The Essential Oil Isolated from Artemisia capillaris Prevents LPS-Induced Production of NO and PGE2 by Inhibiting MAPK-Mediated Pathways in RAW 264.7 Macrophages

Artemisia capillaris (A. capillaris) is used in traditional Korean herbal medicine for its believedanti-inflammatory activities. Previous studies have suggested that the essential oil of A. capillaris contains the active components responsible for its pharmacological effect, even though the mechanism for its action is unclear. This study examined the inhibitory effects of the essential oil of A. capillaris on the lipopolysaccharide (LPS)-induced production of nitric oxide (NO) and prostaglandin E₂ (PGE₂). The essential oil significantly inhibited the production of NO in the LPS-stimulated RAW 264.7 macrophages, which was mediated by the down-regulation of inducible NO synthase (iNOS) expression but not by its direct cytotoxic activity. The essential oil also blocked the secretion of PGE₂ and the expression of cyclooxygenase-2 (COX-2) in the LPS-stimulated cells. Western blot analysis showed that the essential oil inhibited the phosphorylation of IκB-α, nuclear translocation of p65, and subsequent activation of NF-κB. In addition, the essential oil suppressed the LPS-stimulated activation of mitogen-activated protein kinases (MAPKs) as well as the AP-1 DNA-binding activity. Moreover, MAPK inhibitors significantly reduced the LPS-induced production of NO and PGE₂. Collectively, we suggest that the oil inhibits the expression and production of inflammatory mediators by blocking the MAPK-mediated pathways and inhibiting the activation of NF-κB and AP-1.     

Echinocystic Acid Inhibits IL-1β-Induced COX-2 and iNOS Expression in Human Osteoarthritis Chondrocytes

Echinocystic acid (EA), a pentacyclic triterpene isolated from the fruits of Gleditsia sinensis Lam, displays a range of pharmacological activities including anti-inflammatory and antioxidant effects. However, the effect of EA on IL-1β-stimulated osteoarthritis chondrocyte has not been reported. The purpose of this study was to assess the effects of EA on IL-1β-stimulated human osteoarthritis chondrocyte. Chondrocytes were stimulated with IL-1β in the absence or presence of EA. NO and PGE₂ production were measured by Griess reagent and ELISA. The expression of COX-2, iNOS, nuclear factor-κB (NF-κB), inhibitory kappa B (IκBα), c-Jun N-terminal kinase (JNK), p38, and extracellular signal-regulated kinase (ERK) were detected by Western blot analysis. The results showed that EA suppressed IL-1β-induced collagenase-3 (MMP-13), NO, and PGE₂ production in a dose-dependent manner. IL-1β up-regulated the expression of COX-2 and iNOS, and the increase was inhibited by EA. Furthermore, IL-1β-induced NF-κB and mitogen-activated protein kinase (MAPK) activation were inhibited by EA. In conclusion, EA effectively attenuated IL-1β-induced inflammatory response in osteoarthritis chondrocyte which suggesting that EA may be a potential agent in the treatment of osteoarthritis.     

白细胞介素-10抑制人肾系膜细胞环氧化酶-2表达及其催化的前列腺素E2释放的实验研究

目的:观察IL-10对IL-1β诱导的人系膜细胞(HMC)前列腺素E₂(PGE₂)释放及环氧化酶-2(cyclooxygenase-2,COX-2)基因和蛋白表达的影响。方法:应用放射免疫测定法检测HMC培养上清中PGE₂,应用RT-PCR和Westernblot分别检测COX-2mRNA和蛋白水平。结果:①IL-1β显著上调PGE₂释放及COX-2基因和蛋白的表达(P均＜0.01);②IL-10对基础状态下PGE₂释放及COX-2基因和蛋白表达无明显影响(P＞0.05);③IL-10可呈剂量依赖性地下调IL-1β诱导的PGE₂释放及COX-2mRNA和蛋白表达(P＜001)。结论:IL-10抑制IL-1β诱导的HMCPGE₂释放及COX-2表达,提示IL-10对HMC具有多方面抗炎作用。     

