Atorvastatin reduces lipopolysaccharide-induced expression of cyclooxygenase-2 in human pulmonary epithelial cells

Objective

To explore the effects of atorvastatin on expression of cyclooxygenase-2 (COX-2) in human pulmonary epithelial cells (A549).

Methods

A549 cells were incubated in DMEM medium containing lipopolysaccharide (LPS) in the presence or absence of atorvastatin. After incubation, the medium was collected and the amount of prostaglandin E₂ (PGE₂) was measured by enzyme-linked immunosorbent assay (ELISA). The cells were harvested, and COX-2 mRNA and protein were analyzed by RT-PCR and western-blot respectively.

Results

LPS increased the expression of COX-2 mRNA and production of PGE₂ in a dose- and time-dependent manner in A549. Induction of COX-2 mRNA and protein by LPS were inhibited by atorvastatin in a dose-dependent manner. Atorvastatin also significantly decreased LPS-induced production of PGE₂. There was a positive correlation between reduced of COX-2 mRNA and decreased of PGE₂ (r = 0.947, P < 0.05).

Conclusion

Atorvastatin down-regulates LPS-induced expression of the COX-2 and consequently inhibits production of PGE₂ in cultured A549 cells.     

High-performance liquid chromatography of p-nitrophenacyl esters of selected prostaglandins on silver ion-loaded microparticulate cation-exchange resin

A silver ion-loaded microparticulate cation-exchange resin column has been used for high-performance liquid chromatographic (HPLC) separation of the p-nitrophenacyl esters of several series of closely related prostaglandins: 8-iso-PGE₂, 11-epi-PGE₂, 5-trans-PGE₂, PGE₂, PGF_1α, PGE₁, and PGF_2α, PGA₂ and PGB₂; 15 (R)-methyl-PGE₂ and 15 (S)-methyl-PGE₂; 5-trans-PGA₂ and PGA₂; and 5-trans-PGF_2α and PGF_2α. The properties of this column are compared with those of silica-gel and reversed-phase columns.     

Antinociceptive Effect of Tetrandrine on LPS-Induced Hyperalgesia via the Inhibition of IKKβ Phosphorylation and the COX-2/PGE2 Pathway in Mice

Tetrandrine (TET) is a bisbenzylisoquinoline alkaloid that is isolated from the Stephania Tetrandra. It is known to possess anti-inflammatory and immunomodulatory effects. We have shown that TET can effectively suppress the production of bacterial lipopolysaccharide (LPS)-induced inflammatory mediators, including cyclooxygenases (COXs), in macrophages. However, whether TET has an antinociceptive effect on LPS-induced hyperalgesia is unknown. In the present study, we investigated the potential antinociceptive effects of TET and the mechanisms by which it elicits its effects on LPS-induced hyperalgesia. LPS effectively evoked hyperalgesia and induced the production of PGE₂ in the sera, brain tissues, and cultured astroglia. TET pretreatment attenuated all of these effects. LPS also activated inhibitor of κB (IκB) kinase β (IKKβ) and its downstream components in the IκB/nuclear factor (NF)-κB signaling pathway, including COX-2; the increase in expression levels of these components was significantly abolished by TET. Furthermore, in primary astroglia, knockdown of IKKβ, but not IKKα, reversed the effects of TET on the LPS-induced increase in IκB phosphorylation, P65 phosphorylation, and COX-2. Our results suggest that TET can effectively exert antinociceptive effects on LPS-induced hyperalgesia in mice by inhibiting IKKβ phosphorylation, which leads to the reduction in the production of important pain mediators, such as PGE₂ and COX-2, via the IKKβ/IκB/NF-κB pathway.     

