iNOS mRNA Research Articles

Regulatory mechanisms of kaempferol on iNOS expression in RINm5F β-cells under exposure to interleukin-1β

Proinflammatory cytokines and NO play crucial roles in islet β-cells dysfunction. Though anti-inflammatory effects of kaempferol were revealed in several studies, the detailed mechanisms remain unclear. This study explored protective actions of kaempferol in interleukin-1β-treated RINm5F β-cells. Kaempferol significantly inhibited NO generation, iNOS protein, and iNOS mRNA level. Promoter study, EMSA, and κB-dependent reporter assay showed that kaempferol inhibited NF-κB-mediated iNOS gene transcription. Also, we found that kaempferol accelerated iNOS mRNA instability in iNOS 3′-UTR construct and actinomycin D chase studies. Additionally, kaempferol reduced iNOS protein stability in cycloheximide chase study and it inhibited NOS enzyme activity. Kaempferol inhibited ROS generation and preserved cell viability, and it improved insulin release. These findings suggest that kaempferol appears to be helpful in protecting islet β-cells, thereby supports kaempferol as a supplementary therapeutic candidate in inhibiting the incidence and progression of diabetes mellitus.

Microglial immune regulation by epigenetic reprogramming through histone H3K27 acetylation in neuroinflammation.

Epigenetic reprogramming is the ability of innate immune cells to form memories of environmental stimuli (priming), allowing for heightened responses to secondary stressors. Herein, we explored microglial epigenetic marks using the known inflammagen LPS as a memory priming trigger and Parkinsonian-linked environmental neurotoxic stressor manganese (Mn) as the secondary environmental trigger. To mimic physiological responses, the memory priming trigger LPS treatment was removed by triple-washing to allow the cells' acute inflammatory response to reset back before applying the secondary insult. Our results show that after the secondary Mn insult, levels of key proinflammatory markers, including nitrite release, iNOS mRNA and protein expression, Il-6, Il-α and cytokines were exaggerated in LPS-primed microglia. Our paradigm implies primed microglia retain immune memory that can be reprogrammed to augment inflammatory response by secondary environmental stress. To ascertain the molecular underpinning of this neuroimmune memory, we further hypothesize that epigenetic reprogramming contributes to the retention of a heightened immune response. Interestingly, Mn-exposed, LPS-primed microglia showed enhanced deposition of H3K27ac and H3K4me3 along with H3K4me1. We further confirmed the results using a PD mouse model (MitoPark) and postmortem human PD brains, thereby adding clinical relevance to our findings. Co-treatment with the p300/H3K27ac inhibitor GNE-049 reduced p300 expression and H3K27ac deposition, decreased iNOS, and increased ARG1 and IRF4 levels. Lastly, since mitochondrial stress is a driver of environmentally linked Parkinson's disease (PD) progression, we examined the effects of GNE-049 on primary trigger-induced mitochondrial stress. GNE-049 reduced mitochondrial superoxide, mitochondrial circularity and stress, and mitochondrial membrane depolarization, suggesting beneficial consequences of GNE-049 on mitochondrial function. Collectively, our findings demonstrate that proinflammatory primary triggers can shape microglial memory via the epigenetic mark H3K27ac and that inhibiting H3K27ac deposition can prevent primary trigger immune memory formation and attenuate subsequent secondary inflammatory responses.

M2 macrophage-derived exosomal lncRNA NR_028113.1 promotes macrophage polarization possibly by activating the JAK2/STAT3 signaling pathway

