iNOS mRNA Expression Research Articles

Anti-proliferation and anti-inflammation effects of corilagin in rheumatoid arthritis by downregulating NF-κB and MAPK signaling pathways

Ethnopharmacological relevanceThe dried aboveground part of Geranium Wilfordii Maxim. (G. Wilfordii) is a traditional Chinese herbal medicine named lao-guan-cao. It has long been used for dispelling wind-dampness, unblocking meridians, and stopping diarrhea and dysentery. Previous investigations have revealed that 50% ethanolic extract of G. Wilfordii has anti-inflammatory and anti-proliferation activities on TNF-α induced murine fibrosarcoma L929 cells. Corilagin (COR) is a main compound in G. Wilfordii with the content up to 1.69 mg/g. Pharmacology study showed that COR has anti-inflammatory, anti-tumor, anti-microorganism, anti-oxidant, and hepatoprotective effects. However, there is no any investigation on its anti-proliferation and anti-inflammation effects in rheumatoid arthritis (RA). Aim of the studyThe present study aimed to evaluate the potential pharmacological mechanisms of anti-proliferation and anti-inflammation effects of COR in RA. Materials and methodsIn vitro, MH7A cells model induced by IL-1β was used. The anti-proliferation activity of COR was assessed by Cell Counting Kit-8 (CCK-8) assay, and the anti-migration and anti-invasion activity of COR was determined by wound healing assay and transwell assay, respectively. Furthermore, apoptosis assay by flow cytometer was used to measure the pro-apoptotic effect of COR. The mRNA expressions of Bax, Bcl-2, IL-6, IL-8, MMP-1, MMP-2, MMP-3, MMP-9, COX-2, and iNOS were measured by qRT-PCR, and related protein were further verified by ELISA kits or Western blot. Moreover, protein levels associated with NF-κB and MAPK signaling pathways of p65, P-p65, IκBα, P-IκBα, ERK1/2, P-ERK1/2, JNK, P-JNK1/2/3, p38, and P-p38 were determined by Western blot. The nuclear translocation of NF-κB-p65 was detected by immunofluorescent staining. In vivo, adjuvant-induced arthritis (AIA) rat model was used, and the body weight, paw swelling, and arthritis score during the entire period were measured. Histopathological analysis of joints of synovial tissues was also determined. The expression of pro-inflammatory cytokines in serum including IL-6, TNF-α, IL-1β, and IL-17 were measured. ResultsThe in vitro results showed that COR could dose-dependently inhibit the proliferation, migration, and invasion of IL-1β-induced MH7A cells, as well as promote its apoptosis. Moreover, it also suppressed the over-expression of Bcl-2, IL-6, IL-8, MMP-1, MMP-2, MMP-3, MMP-9, COX-2, and iNOS while up-regulated the level of Bax. Besides, the ratios of P-p65/p65, P-IκBα/IκBα, P-ERK/ERK, P-JNK/JNK, and P-p38/p38 were decreased, and the nuclear translocation of p65 induced by IL-1β was blocked by COR. In vivo results indicated that COR significantly reduced the paw swelling and arthritis score in AIA rats, and inhibited synovial tissue hyperplasia and erosion, as well as inflammatory cells infiltration. It also decreased the serum pro-inflammatory cytokines (IL-6, TNF-α, IL-1β, and IL-17) production. ConclusionThese results revealed that COR exerted anti-rheumatoid arthritis effect, and its underlying mechanisms may be related to inhibiting the proliferation, migration, and invasion of synovial fibroblasts, enhancing cell apoptosis, and suppressing inflammatory responses via downregulating NF-κB and MAPK signaling pathways.

Protective potential of ethylacetate extract of Abrus precatorius (Linn) seeds against HCl/EtOH-induced gastric ulcer via pro-inflammatory regulation: In vivo and in silico study