NF-κB/IκB信号通路在IL-1β诱导的肾系膜细胞环氧化酶-2表达中的作用

目的:探讨核因子-κB(NF-κB)/IκB信号通路在肾小球系膜细胞环氧化酶-2(COX-2)表达中的作用。方法:放射免疫测定法检测系膜细胞培养上清中PGE₂水平;RT-PCR和Westernblot检测COX-2表达;凝胶电泳迁移率(EMSA)和Westernblot检测肾小球系膜细胞中NF-κB活化、p65亚基核转位以及IκBα和IκBβ的降解。结果:IL-1β显著上调系膜细胞PGE₂释放和COX-2表达,PDTC显著抑制IL-1β诱导的COX-2表达及PGE₂释放;IL-1β诱导系膜细胞NF-κB活化,p65核转位及胞浆内IκBα和IκBβ的降解。结论:IL-1β通过NF-κB/IκB信号转导通路诱导肾小球系膜细胞中COX-2表达。     

Triggering of DC migration by dengue virus stimulation of COX‐2‐dependent signaling cascades in vitro highlights the significance of these cascades beyond inflammation

A term “bone‐breaking fever” is used in Chinese medicine to describe the symptoms of patients infected with dengue virus (DV). We examined the significance of the COX‐prostaglandin pathway in human DC infected by DV. We show that DV infection induced the expression of COX‐2 and the production of prostaglandin E₂ (PGE₂) in DC, and stimulated the DNA binding of NF‐κB and the kinase activity of both IκBα kinase (IKK) α and β. DV infection also activated MAPK and AP‐1 signaling. Both IκBα kinase‐NF‐κB and MAPK‐AP‐1 were upstream of COX‐2 activation. Our investigation into the significance of COX‐2‐PGE₂ pathway also revealed that DV infection enhances DC migration by inducing CC chemokine receptor 7 (CCR7) expression, and that blocking COX‐2 or MAPK activity suppresses DV‐induced DC migration. Our data also suggest that PGE₂ can induce CCR7 expression on DC and that antagonists of the PGE₂ receptors EP2 and EP4 suppress DV‐induced DC migration. We further show that the increased CCR7 expression was observed in both DV‐infected and bystander DC, suggesting the presence of secondary effects in inducing CCR7 expression. Collectively, this study reveals not only the pathways involved in COX‐2 synthesis in DV‐infected DC but also the autocrine action of PGE₂ on the migration of DV‐infected DC.     

Pinoresinol from the fruits of Forsythia koreana inhibits inflammatory responses in LPS-activated microglia

The activation of microglia plays an important role in a variety of brain disorders by the excessive production of inflammatory mediators such as nitric oxide (NO), prostaglandin E₂ (PGE₂) and proinflammatory cytokines. We investigated here whether pinoresinol isolated from the fruits of Forsythia koreana Nakai inhibits the inflammatory responses in LPS-activated microglia. Pinoresinol inhibited the production of NO, PGE₂, TNF-α, IL-1β and IL-6 in LPS-activated primary microglia. Also, pinoresinol attenuated mRNA and protein levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2) and proinflammatory cytokines in LPS-activation. However, most of these inhibitory effects of pinoresinol have been mediated by extracellular-signal-regulated kinase (ERK) 1/2 mitogen-activated protein kinase (MAPK) phosphorylation and the NF-κB dependent. The results suggest that pinoresinol attenuates inflammatory responses of microglia and could be potentially useful in modulation of inflammatory status in brain disorders.     

<Emphasis Type="Italic">Toxoplasma gondii</Emphasis> induces prostaglandin E<Subscript>2</Subscript> synthesis in macrophages via signal pathways for calcium-dependent arachidonic acid production and PKC-dependent induction of cyclooxygenase-2

