Inducible Nitric Oxide Synthase Protein Research Articles

The Role of Hydrogen Sulfide in iNOS and APP Localization and Expression in Neurons and Glial Cells Under Traumatic Effects: An Experimental Study with Bioinformatics Analysis and Biomodeling

Hydrogen sulfide (H2S) donors are emerging as promising candidates for neuroprotective agents. However, H2S-dependent neuroprotective mechanisms are not yet fully understood. We have demonstrated that an H2S donor (sodium sulfide, Na2S) reduces the expression of inducible NO synthase (iNOS) and amyloid-beta precursor protein (APP) in damaged neural tissue at 24 h and 7 days following traumatic brain injury (TBI). The application of aminooxyacetic acid (AOAA), an inhibitor of cystathionine β-synthase (CBS), produced the opposite effect. Seven days after TBI, iNOS expression was observed not only in the cytoplasm but also in some neuronal nuclei, while APP was exclusively localized in the cytoplasm and axons of damaged neurons. It was also shown that iNOS and APP were present in the cytoplasm of mechanoreceptor neurons (MRNs) in the crayfish, in axons, as well as in certain glial cells 8 h after axotomy. Na2S and AOAA had opposing effects on axotomized MRNs and ganglia in the ventral nerve cord (VNC). Multiple sequence alignments revealed a high degree of identity among iNOS and APP amino acid residues in various vertebrate and invertebrate species. In the final stage of this study, biomodeling identified unique binding sites for H2S, hydrosulfide anion (HS−), and thiosulfate (S2O32−) with iNOS and APP.

Two New Steroidal Saponins with Potential Anti-Inflammatory Effects from the Aerial Parts of Gnetum formosum Markgr.

Gnetum formosum Markgr., a member of the Gnetaceae family, is distributed in Vietnam. This plant remains a botanical enigma with an unexplored diversity of chemical constituents and pharmacological effects. In this study, two new steroidal saponins, namely gnetumosides A (1) and B (2), were isolated from the aerial parts of G. formosum. Their chemical structures were elucidated using spectroscopic techniques, including high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) and NMR, along with chemical hydrolysis and comparison with the reported literature. The potential anti-inflammatory effects of the isolated compounds were evaluated by measuring lipopolysaccharide-stimulated nitric oxide (NO) production in murine macrophage cells. Notably, compound 1 exhibited the most potent inhibitory activity (IC50 = 14.10 ± 0.75 µM), comparable to dexamethasone. Additionally, the mechanisms underlying the observed anti-inflammatory effects were investigated through molecular docking and molecular dynamics simulations on inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) proteins. This study is the first to investigate the chemical constituents and pharmacological effects of G. formosum.

Effect of macrophage-to-myofibroblast transition on silicosis.

The aim was to explore the effect of macrophage polarization and macrophage-to-myofibroblast transition (MMT) in silicosis. Male Wistar rats were divided into a control group and a silicosis group developed using a HOPE MED 8050 dynamic automatic dusting system. Murine macrophage MH-S cells were randomly divided into a control group and an SiO2 group. The pathological changes in lung tissue were observed using hematoxylin and eosin (HE) and Van Gieson (VG) staining. The distribution and location of macrophage marker (F4/80), M1 macrophage marker (iNOS), M2 macrophage marker (CD206), and myofibroblast marker (α-smooth muscle actin [α-SMA]) were detected using immunohistochemical and immunofluorescent staining. The expression changes in iNOS, Arg, α-SMA, vimentin, and type I collagen (Col I) were measured using Western blot. The results of HE and VG staining showed obvious silicon nodule formation and the distribution of thick collagen fibers in the lung tissue of the silicosis group. Macrophage marker F4/80 increased gradually from 8 to 32 weeks after exposure to silica. Immunohistochemical and immunofluorescent staining results revealed that there were more iNOS-positive cells and some CD206-positive cells in the lung tissue of the silicosis group at 8 weeks. More CD206-positive cells were found in the silicon nodules of the lung tissues in the silicosis group at 32 weeks. Western blot analysis showed that the expressions of Inducible nitric oxide synthase and Arg protein in the lung tissues of the silicosis group were upregulated compared with those of the control group. The results of immunofluorescence staining showed the co-expression of F4/80, α-SMA, and Col I, and CD206 and α-SMA were co-expressed in the lung tissue of the silicosis group. The extracted rat alveolar lavage fluid revealed F4/80+α-SMA+, CD206+α-SMA+, and F4/80+α-SMA+Col I+ cells using immunofluorescence staining. Similar results were also found in MH-S cells induced by SiO2. The development of silicosis is accompanied by macrophage polarization and MMT.

