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.     

Prostaglandin E2 upregulates survivin expression via the EP1 receptor in hepatocellular carcinoma cells

AimsCyclooxygenase-2 (COX-2)-controlled production of prostaglandin E₂ (PGE₂) has been implicated in cell growth and metastasis in many cancers. Recent studies have found that COX-2 is co-expressed with survivin in many cancers. Survivin is a member of the inhibitor-of-apoptosis protein family. Some COX-2 inhibitors (e.g., celecoxib) can reduce the expression of survivin. However, little is known about the mechanism of PGE₂-mediated expression of survivin. This study was designed to uncover the effect of PGE₂ on survivin expression in hepatocellular carcinoma cells.Main methodsThe effects of PGE₂ and EP1 agonist on survivin expression were examined in HUH-7 and HepG2 cells. Plasmid transfection and EP1 siRNA were used to regulate the expression of COX-2 and the EP1 receptor protein.Key findingsPGE₂ treatment increased survivin expression 2.3-fold. COX-2 overexpression resulted in a similar level of survivin upregulation. However, this effect was suppressed by treatment with celecoxib. EP1 receptor transfection or treatment with a selective EP1 agonist mimicked the effect of PGE₂ treatment. Conversely, the PGE₂-induced upregulation of survivin was blocked by treatment with a selective EP1 antagonist or siRNA against the EP1 receptor. The phosphorylation of EGFR and Akt were elevated in EP1 agonist-treated cells, and both EGFR and PI3K inhibitors suppressed the upregulation of survivin induced by PGE₂ or EP1 agonist.SignificancePGE₂ regulates survivin expression in hepatocellular carcinoma cells through the EP1 receptor by activating the EGFR/PI3K pathway. Targeting the PGE₂/EP1/survivin signaling pathway may aid the development of new therapeutic strategies for both the prevention and treatment of this cancer.     

Celecoxib increases retinoid sensitivity in human colon cancer cell lines

Retinoid resistance has limited the clinical application of retinoids as differentiation-inducing and apoptosis-inducing drugs. This study was designed to investigate whether celecoxib, a selective COX-2 inhibitor, has effects on retinoid sensitivity in human colon cancer cell lines, and to determine the possible mechanism of said effects. Cell viability was measured using the MTT assay. Apoptosis was detected via Annexin-V/PI staining and the flow cytometry assay. PGE₂ production was measured with the ELISA assay. The expression of RARβ was assayed via western blotting. The results showed that celecoxib enhanced the inhibitory effect of ATRA in both COX-2 high-expressing HT-29 and COX-2 low-expressing SW480 cell lines. Further study showed the ATRA and celecoxib combination induced greater apoptosis, but that the addition of PGE₂ did not affect the enhanced growth-inhibitory and apoptosis-inducing effects of the combination. Moreover, NS398 (another selective COX-2 inhibitor) did not affect the inhibitory effects of ATRA in the two cell lines. Western blotting showed that the expression of RARβ in HT-29 cell lines was increased by celecoxib, but not by NS398, and that the addition of PGE₂ did not affect the celecoxib-induced expression of the retinoic acid receptor beta. In conclusion, celecoxib increased the expression of RARβ and the level of cellular ATRA sensitivity through COX-2-independent mechanisms. This finding may provide a potential strategy for combination therapy.     

Effects of nimesulide, a preferential cyclooxygenase-2 inhibitor, on carrageenan-induced pleurisy and stress-induced gastric lesions in rats

Intrapleural injection of carrageenan in rats increased prostaglandin E₂ (PGE₂) production and induced newly synthesized cyclooxygenase-2 (COX-2) in pleural exudate cells without affecting COX-1 levels. Nimesulide, a preferential inhibitor of COX-2, reduced pleural PGE₂ production and was almost as active as indomethacin and 10 times more active than ibuprofen. Only COX-1, and no COX-2, was detected in gastric mucosal cells, and PGE₂ concentration of gastric mucosa was significantly decreased by indomethacin and ibuprofen. The decrease in gastric PGE₂ production induced by indomethacin and ibuprofen was enhanced in stressed rats, resulting in aggravation of stress-induced gastric lesions at anti-inflammatory doses. However, nimesulide did not produce stress-induced gastric lesions even at 30 times the anti-inflammatory dose. This supports the hypothesis that inhibition of COX-1 causes unwanted side effects and inhibition of COX-2 produces anti-inflammatory effects.     