BackgroundGastric ulcer is the most common gastrointestinal disorder of the stomach which has become global health challenge. Management with natural therapeutic agents such as Abrus precatorius seed extract through regulation of pro-inflammatory cytokines messenger ribonucleic acid (mRNA) expression would provide relief to patients with ulcer. However, there are several reports on pharmacological efficacy of A. precatorius seed. HypothesisUp-regulation of pro-inflammatory genes and elevation of the inflammatory cytokines are significant mechanism of ulcer pathogenesis. This work sought to investigate the anti-ulcerative efficacy of ethylacetate extract of Abrus precatorius seed in hydrochloric acid/Ethanol (HCl/EtOH)-induced ulcerative rats via regulation of inflammatory genes. MethodsExperimental animals were administered 100 and 200 mg/kg b.w of EEAPS, diclofenac and cimetidine (50 mg/kg b.w) for period of 8 days. One hour after the last administration, EtOH/HCl was administered orally to induce gastric injury. Stomach ulceration score, histological changes and messenger ribonucleic acid (mRNA) expression were evaluated using standard procedures. The bioactive compounds present in EEAPS were identified using high performance liquid chromatography (HPLC) followed by molecular docking against cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), nuclear factor kappa (NF-KB), interleukin-1β (IL-1β) and Tumor Necrosis Factor alpha (TNF-α) proteins. ResultsExposure of rats to EtOH/HCl significantly (P < 0.05) elevates stomach ulceration scores and up-regulation of COX-2, INOS, IL-1β, NF-kb and TNF-α mRNA expression compared to normal control rats. The alterations were reversed in animals pre-treated with EEAPS and standard drugs, 200 mg/kg b. w of EEAPS showed the highest ameliorative efficacy. HPLC analysis of EEAPS revealed the presence of 5 compounds which showed varying degrees of binding affinities when docked against target proteins. Apigenin showed the highest binding affinity (-9.6 kcal/mol) against iNOS. The results of the in silico pharmacokinetic and drug likeness properties of the compounds reveals that, these compounds are potential drug candidates. ConclusionAbrus precatorius seed might be a source of new drugs and natural remedy against gastric injury through inhibition of inflammatory reactions.

Constitutive expression of inducible nitric oxide synthase in healthy rat urothelium?

Background Contrasting findings have been reported regarding a possible constitutive expression of inducible nitric oxide synthase (iNOS) in a normal mammalian bladder. The current study was designed to further investigate such putative iNOS expression. Materials and methods The experiments were conducted with paraffin-embedded archival material from the urinary bladder of 6 normal, male Sprague–Dawley rats. In addition, two normal female mice (C57BL/6) were sacrificed and the urinary bladders were harvested. The occurrence of iNOS mRNA was examined by the RNAScope in situ hybridization method. Protein expression of iNOS and 3-nitrotyrosine (the latter used as an indicator of oxidative stress) was investigated by immunohistochemistry. Results No expression of iNOS mRNA was observed in the bladder tissue. iNOS protein and 3-nitrotyrosine were strongly expressed in the urothelium. iNOS was also expressed perinuclearly in the detrusor. Conclusions Although the RNAScope methodology could not demonstrate mRNA for iNOS in the normal urinary bladder, the results by immunohistochemistry strongly suggest the occurrence of iNOS in particular, in the urothelium. Positive reactivity for 3-nitrotyrosine may indicate ongoing oxidative stress of the urothelium. The finding of perinuclear iNOS immunoreactivity could suggest an intracrine signaling function by iNOS to the nucleus.

Mechanism of PI3K/AKT/mTOR signaling pathway for mediating anti-inflammatory and anti-oxidant effects of chrysin: a protein microarray-based study

To investigate the mechanism of PI3K/AKT/mTOR signaling pathway for mediating the anti-inflammatory and anti-oxidant effects of chrysin. RAW264.7 cells were treated with different concentrations of chrysin for 24 h, and the changes in cell viability were detected using CCK-8 method. The cells with or without chrysin pretreatment for 2 h were stimulated with lipopolysaccharide (LPS) for different lengths of time, and the related signal molecules were screened using protein chip technique. In cells pretreated with chrysin for 2 h followed by LPS stimulation for 18 h, the release of IL-6, MCP-1 and TNF-α by the cells was detected with ELISA, and NO production was examined using Griess method, and ROS level was determined using DCFH-DA. The effects of chrysin, LPS, and their combination on the mRNA expressions of iNOS and COX-2 were detected using RT-PCR; Western blotting was performed to examine the changes in cellular expressions of p-AKT, p-PRAS40, p-mTOR, mTOR, p-P70S6k, p-S6RP and S6RP following the treatments with LPS, N-Acetyl-L-cysteine, and chrysin, alone or in combinations. Chrysin below 60 μg/mL did not significantly affect the viability of RAW264.7 cells (P>0.05). Chrysin treatment significantly reduced the release of IL-6, MCP-1, and TNF-α and the level of NO (P < 0.01), and inhibited the mRNA and protein expressions of iNOS and COX-2 (P < 0.01) in the cells. The results of protein chip screening suggested that LPS could activate the AKT/mTOR pathway, which was significantly inhibited by chrysin pretreatment, and the results were verified by Western blotting (P < 0.01). Chrysin treatment significantly reduced the generation of endogenous ROS, and treatment with N-Acetyl-L-cysteine to eliminate intracellular ROS obviously reduced the expressions of iNOS and COX-2 (P < 0.05) and blocked the AKT/mTOR pathway (P < 0.05). Chrysin can inhibit the synthesis of the upstream signaling molecule ROS to inhibit the activation of AKT/mTOR signaling pathway, regulate the translation process of ribosomes, down-regulate the synthesis and release of pro-inflammatory cytokines and inflammatory mediators, and thus produce anti-inflammatory effects.