To explore the effect of M2 macrophage-derived exosomal lncRNA NR_028113.1 on macrophage polarization and its possible mechanism. Bone marrow-derived macrophages (BMDMs) from BALB/c mice were isolated and cultured in vitro. After IL-4 treatment to induce M2 macrophage polarization, exosomes (M2-exo) were extracted from the supernatant of M2 macrophages and identified. The expression of lncRNA in M2-exo was detected by qRT-PCR. BMDMs were co-cultured with M2-exo (100 μg/mL) or PBS for 48 h, and the changes in cellular expression levels of Arg1, YM-1, FIZZ1, iNOS and TNF-α were detected using qRT-PCR and Western blotting. The percentage of CD206+ cells was analyzed using flow cytometry, and the phosphorylation levels of JAK2/STAT3 proteins were detected using Western blotting. A lncRNA smart silencer was designed to specifically inhibit the expression of lncRNA NR_028113.1 in the M2 macrophages, from which exosomes were extracted and co-cultured with BMDMs for 48 h. The mRNA expression levels of Arg1, YM-1, FIZZ1, iNOS and TNF-α, CD206+ cell percentage and the phosphorylation levels of JAK2/STAT3 proteins were detected using qRT-PCR, flow cytometry and Western blotting. LncRNA NR_028113.1 was highly expressed in the exosomes of M2 macrophages (P < 0.05). Co-culture with M2-exo significantly increased mRNA expressions of M2 macrophage marker genes Arg1, YM-1 and FIZZ1 (P < 0.05), lowered the expressions of iNOS and TNF-α (P < 0.05), and increased CD206+ cell percentage and JAK2/STAT3 protein phosphorylation level in BMDMs (P < 0.05). After inhibiting the expression of lncRNA NR_028113.1 in M2 macrophages, the extracted M2-exo caused significant down-regulation of the mRNA expressions of Arg1, YM-1 and FIZZ1 and up-regulation of iNOS and TNF-α mRNA (P < 0.05), resulting also in signi-ficantly reduced CD206+ cell percentage and lowered phosphorylation levels of JAK2/STAT3 proteins in co-cultured BMDM (P < 0.05). M2 macrophage-derived exosomal lncRNA NR_028113.1 can significantly promote M2 polarization of macrophages possibly by activating the JAK2/STAT3 signaling pathway.

Immunomodulatory Effects of Endodontic Sealers: A Systematic Review.

Inflammation is a crucial step prior to healing, and the regulatory effects of endodontic materials on the immune response can influence tissue repair. This review aimed to answer whether endodontic sealers can modulate the immune cells and inflammation. An electronic search in Scopus, Web of Science, PubMed, and Google Scholar databases were performed. This systematic review was mainly based on PRISMA guidelines, and the risk of bias was evaluated by SYRCLEs and the Modified CONSORT checklist for in vivo and in vitro studies, respectively. In total, 28 articles: 22 in vitro studies, and six in vivo studies were included in this systematic review. AH Plus and AH 26 can down-regulate iNOS mRNA, while S-PRG sealers can down-regulate p65 of NF-κB pathways to inhibit the production of TNF-α, IL-1, and IL-6. In vitro and in vivo studies suggested that various endodontic sealers exhibited immunomodulatory impact in macrophages polarization and inflammatory cytokine production, which could promote healing, tissue repair, and inhibit inflammation. Since the paradigm change from immune inert biomaterials to bioactive materials, endodontic materials, particularly sealers, are required to have modulatory effects in clinical conditions. New generations of endodontic sealers could hamper detrimental inflammatory responses and maintain periodontal tissue, which represent a breakthrough in biocompatibility and functionality of endodontic biomaterials.

Anti-inflammatory Quinoline Alkaloids from the Roots of Waltheria indica

Sixteen new quinoline alkaloids (1a-7, 8a, 9, 10, 13-15, 17, and 21) and 10 known analogs (8b, 11, 12, 16, 18-20, and 22-24), along with three known cyclopeptide alkaloids (25-27), were isolated from the roots of Waltheria indica. The structures of the new compounds were elucidated by detailed NMR and circular dichroism with computational support and mass spectrometry data interpretation. Anti-inflammatory potential of isolates was evaluated based on inhibition of lipopolysaccharide (LPS)-induced nitric oxide (NO) production and tumor necrosis factor-alpha (TNF-α)-induced nuclear factor kappa B (NF-κB) activity with cell culture models. In the absence of cell growth inhibition, compounds 6, 8a, 9-11, 13, 21, and 24 reduced TNF-α-induced NF-κB activity with IC50 values ranging from 7.1 to 12.1 μM, comparable to the positive control (BAY 11-7082, IC50 = 9.7 μM). Compounds 6, 8a, 8b, and 11 showed significant NO-inhibitory activity with IC50 values ranging from 11.0 to 12.8 μM, being more active than the positive control (l-NMMA, IC50 = 22.7 μM). Structure-activity relationships indicated that NO inhibitory activity was significantly affected by C-8 substitution. Inhibition of LPS-induced nitric oxide synthase (iNOS) by 8b [(5S)-waltherione M, IC50 11.7 ± 0.8 μM] correlated with inhibition of iNOS mRNA expression. The biological potential of W. indica metabolites supports the traditional use of this plant for the treatment of inflammatory-related disorders.

Nootkatone Supplementation Attenuates Carbon Tetrachloride Exposure-Induced Nephrotoxicity in Mice.