Synthesis,biological evaluation,and docking analysis of a novel family of 1-methyl-1H-pyrrole-2,5-diones as highly potent and selective cyclooxygenase-2 (COX-2) inhibitors

As a continuous research for discovery of new COX-2 inhibitors, we present the simple chemical synthesis, in vitro biological screening, and molecular docking study of 1H-pyrrole-2,5-dione derivatives. New synthetic compounds were evaluated for the inhibitory activities on LPS-induced PGE₂ production in RAW 264.7 macrophage cells as well as the COX-1 and COX-2 inhibitory potency. Among them, compound 9d (MPO-0029) was identified as more potent and selective COX-2 inhibitor [PGE₂ IC₅₀ = 8.7 nM, COX-2 IC₅₀ = 6.0 nM; COX-2 selectivity index (SI) = >168] than celecoxib. Molecular docking experiments were further performed against COX-2 and COX-1 isozymes to determine their probable binding models. Results of molecular docking studies revealed that compound 9d (MPO-0029) has stronger binding interaction with COX-2 than with COX-1 isozyme, and provided successfully complementary theoretical support for the obtained experimental biological data.     

Inhibitory effects of triarylpyrazole derivatives on LPS-induced nitric oxide and PGE2 productions in murine RAW 264.7 macrophages

In this article, a series of 22 triarylpyrazole derivatives were evaluated for in vitro antiinflammatory activity as inhibitors of nitric oxide (NO) and prostaglandin E₂ (PGE₂) release induced by lipopolysaccharide (LPS) in murine RAW 264.7 macrophages. The synthesized compounds 1a-h, 2a-f and 3a-h were first examined for their cytotoxicity for determination of the non-toxic concentration for antiinflammatory screening, so that the inhibitory effects against NO and PGE₂ production were not caused by non-specific cytotoxicity. Compounds 1h and 2f were the most active PGE₂ inhibitors with IC₅₀ values of 2.94 μM and 4.21 μM, respectively. Western blotting and cell-free COX-2 screening revealed that their effects were due to inhibition of COX-2 protein expression. Moreover, compound 1h exerted strong inhibitory effect on the expression of COX-2 mRNA in LPS-induced murine RAW 264.7 macrophages.     

Prostaglandin E2 production in astrocytes: regulation by cytokines, extracellular ATP, and oxidative agents

Upregulation and activation of phospholipases A₂ (PLA₂) and cyclooxygenases (COX) leading to prostaglandin E₂(PGE₂) production have been implicated in a number of neurodegenerative diseases. In this study, we investigated PGE₂ production in primary rat astrocytes in response to agents that activate PLA₂ including pro-inflammatory cytokines (IL-1β, TNFα and IFNγ), the P2 nucleotide receptor agonist ATP, and oxidants (H₂O₂ and menadione). Exposure of astrocytes to cytokines resulted in a time-dependent increase in PGE₂ production that was marked by increased expression of secretory sPLA₂ and COX-2, but not COX-1 and cytosolic cPLA₂. Although astrocytes responded to ATP or phorbol ester (PMA) with increased cPLA₂ phosphorylation and arachidonic acid release, ATP or PMA only caused a small increase in levels of PGE₂. However, when astrocytes were first treated with cytokines, further exposure to ATP or PMA, but not H₂O₂ or menadione, markedly increased PGE₂ production. These results suggest that ATP release during neuronal excitation or injury can enhance the inflammatory effects of cytokines on PGE₂ production and may contribute to chronic inflammation seen in Alzheimer's disease.     

Lactobacillus fermentum ZYL0401 Attenuates Lipopolysaccharide-Induced Hepatic TNF-α Expression and Liver Injury via an IL-10- and PGE2-EP4-Dependent Mechanism