Effect of Prostaglandins on Hepatic Cyclic Nucleotide Concentration,Carbohydrate and Lipid Metabolism

The effects of exogenous prostaglandin E₁ (PGE₁) or prostaglandin E₂ (PGE₂) were studied in the isolated perfused rat liver and in the intact canine liver in order to determine the possible physiological role of prostaglandins on hepatic carbohydrate and lipid metabolism. The data indicate that PGE₁ and PGE₂ did not stimulate cyclic AMP (cAMP) and cyclic GMP (cGMP) concentrations in intact dog liver and PGE₁ failed to stimulate cAMP or cGMP in fed or fasted perfused rat liver. PGE₁ did not promote hyperglycemia, glycogenolysis, lipolysis, or prevent epinephrine-induced hyperglycemia in the isolated perfused rat liver. Other known glycogenolytic agents including glucagon and epinephrine increased cAMP and glycogenolysis in the same perfusion system. This study does not support a physiologic role for PGE₁ on hepatic glycogenolysis or lipolysis. If PGE₁ subsequently is found to influence other metabolic parameters such as lipogenesis, gluconeogenesis, ureogenesis or amino acid transport in isolated perfused liver, such alterations would probably occur independent of changes in cyclic nucleotide activity.     

Diallyl disulfide inhibits proliferation and transdifferentiation of lung fibroblasts through induction of cyclooxygenase and synthesis of prostaglandin E2

Platelet-derived growth factor-BB (PDGF-BB) and transforming growth factor-β1 (TGF-β1) are critically involved in idiopathic pulmonary fibrosis by inducing the proliferation and transdifferentiation of lung fibroblasts. In the present study, we examined the impact of diallyl disulfide (DADS), a garlic-derived compound, on such pathological conditions. DADS showed profound inhibitory effects on the PDGF-BB-induced proliferation of human and mouse lung fibroblasts. DADS also abrogated the TGF-β1-induced expression of α-smooth muscle actin, type I collagen and fibronectin. Following treatment with DADS, the expression of cyclooxygenase-2 (COX-2) and the synthesis of prostaglandin E₂ (PGE₂) were found to be markedly enhanced, which in turn led to elevated cAMP levels in lung fibroblasts. Notably, the effect of DADS was largely abolished in the presence of either COX inhibitor indomethacin or siRNA-targeting COX-2, or in the absence of the PGE₂ receptor EP2, supporting an essential role for the COX-2–PGE₂–cAMP autocrine loop. Furthermore, we demonstrated that the upregulated expression of COX-2 was a result of increased level of histone 3 acetylation at COX-2 locus in DADS-treated cells. Together, these results suggest that DADS, by inducing COX-2 expression, may have therapeutic potential in treating lung fibrosis.     

Feedback amplification of fibrosis through matrix stiffening and COX-2 suppression

Tissue stiffening is a hallmark of fibrotic disorders but has traditionally been regarded as an outcome of fibrosis, not a contributing factor to pathogenesis. In this study, we show that fibrosis induced by bleomycin injury in the murine lung locally increases median tissue stiffness sixfold relative to normal lung parenchyma. Across this pathophysiological stiffness range, cultured lung fibroblasts transition from a surprisingly quiescent state to progressive increases in proliferation and matrix synthesis, accompanied by coordinated decreases in matrix proteolytic gene expression. Increasing matrix stiffness strongly suppresses fibroblast expression of COX-2 (cyclooxygenase-2) and synthesis of prostaglandin E₂ (PGE₂), an autocrine inhibitor of fibrogenesis. Exogenous PGE₂ or an agonist of the prostanoid EP2 receptor completely counteracts the proliferative and matrix synthetic effects caused by increased stiffness. Together, these results demonstrate a dominant role for normal tissue compliance, acting in part through autocrine PGE₂, in maintaining fibroblast quiescence and reveal a feedback relationship between matrix stiffening, COX-2 suppression, and fibroblast activation that promotes and amplifies progressive fibrosis.     