Nootkatone (NKT), a major ingredient of Alpinia oxyphylla, exhibited potential nephroprotective effects; however, the precise molecular mechanisms remain poorly understood. This study aimed to study the nephroprotective effects of NKT and the underlying mechanisms in a mouse model. Our results showed that NKT pretreatment at the doses of 5, 10, and 20 mg/kg per day for 7 days significantly attenuates carbon tetrachloride (CCl4)-induced increases of serum BUN and CRE and kidney pathology injury. NKT pretreatment also markedly inhibited oxidative stress, inflammatory response, and the activation of caspases-9 and -3 in kidneys of mice exposed to CCl4. Meanwhile, NKT pretreatment downregulated the expression of NOX4, IL-1β, IL-6, and TNF-α proteins and NO levels in the kidney tissues. Moreover, NKT pretreatment upregulated the expression of Nrf2 and HO-1 mRNAs, and downregulated the expression of NF-κB, IL-1β, IL-6, TNF-α, and iNOS mRNAs in the kidneys of mice, compared to those in the CCl4 alone treatment group. In conclusion, our results reveal that NKT supplementation could protect against CCl4 exposure-induced oxidative stress and inflammatory response in the kidneys by inhibiting NOX4 and NF-κB pathways and activating the Nrf2/HO-1 pathway. Our current study highlights the therapeutic application of NKT for kidney diseases.

Cinnamaldehyde modulates host immunoinflammatory responses in rat ligature-induced periodontitis and peripheral blood mononuclear cell models.

Cinnamaldehyde is a natural product with anti-inflammatory and immune-modulatory properties, known to regulate host responses to bacterial stimuli. This study aimed to investigate the effects of cinnamaldehyde on ligature-induced periodontitis in rats, and its impact on the modulation of human peripheral blood mononuclear cells (PBMC). Male Wistar rats were assigned into three groups:i) control: no ligature+vehicle; ii) ligature: ligature+vehicle; and iii) ligature+cinnamaldehyde (50mg/kg); all treatments by daily oral gavage. After 14days of induced periodontitis, the hemimandibles were collected for bone loss evaluation. The gingival levels of IL-1β, MMP-9 and iNOS mRNA were evaluated. Nitric oxide (NO) was measured in both rat saliva and plasma. PBMC were stimulated with Aggregatibacter actinomycetemcomitans (Aa) in the presence or absence of cinnamaldehyde (5, 20 e 40µM), and cytokine production was quantified in cell supernatant. Proliferating lymphocytes were taken for flow cytometer reading, while culture supernatants were used for IFN-γ and IL-10 assessment. The ligature group had both increased alveolar bone loss and gingival expression of IL-1β, MMP-9 and iNOS compared to the control group. All parameters were attenuated by cinnamaldehyde treatment. Lower salivary but not plasma NO was detected in the cinnamaldehyde compared to the ligature group. Aa-stimulated PBMCs treated with cinnamaldehyde produced less IL-1β; the compound also attenuated lymphocyte proliferation in a dose-dependent manner, as well as cell IL-10 production. Cinnamaldehyde treatment reduced periodontal bone loss, and downregulated key inflammatory mediators and human PBMC responses, pointing to novel potential therapeutic effects of this compound.

Microwave-assisted extract of rhodomyrtone from rhodomyrtus tomentosa leaf: Anti-inflammatory, antibacterial, antioxidant, and safety assessment of topical rhodomyrtone formulation

ABSTRACT Series of works on the biological properties of rhodomyrtone, a major active component of Rhodomyrtus tomentosa leaf has been reported. In this study, rhodomyrtone-rich microwave-assisted extract from R. tomentosa leaf (RT-MAE) before formulation was profiled for phytochemical composition using gas chromatography-mass spectrometry (GC-MS) and targeted for anti-inflammatory activity with topical application. The formulation was assessed for bioactivity, biocompatibility, and safety on human volunteers. The GC-MS spectra indicated a rich content of phytosterols, sesquiterpenes, and acylphloroglucinols. At concentrations 50–600 ng/mL, RT-MAE before formulation treated macrophages showed >86% cell viability. Nitric oxide production significantly decreased by 19.26–88.36% following the treatment with 50–600 ng/mL RT-MAE before formulation (p < 0.001). Moreover, a significant (p < 0.01) concentration-dependent downregulation of lipopolysaccharide-induced IL-6, IL-1β, iNOS, and COX-2 mRNA was observed in RT-MAE before formulation treated macrophages. The extract demonstrated minimum inhibitory (MIC) and minimum bactericidal concentration (MBC) at 0.25–4 and 1–16 μg/mL, respectively. The antioxidant activity of RT-MAE before formulation indicated IC50 of 0.16 and 0.06 ug/mL for DPPH and ABTS assays, respectively. Ex-vivo diffusion of rhodomyrtone from formulation showed steady-state flux 64.935 ng/cm2 h, with >80% viability on HaCaT cells. Safety assessment of formulation containing RT-MAE after formulation was conducted on healthy volunteers. Hematological and biochemical analysis showed no significant difference between test and baseline (p < 0.05). The results suggested that the formulation can be effectively employed as a topical inflammatory agent.