Lipopolysaccharide (LPS) has essential role in the pathogenesis of D-galactosamine-sensitized animal models and alcoholic liver diseases of humans, by stimulating release of pro-inflammatory mediators that cause hepatic damage and intestinal barrier impairment. Oral pretreatment of probiotics has been shown to attenuate LPS-induced hepatic injury, but it is unclear whether the effect is direct or due to improvement in the intestinal barrier. The present study tested the hypothesis that pretreatment with probiotics enables the liver to withstand directly LPS-induced hepatic injury and inflammation. In a mouse model of LPS-induced hepatic injury, the levels of hepatic tumor necrosis factor-alpha (TNF-α) and serum alanine aminotransferase (ALT) of mice with depleted intestinal commensal bacteria were not significantly different from that of the control models. Pre-feeding mice for 10 days with Lactobacillus fermentum ZYL0401 (LF41), significantly alleviated LPS-induced hepatic TNF-α expression and liver damage. After LF41 pretreatment, mice had dramatically more L.fermentum-specific DNA in the ileum, significantly higher levels of ileal cyclooxygenase (COX)-2 and interleukin 10 (IL-10) and hepatic prostaglandin E2 (PGE₂). However, hepatic COX-1, COX-2, and IL-10 protein levels were not changed after the pretreatment. There were also higher hepatic IL-10 protein levels after LPS challenge in LF41-pretreaed mice than in the control mice. Attenuation of hepatic TNF-α was mediated via the PGE₂/E prostanoid 4 (EP4) pathway, and serum ALT levels were attenuated in an IL-10-dependent manner. A COX-2 blockade abolished the increase in hepatic PGE₂ and IL-10 associated with LF41. In LF41-pretreated mice, a blockade of IL-10 caused COX-2-dependent promotion of hepatic PGE₂, without affecting hepatic COX-2levels. In LF41-pretreated mice, COX2 prevented enhancing TNF-α expression in both hepatic mononuclear cells and the ileum, and averted TNF-α-mediated increase in intestinal permeability. Together, we demonstrated that LF41 pre-feeding enabled the liver to alleviate LPS-induced hepatic TNF-α expression and injury via a PGE₂-EP4- and IL-10-dependent mechanism.     

Regulation of prostaglandin E2 production by the superoxide radical and nitric oxide in mouse peritoneal macrophages

The purpose of this study was to elucidate the role of NO and O^-₂ on enzymatic components of cyclooxygenase (COX) pathway in peritoneal macrophages. Activation of murine peritoneal macrophages by lipopolysaccharides (LPS) resulted in time-dependent production of nitric oxide (NO) and prostaglandin E₂ (PGE₂). This stimulation was also accompanied by the production of other reactive oxygen species such as superoxide (O^-₂), and by increased expression of COX-2. Our results provide evidence that O^-₂ may be involved in the pathways that result in arachidonate release and PGE₂ formation by COX-2 in murine peritoneal macrophages stimulated by LPS. However, we were not able to demonstrate that NO participates in the regulation of PG production under our experimental conditions.     

Aspirin inhibits lipopolysaccharide-induced COX-2 expression and PGE2 production in porcine alveolar macrophages by modulating protein kinase C and protein tyrosine phosphatase activity

Aspirin has been demonstrated to be effective in inhibiting COX-2 and PGE₂ in Alveolar macrophages (AMs). However, the mechanisms have not been fully understood. In the present study, we found that pretreatment with aspirin inhibited LPS-induced COX-2 and PGE₂ upregulation, IκBα degradation, NFκB activation and the increase of PKC activity, but elevated LPS-induced the decrease of PTP activity. The PKC inhibitor calphostin C dramatically reduced the COX-2 mRNA and PGE₂ levels, but the PTP inhibitor peroxovanadium (POV) significantly increased the COX-2 mRNA and PGE₂ levels. Furthermore, the PTP inhibitor mitigated the inhibitory effect of aspirin on COX-2 and PGE₂ upregulation and NF-κB activation, whereas the PKC inhibitor enhanced the inhibitory effects of aspirin on the production of COX-2 and PGE₂. Our data indicate a novel mechanism by which aspirin acts as a potent anti-inflammatory agent in alveolus macrophages and ALI. [BMB Reports 2014; 47(1): 45-50]     

