Inhibition Of iNOS Expression Research Articles

Rosemary and neem: an insight into their combined anti-dandruff and anti-hair loss efficacy

Dandruff, a common scalp disorder characterized by flaking dead skin, is often treated with conventional topical products. However, limitations exist due to potential side effects and high costs. Therefore, searching for natural, cost-effective solutions for dandruff and hair loss is crucial. Rosemary herb and neem tree, both cultivated in Egypt, possess well-documented anti-inflammatory properties derived from their rich phenolic phytoconstituents. This study formulated a standardized combined extract of rosemary and neem (RN-E 2:1) into hair gel and leave-in tonic formats. This extract demonstrated superior efficacy against Malassezia furfur (a causative agent of dandruff) and Trichophyton rubrum (associated with scalp disorders) compared to the conventional antifungal agent, ketoconazole. The combined extract (RN-E 2:1) also exhibited potent anti-inflammatory activity. Additionally, the suppression of iNOS expression is considered concentration-dependent. Quality control verified formulation stability, and ex-vivo studies confirmed effective ingredient penetration into the epidermis, the primary site of fungal presence. Remarkably, both formulations outperformed the standard treatment, minoxidil in hair growth trials. These findings highlight the potential of natural extracts for scalp and hair health.

Oral administration of tilapia hydrolysate peptides ameliorates the immune-related side effects of cyclophosphamide in BALB/c mice

Bioactive peptide derived from fish is a novel and promising immunomodulating agent. However, the use of tilapia peptides as adjuvant therapy to ameliorate chemotherapy-induced immunosuppression in vivo has not been previously studied. In this study, tilapia hydrolysate peptides (THP) with a low molecular weight distribution (<3 kDa) were obtained from tilapia processing byproducts using Flavourzyme hydrolysis. These peptides showed no cytotoxicity to RAW264.7 macrophage cells and splenocytes. THP promoted the proliferation of RAW264.7 macrophage cells under basal conditions and enhanced mitogen-induced splenocyte proliferation. When treated with LPS-induced RAW264.7 macrophage cells, THP exhibited anti-inflammatory properties by inhibiting iNOS expression and NO production. To evaluate the in vivo immunomodulatory activity of THP, BALB/c mice were assigned to four groups (n = 7) and received either water (Control and CY group) or THP at doses of 400 mg/kg (Low-THP) and 800 mg/kg (High-THP) body weight through oral gavage for total 12 days. All groups, except the control, were injected with 100 mg/kg BW of cyclophosphamide (CY) on day 7. The high dose of THP positively influenced the recovery of peripheral white blood cell counts. Both doses of THP improved the proliferation ability of splenic T cells and immune organ indices. Additionally, the results of pro-inflammatory gene expression and histological examination suggested that both doses of THP alleviated CY-induced spleen damage. Consequently, oral administration of THP effectively ameliorated the immune-related side effects of CY, including reductions of peripheral white blood cell count, inhibition of splenic lymphocytes proliferation, atrophy of the spleen and thymus, and spleen damage.

Alamandine treatment prevents LPS-induced acute renal and systemic dysfunction with multi-organ injury in rats via inhibiting iNOS expression

Sepsis is defined as the dysregulated immune response leading to multi-organ dysfunction and injury. Sepsis-induced acute kidney injury is a significant contributor to morbidity and mortality. Alamandine (ALA) is a novel endogenous peptide of the renin-angiotensin-aldosterone system. It is known for its anti-inflammatory and anti-apoptotic effects, but its functional and vascular effects on sepsis remain unclear. We aimed to investigate the effects of ALA, as a pre- and post-treatment agent, on lipopolysaccharide (LPS)-induced systemic and renal dysfunction and injury in the LPS-induced endotoxemia model in rats via functional, hemodynamic, vascular, molecular, biochemical, and histopathological evaluation. 10 mg/kg intraperitoneal LPS injection caused both hepatic and renal injury, decreased blood flow in several organs, and renal dysfunction at 20 h in Sprague-Dawley rats. Our results showed that ALA treatment ameliorated systemic and renal inflammation, reduced inflammatory cytokines, prevented the enhancement of the mortality rate, reversed vascular dysfunction, corrected decreased blood flows in several organs, and reduced renal and hepatic injury via inhibiting iNOS (inducible nitric oxide synthase) and caspase expressions in the kidney. In addition, expressions of different ALA-related receptors showed alterations in this model, and ALA treatment reversed these alterations. These data suggest that ALA's systemic and renal protective effects are achieved through its anti-inflammatory, anti-pyroptotic, and anti-apoptotic effects on hemodynamic and vascular functions via reduced iNOS expression.