5α-Epoxyalantolactone from Inula macrophylla attenuates cognitive deficits in scopolamine-induced Alzheimer’s disease mice model

Alzheimer’s disease (AD) is a complex neurodegenerative condition. 5α-epoxyalantolactone (5α-EAL), a eudesmane-type sesquiterpene isolated from the herb of Inula macrophylla, has various pharmacological effects. This work supposed to investigate the improved impact of 5α-EAL on cognitive impairment. 5α-EAL inhibited the generation of nitric oxide (NO) in BV-2 cells stimulated with lipopolysaccharide (LPS) with an EC50 of 6.2 μM. 5α-EAL significantly reduced the production of prostaglandin E2 (PGE2) and tumor necrosis factor-α (TNF-α), while also inhibiting the production of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) proteins. The ability of 5α-EAL to penetrate the blood–brain barrier (BBB) was confirmed via a parallel artificial membrane permeation assay. Scopolamine (SCOP)-induced AD mice model was employed to assess the improved impacts of 5α-EAL on cognitive impairment in vivo. After the mice were pretreated with 5α-EAL (10 and 30 mg/kg per day, i.p.) for 21 days, the behavioral experiments indicated that the administration of the 5α-EAL could alleviate the cognitive and memory impairments. 5α-EAL significantly reduced the AChE activity in the brain of SCOP-induced AD mice. In summary, these findings highlight the beneficial effects of the natural product 5α-EAL as a potential bioactive compound for attenuating cognitive deficits in AD due to its pharmacological profile.Graphical

Serum-Derived Macrophage-Activating Factor Exhibits Anti-Tumor Activity via M2-to-M1 Macrophage Reprogramming

Many anti-tumor effects of group-specific component-derived macrophage-activating factors (GcMAFs) have been reported; however, the specific mechanisms remain unclear. Controlling tumor-associated macrophages (TAMs) in the tumor microenvironment is essential for cancer treatment. This study assessed the role of GcMAF in macrophage activation, elucidated the mechanisms by which it exerts its anti-tumor effects, and determined its effects on TAMs in the tumor microenvironment. GcMAF-stimulated RAW264.7 macrophages and EMT6 breast tumor cells were co-cultured in a 0.4 µm pore cell culture insert, and gene and protein expression and cell viability were evaluated. DNA microarray analysis of GcMAF-stimulated RAW264.7 cells was conducted. The induction of M2 RAW264.7 cells by interleukin (IL)-4 and IL-13 was analyzed. GcMAF stimulation increased the tumor necrosis factor-α and inducible nitric oxide synthase mRNA and protein levels in RAW264.7 cells but decreased the viability of co-cultured EMT6 cells. Although the details of the receptor or signal pathway of GcMAF are still unclear, these results were confirmed in the M2 RAW264.7 cells, suggesting that GcMAF exerts anti-tumor effects by inducing the differentiation of macrophages into the M1 type and reprogramming M2 macrophages to the M1 type. The anti-tumor activity of GcMAF via M2-to-M1 macrophage reprogramming could aid in developing novel cancer immunotherapies.

Vildagliptin ameliorates intrapulmonary vasodilatation and angiogenesis in chronic common bile duct ligation-induced hepatopulmonary syndrome in rat

