NOS Inhibitor Research Articles

Modulation of intracellular calcium activity in interstitial cells of Cajal by inhibitory neural pathways within the internal anal sphincter.

The internal anal sphincter (IAS) functions to maintain continence. Previous studies utilizing mice with cell-specific expression of GCaMP6f revealed two distinct subtypes of intramuscular interstitial cells of Cajal (ICC-IM) with differing Ca2+ activities in the IAS. The present study further examined Ca2+ activity in ICC-IM and its modulation by inhibitory neurotransmission. The spatiotemporal properties of Ca2+ transients in Type II ICC-IM mimicked those of smooth muscle cells (SMCs), indicating their joint participation in the "SIP" syncytium. Electrical field stimulation (EFS; atropine present) abolished localized and whole cell Ca2+ transients in Type I and II ICC-IM. The purinergic antagonist MRS2500 did not abolish EFS responses in either cell type, whereas the nitric oxide synthase (NOS) inhibitor NG-nitro-l-arginine (l-NNA) abolished responses in Type I but not Type II ICC-IM. Combined antagonists abolished EFS responses in Type II ICC-IM. In both ICC-IM subtypes, the ability of EFS to inhibit Ca2+ release was abolished by l-NNA but not MRS2500, suggesting that the nitrergic pathway directly inhibits ICC-IM by blocking Ca2+ release from intracellular stores. Since inositol (1,4,5)-trisphosphate receptor-associated cGMP kinase substrate I (IRAG1) is expressed in ICC-IM, it is possible that it participates in the inhibition of Ca2+ release by nitric oxide. Platelet-derived growth factor receptor α (PDGFRα)+ cells but not ICC-IM expressed P2Y1 receptors (P2Y1R) and small-conductance Ca2+-activated K+ channels (SK3), suggesting that the purinergic pathway indirectly blocks whole cell Ca2+ transients in Type II ICC-IM via PDGFRα+ cells. This study provides the first direct evidence for functional coupling between inhibitory motor neurons and ICC-IM subtypes in the IAS, with contractile inhibition ultimately dependent upon electrical coupling between SMCs, ICC, and PDGFRα+ cells via the SIP syncytium.NEW & NOTEWORTHY Two intramuscular interstitial cells of Cajal (ICC-IM) subtypes exist within the internal anal sphincter (IAS). This study provides the first evidence for direct coupling between nitrergic motor neurons and both ICC-IM subtypes as well as indirect coupling between purinergic inputs and Type II ICC-IM. The spatiotemporal properties of whole cell Ca2+ transients in Type II ICC-IM mimic those of smooth muscle cells (SMCs), suggesting that ICC-IM modulate the activity of SMCs via their joint participation in a SIP syncytium (SMCs, ICC, and PDGFRα+ cells).

Rare oxoisoaporphine alkaloids from Menispermum dauricum with potential anti-inflammatory activity

Eleven alkaloids including four previously undescribed oxoisoaporphine alkaloids, menisoxoisoaporphines A-D (1–4), four known analogues (5–8), and three aporphine alkaloids (9–11), were isolated and identified from the rhizomes of Menispermum dauricum. Their structures were elucidated by extensive spectroscopic data and single-crystal X-ray diffraction analyses. Among them, compounds 1 and 4 were the first samples of oxoisoaporphine with C-6 isopentylamino moiety, and 2 was a rare C-4 methylation product of oxoisoaporphine alkaloid. The in vitro anti-inflammatory activity of compounds 1–11 was performed by evaluating the inhibition of NO level in LPS-induced RAW264.7 macrophages. Among them, compound 4 exhibited the most potent NO inhibition activity with an IC50 value of 1.95 ± 0.33 μM. The key structure-activity relationships of those oxoisoaporphine alkaloids for anti-inflammatory effects have been summarized.

Chromones and biflavonoids from Garcinia pedunculata and Garcinia nujiangensis and their anti-inflammatory activity

Plants of the Garcinia genus were rich in structurally diverse and naturally bioactive components, while limited studies have been reported for Garcinia pedunculata Roxb. and G. nujiangensis C. Y. Wu & Y. H. Li. Four previously undescribed compounds including three chromones, garpedunchromones A-C (1–3), and one biflavonoid, nujiangbiflavone A (14), along with fifteen known analogs (4–13, 15–19) were isolated from G. pedunculata and G. nujiangensis. The structures of the isolated compounds were determined based on their HRESIMS data, extensive NMR spectroscopic analyses, and ECD calculations. The chromone derivatives were isolated from Garcinia for the first time. Compound 14 was a rare biflavonoid with C-3─C-6″ linkage. The biological evaluation of these isolates against NO production was conducted in the LPS-induced RAW 264.7 cells, resulting in the identification of a series of potent NO inhibitors, of which garpedunchromone B (2) was the most active with an IC50 value of 18.11 ± 0.96 μM. In the network pharmacology studies, the potential targets of compounds and inflammation were obtained from PharmMapper and GeneCards database. GO and KEGG enrichment analysis revealed that the overlapped targets were closely related to the major pathogenic processes linked to inflammation. Garpedunchromone B and proteins binding sites were being predicted.