The inhibition of 2-arachidonoyl-glycerol (2-AG) biosynthesis,rather than enhancing striatal damage,protects striatal neurons from malonate-induced death: a potential role of cyclooxygenase-2-dependent metabolism of 2-AG

The cannabinoid CB₂ receptor, which is activated by the endocannabinoid 2-arachidonoyl-glycerol (2-AG), protects striatal neurons from apoptotic death caused by the local administration of malonate, a rat model of Huntington''s disease (HD). In the present study, we investigated whether endocannabinoids provide tonic neuroprotection in this HD model, by examining the effect of O-3841, an inhibitor of diacylglycerol lipases, the enzymes that catalyse 2-AG biosynthesis, and JZL184 or OMDM169, two inhibitors of 2-AG inactivation by monoacylglycerol lipase (MAGL). The inhibitors were injected in rats with the striatum lesioned with malonate, and several biochemical and morphological parameters were measured in this brain area. Similar experiments were also conducted in vitro in cultured M-213 cells, which have the phenotypic characteristics of striatal neurons. O-3841 produced a significant reduction in the striatal levels of 2-AG in animals lesioned with malonate. However, surprisingly, the inhibitor attenuated malonate-induced GABA and BDNF deficiencies and the reduction in Nissl staining, as well as the increase in GFAP immunostaining. In contrast, JZL184 exacerbated malonate-induced striatal damage. Cyclooxygenase-2 (COX-2) was induced in the striatum 24 h after the lesion simultaneously with other pro-inflammatory responses. The COX-2-derived 2-AG metabolite, prostaglandin E₂ glyceryl ester (PGE₂-G), exacerbated neurotoxicity, and this effect was antagonized by the blockade of PGE₂-G action with AGN220675. In M-213 cells exposed to malonate, in which COX-2 was also upregulated, JZL184 worsened neurotoxicity, and this effect was attenuated by the COX-2 inhibitor celecoxib or AGN220675. OMDM169 also worsened neurotoxicity and produced measurable levels of PGE₂-G. In conclusion, the inhibition of 2-AG biosynthesis is neuroprotective in rats lesioned with malonate, possibly through the counteraction of the formation of pro-neuroinflammatory PGE₂-G, formed from COX-2-mediated oxygenation of 2-AG. Accordingly, MAGL inhibition or the administration of PGE₂-G aggravates the malonate toxicity.     

Protective effects of 16,16-dimethyl PGE2 on the liver and kidney

The ability of subcutaneous 16,16-dimethyl PGE₂ to protect the liver and the kidney against damage induced by carbon tetrachloride and ANIT (alpha-napthylisothiocyanate) was examined. Rats were given 5–75 μg/kg of 16,16-dimethyl PGE₂ 24 and 0.5 hrs before challenge with 1 ml of oral carbon tetrachloride with an additional prostaglandin dose 6 hrs later. Twenty-four hrs after carbon tetrachloride animals were sacrificed by decapitation. 16,16-Dimethyl PGE₂ partially prevented fat accumulation and necrosis in the liver with complete or partial reduction in the SGPT caused by the hepatotoxin. Higher doses of carbon tetrachloride (1.5 ml) caused elevation in BUN and uric acid also; these changes were prevented by 16,16-dimethyl PGE₂ even when doses of the prostaglandin were too low to protect against liver necrosis. Elevated serum bilirubin observed 48 hrs after oral ANIT (30 mg/kg) was prevented by 100 μg/kg of 16,16-dimethyl PGE₂ given 24 and 0.5 hrs prior to the challenge with additional doses 6 and 24 hrs after ANIT. Higher doses of oral ANIT (200 mg/kg) when combined with small doses of carbon tetrachloride (0.25 ml per rat) resulted in elevated BUN and uric acid levels in the serum although neither compound produced these changes when given alone. 16,16-Dimethyl PGE₂ (75 μg/kg) administered by the same schedule as used for protection against ANIT resulted in normalization of these parameters in the absence of significant liver protection. Thus, it appears that 16,16-dimethyl PGE₂ can protect the liver against necrosis induced by moderate amounts of carbon tetrachloride and ANIT. At higher doses of these hepatotoxins, the liver is not protected by prostaglandins. Elevation of BUN and uric acid is observed under these conditions, however, and can be prevented by 16,16-dimethyl PGE₂.     