IntroductionExperimental hepatopulmonary syndrome (HPS) is best reproduced in the rat common bile duct ligation (CBDL) model. Vildagliptin (Vild) is an anti-hyperglycemic drug that exerts beneficial anti-inflammatory, anti-oxidant and anti-fibrotic effects. Therefore, the present search aimed to explore the possible effectiveness of Vild in CBDL-induced HPS model. MethodsFour groups of male Wistar rats which weigh 220–270 g were used, including the normal control group, the sham control group, the CBDL group and CBDL+Vild group. The first three groups received i.p. saline, while the last group was treated with i.p. Vild (10 mg/kg/day) from the 15th to 28th day of the experiment. ResultsCBDL decreased the survivability and body weight of rats, increased diameter of the pulmonary vessels, and altered the arterial blood gases and the liver function parameters. Additionally, it increased the pulmonary expressions of endothelin-1 (ET-1) and tumor necrosis factor-α (TNF-α) mRNA as well as endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS) and vascular endothelial growth factor-A (VEGF-A) proteins. The CBDL rats also exhibited elevation of the pulmonary interleukin-6 (IL-6), dipeptidyl peptidase-4 (DPP-4) and nitric oxide (NO) levels along with reduction of the pulmonary total anti-oxidant capacity and glucagon-like peptide-1 (GLP-1) levels. Vild mitigated these alterations and improved the histopathological abnormalities caused by CBDL. ConclusionVild effectively attenuated CBDL-induced HPS through its anti-oxidant and anti-inflammatory effects along with its modulatory effects on ET-1/NOS/NO and TNF-α/IL-6/VEGF-A signaling implicated in the regulation of intrapulmonary vasodilatation and angiogenesis, respectively.

Selected cannabis cultivars modulate glial activation: in vitro and in vivo studies

IntroductionMultiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system characterized by neuroinflammation, demyelination and axonal loss. Cannabis, an immunomodulating agent, is known for its ability to treat MS effectively. However, due to variations in the profile of secondary metabolites, especially cannabinoids, among cannabis cultivars, the effectiveness of cannabis treatment can vary, with significant variability in the effects on different biological parameters. For screening available cultivars, cellular in vitro as well as pre-clinical in vivo assays, are required to evaluate the effectiveness of the wide range of chemical variability that exists in cannabis cultivars. This study evaluated comparatively three chemically diverse cannabis cultivars, CN2, CN4 and CN6, containing different ratios of phytocannabinoids, for their neuroinflammatory activity in MS model.Materials and methodsIn vitro experiments were performed with lipopolysaccharide (LPS)-activated BV-2 microglia and primary glial cells to evaluate the effect of different cannabis cultivars on nitric oxide (NO) and inflammatory cytokines, as well as inducible nitric oxide synthase (iNOS) protein expression. An in vivo experiment using the experimental autoimmune encephalomyelitis (EAE) MS model was conducted using Myelin oligodendrocyte glycoprotein (MOG) as the activating peptide. The cannabis extracts of the cultivars CN2, CN4, CN6 or vehicle, were intraperitoneally injected with clinical scores given based on observed symptoms over the course of study. At the end of the experiment, the mice were sacrificed, and splenocyte cytokine secretion was measured using ELISA. Lumbar sections from the spinal cord of treated MS mice were evaluated for microglia, astrocytes and CD4+ cells.ResultsExtracts of the CN2 cultivar contained tetrahydrocannabinolic acid (THCA) and tetrahydrocannabinol (THC) without cannabidiol (CBD), and a number of monoterpenes. CN4 contained cannabidiolic acid (CBDA) and tetrahydrocannabidiolic acid (THCA), with significant amounts of THC: CBD in a 1:1 ratio, as well as sesquiterpenes and some monoterpenes; and CN6 contained primarily CBDA and THCA, as well as THC and CBD in a 2:1 ratio, with some sesquiterpenes and no monoterpenes. All extracts were not cytotoxic in glial cells up to 50 µg/ml. Dose dependent inhibition of LPS-induced BV2 as well as primary microglial NO secretion confirmed the anti-inflammatory and anti-oxidative activity of the three cannabis cultivars. CN2 but not CN4 reduced both astrocytosis and microglial activation in lumbar sections of EAE mice. In contrast, CN4 but not CN2 significantly decreased the secretion of TNFα and Interferon γ (IFNγ) in primary splenocytes extracted from EAE mice.ConclusionsWhile both cannabis cultivars, CN2 and CN4, significantly reduced the severity of the clinical signs throughout the course of the study, they modulated different inflammatory mediators and pathways, probably due to differences in their phytocannabinoid composition. This demonstrates the differential potential of cannabis cultivars differing in chemotype to regulate neuroinflammation and their potential to treat MS.