Photobiomodulation Inhibits Ischemia-Induced Brain Endothelial Senescence via Endothelial Nitric Oxide Synthase.

Recent research suggests that photobiomodulation therapy (PBMT) positively impacts the vascular function associated with various cerebrovascular diseases. Nevertheless, the specific mechanisms by which PBMT improves vascular function remain ambiguous. Since endothelial nitric oxide synthase (eNOS) is crucial in regulating vascular function following cerebral ischemia, we investigated whether eNOS is a key element controlling cerebrovascular function and the senescence of vascular endothelial cells following PBMT treatment. Both rat photothrombotic (PT) stroke and in vitro oxygen-glucose deprivation (OGD)-induced vascular endothelial injury models were utilized. We demonstrated that treatment with PBMT (808 nm, 350 mW/cm2, 2 min/day) for 7 days significantly reduced PT-stroke-induced vascular permeability. Additionally, PBMT inhibited the levels of endothelial senescence markers (senescence green and p21) and antiangiogenic factor (endostatin), while increasing the phospho-eNOS (Ser1177) in the peri-infarct region following PT stroke. In vitro study further indicated that OGD increased p21, endostatin, and DNA damage (γH2AX) levels in the brain endothelial cell line, but they were reversed by PBMT. Intriguingly, the beneficial effects of PBMT were attenuated by a NOS inhibitor. In summary, these findings provide novel insights into the role of eNOS in PBMT-mediated protection against cerebrovascular senescence and endothelial dysfunction following ischemia. The use of PBMT as a therapeutic is a promising strategy to improve endothelial function in cerebrovascular disease.

Hypoxia-induced NOS1 as a therapeutic target in hypercholesterolemia-related colorectal cancer

BackgroundIt is well established that hypercholesterolemia increases the risk of atherosclerosis, especially because it reduces the availability of nitric oxide (NO). However, the relationship between hypercholesterolemia and NO in regulating colorectal cancer development and progression remains unknown.MethodsWe conducted bioinformatics analysis, qRT-PCR, ChIP-qPCR assays, luciferase report assays, clonogenic survival assays, and multiple mouse models to investigate the function and mechanism of hypercholesterolemia in regulating NO signaling. Additionally, NOS inhibitors were used to evaluate the potential of therapeutic strategy in anti-tumor response.ResultsHere, we show that oxidized low-density lipoprotein (oxLDL) cholesterol and its receptor LOX-1 are essential for hypercholesterolemia-induced colorectal tumorigenesis. Mechanically, the oxLDL promotes the oxidant stress-dependent induction of hypoxia signaling to transcriptionally up-regulate NO synthase (NOS) especially NOS1 expression in colorectal cancer (CRC) cells. More importantly, our results suggested that selective inhibition of NOS1 with its specific inhibitor Nω-Propyl-L-arginine is a suitable therapeutic strategy for hypercholesterolemia-related CRC with both efficacy and toxicity reduction.ConclusionsOur findings established that hypercholesterolemia induces the oxidant stress-dependent induction of hypoxia signaling to transcriptionally up-regulate NOS1 expression in CRC cells, and the clinically applicable NOS1 inhibitor Nω-Propyl-L-arginine represents an effective therapeutic strategy for hypercholesterolemia-related CRC.

The Pathway-Selective Dependence of Nitric Oxide for Long-Term Potentiation in the Anterior Cingulate Cortex of Adult Mice.