Cellular senescence involves an intracrine prostaglandin E2 pathway in human fibroblasts

Cyclooxygenase 2 and release of prostaglandin E₂ are involved in many responses including inflammation and are upregulated during cellular senescence. However, little is known about the role of lipid inflammatory mediators in senescence. Here, we investigated the mechanism by which the COX-2/PGE₂ axis induces senescence. Using the NS398 specific inhibitor of COX-2, we provide evidence that reactive oxygen species by-produced by the COX-2 enzymatic activity are negligible in front of the total senescence-associated oxidative stress. We therefore investigated the role of PGE₂ by invalidating the PGE₂ synthases downstream of COX-2, or the specific PGE₂ receptors, or by applying PGE₂ or specific agonists or antagonists. We evaluated the effect on senescence by evaluating the senescence-associated proliferation arrest, the percentage of senescence-associated β-galactosidase-positive cells, and the expression of senescent molecular markers such as IL-6 and MCP1. We show that PGE₂ acting on its EP specific receptors is able to induce both the onset of senescence and the maintenance of the phenotype. It did so only when the PGE₂/lactate transporter activity was enhanced, indicating that PGE₂ acts on senescence more via the pool of intracellular EP receptors than via those localized at the cell surface. Treatment with agonists, antagonists and silencing of the EP receptors by siRNA revealed that EP3 was the most involved in transducing the intracrine effects of PGE₂. Immunofluorescence experiments confirmed that EP3 was more localized in the cytoplasm than at the cell surface. Taken together, these results suggest that COX-2 contributes to the establishment and maintenance of senescence of normal human fibroblasts via an independent-ROS and a dependent-PGE₂/EPs intracrine pathway.     

Synergistic Effect of Interleukin-1β and Tumor Necrosis Factor α on PGE2Production by Articular Chondrocytes Does Not Involve PLA2Stimulation

This study investigates the ways in which two proinflammatory cytokines, tumor necrosis factor α (TNF) and interleukin-1β (IL1), cause increased production of prostaglandin E₂(PGE₂) in rabbit articular chondrocytes (RAC). Rabbit articular chondrocytes in primary culture were incubated with IL1, TNF, or both. Arachidonic acid (AA) release, PGE₂production, and the activities of cytosolic phospholipase A₂(cPLA₂), secreted phospholipase A₂(sPLA₂), and cyclooxygenase (COX) were measured. The mRNA levels of cPLA₂, sPLA₂, and COX-2 were also measured by Northern blotting, using specific complementary DNA probes. Incubation of IL1-stimulated RAC with TNF further increased PGE₂production. This synergy did not involve PLA₂stimulation, as there were no increases in AA release, cPLA₂and sPLA₂activities, or mRNA. In contrast, TNF increased the effect of IL1 on COX-2 activity and mRNA level. These results show that TNF and IL1 act in synergy in PGE₂production in articular chondrocytes. As sPLA₂and cPLA₂do not seem to be involved, COX-2 appears to be the best target for a specific anti-inflammatory strategy against cartilage degradation.     

Differential effect of chondroitin-4-sulfate on the immediate and delayed prostaglandin E2 release from osteoblasts

The present study examines the effect of chondroitin-4-sulfate (C4S) on the immediate (non-inflammatory conditions) and the delayed (inflammatory conditions) prostaglandin E₂ (PGE₂) release from rat calvarial osteoblasts. An immediate low release of PGE₂ was induced by PAF, phorbol ester and arachidonic acid but not by IL1β, TNF-α and LPS whereas a delayed high release of PGE₂ was induced by the inflammatory agents IL1β, TNF-α and LPS but not by PAF, phorbol ester and arachidonic acid. C4S had no effect on the immediate PGE₂ release but inhibited the delayed release of PGE₂. IL1β, TNF-α and LPS enhanced the expression of COX-2 and mPGES1 whereas phorbol ester enhanced COX-2 expression only. PAF and arachidonic acid had no effect on the expression of COX-2 and mPGES1. C4S inhibited the enhanced expression of COX-2 and mPGES1 but had no effect on the IL1β-induced decrease of I-κBα and nuclear translocation of NF-κB. These results indicate that the beneficial effects of C4S in bone inflammatory diseases might be due to a specific inhibition of the delayed high PGE₂ release from osteoblasts.     