FJU-C28 inhibits the endotoxin-induced pro-inflammatory cytokines expression via suppressing JNK, p38 MAPK and NF-κB signaling pathways.

Despite marked improvements in supportive care, the mortality rate of acute respiratory distress syndrome due to the excessive inflammatory response caused by direct or indirect lung injury induced by viral or bacterial infection is still high. In this study, we explored the anti‐inflammatory effect of FJU‐C28, a new 2‐pyridone‐based synthetic compound, on lipopolysaccharide (LPS)‐induced inflammation in vitro and in vivo models. FJU‐C28 suppressed the LPS‐induced mRNA and protein expression of iNOS, COX2 and proinflammatory cytokines. The cytokine protein array results showed that LPS stimulation enhanced the secretion of IL‐10, IL‐6, GCSF, Eotaxin, TNFα, IL‐17, IL‐1β, Leptin, sTNF RII, and RANTES. Conversely, the LPS‐induced secretion of RANTES, TIMP1, IL‐6, and IL‐10 was dramatically suppressed by FJU‐C28. FJU‐C28 suppressed the LPS‐induced expression of RANTES, but its parental compound FJU‐C4 was unable to diminish RANTES in cell culture media or cell lysates. FJU‐C28 blocked the secretion of IL‐6 and RANTES in LPS‐activated macrophages by regulating the activation of JNK, p38 mitogen‐activated protein kinase (MAPK) and nuclear factor‐κB (NF‐κB). FJU‐C28 prevented the LPS‐induced decreases in lung function including vital capacity (VC), lung compliance (C chord), forced expiratory volume at 100 ms (FEV100), and forced vital capacity (FVC) in mice with LPS‐induced systemic inflammatory responses. FJU‐C28 also reduced neutrophil infiltration in the interstitium, lung damage and circulating levels of IL‐6 and RANTES in mice with systemic inflammation. In conclusion, these findings suggest that FJU‐C28 possesses anti‐inflammatory activities to prevent endotoxin‐induced lung function decrease and lung damages by down‐regulating proinflammatory cytokines including IL‐6 and RANTES via suppressing the JNK, p38 MAPK and NF‐κB signaling pathways.

Resistance Against Leishmania major Infection Depends on Microbiota-Guided Macrophage Activation.

Innate immune cells present a dual role during leishmaniasis: they constitute the first line of host defense but are also the main host cells for the parasite. Response against the infection that results in the control of parasite growth and lesion healing depends on activation of macrophages into a classical activated phenotype. We report an essential role for the microbiota in driving macrophage and monocyte-derived macrophage activation towards a resistance phenotype against Leishmania major infection in mice. Both germ-free and dysbiotic mice showed a higher number of myeloid innate cells in lesions and increased number of infected cells, mainly dermal resident and inflammatory macrophages. Despite developing a Th1 immune response characterized by the same levels of IFN-γ production as the conventional mice, germ-free mice presented reduced numbers of iNOS+ macrophages at the peak of infection. Absence or disturbance of host microbiota impaired the capacity of bone marrow-derived macrophage to be activated for Leishmania killing in vitro, even when stimulated by Th1 cytokines. These cells presented reduced expression of inos mRNA, and diminished production of microbicidal molecules, such as ROS, while presenting a permissive activation status, characterized by increased expression of arginase I and il-10 mRNA and higher arginase activity. Colonization of germ-free mice with complete microbiota from conventional mice rescued their ability to control the infection. This study demonstrates the essential role of host microbiota on innate immune response against L. major infection, driving host macrophages to a resistance phenotype.

Potentilla discolor ameliorates LPS-induced inflammatory responses through suppressing NF-κB and AP-1 pathways

Potentilla discolor Bunge (PD) is a traditional Chinese medicine which has been widely used for the treatment of various inflammatory diseases (e.g., diarrhea, fever and furuncle). However, few studies focused on its effect on classical inflammation. This study aimed to investigate the anti-inflammatory effect and potential mechanism of the ethanol extract of the whole herbs of PD (EPD) in lipopolysaccharide (LPS)-induced inflammatory models. The obtained results showed that EPD decreased supernatant NO, tumor necrosis factor-α (TNF-α) and monocyte chemoattractant protein-1 (MCP-1) in LPS-activated RAW264.7 cells and mouse peritoneal macrophages. Moreover, its effect on NO was attributed to the suppression of iNOS expression rather than its activity. At the transcriptional level, EPD suppressed iNOS, TNF-α and MCP-1 mRNA expressions in LPS-stimulated RAW264.7 cells. Further study showed that EPD didn’t affect the phosphorylation and degradation of IκBα, but yet impeded the nuclear translocation of p65 to inhibit NF-κB activation. Meanwhile, it also prevented JNK, ERK1/2 and p38 phosphorylation to dampen the activation of AP-1. In endotoxemia mouse model, EPD not only decreased interleukin-6, TNF-α and MCP-1 levels in serum, but also potently ameliorated diarrhea. These findings provide the theoretical basis for PD to treat inflammatory diseases, especially intestinal inflammation.