Nitric oxide (NO) is a key diffusible messenger in the mammalian brain. It has been proposed that NO may diffuse in retrograde into presynaptic terminals, contributing to the induction of hippocampal long-term potentiation (LTP). Here, we present novel evidence that NO is selectively required for the synaptic potentiation of the interhemispheric projection in the anterior cingulate cortex (ACC). Unilateral low-frequency stimulation (LFS) induced a short-term synaptic potentiation on the contralateral ACC through the corpus callosum (CC). The use of the antagonists of the NMDA receptor (NMDAR), or the inhibitor of the L-type voltage-dependent Ca2+ channels (L-VDCCs), blocked the induction of this ACC-ACC potentiation. In addition, the inhibitor of NO synthase, or inhibitors for its downstream signaling pathway, also blocked this ACC-ACC potentiation. However, the application of the NOS inhibitor blocked neither the local electric stimulation-induced LTP nor the stimulation-induced recruitment of silent responses. Our results present strong evidence for the pathway-selective roles of NO in the LTP of the ACC.

Luminal flow-stimulated superoxide participates in connecting tubule-glomerular feedback

Increasing NaCl concentration in the connecting tubule (CNT) dilates the afferent arteriole (Af-Art) through the connecting tubule-glomerular feedback (CNTGF). CNTGF is mediated by Na+ transport via epithelial Na+ channels (ENaC) in the CNT. It is unknown if CNTGF is mainly activated by NaCl concentration or sodium delivery or if there is an independent luminal flow effect. Previous studies showed that luminal flow stimulates superoxide (O2−), and O2− can activate ENaC. Thus we hypothesized that increasing luminal flow in the CNT induces CNTGF via O2− generation and ENaC activation. Rabbit Af-Arts with the adjacent CNTs were microdissected and perfused in vitro. Protocols consist of two consecutive dose-response curves to flow rate in the same preparation. When the CNT was perfused with 5mM NaCl, increasing flow rates from 5 to 10, 20, and 40 nL/min caused flow rate-dependent Af-Art dilatation (p<0.01). Adding an ENaC blocker benzamil to the perfusate blocked flow-induced Af-Art dilatation, indicating a CNTGF response. Using mathematical modeling, an increased tubular flow controlled by NaCl delivery into the CNT independently increases CNTGF vasodilation. However, when CNT was perfused with 0 mM NaCl, increasing the flow rate did not induce CNTGF vasodilation. The O2− dismutase mimetic tempol reduced flow-induced CNTGF. In the presence of L-NAME (NOS inhibitor), the reduced CNTGF mediated by tempol was unaffected. We concluded that increasing luminal flow in CNT induces CNTGF activation via ENaC, partly due to flow-stimulated O2− production and independently of NOS. 1K01HL155235. 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.

Enhancing the nitric oxide inhibitory activity using a combination of plant essential oils and mixture design approach

The synergistic effects of essential oils (EOs) from three aromatic plant species, Foeniculum vulgare subsp. piperitum (C.Presl) Bég. (FV), Origanum heracleoticum L. (OH) and Lavandula austroapennina N.G.Passal., Tundis & Upson. (LA), were evaluated for their inhibitory properties on nitric oxide production in RAW 264.7 macrophages stimulated with lipopolysaccharide (LPS). We utilized a Design of Experiments (DoE) methodology to optimize a formulation by combining three Essential Oils (EOs), while simultaneously taking into account two response variables, maximization of NO inhibition with minimum cytotoxicity. The optimal blend of components was predicted, and the statistical outcome's efficacy was experimentally verified. The combination corresponding to 87.7 % FV, 12.3 % LA and 0.0 % OH showed high inhibitory effect (76.3 %) with negligible cytotoxicity (4.5 %). This research provides new information on the interactions among fennel, oregano and lavender essential oils and shows how they can synergistically inhibit in vitro LPS-induced NO production.

Divergent Vasodilator Mechanisms to Endothelial-Dependent Agonists with Advancing Age