In this study, the intracellular signaling pathway of PGE₂ synthesis in macrophages (RAW264.7) induced by Toxoplasma gondii was investigated. The T. gondii-induced PGE₂ production in macrophages increased in a time-dependent manner, as PGE₂ induction began at 4 h, peaked at 12 h, and then plateaued at a high level. COX-2 mRNA in macrophages was detectable as early as 4 h after treatment; the maximal expression was observed at 8 h. The earliest induction of COX-2 protein occurred at 4 h and peaked at 16 h; meanwhile, COX-1 mRNA level and protein production remained unchanged throughout. Indomethacin and nimesulide inhibited tachyzoite-induced PGE₂ production and COX-2 mRNA expression in macrophages but they had no significant effect on COX-2 protein expression. EGTA, TFP and BAPTA/AM inhibited both arachidonic acid (AA) and PGE₂ production without effecting COX-2 protein expression, but verapamil inhibited neither AA nor PGE₂ production. H7 was found to inhibit PGE₂ production, and COX-2 mRNA expression and protein expression by tachyzoite or LPS stimulated macrophages in a dose-dependent manner. Our results demonstrate that T. gondii induces PGE₂ biosynthesis in RAW264.7 macrophages by regulating AA production through a calcium-dependent pathway and induction of COX-2 expression by a PKC-dependent pathway.     

Comparative Study of Normal and Rheumatoid Arthritis Fibroblast-Like Synoviocytes Proliferation under Cyclic Mechanical Stretch: Role of Prostaglandin E2

Fibroblast-like synoviocytes (FLSs) are one of the main contributors of prostaglandin E₂ (PGE₂) in the hyperplastic synovium of rheumatoid arthritis (RA) patients. cyclooxygenase-2 (COX-2)/PGE₂ pathway is involved in the proliferation of several cell types. We have previously shown that mechanical stretch affects COX-2 and PGE₂ production in human RA FLSs; however, its role in cell proliferation remains to be elucidated. In this study, a comparison is drawn between human RA and normal FLSs to understand the role of mechanical stretch and PGE₂ on the proliferation of FLSs. The results showed that physiological level (6%, 1 Hz) of cyclic mechanical stretch significantly decreased the proliferation of RA FLSs but not normal FLSs, while the induction of apoptosis was not observed by stretch in either RA or normal FLSs. IL-1β (5 ng/ml)-induced COX-2/PGE₂ levels are downregulated by stretch in RA FLSs only. Further investigation showed that high concentration (100 and 500 ng/ml) of PGE₂ significantly induced cell proliferation only in RA FLSs, and this induction failed to be suppressed by stretch. In conclusion, this study demonstrated that elevated levels of PGE₂ in the synovial cavity are involved in the proliferation of RA FLSs, and cyclic mechanical stretch regulates the RA synovial hyperplasia.     

Neuronal overexpression of cyclooxygenase-2 does not alter the neuroinflammatory response during brain innate immune activation

Neuroinflammation is a critical component in the progression of several neurological and neurodegenerative diseases and cyclooxygenases (COX)-1 and -2 are key regulators of innate immune responses. We recently demonstrated that COX-1 deletion attenuates, whereas COX-2 deletion enhances, the neuroinflammatory response, blood–brain barrier permeability and leukocyte recruitment during lipopolysaccharide (LPS)-induced innate immune activation. Here, we used transgenic mice, which overexpressed human COX-2 via neuron-specific Thy-1 promoter (TgCOX-2), causing elevated prostaglandins (PGs) levels. We tested whether neuronal COX-2 overexpression affects the glial response to a single intracerebroventricular injection of LPS, which produces a robust neuroinflammatory reaction. Relative to non-transgenic controls (NTg), 7 month-old TgCOX-2 did not show any basal neuroinflammation, as assessed by gene expression of markers of inflammation and oxidative stress, neuronal damage, as assessed by Fluoro-JadeB staining, or systemic inflammation, as assessed by plasma levels of IL-1β and corticosterone. Twenty-four hours after LPS injection, all mice showed increased microglial activation, as indicated by Iba1 immunostaining, neuronal damage, mRNA expression of cytokines (TNF-α, IL-6), reactive oxygen expressing enzymes (iNOS and NADPH oxidase subunits), endogenous COX-2, cPLA₂ and mPGES-1, and hippocampal and cortical IL-1β levels. However, the increases were similar in TgCOX-2 and NTg. In NTg, LPS increased brain PGE₂ to the levels observed in TgCOX-2. These results suggest that PGs derived from neuronal COX-2 do not play a role in the neuroinflammatory response to acute activation of brain innate immunity. This is likely due to the direct effect of LPS on glial rather than neuronal cells.     