Biochanin A mitigates ulcerative colitis and intestinal inflammation in mice by inhibiting MAPK/NF-kB (p65) axis.

Ulcerative colitis (UC) is a chronic problem of the intestine and relapsing in nature. Biochanin A is a nature-derived isoflavonoid and has numerous bioactivities. However, its role against UC and intestinal inflammation remains obscure. We aimed to comprehensively explore the pharmacological effect of biochanin A in alleviating colitis and to evaluate the potential mechanisms. Initially, we explored the anti-inflammatory action of biochanin A (15, 30, and 60 μM) by employing lipopolysaccharide (LPS)-activated RAW 264.7 cells. In RAW 264.7 cells under LPS stimulation, biochanin A inhibited the elevation of reactive oxygen species (ROS) (p < 0.0001), interleukin (IL)-1β(p < 0.0001), IL-18 (p < 0.01), and tumor necrosis factor (TNF)-α (p < 0.01) release, nitrite production (p < 0.0001), and the expression of inducible nitric oxide synthase (iNOS)and cyclooxygenase-2 (COX-2)proteins. Next, we studied the effectiveness of biochanin A (20 and 40 mg/kg) in mouse colitis induced with dextran sulfate sodium (DSS)by assessing colon length, disease activity index (DAI)scoring, and performing colonoscopy and histological analysis. The pro-inflammatory cytokines were estimated using ELISA. Western blot studies were performed to assess underlying mechanisms. In mice, biochanin A treatment alleviated DAI score (p < 0.0001), restored colon length (p < 0.05) and morphology, and re-established colon histopathology. Biochanin A affects the phosphorylation of proteins associated with NF-κB (p65) and mitogen-activated protein kinase (MAPK)axis and regulates colonic inflammation by reducing the expression of inflammatory cytokines and myeloperoxidase (MPO)activity. Altogether, our findings support the idea that the anticolitis potential of biochanin A is allied with anti-inflammatory activity by inhibiting the MAPK/NF-κB(p65) axis. Hence, biochanin A may be an alternative option to alleviate the risk of colitis.

Cinnamaldehyde Protects against P. gingivalis Induced Intestinal Epithelial Barrier Dysfunction in IEC-6 Cells via the PI3K/Akt-Mediated NO/Nrf2 Signaling Pathway.

Porphyromonas gingivalis (Pg), a Gram-negative oral pathogen, promotes and accelerates periodontitis-associated gut disorders. Intestinal epithelial barrier dysfunction is crucial in the pathogenesis of intestinal and systemic diseases. In this study, we sought to elucidate the protective role of cinnamaldehyde (CNM, an activator of Nrf2) against P. gingivalis (W83) and Pg-derived lipopolysaccharide (Pg-LPS) induced intestinal epithelial barrier dysfunction via antioxidative mechanisms in IEC-6 cells. IEC-6 (ATCC, CRL-1592) cells were pretreated with or without CNM (100 µM), in the presence or absence of P. gingivalis (strain W83, 109 MOI) or Pg-LPS (1, 10, and 100 µg/mL), respectively, between 0-72 h time points by adopting a co-culture method. Intestinal barrier function, cytokine secretion, and intestinal oxidative stress protein markers were analyzed. P. gingivalis or Pg-LPS significantly (p < 0.05) increased reactive oxygen species (ROS) and malondialdehyde (MDA) levels expressing oxidative stress damage. Pg-LPS, as well as Pg alone, induces inflammatory cytokines via TLR-4 signaling. Furthermore, infection reduced Nrf2 and NAD(P)H quinone dehydrogenase 1 (NQO1). Interestingly, inducible nitric oxide synthase (iNOS) protein expression significantly (p < 0.05) increased with Pg-LPS or Pg infection, with elevated levels of nitric oxide (NO). CNM treatment suppressed both Pg- and Pg-LPS-induced intestinal oxidative stress damage by reducing ROS, MDA, and NO production. Furthermore, CNM treatment significantly upregulated the expression of tight junction proteins via increasing the phosphorylation levels of PI3K/Akt/Nrf2 suppressing inflammatory cytokines. CNM protected against Pg infection-induced intestinal epithelial barrier dysfunction by activating the PI3K/Akt-mediated Nrf2 signaling pathway in IEC-6 cells.