Introduction: Microvascular function is maintained by a number of vasodilating factors, with nitric oxide (NO) and endothelial hyperpolarizing factors (EDHF) predominating. Advancing age is an independent risk factor in the development of cardiovascular disease, and NO-mediated vasodilation declines, or is unchanged, depending on the vascular bed examined, number of cardiovascular risk factors, or specific endothelial-dependent agonist used. Mechanisms contributing to cutaneous microvascular dysfunction with age have been attributed largely to reductions in NO signaling, but the role of EDHF-mediated dilation, particularly to acetylcholine (ACh), remains unclear. The purpose of this study was to determine the endothelial mechanisms contributing to age-induced cutaneous microvascular function and compare cutaneous microvascular function responses to those from isolated adipose arteriole preparations. We hypothesized that cutaneous microvascular vasodilation to ACh in older adults will be mediated primarily by EDHFs relative to young adults and that isolated adipose arteriole preparations will corroborate these findings. Methods: Four intradermal microdialysis fibers were placed in the skin of the ventral forearm of 9 young (3F/6M; 29±7 yrs) and 3 older adults (3F/0M; 59±11 yrs) for infusion of Ringer’s solution; L-NAME (10 mM), an NO synthase inhibitor; tetraethylammonium chloride (TEA; 50 mM), a non-specific K+ channel inhibitor; and a combination of L-NAME and TEA. Laser Doppler flowmetry and brachial mean arterial pressure were measured during infusion of ACh (10 mM and 100 mM) and in response to local heating to 39°C. Maximal vasodilation was assessed with heating to 43°C and infusion of sodium nitroprusside (28 mM). Adipose resistance arterioles for pressure myography were obtained from a gluteal biopsy or from surgical discard tissue, which were pre-constricted (ET-1), and ACh responses (10−9–10−4 M) were assessed in the presence of the above inhibitors. Cutaneous vascular conductance (CVC) and isolated arteriole responses are presented as % maximum vasodilation. Results: In response to local heating in young adults, L-NAME reduced vasodilator magnitude (Ringers 69.0±18.1% vs. L-NAME 30.3±12.1%; p< 0.05) while TEA did not have any effect (69±18% vs. 49±28%; p=0.20). Combined L-NAME and TEA reduced vasodilation (69±18% vs. 19±6%; p<0.05). Conversely, vasodilator magnitude to ACh was not different across the Ringers, L-NAME, or TEA sites (p=0.93-0.94), while combined inhibition of NOS and K+ channels with L-NAME and TEA, respectively, reduced vasodilator responsiveness (p<0.05). In isolated resistance arterioles, there were no differences in %max dilation to ACh across inhibitor sites (p=0.19) in young adults. In older adults, neither independent nor combined inhibition of NOS or K+ channels impacted vasodilator responses to local heating, while vasodilator magnitude to ACh was most impacted by combined inhibition with L-NAME and TEA (83±14% vs. 32±15%; p<0.05). Conclusion: Our data demonstrate that vasodilator plasticity in response to local heating is divergent with advancing age, relying on non-NO or K+ mechanisms relative to young adults, while NO and K+ mechanisms are suffcient to maintain ACh-mediated vasodilation. These findings further support the concept of diversity in vasodilator mechanisms within the microvasculature. NHLBI K99-HL161491 (WEH), K08-HL150340 (JJM), R01-HL160752 (JKF), R01-HL133029 (AB), and NICHD R01-HD099340 (MJD). 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.

Nitrergic inhibition of sympathetic arteriolar constrictions in the female rodent urethra.

During the urine storage phase, tonically contracting urethral musculature would have a higher energy consumption than bladder muscle that develops phasic contractions. However, ischaemic dysfunction is less prevalent in the urethra than in the bladder, suggesting that urethral vasculature has intrinsic properties ensuring an adequate blood supply. Diameter changes in rat or mouse urethral arterioles were measured using a video-tracking system. Intercellular Ca2+ dynamics in arteriolar smooth muscle (SMCs) and endothelial cells were visualised using NG2- and parvalbumin-GCaMP6 mice, respectively. Fluorescence immunohistochemistry was used to visualise the perivascular innervation. In rat urethral arterioles, sympathetic vasoconstrictions were predominantly suppressed by α,β-methylene ATP (10μM) but not prazosin (1μM). Tadalafil (100nM), a PDE5 inhibitor, diminished the vasoconstrictions in a manner reversed by N-ω-propyl-l-arginine hydrochloride (l-NPA, 1μM), a neuronal NO synthesis (nNOS) inhibitor. Vesicular acetylcholine transporter immunoreactive perivascular nerve fibres co-expressing nNOS were intertwined with tyrosine hydroxylase immunoreactive sympathetic nerve fibres. In phenylephrine (1μM) pre-constricted rat or mouse urethral arterioles, nerve-evoked vasodilatations or transient SMC Ca2+ reductions were largely diminished by l-nitroarginine (l-NA, 10μM), a broad-spectrum NOS inhibitor, but not by l-NPA. The CGRP receptor antagonist BIBN-4096 (1μM) shortened the vasodilatory responses, while atropine (1μM) abolished the l-NA-resistant transient vasodilatory responses. Nerve-evoked endothelial Ca2+ transients were abolished by atropine plus guanethidine (10μM), indicating its neurotransmitter origin and absence of non-adrenergic non-cholinergic endothelial NO release. In urethral arterioles, NO released from parasympathetic nerves counteracts sympathetic vasoconstrictions pre- and post-synaptically to restrict arteriolar contractility. KEY POINTS: Despite a higher energy consumption of the urethral musculature than the bladder detrusor muscle, ischaemic dysfunction of the urethra is less prevalent than that of the bladder. In the urethral arterioles, sympathetic vasoconstrictions are predominately mediated by ATP, not noradrenaline. NO released from parasympathetic nerves counteracts sympathetic vasoconstrictions by its pre-synaptic inhibition of sympathetic transmission as well as post-synaptic arteriolar smooth muscle relaxation. Acetylcholine released from parasympathetic nerves contributes to endothelium-dependent, transient vasodilatations, while CGRP released from sensory nerves prolongs NO-mediated vasodilatations. PDE5 inhibitors could be beneficial to maintain and/or improve urethral blood supply and in turn the volume and contractility of urethral musculature.