Compressive Force Induces Osteoclast Differentiation via Prostaglandin E2 Production in MC3T3-E1 Cells

In orthodontic tooth movement, prostaglandin E₂ (PGE₂) released from osteoblasts can alter the normal process of bone remodeling. We previously showed that compressive force (CF) controls bone formation by stimulating the production of PGE₂ and Ep2 and/or Ep4 receptors in osteoblasts. The present study was undertaken to examine the effect of CF on the production of PGE₂, cyclooxygenase-2 (COX-2), macrophage colony-stimulating factor (M-CSF), receptor activator of NF-κB ligand (RANKL), and osteoprotegerin (OPG) using osteoblastic MC3T3-E1 cells and to examine the indirect effect of CF on osteoclast differentiation using RAW264.7 cells as osteoclast precursors. MC3T3-E1 cells were cultured with or without continuous CF (1.0 or 3.0 g/cm²) for 24 hr, and PGE₂ production was determined using ELISA. The expression of COX-2, M-CSF, RANKL, and OPG genes and proteins was determined using real-time PCR and ELISA, respectively. Osteoclast differentiation was estimated using tartrate-resistant acid phosphatase (TRAP) staining of RAW 264.7 cells cultured for 10 days with conditioned medium from CF-treated MC3T3-E1 cells and soluble RANKL. As CF increased, PGE₂ production and the expression of COX-2, M-CSF, and RANKL increased, whereas OPG expression decreased. The number of TRAP-positive cells increased as CF increased. Celecoxib, a specific inhibitor of COX-2, blocked the stimulatory effect of CF on TRAP staining and the production of PGE₂, M-CSF, RANKL, and OPG. These results suggest that CF induces osteoclast differentiation by increasing M-CSF production and decreasing OPG production via PGE₂ in osteoblasts.     

Cholera Toxin Induces a Shift from Inactive to Active Cyclooxygenase 2 in Alveolar Macrophages Activated by Mycobacterium bovis BCG

Intranasal vaccination stimulates formation of cyclooxygenases (COX) and release of prostaglandin E₂ (PGE₂) by lung cells, including alveolar macrophages. PGE₂ plays complex pro- or anti-inflammatory roles in facilitating mucosal immune responses, but the relative contributions of COX-1 and COX-2 remain unclear. Previously, we found that Mycobacterium bovis BCG, a human tuberculosis vaccine, stimulated increased release of PGE₂ by macrophages activated in vitro; in contrast, intranasal BCG activated no PGE₂ release in the lungs, because COX-1 and COX-2 in alveolar macrophages were subcellularly dissociated from the nuclear envelope (NE) and catalytically inactive. This study tested the hypothesis that intranasal administration of BCG with cholera toxin (CT), a mucosal vaccine component, would shift the inactive, NE-dissociated COX-1/COX-2 to active, NE-associated forms. The results showed increased PGE₂ release in the lungs and NE-associated COX-2 in the majority of COX-2⁺ macrophages. These COX-2⁺ macrophages were the primary source of PGE₂ release in the lungs, since there was only slight enhancement of NE-associated COX-1 and there was no change in COX-1/COX-2 levels in alveolar epithelial cells following treatment with CT and/or BCG. To further understand the effect of CT, we investigated the timing of BCG versus CT administration for in vivo and in vitro macrophage activations. When CT followed BCG treatment, macrophages in vitro had elevated COX-2-mediated PGE₂ release, but macrophages in vivo exhibited less activation of NE-associated COX-2. Our results indicate that inclusion of CT in the intranasal BCG vaccination enhances COX-2-mediated PGE₂ release by alveolar macrophages and further suggest that the effect of CT in vivo is mediated by other lung cells.     