Honokiol Inhibits Non-Small Cell Lung Cancer Cell Migration by Targeting PGE2-Mediated Activation of β-Catenin Signaling

Lung cancer remains a leading cause of death due to its metastasis to distant organs. We have examined the effect of honokiol, a bioactive constituent from the Magnolia plant, on human non-small cell lung cancer (NSCLC) cell migration and the molecular mechanisms underlying this effect. Using an in vitro cell migration assay, we found that treatment of A549, H1299, H460 and H226 NSCLC cells with honokiol resulted in inhibition of migration of these cells in a dose-dependent manner, which was associated with a reduction in the levels of cyclooxygenase-2 (COX-2) and prostaglandin E₂ (PGE₂). Celecoxib, a COX-2 inhibitor, also inhibited cell migration. Honokiol inhibited PGE₂-enhanced migration of NSCLC cells, inhibited the activation of NF-κB/p65, an upstream regulator of COX-2, in A549 and H1299 cells, and treatment of cells with caffeic acid phenethyl ester, an inhibitor of NF-κB, also inhibited migration of NSCLC cells. PGE₂ has been shown to activate β-catenin signaling, which contributes to cancer cell migration. Therefore, we checked the effect of honokiol on β-catenin signaling. It was observed that treatment of NSCLC cells with honokiol degraded cytosolic β-catenin, reduced nuclear accumulation of β-catenin and down-regulated matrix metalloproteinase (MMP)-2 and MMP-9, which are the down-stream targets of β-catenin and play a crucial role in cancer cell metastasis. Honokiol enhanced: (i) the levels of casein kinase-1α, glycogen synthase kinase-3β, and (ii) phosphorylation of β-catenin on critical residues Ser⁴⁵, Ser^33/37 and Thr⁴¹. These events play important roles in degradation or inactivation of β-catenin. Treatment of celecoxib also reduced nuclear accumulation of β-catenin in NSCLC cells. FH535, an inhibitor of Wnt/β-catenin pathway, inhibited PGE₂-enhanced cell migration of A549 and H1299 cells. These results indicate that honokiol inhibits non-small cell lung cancer cells migration by targeting PGE₂-mediated activation of β-catenin signaling.     

An accessory role for ceramide in interleukin-1β induced prostaglandin synthesis

Interleukin-1 (IL-1) is a potent inducer of prostaglandin E₂ (PGE₂) synthesis. We previously showed that ceramide accumulates in fibroblasts treated with IL-1 and that it enhances IL-1-induced PGE₂ production. The present study was undertaken to determine the mechanism(s) by which ceramide and IL-1 interact to enhance PGE₂ production by examining their respective effects on the rate-limiting enzymes in PGE₂ synthesis, cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), and cytosolic phospholipase A₂ (cPLA₂). IL-1-induced PGE₂ synthesis required 8 h even though COX-1 was constitutively expressed (both mRNA and protein) and enzymatically active in untreated cells. Conversely, COX-2 mRNA was barely detectable in untreated cells but within 2 h, ceramide or IL-1 alone induced a 5 and 20 fold increase in COX-2 mRNA, respectively. However, IL-1 induced COX-2 protein synthesis was only detectable 6-7 h after maximal COX-2 mRNA induction; COX-2 protein accumulation was not induced by ceramide alone. Ceramide however, reduced the length of time required for IL- 1 to induce COX-2 protein accumulation and increased COX-2 protein accumulation. IL-1 induced a 15 fold increase in COX-1 mRNA including an alternatively spliced form of COX-1. IL-1, but not ceramide induced cPLA₂ mRNA and protein expression which corresponded with the initiation of PGE₂ synthesis. These observations indicate that, (1) while either ceramide or IL-1 rapidly induced COX-2 mRNA, COX-2 protein only accumulated in IL- 1 treated cells after a delay of 6-7 h, (2) IL-1-induced PGE₂ synthesis required both COX-2 and cPLA₂ protein synthesis and, (3) ceramide enhanced (temporally and quantitatively) IL-1-induced COX-2 protein accumulation resulting in enhanced PGE₂ production.     

Celecoxib Ameliorates Portal Hypertension of the Cirrhotic Rats through the Dual Inhibitory Effects on the Intrahepatic Fibrosis and Angiogenesis

Background

Increased intra-hepatic resistance to portal blood flow is the primary factor leading to portal hypertension in cirrhosis. Up-regulated expression of cyclooxygenase-2 (COX-2) in the cirrhotic liver might be a potential target to ameliorate portal hypertension.

Objective

To verify the effect of celecoxib, a selective inhibitor of COX-2, on portal hypertension and the mechanisms behind it.