Effects of Beraprost with or without NOS Inhibition on Plasma Aldosterone and Hemodynamics in Healthy Cats.

The aim of the study was to evaluate the hemodynamic and RA system effects of the oral administration of the clinical dose of beraprost for feline CKD in healthy cats, and also to examine whether NOS inhibition reversed them. A placebo-controlled pharmacological sequential design study was carried out to assess the plasma aldosterone and renin concentrations (PAC and PRC), blood pressure, heart rate, and exploratorily to estimate renal plasma flow (RPF) and renal vascular resistance (RVR) with simplified methods. Beraprost reduced PAC when compared to the placebo (p < 0.05); this was reversed when NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME) was added to the beraprost treatment (p < 0.01). No differences in the PRC or hemodynamic parameters were detected between beraprost and the placebo. The correlation ratios (η2) showed opposite relationships between beraprost and the added L-NAME effects on PAC, mean blood pressure (MBP), heart rate, estimated RPF (p < 0.001), estimated RVR (p < 0.01), and PRC (p < 0.05). In healthy cats, the clinical dose of beraprost suppresses PAC, which can be reversed by the inhibition of NOS.

In Vitro Determination of Nitric Oxide Synthase Inhibition, Antioxidant Capacity and Phenolic Content of Various Natural Products (Bee and Herbal Products).

It is obvious that the oxidation process is an undeniable fact and when it comes to aging, one of the first solutions that come to mind is natural products. When it comes to natural products, both plants and bee products play an important, almost combative role against oxidation. For this purpose, natural products of both plant and animal origin were considered together in our study: Linden, green tea, aronia, wild grapes, myrtle, blueberries and basil, honey, pollen and propolis. Total phenolic content values of the extracts ranged between 49.28 and 3859.06 mg gallic acid equivalent/100 g, and propolis, green tea, chestnut flower and aronia samples were found to have the highest values. When looking at the NOS inhibition potential, it was determined that propolis, pollen and aronia samples had the highest percentage inhibition values of 98.11, 92.29, 83.44, respectively. Antioxidant activities of methanolic extracts were investigated using iron(III) reducing/antioxidant capacity (FRAP), 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity test and NOS inhibition tests. The phenolic composition of methanolic extracts was tested using the RP-HPLC-UV (high-performance liquid chromatographic method with ultraviolet) method with 19 phenolic standards.

Exploring the therapeutic potential of pomegranate juice for uterine relaxation

ABSTRACT Background The effects of pomegranate juice (PJ) and its components on uterine smooth muscle are unknown. Hence, this study unequivocally demonstrates that pomegranate juice (PJ) significantly impacts myometrial function, providing crucial insights into its relaxant properties and their potential therapeutic applications for uterine-related disorders. Research design and methods Rat uterine smooth muscle horn strips were suspended in Krebs solution organ baths. Contractions were measured isometrically using a transducer (AD instrument Australia). The effects of PJ were evaluated on contractile activity elicited by potassium chloride (KCl 60 Mm) depolarization. Inhibitors of nitric oxide (L-NAME 3 X 10−4), guanylate cyclase (methylene blue 1 X 10−5), and Prostaglandin I2 (indomethacin 3 X 10−5), as well as Potassium Channels blockers, were determined. Results The juice at concentrations from 1.5–5 mg/ml significantly decreased the rat uterine horn contraction induced by KCl. The NO, cGMP, and PGI2 inhibitors did not block the relaxation response. Furthermore, the PGI2 inhibitor significantly enhanced the relaxation effects; K+ channel blockers had no inhibitory effects on the relaxation responses. Contrarily, GLIB improved considerably relaxation. Conclusion Research suggests pomegranate juice’s active ingredient may reduce uterine contractions and treat uterotonic disorders, potentially preventing preterm birth and dysmenorrhea. Further research is needed to determine its mechanism of action. Trial registration Code: AEC-013