Antiviral Effect of Hyunggaeyungyo-Tang on A549 Cells Infected with Human Coronavirus.

Background Herbal medicine is widely recommended to treat viral infectious diseases. Over 123,000,000 individuals have been infected with the coronavirus since a worldwide pandemic was declared in March 2020. We conducted this research to confirm the potential of herbal medicine as a treatment for coronavirus. Methods We infected the A549 cell line with betacoronavirus OC43 and then treated it with 100 μg/mL Hyunggaeyungyo-tang (HGYGT) or distilled water with a control of HGYGT. We measured the mRNA expression levels of proinflammatory cytokines and interferon stimulated genes (ISGs) to confirm the effectiveness of HGYGT upon coronavirus infection. Results We found that the effects of HYGYT decrease the expression level of pPKR, peIF2α, IFI6, IFI44, IFI44L, IFI27, IRF7, OASL, and ISG15 when administered to cells with coronavirus infection. The expressions of IL-1, TNF-α, COX-2, NF-κB, iNOS, and IKK mRNA were also significantly decreased in the HGYGT group than in the control group. Conclusion Through the reduction of the amount of coronavirus RNA, our research indicates that HGYGT has antiviral effects. The reduction of IKK and iNOS mRNA levels indicate that HGYGT reduces coronavirus RNA expression and may inhibit the replication of coronavirus by acting on NF-kB/Rel pathways to protect oxidative injury. In addition, decreases in mRNA expression levels of proinflammatory cytokines indicate that the HGYGT may relieve the symptoms of coronavirus infections.

Sphagneticola trilobata (L.) Pruski-derived kaurenoic acid prevents ovalbumin-induced asthma in mice: Effect on Th2 cytokines, STAT6/GATA-3 signaling, NFκB/Nrf2 redox sensitive pathways, and regulatory T cell phenotype markers

Ethnopharmacological relevanceSphagneticola trilobata (L.) Pruski is used in traditional medicine in Brazil for inflammatory diseases treatment including asthma. The diterpene kaurenoic acid (KA) is one of its active compounds, but whether KA activity could explain the traditional use of S. trilobata in asthma is unknown. AimInvestigate KA effect and mechanisms in asthma. MethodsExperimental asthma was induced by ovalbumin immunization and challenge in male Swiss mice. KA (0.1–10 mg/kg, gavage) was administered 1 h before the ovalbumin challenge. Total leukocytes, eosinophil, and mast cell were counted in bronchoalveolar lavage fluid (BALF), and lung histopathology was performed. Lung mRNA expression of Th2 and regulatory T cells markers, and BALF type 2 cytokine production were quantitated. NFκB activation and oxidative stress-related components in pulmonary tissue were measured. ResultsKA inhibited the migration of total leukocytes and eosinophils to BALF, reduced lung histopathology (inflammatory cells and mast cells), mRNA expression of IL-33/ST2, STAT6/GATA-3 and NFκB activation in the lung, and reduced IL-33, IL-4, IL-5 production in the BALF. KA also reduced the mRNA expression of iNOS and gp91phox, and superoxide anion production accompanied by the induction of Nrf2, HO-1 and NQO1 mRNA expression, thus, exerting an antioxidant effect. Finally, KA induced nTreg-like and Tr1-like, but not Th3-like markers of suppressive T cell phenotypes in the lung tissue. ConclusionKA prevents antigen-induced asthma by down-regulating Th2 and NFκB/cytokine-related pathways, and up-regulating Nrf2 and regulatory T cells’ markers. Thus, explaining the ethnopharmacological use of S. trilobata for the treatment of lung inflammatory diseases.