Anti-Inflammatory Effects of Moringa Oleifera Lam Leaf Extract in Lipopolysaccharide-Activated Microglia

Moringa oleifera Lam has been used as a medicinal plant in many Asian countries including Thailand. The plant has a variety of pharmacological effects such as antioxidant, anti-inflammation, hypolipidemic, anti-infection, anticancer, and antidiabetic. The leaves and young pods, which contain various nutritional compounds including proteins, fatty acids, and vitamins, are widely consumed. In this study, we investigated the effects of hexane, ethyl acetate, methanol and ethanol crude extracts of M. oleifera Lam leaves on the viability of microglial cells and on the lipopolysaccharide-induced microglial activation. which is widely used as a model for neuroinflammation. Our findings suggested that all of the crude extracts at concentrations ranging from 10–9 to 10–5 g/mL did not significantly affect the viability of microglial cells and exhibited similar effects. Subsequently, we focused on ethanol extract for investigation of the molecular mechanism involving neuroinflammation. We found that the ethanol extract of M. oleifera Lam leaves at the concentrations of 10–9 to 10–5 g/mL significantly reduced inducible nitric oxide synthase (iNOS) protein expression and nitric oxide (NO) production, accompanied by the reduction of phospho-NF-κB and phospho-IκBα expressions. The results suggested that the ethanol extract of M. oleifera Lam leaves reduced NO production through reduction of iNOS protein expression following the inhibition of NF-κB/IκBα signaling pathways. Our study underscored the therapeutic potential of M. oleifera Lam leaves extract in neurodegenerative diseases associated with neuroinflammation. HIGHLIGHTS The ethanol extract of M. oleifera Lam leaves dose-dependently reduced NO production in LPS-activated microglia The reduction in NO production correlated with the reduction in iNOS protein expression The extract inhibited the expression of NF-kB/I-kBα protein expression in a dose-dependent manner The ethanol extract of oleifera Lam leaves exhibited anti-inflammatory effect via the inhibition of NF-kB/I-kBα signaling pathway GRAPHICAL ABSTRACT

Suppression of Inflammation in Adipocyte-Macrophage Coculture by Passion Fruit Seed Extract: Insights into the p38 and NF-ҡB Pathway.

Obesity, which is characterized by chronic low-grade inflammation, involves the infiltration of immune cells into adipose tissue, leading to the secretion of inflammatory cytokines and subsequent inflammation. Therefore, the aim of this study was to examine the potential of passion fruit seed extract (PSEE) in mitigating lipopolysaccharide (LPS)-induced inflammation in a coculture system comprising macrophages and adipocytes. PSEE demonstrated significant reductions in reactive oxygen species (ROS) and nitric oxide (NO) levels, primarily achieved through the downregulation of inducible nitric oxide synthase (iNOS) protein expression in LPS-induced adipocyte-macrophage cocultures. Furthermore, PSEE effectively suppressed the secretion of TNF-α and IL-1β by attenuating the gene expression of these cytokines, as well as other inflammation-related genes such as MMP-2, IL-6, and MCP-1. Notably, PSEE exhibited potent inhibitory effects on the p38 and NF-κB signaling pathways, thus alleviating inflammation in the LPS-induced adipocyte-macrophage cocultures. Additionally, PSEE led to a decrease in the expression of ACC, HSL, and FaSN, while aP2 and ATGL showed increased expression in LPS-induced cocultured macrophages and adipocytes. These findings suggest that passion fruit seed extract effectively combats inflammation by suppressing the p38 and NF-κB signaling pathways, resulting in reduced levels of proinflammatory cytokines, NO, and ROS production.

Bioactive cytochalasans from the desert soil-derived fungus Chaetomium madrasense 375 obtained via a chemical engineering strategy.