Abstract 7459: iNOS inhibition through L-NMMA reveals immunostimulatory effects in obesity-associated triple-negative breast cancer (TNBC) tumors

Abstract Purpose of study: This study examines how obesity, an inflammatory condition, impacts treatment outcomes in triple-negative breast cancer (TNBC). Obesity fosters an unfavorable tumor microenvironment (TME) marked by immune suppression, metabolic shifts, and protumoral cytokine release. Elevated nitric oxide (NO) levels linked with obesity compromise vascular integrity, promoting breast cancer metastasis. This study examines whether combining standard chemotherapy (docetaxel) with NOS inhibition via L-NMMA could reshape the TME and bolster antitumor effects in TNBC within an obese mouse model. Experimental Procedure: C57bl/6 mice, a TNBC syngeneic mouse model, were fed a high-fat diet (HFD) or normal diet (ND) for 10 weeks. Tumors were induced orthotopically via E0771 cell line injection into the mammary fat pad. Upon reaching 100 mm^3, mice were randomized into vehicle and L-NMMA treatment groups. Digital spatial transcriptomics using NanoString™ GeoMx and nCounter platforms were conducted on FFPE tumor tissue sections from ND, HFD, and HFD+LNMMA treated mice. A panel targeting 64 protein markers aided in immune cell phenotyping and TME assessment. Results: HFD-induced tumors exhibited increased growth, elevated pro-inflammatory cytokine levels, and heightened iNOS expression. Unsupervised hierarchical clustering after nCounter analysis revealed a distinct protein expression signature in HFD tumors associated with T cell infiltration and exhaustion, as evidenced by the upregulation of CD3, CD4, PD-1/PD-L1, FOXP3, and CTLA 4. Markers for identifying immunosuppressed macrophages (F4/80 and CD163) were also elevated. Remarkably, LNMMA treatment on HFD tumors led to reduced tumor growth and lower expression levels of these markers, alongside a downregulation of survival/proliferation markers such as p53, Ki67, and key members of the Pi3k signaling pathway such as phospoAktSer473. Conclusions: Our findings indicate a distinct immunomodulatory mechanism of iNOS inhibition in HFD tumors, which displayed expression profiles marked by heightened immunosuppressive markers when compared to ND tumors. LNMMA treatment reversed this phenotype, aligning the profile closer to ND tumors. These discoveries underscore the potential of inhibiting iNOS to reshape the immune tumor microenvironment (TME) and modulate pathways linked to cell proliferation and survival. This presents a promising avenue for enhancing the effectiveness of treatments for triple-negative breast cancer (TNBC). Citation Format: Ivonne Uzair, Kai Sun, Seun Hynes, Wei Qian, Jianying Zhou, Karina Ortega Martinez, Liliana Guzman, Polina Matre, Jenny Chang. iNOS inhibition through L-NMMA reveals immunostimulatory effects in obesity-associated triple-negative breast cancer (TNBC) tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7459.

Discovery of potential components characteristic by conjugated enone from the branches and leaves of Croton lauioides with anti-neuroinflammatory activity via regulating the NF-κB pathway

In this study, the chemical composition and pharmacological activity of Croton lauioides were investigated for the first time. The bioactive and HPLC-UV guided isolation led to the discovery of twenty-three conjugated enone-type components (1–23), including nine previously unknown sesquiterpenoid derivatives (1–4, 9–10, 12–14). Notably, compounds 1 and 12 are epoxides containing an endoperoxide bridge (1) or a unique dioxaspiro core (12), respectively. Compounds 2–7 are non-benzenoid aromatics featuring a tropone function, while 9–11 possess a rare rearranged scaffold with tropone shift into benzene. Extensive characterization was performed using NMR spectra, HRESIMS data, and electronic circular dichroism (ECD) calculations. Furthermore, we evaluated the bioactivities of all isolated compounds against neuroinflammation in LPS-stimulated BV-2 microglial cells. Remarkably, most sesquiterpenoid derivatives exhibited significant NO inhibit activities, and compound 5 showed the most potent effect with an IC50 value of 0.14 ± 0.04 μM. Structure-activity relationship (SAR) analysis revealed that sesquiterpenoids modified with endocyclic enone conjugation may serve as a key pharmacophore for NO inhibition, particularly involving aromatic tropone moiety. The qPCR and Western blot results demonstrated that 5 exerted an inhibitory effect on the mRNA levels of iNOS, TNF-α and COX-2 in a time-dependent manner, as well as suppressed the protein expression of iNOS, TNF-α, COX-2. In mechanism, 5 could prevented activation of NF-κB pathway by suppressing phosphorylation of p65 and IκB-α. These findings revealed C. lauioides might be a promising resource for drug candidate development targeting neuroinflammation.