Methods

Cirrhotic liver model of rat was established by peritoneal injection of thiacetamide (TAA). 36 rats were randomly assigned to control, TAA and TAA+celecoxib groups. Portal pressures were measured by introduction of catheters into portal vein. Hepatic fibrosis was assessed by the visible hepatic fibrotic areas and mRNAs for collagen III and α-SMA. The neovasculature was determined by hepatic vascular areas, vascular casts and CD31 expression. Expressions of COX-2, vascular endothelial growth factor (VEGF), VEGF receptor-2 (VEGFR-2) and related signal molecules were quantitated.

Results

Compared with TAA group, the portal pressure in TAA+celecoxib group was significantly decreased by 17.8%, p<0.01. Celecoxib treatment greatly reduced the tortuous hepatic portal venules. The data of fibrotic areas, CD31expression, mRNA levels of α-SMA and collagen III in TAA+celecoxib group were much lower than those in TAA group, p<0.01. Furthermore, the up-regulation of hepatic mRNA and protein levels of VEGF, VEGFR-2 and COX-2 induced by TAA was significantly inhibited after celecoxib treatment. The expressions of prostaglandin E2 (PGE2), phosphorylated extracellular signal-regulated kinase (p-ERK), hypoxia-inducible factor-1α (HIF-1α), and c-fos were also down-regulated after celecoxib treatment.

Conclusions

Long term administration of celecoxib can efficiently ameliorate portal hypertension in TAA rat model by its dual inhibitory effects on the intrahepatic fibrosis and angiogenesis. The anti-angiogenesis effect afforded by celecoxib may attribute to its modulation on VEGF/VEGFR-2 through the down-regulation of integrated signal pathways involving PGE2- HIF-1α- VEGF and p-ERK- c-fos- VEGFR-2.     

Elucidation of the role of COX-2 in liver fibrogenesis using transgenic mice

Hepatic COX-2 overexpression is sufficient to induce hepatitis, but its role on liver fibrosis remains unknown. We aim to elucidate possible biological effects of COX-2 in liver fibrosis using both gain-of-function and loss-of-function mouse models. COX-2 transgenic (TG) mice that specifically overexpress the human COX-2 cDNA in the liver, knockout (KO), and wild type (WT) mice were studied in two different murine fibrosis models induced by carbon tetrachloride (CCl₄) injection or methionine and choline-deficient (MCD) diet. Liver injury was assessed by serum ALT and bilirubin levels and histological examination. Hepatic collagen content was determined by picrosirius red stain morphometry assay and quantitation of hydroxyproline. Hepatic stellate cell (HSC) activation was determined by immunohistochemical analysis of α-smooth muscle actin (α-SMA). mRNA expression of fibrogenic genes was assayed by real-time quantitative PCR. COX-2 protein was overexpressed in the liver of TG mice compared with WT littermates. CCl₄ or MCD-induced liver fibrotic injury was equally severe in TG and WT mice, as demonstrated by similar elevated levels of hepatic collagen contents. Enhanced COX-2 expression in TG liver did not affect HSC activation and fibrogenic gene expression upon CCl₄ or MCD treatment. Importantly, CCl₄-treated KO mice did not show significant difference in liver fibrotic damage and fibrogenic gene expression compared with the WT counterparts. This is the first report on the effect of COX-2 in liver fibrosis based on genetic mouse models. The results suggest that COX-2 does not appear to mediate the development of liver fibrosis.     

Prostaglandin E2 synthesis in cartilage explants under compression: mPGES-1 is a mechanosensitive gene