Heat shock proteins took part in oxidative stress-mediated inflammatory injury via NF-κB pathway in excess manganese-treated chicken livers

Manganese (Mn) is an essential metal in humans and animals. However, excess Mn entered environment due to the wide application of Mn in industry and agriculture, and became an environmental pollutant. Exposure to high doses of Mn is toxic to humans and animals (including chickens). Liver is a target organ of Mn poisoning. Nevertheless, there were few studies on whether Mn poisoning damages chicken livers and poisoning mechanism of Mn in chicken livers. Herein, the aim of this study was to explore if oxidative stress, heat shock proteins (HSPs), and inflammatory response were involved in the mechanism of Mn poisoning-caused damage in chicken livers. A chicken Mn poisoning model was established. One hundred and eighty chickens were randomly divided into one control group (containing 127.88 mg Mn kg−1) and three Mn-treated groups (containing 600, 900, and 1800 mg Mn kg−1, respectively). Histomorphological structure was observed via microstructure and ultrastructure. Spectrophotometry was used to detect total antioxidant capacity (T-AOC) and inducible nitric oxide synthase (iNOS) activity, as well as nitric oxide (NO) content. And qRT-PCR was performed to measure mRNA expression of inflammatory genes (nuclear factor kappa B (NF-κB), tumor necrosis factor α (TNF-α), cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2), and iNOS) and heat shock protein (HSP) genes (HSP27, HSP40, HSP60, HSP70, and HSP90). Multivariate correlation analysis, principal component analysis, and cluster analysis were used to demonstrate the reliability of mechanism of Mn poisoning in our experiment. The results indicated that excess Mn led to inflammatory injury at three contents and three time points. Meanwhile, we found that NO content, iNOS activity, and NF-κB, TNF-α, COX-2, PGE2, and iNOS mRNA expression increased after Mn treatment, meaning that exposure to Mn induced inflammatory response via NF-κB pathway in chicken livers. Moreover, excess Mn decreased T-AOC activity, indicating that Mn exposure caused oxidative stress. Furthermore, mRNA expression of above five HSP genes was up-regulated during Mn exposure. Oxidative stress triggered the increase of HSPs and the increase of HSPs mediated inflammatory response induced by Mn. In addition, there were time- and dose-dependent effects on Mn-caused chicken liver inflammatory injury. Taken together, HSPs participated in oxidative stress-mediated inflammatory damage caused by excess Mn in chicken livers via NF-κB pathway. For the first time, we found that oxidative stress can trigger HSP70 and HSPs can trigger poisoning-caused inflammatory damage, which needs to be further explored. This study provided a new insight into environmental pollutants and a reference for further study on molecular mechanisms of poisoning.

Temporal dynamics of nitric oxide wave in early vasculogenesis.

Endothelium-derived nitric oxide (NO) is a mediator of angiogenesis. However, NO-mediated regulation of vasculogenesis remains largely unknown. In the present study, we show that the inhibition of NO significantly attenuated endothelial migration, ring formation, and tube formation. The contribution of nitric oxide synthase (NOS) enzymes during early vasculogenesis was assessed by evaluating endothelial NOS (eNOS) and inducible NOS (iNOS) mRNA expression during HH10-HH13 stages of chick embryo development. iNOS but not eNOS was expressed at HH12 and HH13 stages. We hypothesized that vasculogenic events are controlled by NOS-independent reduction of nitrite to NO under hypoxia during the very early phases of development. Semi-quantitative polymerase chain reaction analysis of hypoxia-inducible factor-1α (HIF-1α) showed higher expression at HH10 stage, after which a decrease was observed. This observation was in correlation with the nitrite reductase (NR) activity at HH10 stage. We observed a sodium nitrite-induced increase in NO levels at HH10, reaching a gradual decrease at HH13. The possible involvement of a HIF/NF-κB/iNOS signaling pathway in the process of early vasculogenesis is suggested by the inverse relationship observed between nitrite reduction and NOS activation between HH10 and HH13 stages. Further, we detected that NR-mediated NO production was inhibited by several NR inhibitors at the HH10 stage, whereas the inhibitors eventually became less effective at later stages. These findings suggest that the temporal dynamics of the NO source switches from NR to NOS in the extraembryonic area vasculosa, where both nitrite reduction and NOS activity are defined by hypoxia.

The effect of intranasal administration of an IL-1b antagonist (RAIL) on the state of the nitroxydergic system of the brain during modeling of acute cerebrovascular accident