Da-Yuan-Yin decoction polyphenol fraction attenuates acute lung injury induced by lipopolysaccharide

Context Da-Yuan-Yin is a Chinese traditional prescription. Objective This study explores the therapeutic effects of the Da-Yuan-Yin decoction polyphenol fraction (DYY-4) on acute lung injury (ALI) in mice induced by lipopolysaccharide (LPS). Materials and methods The mice (n = 10) were orally administrated with DYY-4 (15, 30, and 60 mg/kg) or DXM (5 mg/kg), half an hour after LPS (2 mg/kg) instilled intratracheally. The protein content and the levels of inflammatory factors, the levels of complements, the mRNA expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), the level of myeloperoxidase (MPO) and superoxide dismutase (SOD), the expression of the IkB kinase (IKK) and nuclear factor-kappa B (NF-κB), the lung wet-to-dry weight (W/D) ratio and lung tissue were evaluated, 24 h after LPS challenge. Network pharmacology predicted potential targets. Results DYY-4 (30, 60 mg/kg, p < 0.01, p < 0.01) decreased the lung W/D ratio, total protein concentration, the levels of C3, C3c and C5a, the levels of TNF-α, IL-6, and IL-1β, while increased the levels of IL-4 and IL-10. DYY-4 (60 mg/kg) decreased the levels of C5aR1, C5b-9 and COX-2 mRNA (p < 0.05), the levels of MPO and iNOS mRNA, the activation of the IKK/NF-κB pathway (p < 0.01), and increased the levels of IL-13 and SOD (p < 0.01). DYY-4 (60 mg/kg) relieved the lung tissue pathological changes and reduced the C3c deposition. Discussion and conclusions Network pharmacology combined with animal experiments revealed the targets of DYY-4 alleviating ALI.

Mitogen-activated protein kinase inhibitor PD98059 improves neuroimmune dysfunction in experimental autoimmune encephalomyelitis in SJL/J mice through the inhibition of nuclear factor-kappa B signaling in B cells

Multiple sclerosis (MS) is a severe autoimmune disease leading to demyelination, followed by consequent axonal degeneration, causing sensory, motor, cognitive, and visual symptoms. Experimental autoimmune encephalomyelitis (EAE) is the most well-studied animal model of MS. Most current MS treatments are not completely effective, and severe side effects remain a great challenge. In this study, we report the therapeutic efficacy of PD98059, a potent mitogen-activated protein kinase inhibitor, on proteolipid protein (PLP)139–151-induced EAE in SJL/J mice. Following the induction of EAE, mice were intraperitoneally treated with PD98059 (5 mg/kg for 14 days) daily from day 14 to day 28. This study investigated the effects of PD98059 on C-C motif chemokine receptor 6 (CCR6), CD14, NF-κB p65, IκBα, GM-CSF, iNOS, IL-6, TNF-α in CD45R+ B lymphocytes using flow cytometry. Furthermore, we analyzed the effect of PD98059 on CCR6, CD14, NF-κB p65, GM-CSF, iNOS, IL-6, and TNF-α mRNA and protein expression levels using qRT-PCR analysis in brain tissues. Mechanistic investigations revealed that PD98059-treated in mice with EAE had reduced CD45R+CCR6+, CD45R+CD14+, CD45R+NF-κB p65+, CD45R+GM-CSF+, CD45R+iNOS+, CD45R+IL-6+, and CD45R+TNF-α+ cells and increased CD45R+IκBα+ cells compared with vehicle-treated control mice in the spleen. Moreover, downregulation of CCR6, CD14, NF-κB p65, GM-CSF, iNOS, IL-6, and TNF-α mRNA expression level was observed in PD98059-treated mice with EAE compared with vehicle-treated control mice in the brain tissue. The results of this study demonstrate that PD98059 modulates inflammatory mediators through multiple cellular mechanisms. The results of this study suggest that PD98059 may be pursued as a therapeutic agent for the treatment of MS.