Investigation of the Effects of Opioids on Microglial Nitrite and Nitric Oxide Synthase (iNOS) Production and Phagocytosis during Inflammation.

This study aimed to investigate how opioids affect phagocytosis and microglial nitrite and nitric oxide synthase (iNOS) production during inflammation. Opioids are a group of chemicals that are naturally found in the opium poppy plant and exert a variety of effects on the brain, including pain alleviation in some cases. They are commonly used in surgery and perioperative analgesia. However, research on the impact of opioids on microglial inflammatory factor production and phagocytosis is limited. This study was designed to investigate the effects of opioids on inducible nitric oxide synthase (iNOS) activity and nitric oxide (NO) generation. Moreover, the influence of opioids on the engulfment of C8-B4 microglial cells after stimulation with LPS was also examined. C8-B4 mouse microglial cells were exposed to various concentrations of opioids after stimulation with lipopolysaccharide (LPS) and interferon-γ (IFN-γ). Nitrite production was assayed. The iNOS and Cox-2 were determined by Western blotting, and fluorescent immunostaining was performed to assess the percentage of microglia that engulfed fluorescent microspheres in total microglia cultivating with opioids after being activated by LPS. After LPS and IFN-γ stimulation, microglia produced lower amounts of nitric oxide (NO) production with buprenorphine, salvinorin A, and naloxone (P<0.05). When combined with naloxone, no significant differences were found than buprenorphine. It was observed that buprenorphine and salvinorin A could suppress iNOS expression activated by LPS and IFN-γ. Phagocytosis was greatly increased after LPS stimulation, and a significant increase was observed after adding salvinorin A. Buprenorphine and salvinorin A were found to reduce NO production and iNOS induction in microglial cells activated by LPS and IFN-γ. Salvinorin A promoted the phagocytosis of microglia cells treated by LPS.

LP342, a novel histone deacetylase inhibitor, decreases nitro-oxidative stress, mitochondrial dysfunction and hepatic ischaemia/reperfusion injury in mice.

Some histone deacetylase (HDAC) isoforms contribute to ischaemia/reperfusion (IR) injury (IRI). Here, we examined whether LP342, the lead candidate of a new generation of hydrazide-based HDAC inhibitors (HDACi), decreases hepatic IRI. IR was induced by clamping blood vessels to ~70% of the livers of mice for 1 h. At 6 h after reperfusion, ALT markedly increased, and wide-spread necrosis, leukocyte infiltration, and apoptosis occurred. LP342 treatment (1 mg/kg, ip) at 20 h or 1 h before ischaemia markedly decreased IRI whereas LP342 treatment upon reperfusion was marginally protective. Nitro-oxidative stress, c-Jun-N-terminal kinase (JNK) activation, and mitochondrial dysfunction contribute to IRI. 4-Hydroxynonenal, 3-nitrotyrosine, inducible nitric oxide synthase (iNOS), JNK activation and Sab binding increased markedly after IR, which LP342 blunted. LP342 also induced thioredoxin-1 expression before and after IR. LP342 also decreased mitochondrial depolarisation as detected by intravital microscopy at 2 h after IR. Lastly, LP342 increased acetylation of both histone-3 (class I HDAC substrate) and NFκB p65 but not tubulin (class II HDAC substrate) before and after IR. This novel HDACi protects against IRI most likely by epigenetic upregulation of antioxidant proteins and post-translational modifications of NFκB thus inhibiting iNOS expression and inflammatory responses.

Inhibition of NO Production in LPS-Stimulated Primary Rat Glial Cells by Gnidilatimonoein and Extract of Daphne mucronata.