Knee osteoarthritis (OA) results, at least in part, from overloading and inflammation leading to cartilage degradation. Prostaglandin E2 (PGE₂) is one of the main catabolic factors involved in OA. Its synthesis is the result of cyclooxygenase (COX) and prostaglandin E synthase (PGES) activities whereas NAD+-dependent 15 hydroxy prostaglandin dehydrogenase (15-PGDH) is the key enzyme implicated in the catabolism of PGE₂. For both COX and PGES, three isoforms have been described: in cartilage, COX-1 and cytosolic PGES are constitutively expressed whereas COX-2 and microsomal PGES type 1 (mPGES-1) are inducible in an inflammatory context. COX-3 (a variant of COX-1) and mPGES-2 have been recently cloned but little is known about their expression and regulation in cartilage, as is also the case for 15-PGDH. We investigated the regulation of the genes encoding COX and PGES isoforms during mechanical stress applied to cartilage explants. Mouse cartilage explants were subjected to compression (0.5 Hz, 1 MPa) for 2 to 24 hours. After determination of the amount of PGE₂ released in the media (enzyme immunoassay), mRNA and proteins were extracted directly from the cartilage explants and analyzed by real-time RT-PCR and western blotting respectively. Mechanical compression of cartilage explants significantly increased PGE₂ production in a time-dependent manner. This was not due to the synthesis of IL-1, since pretreatment with interleukin 1 receptor antagonist (IL1-Ra) did not alter the PGE₂ synthesis. Interestingly, COX-2 and mPGES-1 mRNA expression significantly increased after 2 hours, in parallel with protein expression, whereas COX-3 and mPGES-2 mRNA expression was not modified. Moreover, we observed a delayed overexpression of 15-PGDH just before the decline of PGE₂ synthesis after 18 hours, suggesting that PGE₂ synthesis could be altered by the induction of 15-PGDH expression. We conclude that, along with COX-2, dynamic compression induces mPGES-1 mRNA and protein expression in cartilage explants. Thus, the mechanosensitive mPGES-1 enzyme represents a potential therapeutic target in osteoarthritis.     

Effects of non-steroidal anti-inflammatory drugs on prostaglandin E2 production by cyclooxygenase-2 from endogenous and exogenous arachidonic acid in rat peritoneal macrophages stimulated with lipopolysaccharide

Lipopolysaccharide (LPS) stimulated prostaglandin E₂ (PGE₂) formation and induction of cyclooxygenase-2 (COX-2) expression without changing the levels of COX-1 protein in rat peritoneal macrophages. Non-steroidal anti-inflammatory drugs (NSAIDs) (nimesulide, indomethacin and ibuprofen) strongly inhibited LPS-stimulated PGE₂ production without any effect on COX-2 protein expression, suggesting that NSAIDs are active in inhibiting the ability of COX-2 to convert arachidonic acid (AA) endogenously released in response to LPS stimulation. Exogenous AA can be converted to PGE₂ by both COX isoforms even in LPS-stimulated macrophages. NSAIDs inhibited PGE₂ production from exogenous AA mediated by both COX-1 and COX-2. However, the two isoforms interacted differentially with different NSAIDs. Furthermore, NSAIDs were distinctly more active in inhibiting PGE₂ production from endogenous AA than that from exogenous AA. These data suggest that PGE₂ production through COX-2 from exogenous AA may not be subject to the same regulatory processes as that from endogenous AA and the two metabolic processes may be differentially sensitive to different NSAIDs.     

β-oxidation modulates metabolic competition between eicosapentaenoic acid and arachidonic acid regulating prostaglandin E2 synthesis in rat hepatocytes–Kupffer cells

The ability of n-3 PUFA to competitively inhibit the use of arachidonic acid (AA) for membrane phospholipid synthesis and prostaglandin E₂ (PGE₂) production has been well demonstrated in single cell models. In the present study, we investigated the metabolic competition between AA and eicosapentaenoic acid (EPA) for PGE₂ synthesis in a rat hepatocyte–Kupffer cell (HPC/KC) co-culture system when the cellular oxidation capacity was enhanced by exogenous l-carnitine. We demonstrate that in the absence of l-carnitine, 1) β-oxidation rates of EPA and AA were comparable in HPCs and in KCs; 2) AA and not EPA was preferentially incorporated into glycerolipids; and 3) addition of EPA significantly decreased AA-dependent PGE₂ synthesis in HPCs and cyclooxygenase-2 (COX-2) expression in co-cultured HPCs/KCs. However, enhancing the cellular oxidation capacity by the addition of l-carnitine 1) significantly increased β-oxidation of EPA in HPCs, but only marginally elevated the oxidation of AA in HPCs and the oxidation of both fatty acids in KCs; 2) decreased the esterification, but did not alter the preferential incorporation of AA into glycerolipids; and 3) alleviated the significant competitive inhibition of AA-dependent PGE₂ synthesis and COX-2 expression by EPA. Taken together, the results strongly suggest that l-carnitine affects competition between AA and EPA in PG synthesis in liver cells by enhancing oxidation of EPA in HPCs. This implies that the beneficial effects of n-3 PUFA, especially EPA, are affected by the cellular oxidation capacity.