Expression of cyclooxygenase-1/-2, microsomal prostaglandin-E synthase-1 and E-prostanoid receptor 2 and regulation of inflammatory mediators by PGE2 in the amoeboid microglia in hypoxic postnatal rats and murine BV-2 cells

This study aimed to investigate the effect of hypoxia on the expression of cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), microsomal prostaglandin-E synthase (mPGES-1), E-prostanoid receptor 2 (EP2) in microglia; and the roles of EP2-cyclic adenosine monophosphate (cAMP) signaling pathway in the prostaglandin E₂ (PGE₂) regulation of inflammatory mediators released by hypoxic BV-2 cells. Immunoexpression of COX-1, COX-2, mPGES-1 and EP2 was localized in the amoeboid microglial cells (AMC), a nascent brain macrophage in the developing brain, as confirmed by double labeling with OX-42 and lectin, specific markers of microglia. AMC emitted a more intense immunofluorescence in hypoxic rats when compared with the matching controls. In postnatal rats subjected to hypoxia, mRNA and protein expression levels of COX-1, COX-2 and mPGES-1 along with tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), inducible nitric-oxide synthase (iNOS) and PGE₂ product in the callosal tissue were significantly increased. The results were shared in the BV-2 cells except for COX-1 mRNA and protein whose levels remained unaltered. Interestingly, treatment with EP2 antagonist AH-6809 resulted in suppression of hypoxia induced EP2, IL-1β and iNOS mRNA and protein expression, TNF-α protein expression and intracellular cAMP level in BV-2 cells. It is suggested that PGE₂ may regulate above inflammatory mediators in the activated microglia via EP2-cAMP signaling pathway in hypoxic conditions.     

The role of PGE2-associated inflammatory responses in gastric cancer development

Accumulating evidence indicates that inflammation plays a critical role in cancer development. Cyclooxygenase-2 (COX-2) is a rate-limiting enzyme for prostanoid biosynthesis, including prostaglandin E₂ (PGE₂), and plays a key role in both inflammation and cancer. It has been demonstrated that inhibition of COX-2 and PGE₂ receptor signaling results in the suppression of tumor development in a variety of animal models. However, the molecular mechanisms underlying COX-2/PGE₂-associated inflammation in carcinogenesis have not yet been fully elucidated. In order to study the role of PGE₂-associated inflammatory responses in tumorigenesis, it is important to use in vivo mouse models that recapitulate human cancer development from molecular mechanisms with construction of tumor microenvironment. We have developed a gastritis model (K19-C2mE mice) in which an inflammatory microenvironment is constructed in the stomach via induction of the COX-2/PGE₂ pathway. We also developed a gastric cancer mouse model (Gan mice) in which the mice develop inflammation-associated gastric tumors via activation of both the COX-2/PGE₂ pathway and Wnt signaling. Expression analyses using these in vivo models have revealed novel mechanisms of the inflammatory responses underlying gastric cancer development. PGE₂-associated inflammatory responses activate epidermal growth factor receptor (EGFR) signaling through the induction of EGFR ligands and ADAMs that release EGFR ligands from the cell membrane. In Gan mice, a combination treatment with EGFR and COX-2 inhibitors significantly suppresses gastric tumorigenesis. Moreover, PGE₂-associated inflammation downregulates tumor suppressor microRNA, miR-7, in gastric cancer cells, which suppresses epithelial differentiation. These results indicate that PGE₂-associated inflammatory responses promote in vivo gastric tumorigenesis via several different molecular mechanisms.     

Prostaglandin production via induction of cyclooxygenase-2 by human gingival fibroblasts stimulated with lipopolysaccharides