In the CNS, glial cells are involved in neuroinflammation and neuropathic pain. The glial cells are activated by a variety of pathological conditions and release pro-inflammatory mediators, including nitric oxide (NO). Overexpression of iNOS (inducible nitric oxide synthase) and extra NO is detrimental to neurophysiology and neuronal viability. This study aimed to examine the effect of Gnidilatimonein isolated from D. mucronata and its leaves extract (as natural phytochemicals) on NO production in the LPS-induced primary glial cells. A preparative HPLC method was used to isolate gnidilatimonoein from leaves ethanolic extract. Various doses of Gnidilatimonoein, the ethanolic extract were applied to primary glial cells inflamed by lipopolysaccharide. A Colorimetric test, an MTT assay, and a RT-PCR analysis were then performed to analyze and compare NO production, cell viability, and iNOS expression. Gnidilatimonoein treatment of pretreated primary glial cells significantly inhibited iNOS expression and decreased NO synthesis. Plant extracts also reduced NO production in inflamed microglial and glial at 0.1-3 mg.mL-1. At these concentrations, none of these compounds exerted a cytotoxic effect, suggesting that their anti-inflammatory effects were not due to the death of cells. This study indicates that D. mucronata and its active compound, Gnidilatimonoein, could have restrained effects on the expression of iNOS on the induced glial cells; however, further investigation is warranted.

Dendritic cell-derived exosomal miR-3064-5p inhibits SIRT6/PCSK9 to protect the blood-brain barrier after subarachnoid hemorrhage.

The protection of the blood-brain barrier (BBB) is the key direction to improving subarachnoid hemorrhage (SAH). Therefore, developing appropriate targeted drugs and therapies has become an urgent task for SAH patients. In this study, we investigated the role of dendritic cells (DCs) exosomal miR-3064-5p in repairing the BBB, providing a new basis for treating SAH. We detected the expression of miR-3064-5p in exosomes secreted by DCs (DCs-exo). AnSAH rat model was constructed by intravascular perforation and characterized by HE and TUNEL-IF staining. We found that overexpression of miR-3064-5p in SAH rats suppressed iNOS expression and promoted the accumulation of tight junction proteins (Occludin, Claudin-3, ZO-1), whereas knockdown of miR-3064-5p exerted the opposite effect. Dual-LUC assay confirmed that miR-3064-5p could target and inhibit SIRT6. Knockdown of SIRT6 inhibited inflammatory cytokine (IL-6, IL-1β, IFN-γ, and TGF-β1) levels and apoptosis. The results of the co-IP assay showed that SIRT6 interacted with PCSK9, and knockdown of SIRT6 suppressed the expression of PCSK9. Moreover, DCs-exo reduced brain edema, upregulated miR-3064-5p and downregulated SIRT6 and PCSK9 in SAH rats. DCs-exo reduced inflammatory factors and increased tight junction proteins in SAH rats. Overexpression of miR-3064-5p enhanced the protective effect of DCs-exo, while overexpression of SIRT6 partially counteracted the effect. This study confirmed that DCs could secrete miR-3064-5p to ameliorate BBB damage after SAH. Mechanistically, miR-3064-5p alleviated BBB damage by targeting and inhibiting SIRT6/PCSk9 signaling pathway.

Icing after skeletal muscle injury with necrosis in a small fraction of myofibers limits inducible nitric oxide synthase-expressing macrophage invasion and facilitates muscle regeneration.

Growing evidence from animal experiments suggests that icing after skeletal muscle injury is harmful to muscle regeneration. However, these previous experimental models yielded massive necrotic myofibers, whereas muscle injury with necrosis in a small myofiber fraction (<10%) frequently occurs in human sports activities. Although macrophages play a proreparative role during muscle regeneration, they exert a cytotoxic effect on muscle cells through an inducible nitric oxide synthase (iNOS)-mediated mechanism. In this study, we established an animal injury model with necrosis limited to a small myofiber fraction and investigated the effect of icing on muscle regeneration with a focus on macrophage-related events. Icing after muscle injury of this model resulted in an enlarged size of regenerating myofibers compared with those in untreated animals. During the regenerative process, icing attenuated the accumulation of iNOS-expressing macrophages, suppressed iNOS expression in the whole damaged muscle, and limited the expansion of the injured myofiber area. In addition, icing increased the ratio of M2 macrophages within the injured site at an earlier time point than that in untreated animals. Following these phenomena in icing-treated muscle regeneration, an early accumulation of activated satellite cells within the damaged/regenerating area occurred. The expression level of myogenic regulatory factors, such as MyoD and myogenin, was not affected by icing. Taken together, our results suggest that icing after muscle injury with necrosis limited to a small fraction of myofibers facilitates muscle regeneration by attenuating iNOS-expressing macrophage invasion, limiting muscle damage expansion, and accelerating the accumulation of myogenic cells which form regenerating myofibers.