Introduction: This study was designed to evaluate the effect activity of RAIL-gel in comparison with Citicoline on nitroxydergic system during acute cerebrovascular accident. Methods: In this study, 80 white nonlinear rats were randomly assigned to 4 groups (20 rats in each): 1) intact; 2) control - untreated with acute cerebrovascular accident (ACVA), examined on the 4th day; 3) animals with ACVA, receiving RAIL, examined on the 18th day; 4) animals with ACVA, treated with Citicoline, examined on the 4th day. The expression of inducible NOS was determined by Western blotting. The nitrosative stress marker, nitrotyrosine, was determined using the ELISE kit NITROTYROSINE (kit no. HK501-02, series 12825k1212-k). To assess the state of iNOS mRNA expression, we used the method of polymerase chain reaction with reverse transcription in real time (RT-PCR). Results: Our research demonstrated that course administration of the RAIL and Citicoline to animals with ACVA for 4 days leads to the stabilization of the parameters of the brain nitroxydergic system. However, Citicoline does not provide a full effect on the shifts of the NO system in the brain. It does not have the proper effect on such an important link in the pathogenesis of ischemic brain damage as nitrosative stress. RAIL leads to a significant decrease in NOS activity due to its inducible form (decrease in the expression of iNOS and iNOS mRNA) and a decrease in NO metabolits, and inhibition of nitrosative stress (decrease in nitrotyrosine). Conclusion: IL-1b antagonist RAIL (Intranal Gel) significantly exceeds Citicoline in terms of the severity of the effect on the nitroxydergic system indicators.

Anti-inflammatory activity of palmitoylethanolamide ameliorates osteoarthritis induced by monosodium iodoacetate in Sprague\u2013Dawley rats

Novel treatment strategies are urgently required for osteoarthritis (OA). Palmitoylethanolamide (PEA) is a naturally occurring fatty acid amide with analgesic and anti-inflammatory effects. We aimed to examine its effect on OA and elucidate the molecular mechanism of actions in monosodium iodoacetate (MIA)-induced OA Sprague–Dawley rats. The experimental animals were divided into normal control group (injected with saline + treated with phosphate-buffered saline (PBS), NOR), control group (injected with MIA + treated with PBS, CON), 50 or 100 mg/kg body weight (BW)/day PEA-treated group (injected with MIA + treated with 50 or 100 mg of PEA/kg BW/day, PEA50 or PEA100), and positive control group (injected with MIA + treated with 6 mg of diclofenac/kg BW/day, DiC). The changes in blood parameters, body parameters, gene expression of inflammatory mediators and cytokines, knee thickness, and joint tissue were observed. Oral administration of PEA had no adverse effects on the BW, liver, or kidneys. PEA reduced knee joint swelling and cartilage degradation in MIA-induced OA rats. The serum levels of leukotriene B4, nitric oxide, tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and prostaglandin E2 considerably reduced in the PEA100 group compared with those in the CON group. In the synovia of knee joints, the mRNA expression of iNOS, 5-Lox, Cox-2, Il-1β, Tnf-α, and Mmp-2, -3, -9, and -13 apparently increased with MIA administration. Meanwhile, Timp-1 mRNA expression apparently decreased in the CON group but increased to the normal level with PEA treatment. Thus, PEA can be an effective therapeutic agent for OA.

Evaluation of IL-1α and IL-1β, COX-2, and iNOS mRNA Expression in Orthodontic Patients Given Chitosan Mouthwash During Treatment with Miniscrew.

ABSTRACTObjective:Chitosan is a biomaterial with antibacterial properties that may benefit from maintaining peri-miniscrew hygiene and preventing inflammation. This study aimed to evaluate the expression of inflammatory-related molecules from the gingival crevicular fluid (GCF) after treatment of 1% chitosan when compared with 0.2% chlorhexidine mouthwash of patients with orthodontic miniscrew.Materials and Methods:A total of 30 subjects were divided into three groups: the first group received mouthwash containing 1% chitosan, the second group 0.2% chlorhexidine digluconate, and the control group received aquadest. The GCF was collected before and after 4 days of rinsing, and relative expressions of IL-1α and IL-1β, cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) were evaluated by real-time qPCR.Results:The expression of IL-1α was the highest in chitosan-treated patients when compared with that of IL-1β in between-groups. Patients receiving chlorhexidine have the highest expression of COX-2 and iNOS when compared with the chitosan and control groups, respectively.Conclusion:A mouthwash containing 1% of chitosan could suppress the expression of inflammatory mediators IL-1β, COX-2, and iNOS.

The Attenuation of TNF-α-mediated Inflammatory Responses in Human Lung Adenocarcinoma Cell Line by Perilla Seed and Seed Meal Extract