The chemical engineering of natural extracts has emerged as an effective strategy for the production of diverse libraries of chemicals, making it integral to drug discovery. A chemical engineering strategy based on the epoxidation and ring-opening reactions was used to prepare diversity-enhanced extracts of Chaetomium madrasense 375. Eleven unnatural cytochalasan derivatives (1-11) with unique functional groups, such as amine and isoxazole, were isolated and characterized from these chemically engineered extracts of C. madrasense 375. The identification of these new structures was accomplished through comprehensive spectroscopic analysis, supplemented by synthetic considerations. Notably, compounds 5 and 13-16 displayed potent phytotoxic effects on Arabidopsis thaliana, while compounds 1, 2, 5, 10, and 12 demonstrated inhibitory activities on LPS-induced NO production in RAW264.7 cells. Among them, compound 1 was found to be able to inhibit the upregulated expression of the inducible nitric oxide synthase (iNOS) protein induced by LPS, while also decreasing the production of pro-inflammatory cytokines (IL-6) and influencing the phosphorylation of p38, ERK1/2, and JNK at 100 μM. Our findings demonstrate that the chemical engineering of natural product extracts can be an efficient technique for the generation of novel bioactive molecules.

Low-Molecular-Weight Hydrolysate from Black Goat Extract Had Antioxidative and Anti-Inflammatory Effects in Macrophage Cells via Inhibition of MAPKs and NF-κB Pathways

This study evaluated the antioxidative and anti-inflammatory effects of a low-molecular-weight hydrolysate derived from black goat extract using alcalase, ProteAX, and trypsin (APT6 &lt; 3 kDa). The APT6 &lt; 3 kDa exhibited high antioxidative activities with estimated contents of 41.24% for antioxidative amino acids and 49.55% for hydrophobic amino acids. APT6 &lt; 3 kDa inhibited nitric oxide and prostaglandin E2 production in RAW 264.7 cells induced by lipopolysaccharide (p&lt;0.05), which was accompanied by the inhibition of inducible nitric oxide synthase and cyclooxygenase-2 protein expression. Additionally, APT6 &lt; 3 kDa downregulated the mRNA expression and production of TNF-α and IL-1β (p&lt;0.05). Mechanistically, these anti-inflammatory effects of APT6 &lt; 3 kDa were shown by downregulating the MAPK and NF-κB pathways in a dose-dependent manner. Overall, 43 peptides were isolated from a certain fraction (F5) of APT6 &lt; 3 kDa, demonstrating high antioxidative and anti-inflammatory activities. These results suggest that identified peptides from APT6 &lt; 3 kDa possess natural antioxidants and hold potential as anti-inflammatory agents.

Photobiomodulation effects on synovial morphology, iNOS gene, and protein expression in a model of acute inflammation.

To evaluate morphological aspects and inducible nitric oxide synthase (iNOS) gene and protein expression in a model of acute inflammation. Thirty-six female Wistar rats were assigned into three groups: control (saline, n = 12), sham (arthritis, n = 12), and PBM (arthritis and photobiomodulation, n = 12). Arthritis induction was performed with 200 μg of intra-articular Zymosan in sham and PBM animals. PBM was performed 24 h after induction with a laser device (λ = 808 nm, 25 mW of nominal power, fluence of 20 J/cm2, beam area of 0.02 mm2, time of 33 s, total energy of 0.825 J) with punctual and single dose application. Morphological analysis of joint structure (HE) and immunohistochemistry (anti-iNOS antibody) were performed on knee samples, and synovial tissue was submitted to RNA extraction, cDNA synthesis and gene expression analysis by quantitative polymerase chain reaction. Statistical analyses were performed with p < 0.05. It was observed an increase in the thickness of the synovial lining epithelium and inflammatory infiltrate in sham compared to PBM. Gene expression analysis showed higher iNOS expression in PBM, and iNOS protein expression decreased in PBM compared to sham. Photobiomodulation decreased inflammation in PBM animals, upregulated iNOS gene expression, however down egulated protein expression compared to sham.

Effect of miR-412-5p-loaded exosomes in H9c2 cardiomyocytes via the MAPK pathway.