Sodium Tungstate (NaW) Decreases Reactive Oxygen Species (ROS) Production in Cells: New Cellular Antioxidant

Diabetic nephropathy (DN) is the leading cause of end-stage renal failure worldwide. Hyperglycemia generates reactive oxygen species (ROS), contributing to diabetic complications, especially in DN. Sodium Tungstate (NaW) is an effective antidiabetic agent for short and long-term treatments of both type 1 and type 2 diabetes models. In this study, we evaluated the effect of NaW on ROS production in bovine neutrophils incubated with platelet-activating factor (PAF) and in HK-2 cells induced by high glucose or H2O2. In addition, we evaluated the effect on iNOS expression in the type 1 diabetic rat model induced with streptozotocin (STZ). NaW inhibited ROS production in PAF-induced bovine neutrophils, and human tubular cells (HK-2) were incubated in high glucose or H2O2. In addition, NaW inhibited iNOS expression in glomeruli and tubular cells in the type 1 diabetic rat. This study demonstrates a new role for NaW as an active antioxidant and its potential use in treating DN.

Deacetylation of IRF8 inhibits iNOS expression and inflammation via SIRT1 in macrophages

AimsDysregulated interferon regulatory factor 8 (IRF8) mediated inducible nitric oxide synthase (iNOS) transcription is crucial to the pathogenesis of several inflammatory disorders. However, the molecular mechanism that control the transcription activity of IRF8 in the regulation of iNOS is not fully elucidated. This study is undertaken to determine whether SIRT1 impacts IRF8 acetylation level in the macrophages. Main methodsThe silver stain, mass spectrum, bone marrow-derived monocytes differentiation, lentiviral transduction, immunoprecipitation and chromatin immunoprecipitation assay were used to investigate the relationship between IRF8 and SIRT1. Key findingsWe demonstrate that deacetylation of IRF8 is induced by lipopolysaccharide (LPS) and suppresses iNOS expression. Macrophages expressing acetylation-defective iNOS are highly septic upon transfer to macrophages cleaned up mice. Mechanistically, deacetylation IRF8 facilitates the binding of silent information regulator 1 (SIRT1) to the iNOS promoter and restricts iNOS transcription. The expression of iNOS was enhanced in the macrophages from SIRT1 conditional knockout mice and the progression of sepsis is more serious. SignificanceThe discovery of the IRF8-SIRT1 interaction that governs iNOS expression may exploit new therapeutic strategies for inflammatory disorders.

Total Synthesis and Anti-inflammatory Activity of Stemoamide-Type Alkaloids Including Totally Substituted Butenolides and Pyrroles

AbstractTotally substituted butenolide including two tetrasubstituted olefins is a distinct structural motif seen in Stemona alkaloids, but efficient methods for its synthesis are not well developed. As an ongoing program aimed at the collective total synthesis of the stemoamide group, we report a stereodivergent method to give either (E)- or (Z)-totally substituted butenolide from the same intermediate. While AgOTf­-mediated elimination via an E1-type mechanism results in the formation of the kinetic (Z)-tetrasubstituted olefin, subsequent TfOH-mediated isomerization gives the thermodynamic (E)-tetrasubstituted olefin. The pyrrole ring is another important structure found in Stemona alkaloids. The direct oxidation of pyrrolidine rings with MnO2 and careful purification gives the pyrrole groups without isomerization of the stereocenter in the lactone group. These two methods enabled us to synthesize a series of stemoamide-type alkaloids including tricyclic, tetracyclic, and pentacyclic frameworks. The anti-inflammatory activities by inhibition of iNOS expression in macrophage cell line RAW264.7 indicate that the most potent anti-inflammatory compounds without cytotoxicity are protostemonines, which consist of pentacyclic frameworks including the totally substituted butenolide.

Duloxetine ameliorates lipopolysaccharide-induced microglial activation by suppressing iNOS expression in BV-2 microglial cells.