The purpose of the present study was to investigate the involvement of cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) in PGE₂ production by human gingival fibroblasts stimulated with lipopolysaccharides (LPS) from periodondopathogenic bacteria. LPS were isolated fromPorphyromonas gingivalis (P. gingivalis), Actinobacillus actinomycetemcomitans (A. actinomycetemcomitans) andEschericia coli (E. coli) by the phenol-water procedure. The three LPS preparations produced PGE₂ up to 48 h in a time-dependent manner in human gingival fibroblasts.P. gingivalis-LPS was the most potent stimulator of PGE₂ production and, to a lesser extent,A. actinomycetemcomitans- andE. coli-LPS. Treatment of the cells with indomethacin, a non selective COX-1/COX-2 inhibitor and NS-398, a selective COX-2 inhibitor, completely depressed PGE₂ production. Treatment of dexamethasone, known to inhibit COX-2 expression, also significantly prevented PGE₂ production. Immunohistochemical staining of COX-2 protein demonstrated that expression of COX-2 protein was increased at 24 h afterP. gingivalis-LPS stimulation, while expression of COX-1 protein was not affected byP. gingivalis-LPS. In order to investigate the regulation of PGE₂ production,P. gingivalis-LPS-stimulated cells were treated with herbimycin A and genistein, both inhibitors of tyrosine kinases. Both the inhibitors significantly inhibited PGE₂ production. Herbimycin A treatment depressed expression of COX-2 protein. These data suggest that human gingival fibroblasts stimulated with LPS from periodontopathogenic bacteria mainly produce PGE₂ not by COX-1, but by COX-2, induction of which may be regulated by tyrosine kinase and that the produced PGE₂ may be involved in the pathogenesis of periodontal diseases.     

Synergism in the repression of COX-2- and TNFα-induction in platelet activating factor-stressed human neural cells

Platelet activating factor (PAF; β-acetyl-γ-O-hexadecyl-l-α-phosphatidylcholine) triggers a rapid pro-inflammatory gene expression program in primary cultures of human neural (HN) cells. Two genes and gene products consistently induced after PAF treatment are the cytosoluble prostaglandin synthase cycloooxygenase-2 (COX-2) and the pro-apoptotic tumor necrosis factor alpha (TNFα). Both of these mediators are associated with the activation of inflammatory signaling, neural cell dysfunction, apoptosis and brain cell death, and both have been found to be up-regulated after brain injury in vivo. In this study we investigated the effects of the non-halogenated synthetic glucocorticoid budesonide epimer R (BUDeR), the novel PAF antagonist LAU-0901, and the electron spin trap and free radical scavenger phenyl butyl nitrone (PBN), upon early COX-2 and TNFα gene activation and prostaglandin E₂ (PGE₂) release in PAF-stressed primary HN cells. The data indicate that these three biochemically unrelated classes of inflammatory repressors act synergistically in modulating PAF-induced up-regulation of COX-2, TNFα, and PGE₂ by quenching oxidative stress or inflammatory signaling, resulting in increased HN cell survival. These, or analogous classes of compounds, may be useful in the design of more effective combinatorial pharmacotherapeutic strategies in the treatment of complex neuro-inflammatory disorders.     

Suppression of Heme Oxygenase-1 by Prostaglandin E2-Protein Kinase A-A-Kinase Anchoring Protein Signaling Is Central for Augmented Cyclooxygenase-2 Expression in Lipopolysaccharide-Stimulated RAW 264.7 Macrophages

Purpose

Prostaglandin (PG) E₂ is an immunomodulatory lipid mediator generated mainly via the cyclooxygenase-2 (COX-2) pathway from arachidonic acid at sites of infection and inflammation. A positive feedback loop of PGE₂ on COX-2 expression is critical for homeostasis during toll-like receptor (TLR)-mediated inflammatory processes. The mechanism of PGE₂-regulated COX-2 expression remains poorly understood. The low-molecular-weight stress protein heme oxygenase-1 (HO-1) contributes to the anti-inflammatory, anti-oxidant and anti-apoptotic response against environmental stress.

Methods

We explored the involvement of HO-1 on PGE₂ regulation of LPS-induced COX-2 expression in RAW 264.7 macrophages.

Results

LPS-induced COX-2 expression in RAW 264.7 macrophages was enhanced by exogenous PGE₂ or cyclic AMP (cAMP) analogue and was suppressed by a COX inhibitor (indomethacin), a protein kinase A (PKA) inhibitor (KT5720), and A kinase anchoring protein (AKAP) disruptors (Ht31 and RIAD). This result suggests that the stimulatory effects of endogenous and exogenous PGE₂ on COX-2 expression are mediated by a cAMP-PKA-AKAP-dependent pathway. The induction of HO-1 was observed in LPS-stimulated RAW 264.7 macrophages. This induction was suppressed by exogenous PGE₂ and enhanced by blockage of the endogenous PGE₂ effect by the PKA inhibitor or AKAP disruptors. In addition, HO-1 induction by the HO activator copper protoporphyrin suppressed LPS-induced COX-2 expression, which was restored by the addition of exogenous PGE₂. The induction of HO-1 inhibited LPS-induced NF-κB p-65 nuclear expression and translocation.