MicroRNAs (miRNAs) are small non-coding RNAs that function in all biological processes. Recent findings suggest that exosomes, which are small vesicles abundantly secreted by various cell types, can transport miRNAs to target cells. Here, we elucidated the effect of miRNA-loaded exosomes on lipopolysaccharide (LPS)-induced inflammation in H9c2 cardiomyocytes. Exosomes were isolated from mesenchymal stem cells (MSC) and loaded with miR-412-5p. Additionally, the effect of the miR-412-5p-loaded exosomes on LPS-induced inflammation in H9c2 cardiomyocytes was evaluated by assessing the levels of nitric oxide (NO), reactive oxygen species (ROS), and prostaglandin E2 (PGE2). The expression of cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), inflammatory cytokines, and mitogen-activated protein kinase (MAPK) signaling factors was evaluated using reverse transcription-quantitative PCR and western blotting. miR-412-5p-loaded exosomes inhibited LPS-induced secretion of inflammatory mediators (NO, PGE2, and ROS), pro-inflammatory cytokines (IL-1β and IL-6), and COX-2 and iNOS expression. Additionally, miR-412-5p-loaded exosomes significantly decreased the expression of MAPK signaling molecules, including p-extracellular signal-regulated kinase (ERK), p-p38, and p-Jun kinase (JNK), in H9c2 cardiomyocytes. These findings showed that miR-412-5p-loaded exosomes ameliorated LPS-induced inflammation in H9c2 cardiomyocytes by inhibiting COX-2 and iNOS expression, inflammatory mediators, and pro-inflammatory cytokines via the MAPK pathway. The findings indicate that miR-412-5p-loaded exosomes may be effective for the prevention of myocardial injury.

Carthamus tinctorius seeds-Taraxacum coreanum combination attenuates scopolamine-induced memory deficit through regulation of inflammatory response and cholinergic function.

There is growing interest in herbal medicines for managing age-related diseases, such as Alzheimer's and Parkinson's. Safflower seeds (Carthamus tinctorius L. seeds, CTS) and dandelions (Taraxacum coreanum, TC) are widely used to treat bone- or inflammation-related diseases in Oriental countries. This study investigated the protective effect of the CTS-TC combination on scopolamine (Sco)-induced memory deficits through inflammatory response and cholinergic function. Moreover, marker components such as serotonin, N-(p-coumaroyl) serotonin, N-feruloylserotonin, chlorogenic acid, and chicoric acid in the CTS-TC combination were analyzed for their potential benefits on memory function. Water extracts of CTS, TC, and the CTS-TC combination at various ratios (4:1, 1:1, and 1:4) (100 mg/kg) were orally administered to mice for 14 days. Sco (1 mg/kg) was intraperitoneally injected into the mice before each behavioral test. T-maze and novel object recognition tests were conducted to monitor behavioral changes after the treatment. Western blotting was performed to detect protein expression. In addition, the presence of 5 biomarkers, serotonin, N-(p-coumaroyl) serotonin, N-feruloylserotonin, chlorogenic acid, and chicoric acid, was analyzed using high-performance liquid chromatography (HPLC). Behavioral tests showed that the CTS-TC combination enhanced memory function in Sco-injected mice. Inflammation-related proteins (inducible nitric oxide synthase, cyclooxygenase-2, and glial fibrillary acidic protein) were downregulated after treatment with the CTS-TC combination. The acetylcholinesterase protein expression was also downregulated. HPLC analysis revealed that N-feruloylserotonin and chicoric acid were the predominant components, followed by N-(p-coumaroyl) serotonin, chlorogenic acid, and serotonin. These findings suggest that the CTS-TC combination protects against Sco-induced memory deficits by inhibiting inflammatory responses and cholinergic dysfunction. N-feruloylserotonin and chicoric acid, along with N-(p-coumaroyl) serotonin, chlorogenic acid, and serotonin, might be biomarkers for the CTS-TC combination, and their effects on memory protection warrant further study.

The Effect of Sildenafil Citrate (Viagra®) on Nasal Olfactory Mucosa of Adult Male Albino Rat: caspase-3 and inducible nitric oxide synthase protein expressions.

The Effect of Sildenafil Citrate (Viagra®) on Nasal Olfactory Mucosa of Adult Male Albino Rat: caspase-3 and inducible nitric oxide synthase protein expressions.