Differences in PGE2 Production between Primary Human Monocytes and Differentiated Macrophages: Role of IL-1β and TRIF/IRF3

Prostaglandin E2 (PGE₂) is induced in vivo by bacterial products including TLR agonists. To determine whether PGE₂ is induced directly or via IL-1β, human monocytes and macrophages were cultured with LPS or with Pam3CSK4 in presence of caspase-1 inhibitor, ZVAD, or IL-1R antagonist, Kineret. TLR agonists induced PGE₂ in macrophages exclusively via IL-1β-independent mechanisms. In contrast, ZVAD and Kineret reduced PGE₂ production in LPS-treated (but not in Pam3CSK4-treated) monocytes, by 30–60%. Recombinant human IL-1β augmented COX-2 and mPGES-1 mRNA and PGE₂ production in LPS-pretreated monocytes but not in un-primed or Pam3CSK4-primed monocytes. This difference was explained by the finding that LPS but not Pam3CSK4 induced phosphorylation of IRF3 in monocytes suggesting activation of the TRIF signaling pathway. Knocking down TRIF, TRAM, or IRF3 genes by siRNA inhibited IL-1β-induced COX-2 and mPGES-1 mRNA. Blocking of TLR4 endocytosis during LPS priming prevented the increase in PGE₂ production by exogenous IL-1β. Our data showed that TLR2 agonists induce PGE₂ in monocytes independently from IL-1β. In the case of TLR4, IL-1β augments PGE₂ production in LPS-primed monocytes (but not in macrophages) through a mechanism that requires TLR4 internalization and activation of the TRIF/IRF3 pathway. These findings suggest a key role for blood monocytes in the rapid onset of fever in animals and humans exposed to bacterial products and some novel adjuvants.     

7beta-hydroxy-epiandrosterone modulation of 15-deoxy-delta12,14-prostaglandin J2, prostaglandin D2 and prostaglandin E2 production from human mononuclear cells

7β-hydroxy-epiandrosterone (7β-OH-EPIA) has been shown to be cytoprotective in various organs including the brain. It has also been shown that prostaglandin D₂ (PGD₂) and its spontaneous metabolite 15-deoxy-Δ^12,14-prostaglandin J₂ (15d-PGJ₂) are also cytoprotective. It is possible that these prostaglandins derived from circulating mononuclear cells may mediate the actions of 7β-OH-EPIA. The aim of this study, therefore, was to ascertain the effect of 7β-OH-EPIA (in the absence or presence of tumour necrosis factor-α (TNF-α)), a pro-inflammatory stimulus, on the biosynthesis of PGD₂, PGE₂ and 15d-PGJ₂ from human mononuclear cells. Prostaglandins were measured by enzyme immunoassay (EIA). 7β-OH-EPIA alone induced a concentration-dependant increase in the production of PGD₂. TNF-α increased PGD₂ levels which were enhanced by 7β-OH-EPIA. 7β-OH-EPIA increased 15d-PGJ₂ levels both in the absence and presence of TNF-α. 7β-OH-EPIA alone had no effect on PGE₂ biosynthesis but suppressed TNF-α-induced PGE₂ circa 50%. 7β-OH-EPIA also increased the level of free arachidonic acid and radiolabelled prostaglandins in cells pre-incubated with radiolabelled arachidonic acid, indicating that the increase may occur via the enhanced release of substrate arachidonic acid. 7β-OH-EPIA did not affect levels of the anti-inflammatory cytokine IL-10 indicating that this is an unlikely mechanism by which 7β-OH-EPIA induces its actions but more likely exerts its effects via the production of cytoprotective prostaglandins.     