It is known that both selective serotonin and serotonin noradrenaline reuptake inhibitors (SSRI, SNRI) are first-line drugs for the treatment of major depressive disorder. It has also been considered that both SSRI and SNRI can improve the symptoms of major depressive disorder by increasing the concentration of monoamine in the synaptic cleft based on the monoamine hypothesis. However, accumulating evidence has indicated that inflammation in the brain may be a key factor in the pathophysiological mechanisms that underlie the development of major depressive disorder. It has been advocated that microglial cells may regulate the inflammatory response under pathological conditions such as major depressive disorder. In this study, we focused on whether duloxetine can ameliorate the inflammatory response induced by lipopolysaccharide (LPS) in BV-2 microglial cells. Our results indicated that duloxetine significantly decreased the NO production induced by LPS. The increase in the protein expression level of iNOS induced by LPS was significantly decreased by treatment with duloxetine. Moreover, the increases in the protein expression levels of phosphorylated-IκBα, phosphorylated-Akt and Akt induced by LPS were also significantly decreased. Unexpectedly, the protein expression levels of other pro-inflammatory factors such as COX-2 and the phosphorylation ratios for various molecules including IκBα and Akt were not changed by treatment with duloxetine. These findings suggest that duloxetine may have an anti-inflammatory effect, which could contribute to its therapeutic effectiveness for major depressive disorder.

Bifidobacterium lactis Inhibits iNOS Expression in LPS-stimulated RAW 264.7 Macrophages

BACKGROUND: Bifidobacterium is a genus of lactic acid bacteria that lives in the large intestine of humans and animals. The health benefits of this genus are well established; however, the anti-inflammatory activity of this genus, specifically Bifidobacterium lactis, has not been well defined. Therefore, in this study, we evaluated anti-inflammatory activity of B. lactis hydrolysates using lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages.METHODS: RAW 264.7 cells were cultured using Dulbecco’s Modified Eagle’s Medium in 5 % CO2 incubator at 37 ℃. One µg/mL of LPS was used to stimulate RAW 264.7 cells. Nitric oxide (NO) production, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) were measured to evaluate anti-inflammatory activity of B. lactis hydrolysates. The cytotoxicity of the inhibitor was also measured in present study through 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay.RESULTS: The results showed that B. lactis hydrolysates at 25–200 μg/mL inhibited NO production. In concentration-dependent manner, B. lactis hydrolysate showed inhibition of iNOS expression. However, no inhibition on COX-2 expression was observed. The MTS assay of the B. lactis hydrolysates showed no side effects on the cell viability at all concentrations.CONCLUSION: The current study revealed that B. lactis hydrolysates possess specific anti-inflammatory effects by inhibiting iNOS expression without cytotoxicity and therefore could potentially be developed as a new iNOS inhibitor.KEYWORDS: Bifidobacterium lactis, macrophages, hydrolysates, iNOS, COX-2

Antitubercular and immunomodulatory activities of Eugenia astringens n-hexane fraction