Computational analysis followed by invitro studies to explore cytokines (TNF-α, IL-6 and IL-1β) inhibition potential of the new natural molecule polonilignan.

Targeting pro-inflammatory cytokines and their production is found to be of therapeutic benefit for the regulation of inflammation in various chronic autoimmune diseases. Our continued efforts to discover small molecular-weight pro-inflammatory cytokine inhibitors resulted in identifying a novel natural lignan molecule named polonilignan, isolated from the culture broth extract of an endophytic fungus Penicillium polonicum. An in silico study (molecular docking, ADME predictions, binding free energy calculation and molecular dynamics simulation) of the polonilignan over the pro-inflammatory cytokines proteins TNF-α, IL-6 and IL-1β was performed using Schrodinger LLC software to understand the binding interactions, drug-like properties, and stability of the ligand-protein complex. Further, in-vitro testing of inhibition of TNF-α, IL-6 and IL-1β by polonilignan was carried out using ELISA and RT-PCR on LPS-induced RAW 264.7 cell lines along with the testing of nitrite production effect (Griess assay) and cytotoxicity (MTT) analysis. Under the computational study, polonilignan revealed good docking scores, binding interactions, and stability under MDS and desirable in silico ADME results over the proteins TNF-α, IL-1β and IL-6. Poloniligan showed significant inhibition of IL-1β, IL-6 and TNF-α with IC50 values of 2.01 μM, 6.59 μM and 42.10 μM, respectively. Also, it reduced the translocation of the NF-κB subunit p65 to the nucleus (confocal microscopy). The mRNA expression levels of pro-inflammatory markers IL-1β, TNF-α and IL-6 levels were lowered significantly (p < .001) by the compound, and the diminution was higher with IL-1β. Further, the lignan was non-cytotoxic and effective in attenuating nitrite release (IC50 48.56 μM). Thus, polonilignan has been identified as a new pan-cytokine and NO inhibitor, it is recommended to optimise a method for the synthesis of this small molecular weight lignan and explore its pharmacokinetic characteristics, toxicity and therapeutic effect under various chronic inflammatory disease models.

Crystallographic and Computational Insights into Isoform-Selective Dynamics in Nitric Oxide Synthase.

In our efforts to develop inhibitors selective for neuronal nitric oxide synthase (nNOS) over endothelial nitric oxide synthase (eNOS), we found that nNOS can undergo conformational changes in response to inhibitor binding that does not readily occur in eNOS. One change involves movement of a conserved tyrosine, which hydrogen bonds to one of the heme propionates, but in the presence of an inhibitor, changes conformation, enabling part of the inhibitor to hydrogen bond with the heme propionate. This movement does not occur as readily in eNOS and may account for the reason why these inhibitors bind more tightly to nNOS. A second structural change occurs upon the binding of a second inhibitor molecule to nNOS, displacing the pterin cofactor. Binding of this second site inhibitor requires structural changes at the dimer interface, which also occurs more readily in nNOS than in eNOS. Here, we used a combination of crystallography, mutagenesis, and computational methods to better understand the structural basis for these differences in NOS inhibitor binding. Computational results show that a conserved tyrosine near the primary inhibitor binding site is anchored more tightly in eNOS than in nNOS, allowing for less flexibility of this residue. We also find that the inefficiency of eNOS to bind a second inhibitor molecule is likely due to the tighter dimer interface in eNOS compared with nNOS. This study provides a better understanding of how subtle structural differences in NOS isoforms can result in substantial dynamic differences that can be exploited in the development of isoform-selective inhibitors.