Estradiol -17-β enhances the formation of {H}-PGF2α from {H}-PGE2 in the uterus isolated from ovariectomized rats

Formation of {³H}-PGF_2α and {³H}-13,14,dihydro-15-keto-PGF_2α from {³H}-PGE₂ by the supernatant of uterine homogenates from estrous and ovariectomized rats, was studied, using the reaction system PGE₂ + NADPH + {³H}-PGE₂ + supernatant. Enzymatic conversion was lower in uterine supernatants from spayed rats than in uterine homogenates of rats at natural estrus.Spayed animals were injected with progesterone (P) or with estradiol-17-β (E₀) at a dose of 1.0 or 50.0 ug. Conversion of {³H}-PGF_2α to {³H}-PGE₂ or to {³H}-13,14,dihydro-15-keto-PGF_2α did not differ in control ovariectomized or ovariectomized rats receiving P or 1.0 ug E₀. However, 50.0 ug E₀ induced a significant oversion after 30 (P < 0.01) and 60 (P < 0.001) min of incubation.It is concluded that E₀, at the 50.0 ug dose, but not the 1.0 ug dose of E₀, nor progesterone, stimulated conversion of {³H}-PGE₂ into {³H}-PGF_2α or {³H}-13, 14,dihydro-15-keto-PGF_2α, presumably through the activity of the enzyme PGE₂-9-keto-reductase.     

Properties of a novel β-glucosidase from Fusarium proliferatum ECU2042 that converts ginsenoside Rg3 into Rh2

A novel β-glucosidase from Fusarium proliferatum ECU2042 (FPG) was successfully purified to homogeneity with a 506-fold increase in specific activity. The molecular mass of the native purified enzyme (FPG) was estimated to be approximately 78.7 kDa, with two homogeneous subunits of 39.1 kDa, and the pI of this enzyme was 4.4, as measured by two-dimensional electrophoresis. The optimal activities of FPG occurred at pH 5.0 and 50 °C, respectively. The enzyme was stable at pH 4.0–6.5 and temperatures below 60 °C, and the deactivation energy (E_d) for FPG was 88.6 kJ mo1⁻¹. Moreover, it was interesting to find that although the purified enzyme exhibited a very low activity towards p-nitrophenyl β-d-glucoside (pNPG), and almost no activity towards cellobiose, a relatively high activity was observed on ginsenoside Rg₃. The enzyme hydrolyzed the 3-C, β-(1 → 2)-glucoside of ginsenoside Rg₃ to produce ginsenoside Rh₂, but did not sequentially hydrolyze the β-d-glucosidic bond of Rh₂. The K_m and V_max values of FPG for ginsenoside Rg₃ were 2.37 mM and 0.568 μmol (h mg protein)⁻¹, respectively. In addition, this enzyme also exhibited significant activities towards various alkyl glucosides, aryl glucosides and several natural glycosides.     

Effects of pro-inflammatory cytokines,lipopolysaccharide and COX-2 mediators on human colonic neuromuscular function and epithelial permeability