Conclusions

AKAP plays an important role in PGE₂ regulation of COX-2 expression, and the suppression of HO-1 by PGE₂-cAMP-PKA-AKAP signaling helps potentiate the LPS-induced COX-2 expression through a positive feedback loop in RAW 264.7 macrophages.     

Increased dietary sodium induces COX2 expression by activating NFκB in renal medullary interstitial cells

High salt diet induces renal medullary cyclooxygenase 2 (COX2) expression. Selective blockade of renal medullary COX2 activity in rats causes salt-sensitive hypertension, suggesting a role for renal medullary COX2 in maintaining systemic sodium balance. The present study characterized the cellular location of COX2 induction in the kidney of mice following high salt diet and examined the role of NFκB in mediating this COX2 induction in response to increased dietary salt. High salt diet (8 % NaCl) for 3 days markedly increased renal medullary COX2 expression in C57Bl/6 J mice. Co-immunofluorescence using a COX2 antibody and antibodies against aquaporin-2, ClC-K, aquaporin-1, and CD31 showed that high salt diet-induced COX2 was selectively expressed in renal medullary interstitial cells. By using NFκB reporter transgenic mice, we observed a sevenfold increase of luciferase activity in the renal medulla of the NFκB-luciferase reporter mice following high salt diet, and a robust induction of enhanced green fluorescent protein (EGFP) expression mainly in renal medullary interstitial cells of the NFκB-EGFP reporter mice following high salt diet. Treating high salt diet-fed C57Bl/6 J mice with selective IκB kinase inhibitor IMD-0354 (8 mg/kg bw) substantially suppressed COX2 induction in renal medulla, and also significantly reduced urinary prostaglandin E₂ (PGE₂). These data therefore suggest that renal medullary interstitial cell NFκB plays an important role in mediating renal medullary COX2 expression and promoting renal PGE₂ synthesis in response to increased dietary sodium.     

Naringenin mitigates titanium dioxide (TiO2)-induced chronic arthritis in mice: role of oxidative stress,cytokines, and NFκB

Objective

To evaluate the effect and mechanisms of naringenin in TiO₂-induced chronic arthritis in mice, a model resembling prosthesis and implant inflammation.

Treatment

Flavonoids are antioxidant and anti-inflammatory molecules with important anti-inflammatory effect. Mice were daily treated with the flavonoid naringenin (16.7–150 mg/kg, orally) for 30 days starting 24 h after intra-articular knee injection of 3 mg of TiO₂.

Methods

TiO₂-induced arthritis resembles cases of aseptic inflammation induced by prosthesis and/or implants. Mice were stimulated with 3 mg of TiO₂ and after 24 h mice started to be treated with naringenin. The disease phenotype, treatment toxicity, histopathological damage, oxidative stress, cytokine expression and NFκB were evaluated after 30 days of treatment.

Results

Naringenin inhibited TiO₂-induced mechanical hyperalgesia (96%), edema (77%) and leukocyte recruitment (74%) without inducing toxicity. Naringenin inhibited histopathological index (HE, 49%), cartilage damage (Toluidine blue tibial staining 49%, and proteoglycan 98%), and bone resorption (TRAP-stained 73%). These effects were accompanied by inhibition of oxidative stress (gp91^phox 93%, NBT 83%, and TBARS 41%) cytokine mRNA expression (IL-33 82%, TNFα 76%, pro-IL-1β 100%, and IL-6 61%), and NFκB activation (100%).

Conclusion

Naringenin ameliorates TiO₂-induced chronic arthritis inducing analgesic and anti-inflammatory responses with improvement in the histopathological index, cartilage damage, and bone resorption.