Abstract Thai perilla (Perilla frutescens) extracts, which contain a substantial quantity of bioactive substances including phenolics and flavonoids, have shown marked anti-inflammatory activities in several investigated models. In the present study, the effect of perilla seed extract (PSE) and seed meal extract (PSME) on TNF-α-induced inflammatory response in human lung adenocarcinoma A549 cells was investigated. The total phenolic and flavonoid contents in PSME was lower than PSE. Markedly, rosmarinic acid was identified as the main constituent in both extracts. However, the DPPH and ABTS assays indicated that the antioxidant capacity of PSME was equal to PSE. Moreover, the iron-binding activity of PSE and PSME were exhibited by complex formation with Fe3+-NTA, indicating that the extracts may inhibit hydroxyl radical production via Fenton reaction. In vitro cytotoxicity analysis showed that both PSE or PSME co-treated with TNF-α, at 24 h exposure, were not toxic to the A549 cells. Interestingly, PSE and PSME dramatically exhibited an anti-inflammatory activity by inhibiting the mRNA expression of pro-inflammatory cytokines, IL-1β, IL-6, IL-8, and TNF-α, but did not influence iNOS and COX-2 mRNA expressions. Moreover, both extracts significantly reduced reactive oxygen species (ROS) production in TNF-α-induced A549 cells. The findings presented in this paper suggest that PSE and PSME could mitigate TNF-α-mediated inflammatory responses via limiting pro-inflammatory cytokine expressions and decreasing ROS production. Thus, perilla seed and seed meal, the by-product of a perilla seed oil cold-pressed extraction process, could be developed as food supplements or functional foods for the prevention of inflammation-induced lung carcinogenesis development. Keywords: Human lung adenocarcinoma cell line, Inflammation, Perilla seed, Perilla seed meal, Tumor necrosis factor-alpha (TNF-α)

Improved immune-enhancing activity of egg white protein ovotransferrin after enzyme hydrolysis.

Ovotransferrin (OTF), an egg protein known as transferrin family protein, possess strong antimicrobial and antioxidant activity. This is because OTF has two iron binding sites, so it has a strong metal chelating ability. The present study aimed to evaluate the improved immune-enhancing activities of OTF hydrolysates produced using bromelain, pancreatin, and papain. The effects of OTF hydrolysates on the production and secretion of pro-inflammatory mediators in RAW 264.7 macrophages were confirmed. The production of nitric oxide (NO) was evaluated using Griess reagent and the expression of inducible nitric oxide synthase (iNOS) were evaluated using quantitative real-time polymerase chain reaction (PCR). And the production of pro-inflammatory cytokines (tumor necrosis factor [TNF]-α and interleukin [IL]-6) and the phagocytic activity of macrophages were evaluated using an ELISA assay and neutral red uptake assay, respectively. All OTF hydrolysates enhanced NO production by increasing iNOS mRNA expression. Treating RAW 264.7 macrophages with OTF hydrolysates increased the production of pro-inflammatory cytokines and the phagocytic activity. The production of NO and pro-inflammatory cytokines induced by OTF hydrolysates was inhibited by the addition of specific mitogen-activated protein kinase (MAPK) inhibitors. In conclusion, results indicated that all OTF hydrolysates activated RAW 264.7 macrophages by activating MAPK signaling pathway.

Diterpenoid glycosides from the flower of Trollius chinensis Bunge and their nitric oxide inhibitory activities

Trolliusditerpenosides A-Q (1–17), seventeen new labdane-diterpenoid glycosides, were isolated from the dried flowers of Trollius chinensis Bunge, a plant that has been commonly used as both an anti-inflammatory folk medicine and a healthcare tea for its therapeutic and anti-viral and antibacterial properties. Their structures were corroborated via comprehensive spectroscopic analysis, ECD calculations, and single-crystal X-ray diffraction analysis. Furthermore, the inhibitory activities on lipopolysaccharide (LPS)-induced NO production in RAW 264.7 macrophages of all compounds (1–17) were evaluated in vitro. Compounds 3, 6, 7, and 11 displayed significant inhibitory activities against NO production, with IC50 values ranging from 1.6 ± 0.1 to 14.4 ± 0.2 μM. In addition, compounds 3, 6, 7, and 11 all down-regulated the mRNA expression of iNOS, COX-2, and IL-1β in RAW 264.7 cells mediated by LPS. These findings not only support the chemical context of genus Trollius but also the exploration of new chemical entities with pharmacological significance from this genus.

Design, synthesis, in vitro determination and molecular docking studies of 4-(1-(tert-butyl)-3-phenyl-1H-pyrazol-4-yl) pyridine derivatives with terminal sulfonamide derivatives in LPS-induced RAW264.7 macrophage cells