BackgroundIn Brazil, the Eugenia genus is traditionally used to treat several types of human diseases, including diabetes, gastric ulcers and rheumatoid arthritis. Previous studies demonstrated that medicinal properties of Eugenia astringens Cambess (Myrtaceae), popularly known as “baguaçu" in Brazil, were associated with anti-inflammatory and antimicrobial activities exhibited by the plant extracts. Anti-tubercular effects of E. astringens have not been elucidated yet. Aim of the studyTo study anti-tubercular and immunomodulatory potential of the E. astringens leaves extracts (syn. E. umbelliflora O. Berg), as well as the chemical profiles of active fractions. MethodsThe plant material was collected in the Parque Nacional da Restinga de Jurubatiba (Rio de Janeiro) and the leaves were dried and extracted with ethanol. The extracts were partitioned, to yield n-hexane (Hex), dichloromethane (DCM), ethyl acetate (EtOAc) and n-butanol (BuOH) fractions. The crude extract and fractions were evaluated for antitubercular effects in broth cultures of M. tuberculosis strains and assessed for cytotoxicity in mammalian cell cultures by the MTT method. The Hex fraction, exhibiting low cytotoxicity and stronger inhibitory effect on mycobacterial growth relative to other fractions, was selected for the further studies. Immunomodulatory effects of the Hex fraction were evaluated in the RAW 264.7 macrophages, stimulated by LPS to produce inflammatory mediators, such as TNF-α, IL-6 and nitric oxide. Additionally, the effects on mitogen-stimulated human lymphocyte functions were estimated by measuring cell proliferation and IFN-γ production. ResultsThe E. astringens Hex fraction inhibited growth of mycobacterial strains both in the bacterial culture and infected macrophages, demonstrating strong anti-tubercular potential. In addition, this fraction suppressed secretion of inflammatory cytokines and reduced production of nitric oxide through inhibition of iNOS expression in the LPS-stimulated macrophages. In the mitogen-stimulated lymphocyte assay, Hex fraction inhibited cell proliferation but did not alter IFN-γ production. GC–MS analyses showed that the Hex fraction is composed by caryophyllene-type, eudesmane-type, cadinene-type, and aromadendrene-type sesquiterpenes; and 28 compounds were identified, including spathulenol, previously described for antitubercular acitivity. It is possible that the overall biological efficiency of the Hex fraction depends on the cumulative and/or synergistic effect of these sesquiterpenes. ConclusionThe Hex fraction of E. astringens leaves showed strong antitubercular and immunomodulatory activities, demonstrating pharmacological potential of the identified compounds for the development of new anti-TB drugs.

In vitro Pro- and Anti-Inflammatory Screening of Pappea capensis Extracts

Pappea capensis has been used traditionally in the Limpopo province of South Africa to treat different diseases caused by viruses or infectious microorganisms such as fungi and bacteria. To date no reports of pro- and anti-inflammatory activity of this plant has been reported so far. The aim of the study was to determine the intro pro- and anti-inflammatory activity of water, ethanoic and methanolic extracts of the pappea capensis wood material. Serial dilutions were prepared at 0, 12.5, 25, 50, 100 and 200 μg/mL from each extract, which were subjected to the determination of the pro- and anti-inflammatory activity in vitro by RAW 264.7 cells using Resveratrol as a reference drug. The in vitro screening results for anti- and pro-inflammatory assays showed an increased nitric oxide (NO) production which correlated with each extract cytotoxicity in LPS-activated macrophages. Resveratrol, a known inhibitor of iNOS expression served as a positive control to the regime. There was a noticeable macrophage activation observed which was extracts concentrations depended. This response to treatment with different concentrations of pappea capensis extracts corresponded with nitric oxide production a similar trend was observed with the analysis of cell viability which predicts the corresponding cytotoxicity of all extracts. However, there was a high nitric oxide production and observable macrophage activation in the presence of water extract A confirmatory test was done to ascertain if this effect is owed to plant constituents or due to endotoxin contamination. Then, nitric oxide production was analyzed in the presence and absence of polymyxin B (PMB) and it can be concluded that polymyxin B counteracts the pro-inflammatory effect suggesting that this activity is possibly a result of endotoxins in this wood part of the plant. The determination of endotoxin as a cofactor perpetrating pro-inflammatory activity tested with polymyxin B was used and indicated the presence of a toxin in water extract. The low anti-inflammatory effect seen in the three extracts at 200 μg/mL are not physiologically active.

Water extract of Cayratia albifolia C.L.Li root relieves zymosan A-induced inflammation by restraining M1 macrophage polarization