The effect of dapagliflozin on myocardial ischemia–reperfusion injury in diabetic rats

The incidence of ischemic heart disease is 2-3 times higher in diabetic patients. However, the effect of dapagliflozin on ischemia-reperfusion myocardial injury in diabetic rats has not been studied. We examined the effects of dapagliflozin on myocardial IR injury in streptozotocin-nicotinamide-induced diabetic rats. Rats were divided into four groups (n = 7 in each group): control, control-dapagliflozin, diabetes, and diabetes-dapagliflozin. Dapagliflozin (1.5 mg/kg/day) was administered concomitantly in drinking water for 2 months. The hearts were perfused in a Langendorff's apparatus at 2 months and assessed before (baseline) and after myocardial IR for the following parameters: left ventricular developed pressure (LVDP), minimum and maximum rates of pressure change in the left ventricle (±dP/dt), endothelial nitric oxide (NO) synthase (eNOS) and inducible NO synthase (iNOS) mRNA expressions, creatine kinase MB (CK-MB) and troponin imyocardial enzyme extravasation, and lactate dehydrogenase. The recovery of LVDP and±dP/dt in diabetic rats was lower than that in controls but near normal after dapagliflozin treatment. Diabetic rats had decreased eNOS expression and increased iNOS expression at baseline and after IR, whereas dapagliflozin normalized these parameters after IR. Compared with controls, cardiac NOx levels were initially lower in diabetic patients but higher after IR. Baseline MDA levels were higher in diabetic rats after IR, whereas cardiac NOx levels decreased after treatment with dapagliflozin. Dapagliflozin protects the diabetic rat heart from ischemia-reperfusion myocardial injury by regulating the expression of eNOS and iNOS and inhibiting cardiac lipid peroxidation.

Pharmacocorrection of Disturbances in the no System in Experimental Chronic Generalized Periodontitis

BACKGROUND: In the light of modern views on the pathogenesis of inflammatory diseases of the oral cavity, a promising direction is the use of agents with metabolitotropic, endothelioprotective, and especially with antioxidant action. AIM: The purpose of this study was to evaluate the effect of a combination of thiotriazoline and L-arginine (1:4) on the parameters of the nitroxidergic system of the blood and periodontium of rats with experimental chronic generalized periodontitis and substantiate further study of this combination. METHODS: Real-time reverse-transcription polymerase chain reaction was used to assess the mRNA expression status of iNOS and nNOS mRNAs. The total content of reduced thiols was also determined by the reaction with Elman’s reagent. RESULTS: We found an increase in the total activity of NOS by 90.01% due to an increase in the expression of iNOS, while a decrease in the expression of its endothelial form was observed (a decrease in the expression of eNOS mRNA by 74.3%) compared with the intact group. An increase in iNOS activity led to an increase in the production of NO, which, under conditions of antioxidant deficiency, is converted into cytotoxic forms (peroxynitrite and nitrosonium ion). CONCLUSIONS: The course administration of Mexidol (250 mg/kg) and, especially, the combination of thiotriazoline and L-arginine (1:4) (200 mg/kg) to animals with CGP, leads to a decrease in the gingival pocket to 6 mm (Mexidol) and to 4 mm against the background almost complete absence of bleeding, swelling, and tooth mobility (combination), and also led to a decrease in iNOS mRNA expression by 65.6% (p &lt; 0.05).

Design, synthesis, and biological evaluation of morpholinopyrimidine derivatives as anti-inflammatory agents.

Here, we outline the synthesis of a few 2-methoxy-6-((4-(6-morpholinopyrimidin-4-yl)piperazin-1-yl)(phenyl)methyl)phenol derivatives and assess their anti-inflammatory activity in macrophage cells that have been stimulated by LPS. Among these newly synthesized morpholinopyrimidine derivatives, 2-methoxy-6-((4-methoxyphenyl)(4-(6-morpholinopyrimidin-4-yl)piperazin-1-yl)methyl)phenol (V4) and 2-((4-fluorophenyl)(4-(6-morpholinopyrimidin-4-yl)piperazin-1-yl)methyl)-6-methoxyphenol (V8) are two of the most active compounds which can inhibit the production of NO at non-cytotoxic concentrations. Our findings also showed that compounds V4 and V8 dramatically reduced iNOS and cyclooxygenase mRNA expression (COX-2) in LPS-stimulated RAW 264.7 macrophage cells; western blot analysis showed that the test compounds decreased the amount of iNOS and COX-2 protein expression, hence inhibiting the inflammatory response. We find through molecular docking studies that the chemicals had a strong affinity for the iNOS and COX-2 active sites and formed hydrophobic interactions with them. Therefore, use of these compounds could be suggested as a novel therapeutic strategy for inflammation-associated disorders.