Novel anti-neuroinflammatory pyranone-carbamate derivatives as selective butyrylcholinesterase inhibitors for treating Alzheimer’s disease

Butyrylcholinesterase (BuChE) and neuroinflammation have recently emerged as promising therapeutic directions for Alzheimer’s disease (AD). Herein, we synthesised 19 novel pyranone-carbamate derivatives and evaluated their activities against cholinesterases and neuroinflammation. The optimal compound 7p exhibited balanced BuChE inhibitory activity (eqBuChE IC50 = 4.68 nM; huBuChE IC50 = 9.12 nM) and anti-neuroinflammatory activity (NO inhibition = 28.82% at 10 μM, comparable to hydrocortisone). Enzyme kinetic and docking studies confirmed compound 7p was a mix-type BuChE inhibitor. Additionally, compound 7p displayed favourable drug-likeness properties in silico prediction, and exhibited high BBB permeability in the PAMPA-BBB assay. Compound 7p had good safety in vivo as verified by an acute toxicity assay (LD50 > 1000 mg/kg). Most importantly, compound 7p effectively mitigated cognitive and memory impairments in the scopolamine-induced mouse model, showing comparable effects to Rivastigmine. Therefore, we envisioned that compound 7p could serve as a promising lead compound for treating AD.

Variations in Proline Content, Polyamine Profiles, and Antioxidant Capacities among Different Provenances of European Beech (Fagus sylvatica L.).

International provenance trials are a hot topic in forestry, and in light of climate change, the search for more resilient beech provenances and their assisted migration is one of the challenges of climate-smart forestry. The main aim of the study was to determine intraspecific variability in European beech (Fagus sylvatica L.) among 11 beech provenances according to total antioxidant capacities estimated by various assays, such as DPPH (2,2-diphenyl-1-picrylhydrazyl), ABTS (2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic) acid), FRAP (ferric reducing antioxidant power) assay, and radical scavenging capacity against nitric oxide (RSC-NO assays), as well as osmolyte content, primarily individual polyamines (putrescine, spermidine, and spermine), and free proline content. Polyamine amounts were quantified by using HPLC coupled with fluorescent detection after dansylation pretreatment. The highest values for radical scavenger capacity assays (ABTS, DPPH, and FRAP) were measured in the German provenances DE47 and DE49. Also, the highest NO inhibition capacity was found in the provenance DE49, while the highest content of proline (PRO), total phenolic content (TPC), and total flavonoid content (TFC) was recorded in DE47. The Austrian AT56 and German provenance DE49 were most abundant in total polyamines. This research underlines the importance of the application of common antioxidant assays as well as osmolyte quantification as a criterion for the selection of climate-ready beech provenances for sustainable forest management.

Why is early-onset atrial fibrillation uncommon in patients with Duchenne muscular dystrophy? Insights from the mdx mouse.

A reduction in both dystrophin and neuronal nitric oxide synthase (NOS1) secondary to microRNA-31 (miR-31) up-regulation contributes to the atrial electrical remodelling that underpins human and experimental atrial fibrillation (AF). In contrast, patients with Duchenne muscular dystrophy (DMD), who lack dystrophin and NOS1 and, at least in the skeletal muscle, have raised miR-31 expression, do not have increase susceptibility to AF in the absence of left ventricular (LV) dysfunction. Here, we investigated whether dystrophin deficiency is also associated with atrial up-regulation of miR-31, loss of NOS1 protein, and increased AF susceptibility in young mdx mice. Echocardiography showed normal cardiac structure and function in 12-13 weeks mdx mice, with no indication by assay of hydroxyproline that atrial fibrosis had developed. The absence of dystrophin in mdx mice was accompanied by an overall reduction in syntrophin and a lower NOS1 protein content in the skeletal muscle and in the left atrial and ventricular myocardium, with the latter occurring alongside reduced Nos1 transcript levels (exons 1-2 by quantitative polymerase chain reaction) and an increase in NOS1 polyubiquitination [assessed using tandem polyubiquitination pulldowns; P < 0.05 vs. wild type (WT)]. Neither the up-regulation of miR-31 nor the substantial reduction in NOS activity observed in the skeletal muscle was present in the atrial tissue of mdx mice. At difference with the skeletal muscle, the mdx atrial myocardium showed a reduction in the constitutive NOS inhibitor, caveolin-1, coupled with an increase in NOS3 serine1177 phosphorylation, in the absence of differences in the protein content of other NOS isoforms or in the relative expression NOS1 splice variants. In line with these findings, transoesophageal atrial burst pacing revealed no difference in AF susceptibility between mdx mice and their WT littermates. Dystrophin depletion is not associated with atrial miR-31 up-regulation, reduced NOS activity, or increased AF susceptibility in the mdx mouse. Compared with the skeletal muscle, the milder atrial biochemical phenotype may explain why patients with DMD do not exhibit a higher prevalence of atrial arrhythmias despite a reduction in NOS1 content.