In the present work, a new series of 4-(1-(tert-butyl)-3-phenyl-1H-pyrazol-4-yl) pyridine possessing terminal ethyl or propyl sulfonamides was designed and synthesized. The cytotoxic effect of the final compounds was measured by applying MTT assay in LPS-Induced RAW264.7 macrophage cells. The final target compounds were screened for their anti-inflammatory effect through their ability to inhibit NO and PGE2 production and cytokines production (TNF-α, IL-6, IL-1β) in LPS-induced RAW264.7 macrophage at 10 μM concentration. Compounds 8d, 9d, and 9k showed the highest inhibitory effect on NO production. Compounds 8d and 9k exhibited high PGE2 inhibition with IC50 values of 3.47, 2.54 μM, respectively. Compounds 8d and 9k exhibited high cytokines inhibition ≥60%. The most potent compounds 8d and 9k were tested to determine their effect on iNOS and COX-2 mRNA expression level. Compound 9k activity on iNOS and COX-2 proteins level, pro-inflammatory mediators and cytokines was determined and showed remarkable inhibition for both proteins level. Compounds 8d, 9k showed high binding affinity to COX-2 active site and exhibited similar binding interactions of the native ligand celecoxib.

CCAAT/enhancer binding protein δ inhibits invasion and metastasis of liver cancer by regulating M1 type macrophages polarization

Objective: To explore the regulation of macrophage polarization and its effects on liver cancer invasion, metastasis and apoptosis by CCAAT/enhancer binding protein δ (CEBPD). Methods: THP-1 stable transfected cells with knockdown CEBPD (shCEBPD) and negative control shNC were constructed by lentviral transfection technique. THP-1 transfected cells were induced into macrophages, lipopolysaccharide (LPS) and interferon γ(IFNγ) by phorbol 12-tetradecanoate 13-acetate (PMA), and then the polarized macrophages were further induced to M1 type. The quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect M1 type macrophage related interleukin 1β (IL-1β) genes, IL-6, tumor necrosis factor α (TNFα), and inducible nitric oxide synthase (iNOS) mRNA expression level. Flow cytometry was used to detect M1 macrophage-specific surface marker CD80 expression levels. M1-induced macrophages were co-cultured with liver cancer MHCC97H cells using Transwell non-contact small sized co-culture dishes. MHCC97H cells invasion and metastasis were detected by Transwell and scratch assay under co-culture conditions, and the MHCC97H cells apoptosis was detected by flow cytometry. Results: The mRNA expression levels of M1 macrophage marker genes iNOS, TNFα, IL-6 and IL-1β in THP-1 derived macrophages were decreased after CEBPD knockdown. M1 macrophage-specific surface marker CD80 expression levels were decreased (23.7% ± 2.1% and 62.5% ± 2.0%, t = 9.58, P < 0.05). THP-1 were co-cultured with MHCC97H in shCEBPD and shNC group, respectively. Compared with shNC group, the invasion [(158.0 ± 3.5) and (75.0 ± 4.5), t = 39.87, P < 0.01] and metastatic ability (54.6% ± 1.5% and 24.3% ± 1.0%, P < 0.01) of MHCC97H cells co-cultured in shCEBPD group were stronger and the apoptosis rate was reduced [(9.4% ± 1.0%) vs. (23.7% ± 1.2%), t = 12.68, P < 0.01]. Conclusion: CEBPD can inhibit the invasion and metastasis and increase the apoptosis by amplifying M1 type macrophages polarization in liver cancer cells.

Role of nitric oxide on zymosan-induced inhibition of crop emptying in chicks

Zymosan, a component of yeast cell walls, reduces feed passage through the digestive tract in chicks (Gallus gallus), although the mechanism mediating this effect is poorly understood. Nitric oxide (NO) is associated with a variety of biological actions including effects on the immune system. In addition, it has been suggested that NO is involved in relaxation of the digestive tract and affects feed passage in mammals. It is therefore possible that NO might be related to zymosan-induced reduction of feed passage in chicks. However, the role of NO on the effect of zymosan feed passage has not been clarified yet. Thus, the purpose of the present study was to investigate whether NO is associated with zymosan-induced alteration of feed passage in chicks. Intraperitoneal (IP) injection of zymosan significantly increased plasma nitrate and nitrite (NOx) concentrations at 6 h after injection. Zymosan-induced elevation of plasma NOx concentration was abolished by co-injection of S-methylisothiourea (SMT), a selective inhibitor for inducible NO synthase (iNOS), indicating that zymosan facilitated the induction of iNOS. Furthermore, because zymosan increased iNOS mRNA expression in the digestive tract, NO is likely associated with the effect of zymosan on the digestive tract. IP injection of NO donors significantly decreased crop emptying rate, suggesting that NO functions as an inhibitor of crop emptying. This result implied that zymosan stimulates NO production by the induction of iNOS in the digestive tract and thereby inhibits crop emptying rate. However, the co-injection of SMT did not attenuate the inhibitory effect of zymosan on crop emptying. The present study provides evidence that some changes in the digestive tract caused by zymosan are mediated by iNOS-induced NO in chicks, but NO does not mediate the effect of zymosan on feed passage through the crop.