BackgroundCayratia albifolia C.L.Li (CAC) is a traditional Chinese herbal medicine used to treat inflammatory diseases. Our laboratory has firstly reported that the water extract from CAC relieved lipopolysaccharide (LPS)-induced inflammation, however stronger evidence is still needed to prove its anti-inflammatory effects and the mechanisms involved are also ambiguous. PurposeThis study sought to provide more evidence for the application of CAC in alleviating infectious inflammation and disclose novel pharmacological mechanisms. MethodsMice were injected with zymA into their paws or peritoneal cavities, and then treated with CAC. ELISA, immunofluorescence and flow cytometry were performed to detect the cytokines (IL-1β, IL-6, TNF-α and IL-10) generation, the cell infiltration, and the CD86 or CD206 expression of macrophages. Then in vitro assays were performed on bone marrow-derived macrophages (BMDMs) and peritoneal macrophages (PMs) to detect their expression of iNOS, arg-1 and the cytokines above. On mechanisms, western blotting (WB), electrophoretic mobility shift assay (EMSA) and flow cytometry were carried out to measure NF-κB transcriptional activity, mitochondrial bioactivity and the mTORC1 activation when BMDMs were stimulated by zymA and treated with CAC. Finally, the chemical components consisted in the extract were analyzed by LC-MS. Results200 mg/kg CAC clearly inhibited zymA induced mouse paw edema and reduced the contents of IL-1β, IL-6 and TNF-α rather than IL-10 in local tissues. CAC also reduced CD86 but not CD206 in macrophages in situ. Through in vitro experiments, it was discovered that CAC reduced the protein and mRNA levels of IL-1β, IL-6 and TNF-α, and also inhibited iNOS expression, but showed no influence on IL-10 and arg-1 in macrophages. We found CAC reduced NF-κB transcriptional activity, down-regulated mitochondrial membrane potential and ROS levels, and inhibited mTORC1 activity. Finally, we identified 15 major compounds in the extract, among which 4-guanidinobutyric acid and kynurenic acid were the most abundant. ConclusionThis study provides further evidence that CAC significantly reduces zymA induced infectious inflammation. In addition, this novel data revealed that CAC restrained M1 rather than promoting M2 macrophages polarization via multi-target inhibitory effects, based on its potentially active components.

Antimycobacterial and anti-inflammatory activities of thiourea derivatives focusing on treatment approaches for severe pulmonary tuberculosis

Tuberculosis (TB) remains a serious public health problem and one of the main concern is the emergence of multidrug-resistant and extensively resistant TB. Hyper-reactive patients develop inflammatory necrotic lung lesions that aggravate the pathology and facilitate transmission of mycobacteria. Treatment of severe TB is a major clinical challenge that has few effective solutions and patients face a poor prognosis, years of treatment and different adverse drug reactions. In this work, fifteen novel and thirty-one unusual thiourea derivatives were synthesized and evaluated in vitro for their antimycobacterial and anti-inflammatory potential and, in silico for ADMET parameters and for structure–activity relationship (SAR). Thioureas derivatives 10, 15, 16, 28 and 29 that had shown low cytotoxicity and high activities were selected for further investigation, after SAR study. These five thioureas derivatives inhibited Mtb H37Rv growth in bacterial culture and in infected macrophages, highlighting thiourea derivative 28 (MIC50 2.0 ± 1.1 and 2.3 ± 1.1 µM, respectively). Moreover, these compounds were active against the hypervirulent clinical Mtb strain M299, in bacterial culture, especially 16, 28 and 29, and in extracellular clumps, highlighting 29, with MIC50 5.6 ± 1.2 µM. Regarding inflammation, they inhibited NO through the suppression of iNOS expression, and also inhibited the production of TNF-α and IL-1β. In silico studies were carried out suggesting that these five compounds could be administered by oral route and have low toxicological effects when compared to rifampicin. In conclusion, our data show that, at least, thiourea derivatives 16, 28 and 29 are promising antimycobacterial and anti-inflammatory agents, and candidates for further prospective studies aiming new anti-TB drugs, that can be used on a dual approach for the treatment of severe TB cases associated with exacerbated inflammation.

Aspartate induces metabolic changes and improves the host's ability to fight against Aeromonas hydrophila infection in Cyprinus carpio

Host innate immunity modulated by metabolites is a powerful strategy that can be used to defend against microbial infections. However, the mechanisms underlying the metabolic processes in controlling bacterial infections remain unexplored. Here, a GC-MS based metabolomics approach was adopted to investigate the metabolic characteristics between the surviving and dying Cyprinus carpio upon Aeromonas hydrophila infection. Aspartate was identified as an essential biomarker whose abundance significantly decreased in the dying group while increasing in the surviving group when compared to the control. Exogenous aspartate increased fish survival rate in a dose-dependent manner. Concurrently, we also found that aspartate regulated the expressions of inflammatory and anti-inflammatory factors, and alleviated oxidative damage based on the evaluations of the activities of antioxidant enzymes and histopathology. Interestingly, the results revealed that exogenous aspartate promoted arginine and proline metabolism and arginine biosynthesis, but attenuated the TCA cycle, thereby enhancing the disease resistance against A. hydrophila. We further demonstrated that aspartate induced iNOS expression and NO production, and L-NAME treatment inhibited iNOS expression and blocked NO production, which significantly increased the cumulative mortality of infecting A. hydrophila. These findings indicated that exogenous aspartate could reprogram C. carpio metabolome and improve the host's ability to fight against A. hydrophila infection by inducing of NO release, and demonstrated the metabolite-mediated immunomodulatory as a therapeutic intervention for A. hydrophila infection.