Alteration of Activated Phenotypes of the Macrophages Treated with Lipopolysaccharide and Interferon- by Sodium Bicarbonate in the Culture Medium

Macrophage is the immune phagocytic cell, playing variety of immunological and inflammatory reactions. The macrophage activation has been extensively studied in vitro using culture media like Dulbecco's modified Eagle’s medium (DMEM) and Ham's F-12 medium (F-12) in response to bacterial infection, tumor development, cytokines and so on. In the study of macrophage activation during co-culture with EL-4 tumor cells, we found different phenotypes of J774.1 macrophage-like cell line in F-12 and DMEM, when treated with lipopolysaccharide (LPS) and interferon-g (IFN- g). Among these phenotypes, nitric oxide (NO) production with corresponding inducible NO synthase (iNOS) expression was remarkable, showing higher in F-12 than in DMEM. Besides, O2-generating activity and production of interleukin-1b (IL-1b) were also higher in F-12 than DMEM, although production of tumor necrosis factor-a (TNF- a) was higher in DMEM than F-12. RT-PCR analysis revealed significantly higher expression of mRNA of iNOS, IL-1 b, IL-18, IkBa in F-12, but higher that of p65 and p105 in DMEM after treatment with LPS + IFN-g, suggesting these differences being induced at the transcriptional levels. Through investigation of critical factor(s) in these culture media that influence the activation phenotypes of the macrophages, we found that sodium bicarbonate (NaHCO3) concentrations in these culture media, 14 mM in Ham's F-12 and 44 mM in DMEM, were the key. Culture medium-induced differences in macrophage activation were also observed in RAW264.7 macrophage-like cell line and in mouse peritoneal macrophages. The recent studies suggested involvement of carrier (SLC) transporter gene expression and subsequent elevation of JAK/STAT signaling cascades in these NaHCO3 responses. Taken together, these results provide evidence for the importance of NaHCO3 in the culture medium in the macrophage activation in vitro, implying important insights to the NaHCO3 concentration in vivo in the body of patients suffering from inflammation, tumor development or immune disorders where macrophage activation is involved.

Microtomographic, histomorphometric, and molecular features show a normal alveolar bone healing process in iNOS-deficient mice along a compensatory upregulation of eNOS and nNOS isoforms.

Inducible nitric oxide synthase (iNOS) is one of the enzymes responsible for the synthesis of nitric oxide (NO), which is an important signaling molecule with effects on blood vessels, leukocytes, and bone cells. However, the role of iNOS in alveolar bone healing remains unclear. This study investigated the role of iNOS in alveolar bone healing after tooth extraction in mice. C57Bl/6 wild type (WT) and iNOS genetically deficient (iNOS-KO) mice were subjected to upper incision tooth extraction, and alveolar bone healing was evaluated by micro-computed tomography (μCT) and histological/histomorphometric, birefringence, and molecular methods. The expression of iNOS had very low control conditions, whereas a significant increase is observed in healing sites of WT mice, where iNOS mRNA levels peak at 7d time point, followed by a relative decrease at 14d and 21d. Regarding bone healing, both WT and iNOS-KO groups showed the usual phases characterized by the presence of clots, granulation tissue development along the inflammatory cell infiltration, angiogenesis, proliferation of fibroblasts and extracellular matrix synthesis, bone neoformation, and remodeling. The overall micro-computed tomography and histomorphometric and birefringence analyses showed similar bone healing readouts when WT and iNOS-KO strains are compared. Likewise, Real-Time PCR array analysis shows an overall similar gene expression pattern (including bone formation, bone resorption, and inflammatory and immunological markers) in healing sites of WT and iNOS-KO mice. Moreover, molecular analysis shows that nNOS and eNOS were significantly upregulated in the iNOS-KO group, suggesting that other NOS isoforms could compensate the absence of iNOS. The absence of iNOS does not result in a significant modulation of bone healing readouts in iNOS-KO mice. The upregulation of nNOS and eNOS may compensate iNOS absence, explaining the similar bone healing outcome in WT and iNOS-KO strains.

Overexpression of FTO alleviates osteoarthritis by regulating the processing of miR-515-5p and the TLR4/MyD88/NF-κB axis.