Chronic colitis is associated with decreased colonic muscle contraction and loss of mucosal barrier function. Pro-inflammatory cytokines and bacterial lipopolysaccharide (LPS) are important in the generation and maintenance of inflammation. While colitis is associated with upregulated COX-2 -derived prostanoids and nitric oxide (NO), the direct activity of pro-inflammatory cytokines on human colonic neuromuscular function is less clear. This study investigated the effects of IBD-associated pro-inflammatory cytokines IL-17, TNF-α, IL-1β and LPS on human colonic muscle strip contractility, alone and following inhibition of COX-2 or nitric oxide production. In addition, human colonic epithelial Caco-2 cell monolayers were treated with LPS or COX-2 mediators including prostaglandins (PGE₂, PGF_2α) or their corresponding ethanolamides (PGE₂-EA or PGF_2α-EA) over 48 h and trans-epithelial electrical resistance used to record permeability changes. Longitudinal muscle strips were obtained from healthy colonic resection margins and mounted in organ baths following IL-17, TNF-α, IL-1β and bacterial LPS incubations in an explant setting over 20 h. Contraction in response to acetylcholine (ACh) was then measured, before and after either COX-2 inhibition (nimesulide; 10⁻⁵ M) or nitric oxide synthase (NOS) inhibition (l-NNA; 10⁻⁴ M). None of the cytokine or LPS explant incubations affected the potency or maximum cholinergic contraction in vitro, and subsequent COX-2 blockade with nimesulide revealed a significant but similar decrease in potency of ACh-evoked contraction in control, LPS and cytokine-incubated muscle strips. Pre-treatment with l-NNA provided no functional differences in the potency or maximum contractile responses to ACh in cytokine or LPS-incubated colonic longitudinal smooth muscle. Only PGE₂ transiently increased Caco-2 monolayer permeability at 24 h, while LPS (10 μg/ml) increased permeability over 24–48 h.These findings indicate that cholinergic contractility in the human colon can be decreased by the blockade of COX-2 generated excitatory prostanoids, but major pro-inflammatory cytokines or LPS do not alter the sensitivity or amplitude of this contraction ex vivo. While PGE₂ transiently increase epithelial permeability, LPS generates a significant and sustained increase in permeability indicative of an important role on barrier function at the mucosal interface.     

Determination of 9α,11β-prostaglandin F2 in human urine and plasma by gas chromatography—mass spectrometry

Sensitive and specific assay methods for 9α,11β-prostaglandin F₂ (9α,11β-PGF₂) by gas chromatography—mass spectrometry with electron impact ionization are described. The mass spectrometric assay for 9α,11β-PGF₂ was based on the use of the methyl ester—dimethylisopropylsilyl ether derivative, and pentadeuterated PGF_2α as a convenient internal standard. The calibration graph for 9α,11β-PGF₂ was linear from 5 pg to 100 ng for both the standard and spiked biological samples. The limit of detection was 50 pg/ml for urine and 25 pg/ml for plasma (signal-to-noise RATIO = 2.3). The method was applied to the determination of 9α,11β-PGF₂ in urine and plasma samples from patients with bronchial asthma.     

Identification and quantitative determination of prostaglandins by high pressure liquid chromatography

High pressure liquid chromatography (HPLC) using 4′ × 1/8″ columns of a pellicular silica support (Corasil-II) allows identification of prostaglandins diastereomers based on their characteristic retention relative to a standard, PGE₂ in this study. Surprisingly this simple method allows separation of PGE₂, PGE₁, and PGE_o (dihydro-PGE₁) or PGF₂α, PGF₁α, and PGF_oα without resort to silver nitrate impregnated stationary phases. Even more subtle distinctions such as that between 13,14-dihydro-PGF₂α, PGF₁α and 5,6- -PGF₂α are possible by HPLC. The differential refractometer detector used throughout can also be used for quantitation. This is illustrated by a study of the relative rates of degradation of natural PGF₂α (an oil at the temperature employed, 41°C) and racemic PGF₂α (mp. 63°) on exposure to air.     

Catabolism of prostaglandin F2α in rat ovary : Differences between ovarian and uterine tissues

The catabolism of prostaglandin F_2α(PGF_2α) in rat ovarian homogenate was studied comparing with uterine homogenate. Two kinds of metabolite were recognized by incubation of PGF_2α with ovarian or uterine homogenate; 13,14-dihydro-15-keto-PGF_2α(15KD-PGF_2α) and 13,14-dihydro-PGF_2α(13,14H₂-PGF_2α) in ovarian homogenate and 15-keto-PGF_2α(15K-PGF_2α) and 15KD-PGF_2α in uterine homogenate. Incubation of 15KD-PGF_2α with ovarian homogenate resulted in the formation of 13,14H₂-PGF_2α but incubation with uterine homogenate did not produce 13,14H₂-PGF_2α. 13,14H₂-PGF_2α was in accord with Rf value of a compound formed by reduction of 15KD-PGF_2α with sodium borohydride.