitro anti-inflammatory profiles against Lipopolysaccharide (LPS)-induced inflammation in RAW 264.7 macrophage cells. The anti-inflammatory activities assay demonstrated that compound 12e was considered as the most effective anti-inflammatory drug, which suppressed the expression of pro-inflammatory mediators including nitric oxide (NO), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α), it also dose-dependently inhibited the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in LPS-induced RAW 264.7 macrophage cells. Furthermore, the results of the Western blot analysis showed a correlation between the inhibition of the Nuclear factor-kappa B (NF-κB) and Mitogen-activated protein kinases (MAPKs) signaling pathways and the suppressive effects of compound 12e on pro-inflammatory cytokines. Molecular docking studies of compound 12e into the COX-2 protein receptor (PDB ID: 5IKQ) active site was performed to rationalize their COX-2 inhibitory potency. The results were found to be in line with the biological findings as they exerted more favorable interactions compared to that of dexamethasone (DXM), explaining their remarkable COX-2 inhibitory activity. The findings revealed that these candidates could be identified as potent anti-inflammatory agents, compound 12e could be a promising drug for the treatment of inflammatory diseases.

Dietary honokiol supplementation improves antioxidant capacity, enhances intestinal health, and modulates cecal microbial composition and function of broiler chickens

Honokiol is a multifunctional polyphenol present in Magnolia officinalis. The effects of honokiol as a supplement in broiler chicken diets, and the underlying mechanisms, remain unclear. Therefore, the aim of the present study was to investigate the effects of honokiol on the growth performance, antioxidant capacity, and intestinal histomorphology of broiler chickens and to explore the underlying mechanisms. In total, 240 one-day-old broilers were randomly allocated to 5 dietary treatments, with 6 replicate pens and 8 birds per pen. Birds were fed a basal diet supplemented with 0 (blank control, BC), 100, 200, or 400 mg/kg honokiol (H100, H200, and H400), or 200 mg/kg bacitracin zinc (PC) for 42 d. The results showed that H200 and H400 increased body weight gain (BWG) and decreased feed conversion ratio (FCR) during the starter period (P < 0.05). H100 and H200 increased total superoxide dismutase (T-SOD) activity in the serum and decreased malondialdehyde (MDA) amount in the jejunum on d 42 (P < 0.05). Moreover, H100 increased villus height-to-crypt depth ratio in both the jejunum and ileum on d 21 (P < 0.05). PCR analysis showed that honokiol upregulated intestinal expression of glutathione peroxidase (GSH-Px) and downregulated intestinal expression of inducible nitric oxide synthase (iNOS) on d 42 (P < 0.05). The Shannon index, which represents the microbial alpha diversity, was reduced for the PC, H200, and H400 groups. Notably, honokiol treatment altered the cecal microbial community structure and promoted the enrichment of several bacteria, including Firmicutes and Lactobacillus. Higher production of short-chain fatty acids was observed in the cecal digesta of H100 birds, accompanied by an enriched glycolysis/gluconeogenesis pathway, according to the functional prediction of the cecal microbiota. This study provides evidence that honokiol improves growth performance, antioxidant capacity, and intestinal health of broiler chickens, possibly by manipulating the composition and function of the microbial community.

Expression of neuronal NO synthase α- and β-isoforms in skeletal muscle of mice.

Knowledge of the primary structure of neuronal NO synthase (nNOS) in skeletal muscle is still conflicting and needs further clarification. To elucidate the expression patterns of nNOS isoforms at both mRNA and protein level, systematic reverse transcription (RT)-PCR and epitope mapping by qualitative immunoblot analysis on skeletal muscle of C57/BL6 mice were performed. The ability of the nNOS isoforms to form aggregates was characterized by native low-temperature polyacrylamide electrophoresis (LT-PAGE). The molecular analysis was focused on the rectus femoris (RF) muscle, a skeletal muscle with a nearly balanced ratio of nNOS α- and β-isoforms. RT-PCR amplificates from RF muscles showed exclusive exon-1d mRNA expression, either with or without exon-μ. Epitope mapping demonstrated the simultaneous expression of the nNOS splice variants α/μ, α/non-μ, β/μ and β/non-μ. Furthermore, immunoblotting suggests that the transition between nNOS α- and β-isoforms lies within exon-3. In LT-PAGE, three protein nNOS associated aggregates were detected in homogenates of RF muscle and tibialis anterior muscle: a 320 kDa band containing nNOS α-isoforms, while 250 and 300 kDa bands consist of nNOS β-isoforms that form homodimers or heterodimers with non-nNOS proteins.