α-Hydroxyisocaproic Acid Decreases Protein Synthesis but Attenuates TNFα/IFNγ Co-Exposure-Induced Protein Degradation and Myotube Atrophy via Suppression of iNOS and IL-6 in Murine C2C12 Myotube.

There is ongoing debate as to whether or not α-hydroxyisocaproic acid (HICA) positively regulates skeletal muscle protein synthesis resulting in the gain or maintenance of skeletal muscle. We investigated the effects of HICA on mouse C2C12 myotubes under normal conditions and during cachexia induced by co-exposure to TNFα and IFNγ. The phosphorylation of AMPK or ERK1/2 was significantly altered 30 min after HICA treatment under normal conditions. The basal protein synthesis rates measured by a deuterium-labeling method were significantly lowered by the HICA treatment under normal and cachexic conditions. Conversely, myotube atrophy induced by TNFα/IFNγ co-exposure was significantly improved by the HICA pretreatment, and this improvement was accompanied by the inhibition of iNOS expression and IL-6 production. Moreover, HICA also suppressed the TNFα/IFNγ co-exposure-induced secretion of 3-methylhistidine. These results demonstrated that HICA decreases basal protein synthesis under normal or cachexic conditions; however, HICA might attenuate skeletal muscle atrophy via maintaining a low level of protein degradation under cachexic conditions.

Lithium inhibits NF-\u03baB nuclear translocation and modulate inflammation profiles in Rift valley fever virus-infected Raw 264.7 macrophages

IntroductionRift Valley fever virus (RVFV) is a zoonotic life-threatening viral infection endemic across sub-Saharan African countries and the Arabian Peninsula; however, there is a growing panic of its spread to non-endemic regions. This viral infection triggers a wide spectrum of symptoms that span from fibril illnesses to more severe symptoms such as haemorrhagic fever and encephalitis. These severe symptoms have been associated with dysregulated immune response propagated by the virulence factor, non-structural protein (NSs). Thus, this study investigates the effects of lithium on NF-κB translocation and RFVF-induced inflammation in Raw 264.7 macrophages.MethodsThe supernatant from RVFV-infected Raw 264.7 cells, treated with lithium, was examined using an ELISA assay kit to measure levels of cytokines and chemokines. The H2DCF-DA and DAF-2 DA florigenic assays were used to determine the levels of ROS and RNS by measuring the cellular fluorescence intensity post RVFV-infection and lithium treatment. Western blot and immunocytochemistry assays were used to measure expression levels of the inflammatory proteins and cellular location of the NF-κB, respectively.ResultsLithium was shown to stimulate interferon-gamma (IFN-γ) production as early as 3 h pi. Production of the secondary pro-inflammatory cytokine and chemokine, interleukin-6 (IL-6) and regulated on activation, normal T cell expressed and secreted (RANTES), were elevated as early as 12 h pi. Treatment with lithium stimulated increase of production of tumor necrosis factor-alpha (TNF-α) and Interleukin-10 (IL-10) in RVFV-infected and uninfected macrophages as early as 3 h pi. The RVFV-infected cells treated with lithium displayed lower ROS and RNS production as opposed to lithium-free RVFV-infected control cells. Western blot analyses demonstrated that lithium inhibited iNOS expression while stimulating expression of heme oxygenase (HO) and IκB in RVFV-infected Raw 264.7 macrophages. Results from immunocytochemistry and Western blot assays revealed that lithium inhibits NF-κB nuclear translocation in RVFV-infected cells compared to lithium-free RVFV-infected cells and 5 mg/mL LPS controls.ConclusionThis study demonstrates that lithium inhibits NF-kB nuclear translocation and modulate inflammation profiles in RVFV-infected Raw 264.7 macrophage cells.