Osteoarthritis (OA) is regarded as the most prevalent chronic joint disease. Fat-mass and obesity-associated gene (FTO) is involved in OA alleviation. This study elucidated the role of FTO in OA and the associated mechanism. We established a cell injury model by stimulating human normal chondrocytes (C28/I2) with lipopolysaccharide (LPS), and measured cell viability, apoptosis, and inflammatory cytokines using CCK-8, flow cytometry, Western blot, and ELISA. TLR4, MyD88, p/t-p65, and p/t-IκBα levels, FTO, COX-2, and iNOS mRNA levels, and m6A methylation levels were measured by Western blot, RT-qPCR, and colorimetry. RNA immunoprecipitation and co-immunoprecipitation were conducted to confirm the interaction between FTO and DGCR8. pri-miR-515-5p process was regulated in an m6A-dependent manner. After predicting the presence of several binding sites between miR-515-5p and TLR4 on Targetscan, we further confirmed their relationship by dual-luciferase assay. OA rat models were established by monosodium iodoacetate injection. The pathological changes in knee joint were observed by HE staining. FTO was diminished in LPS-induced C28/I2 cells. With the increase of LPS concentration, cell viability was repressed, apoptosis rate was increased, and inflammatory markers were promoted, which were annulled by FTO overexpression. FTO interacted with DGCR8 and modulated the pri-miR-515-5p processing in an m6A-dependent manner. miR-515-5p silencing partially averted the inhibitory effect of FTO on LPS-induced cell injury. Given that TLR4 was a direct target of miR-515-5p, miR-515-5p inactivated the MyD88/NF-κB pathway by targeting TLR4. FTO overexpression improved cartilage structure in OA rats, reduced apoptosis, inhibited inflammation in synovial fluid, and repressed the TLR4/MyD88/NF-κB axis. FTO alleviated OA in an m6A-dependent manner via the miR-515-5p/TLR4/MyD88/NF-κB axis.

Ameliorative Effect of Citrus Lemon Peel Extract and Resveratrol on Premature Ovarian Failure Rat Model: Role of iNOS/Caspase-3 Pathway

Premature ovarian failure (POF) is described as a loss of oocytes and the absence of folliculogenesis and is considered an adverse effect of chemotherapeutic drugs, which leads to infertility. Subsequently, the existing inquiry was achieved by exploring the potential suspicious influences of lemon peel extract (LPE), and resveratrol (RES) on cyclophosphamide (CPA) induced-POF. The results showed that CPA-induced POF significantly decreased serum estradiol (E2) and progesterone levels, along with a considerable rise in serum luteinizing hormone (LH) and follicle-stimulating hormone (FSH) levels. Moreover, CPA administration to rats significantly increased the serum level of Malondialdehyde (MDA) and significantly lowered the levels of reduced glutathione (GSH) and superoxide dismutase (SOD); in addition, it increased nuclear factor kappa B (NF-κB) levels, tumor necrosis factor-α (TNF-α), as well as cyclooxygenase 2 (COX-2) with the spread expression of inducible nitric oxide synthase (iNOS) mRNA levels and caspase-3 (Casp3) levels in ovarian tissues versus the control rats. However, treatment with LPE and RES suppressed the triggering of NF- κB pathways, evidenced by a considerable reduction in Casp3 & iNOS mRNA expression level and significant ameliorative effects in all evaluated parameters, as confirmed by the histological and immunohistochemical investigation when comparing the model group. In overall findings, both lemon peel extract and resveratrol can mitigate the adverse effects of CPA-induced POF. Most crucially, its combination therapy is a promising pharmacological agent for this disease.

Regenerative Properties of Polypropylene Mesh Coated with Thiotriazoline and L-arginine

Restoration and strengthening of soft tissue structures of the musculoskeletal system underlie the principles of regenerative medicine to improve treatment results and prevent possible complications. Influencing various parts of the regenerative process, it is possible to correct the recovery time, the morphological properties of the scar, and, as a result, the clinical results of treatment. We have proposed using a polypropylene mesh (PM) impregnated with a metabolitotropic solution to restore damaged and strengthen weakened structures. The work aimed to study the effect of PM with metabolite impregnation on the regeneration process of soft tissues. A series of morphological and biochemical studies were carried out on laboratory animals. The degree of inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) mRNA expression was studied as indicators of tissue ischemia and their trophism in correlation with morphological changes in the zone of mesh implantation. Using a PM with a metabolitotrope increases the expression of eNOS mRNA by 10.8 times, and iNOS mRNA - reduces by 3.6 times. Morphologically coated PM causes a pronounced inflammatory effect, followed by a pronounced proliferation of connective tissue fibers around the mesh elements. Particular attention is drawn to the growth of the microvasculature and the increase in the number of anastomoses - 10.3% of the relative area compared to 8.8% in the group using PM without a metabolite. The metabolitotropic impregnation improves oxygenation and trophism of damaged soft tissues, which helps to stimulate regeneration.