4-methoxycinnamyl p-coumarate reduces neuroinflammation by blocking NF-κB, MAPK, and Akt/GSK-3β pathways and enhancing Nrf2/HO-1 signaling cascade in microglial cells

The active compound, 4-methoxycinnamyl p-coumarate (MCC), derived from the rhizome of Etlingera pavieana (Pierre ex Gagnep) R.M.Sm., has been shown to exert anti-inflammatory effects in several inflammatory models. However, its effects on microglial cells remain elusive. In the current study, we aimed to investigate the anti-neuroinflammatory activities of MCC and determine the potential mechanisms underlying its action on lipopolysaccharide (LPS)-induced BV2 microglial cells. Our results revealed that MCC significantly reduced the secretion of nitric oxide (NO) and prostaglandin E2, concomitantly inhibiting the expression levels of inducible NO synthase and cyclooxygenase-2 mRNA and proteins. Additionally, MCC effectively decreased the production of reactive oxygen species in LPS-induced BV2 microglial cells. MCC also attenuates the activation of NF-κB by suppressing the phosphorylation of IκBα and NF-κB p65 subunits and by blocking the nuclear translocation of NF-κB p65 subunits. Furthermore, MCC significantly reduced the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), and protein kinase B (Akt)/glycogen synthase kinase-3β (GSK-3β). In addition, MCC markedly increased the expression of heme oxygenase-1 (HO-1) by upregulating the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway. Collectively, our findings suggest that the anti-inflammatory activities of MCC could be attributed to its ability to suppress the activation of NF-κB, MAPK, and Akt/GSK-3β while enhancing that of Nrf2-mediated HO-1. Accordingly, MCC has promising therapeutic potential to treat neuroinflammation-related diseases.

Daeshiho-tang attenuates inflammatory response and oxidative stress in LPS-stimulated macrophages by regulating TLR4/MyD88, NF-κB, MAPK, and Nrf2/HO-1 pathways

Daeshiho-tang (DSHT), a traditional herbal formula with diverse pharmacological effects, has shown promise in medicine owing to its anti-hypertensive, anti-diabetic, and anti-inflammatory properties. However, the precise molecular mechanism underlying these effects remains unclear. Thus, we investigated the effect of DSHT on inflammatory response and oxidative stress to understand its molecular mechanism using lipopolysaccharide (LPS)-induced macrophage (RAW 264.7) cells. DSHT decreased the contents of nitric oxide (NO) and prostaglandin E2 (PGE2) through downregulating inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein expressions. DSHT suppressed the LPS-induced TLR4 as well as MyD88, subsequently suppressing the NF-κB activation and the phosphorylation of MAPK (p38, ERK, and JNK). Radical scavenging activity results revealed a dose-dependent response of DSHT with diminished ABTS activity, a hallmark of oxidative stress potential. Furthermore, DSHT enhanced Nrf2 and HO-1 expression in response to LPS. Collectively, our findings indicated that DSHT exert anti-inflammatory effect and regulating oxidative stress by modulating TLR4/MyD88, NF-κB, MAPK, and Nrf2/HO-1 pathways, consequently can provide potential therapeutic strategy for the prevention and treatment of inflammation and oxidative stress-related diseases.

Molecular Networking and Bioassay-Guided Preparation and Separation of Active Extract and Constituents from Vicia tenuifolia Roth.

Molecular networking drove the selection of material from V. tenuifolia organs that targeted active flavonoid glycosides. To optimize the extraction process, the flowers of V. tenuifolia were used to produce an anti-inflammatory extract. The effects of variables-organic solvent ratio; extraction time; and temperature-were investigated by the response of anti-inflammatory activity. Bioactivities-guided experiments helped identify fractions with high total phenolic and flavonoid content as well as antioxidant potential. Furthermore, one new compound (1), 19 first isolated together, and two known compounds were obtained and identified from the active fraction of this plant. Among them, compounds (15 and 22) were first reported for nuclear magnetic resonance (NMR) data from this study. All the isolates were evaluated for their anti-inflammatory capacity throughout, modulating nitric oxide (NO), interleukin (IL)-1β, and IL-8 production. Active compounds were further investigated for their regulation and binding affinity to the inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) proteins by Western blot and in silico approaches, respectively. The findings of this study suggested that the developed extract method, active fraction, and pure components should be further investigated as promising candidates for treating inflammation and oxidation.