Effects of rearing system and antibiotic treatment on immune function, gut microbiota and metabolites of broiler chickens

BackgroundIn China, cage systems with a high space utilization have gradually replaced ground litter systems, but the disease incidence of chickens in cages is higher. Broilers in the ground litter pens may be stimulated by more environmental microbes during the growth process and show strong immune function and status, but knowledge of which microbes and their metabolites play an immunomodulatory role is still limited. This study aimed to explore the differences and correlations in the immune function, gut microbiota and metabolites and the importance of gut microbiota of broilers raised in cages and ground litter pens.MethodsThe experiment involved a 2 × 2 factorial arrangement, with rearing systems (cages or ground litter pens) and antibiotic treatment (with or without broad-spectrum antibiotics in drinking water) as factors.ResultsThe results showed that, compared with the cage group, the ground litter broilers had stronger nonspecific immune function (Macrophages% and NO in blood), humoral immune function (IgG in blood, LPS stimulation index in ileum) and cellular immune function (T%, Tc%, ConA stimulation index and cytokines in blood). Antibiotic (ABX) treatment significantly reduced nonspecific immune function (Macrophages% and NO in blood, iNOS and Mucin2 mRNA expression in ileum), humoral immune function (IgG in blood and sIgA in ileum) and cellular immune function (T% and cytokines in blood, Th and Tc ratio, TLRs and cytokines mRNA expression in ileum). Furthermore, the ground litter broilers had higher α diversity of microbiota in ileum. The relative abundance of Staphylococcus, Jeotgalicoccus, Jeotgalibaca and Pediococcus in the ileum of ground litter broilers were higher. ABX treatment significantly reduced the α diversity of ileal microbiota, with less Chloroplast and Mitochondria. In addition, the levels of acetic acid, isobutyric acid, kynurenic acid and allolithocholic acid in the ileum of ground litter broilers were higher. Spearman correlation analysis showed that Jeotgalibaca, Pediococcus, acetic acid, kynurenic acid and allolithocholic acid were related to the immune function.ConclusionsThere were more potential pathogens, litter breeding bacteria, short-chain fatty acids, kynurenine, allolithocholic acid and tryptophan metabolites in the ileum of broilers in ground litter pens, which may be the reason for its stronger immune function and status.

2-Aryl Propionic Acid Amide Modification of Naproxen and Ibuprofen Dimers for Anti-neuroinflammatory Activity in BV2 mouse Microglial Cells

Inflammation is a common link in the pathophysiology of many neurological illnesses, including Alzheimer’s disease. Activated glial cells contribute to neuroinflammation by producing pro-inflammatory mediators. Naproxen and ibuprofen are nonsteroidal anti-inflammatory drugs with 2-aryl(s) propionic acid as a common pharmacophore. Here we designed a small series of naproxen and ibuprofen amide dimers and tested their effects on the expression of inducible nitric oxide synthase (iNOS), a neuroinflammatory enzyme in lipopolysaccharide (LPS)-stimulated BV2 mouse microglial cells. Of note, treatment with CNU 019, 020, 021, 023, 024, and 027 at 10 M markedly inhibited the LPS-induced iNOS expression in BV2 cells. CNU 024 was tested further at different concentrations to regulate the LPS-induced iNOS expression in BV2 cells. Treatment with CNU 024 at 5, 10, or 20 M dose-dependently suppressed the LPS-induced iNOS protein and mRNA expression levels in BV2 cells, in which maximal inhibition was seen at 20 M. CNU 024 treatment at doses tested further led to a concentration-dependent inhibition of the LPS-induced phosphorylation (activation) of p38 mitogen-activated protein kinase (MAPK) without influencing its total protein expression in BV2 cells, but it did not affect the LPS-induced activation of c-jun N-terminal kinase-1/2 and extracellular signal-regulated kinases-1/2 in these cells. In summary, our results demonstrate that CNU 024 inhibits the LPS-induced iNOS expression in BV2 cells, partly mediated by the inhibition of p38 MAPK. This work shows that CNU 024 could be a valuable ligand for further development as a potential drug candidate for treating neuroinflammatory pathologies.