Rhein, the metabolite of diacerhein, reduces the proliferation of osteoarthritic chondrocytes and synoviocytes without inducing apoptosis.

OBJECTIVE: The aim of this study was to determine the effects of pharmacologically relevant concentrations of rhein (1,8-dihydroxy-3-carboxyanthraquinone) on the cell proliferation rate of human chondrocytes and synoviocytes. METHODS: Cultures of human osteoarthritic synoviocytes and chondrocytes were incubated with 10(-6), 10(-5), and 10(-4) M rhein. [3H]thymidine incorporation was used to determine rhein proliferative effects after incubation periods of 24 h, 48 h, and 1 week. The cytotoxicity of the drug was assayed with a nonradioactive assay kit. Nuclear extracts were used to detect variations in cell-cycle proteins (p21, p27, and cyclin D1) by Western blotting. The effect of rhein on apoptosis was investigated by measurement of caspase-3/7 activity and DNA fragmentation. RESULTS: Rhein was found to downregulate the proliferation rate of both chondrocytes and synoviocytes, two-fold for 10(-5) M rhein and five- to six-fold for 10(-4) M rhein. No cytotoxicity of the drug was observed. Rhein (10(-4) M) decreased caspase-3/7 activity and did not induce DNA fragmentation. Western blots showed that 10(-4) M rhein increased the expression of p21 and/or p27, but not that of cyclin D1. CONCLUSIONS: Rhein has previously been shown to reduce the interleukin (IL)-1beta deleterious effects on osteoarthritis (OA) cartilage through inhibition of the expression of degrading enzymes. Here, rhein was also found to inhibit proliferation of both synoviocytes and chondrocytes, suggesting that the drug may decrease the development of the inflammatory synovial tissue that accompanies joint pathologies. Both its anti-catabolic and anti-proliferative effects may explain its beneficial effect in the treatment of joint diseases.

Modulatory effect of rhein on IL-1alpha-induced responses in human chondrocytes: a comparative study between antibody microarrays and specific ELISAs.

The present work aimed to take advantage of the screening capacity of protein arrays to search for additional targets of rhein in interleukin (IL)-1-stimulated chondrocytes. Primary cultures of chondrocytes from osteoarthritic (OA) patients were stimulated for 24 and 48 h with 1 ng/ml of IL-1alpha, in the presence or absence of 10(-5) M of rhein. Culture supernatants were analyzed with arrays membranes consisting of 120 antibodies directed against cytokines, chemokines, and angiogenic or growth factors and were controlled for 8 proteins by specific immuno-enzymatic assays (ELISA). Protein arrays showed that several CC or CXC chemokines, the growth factor GM-CSF, the cytokines IL-6, IL-7 and IL-10 (but unexpectedly not IL-1beta or TNFalpha) and the adhesion molecule ICAM-1 were induced maximally by IL-1alpha. In IL-1-stimulated chondrocytes, rhein reduced slightly the production of MCP-1 and increased those of IL-1Ra, of the cytokine receptors sgp130, IL-6R, sTNFR I and R II, but also of some chemokines or ICAM-1. Specific ELISAs confirmed the effect of rhein on MCP-1, IL-1Ra, sgp130, IL-6R and sTNFR II but was discrepant for GROalpha and were always more sensitive than protein arrays to detect IL-1 effects such as IL-1Ra and TNFalpha release. The present data show that rhein modulated some IL-1-induced responses contributing possibly to its chondroprotective (IL-1Ra, MCP-1) or cytokine modifying (sTNFR II, sgp130) properties, but that protein arrays were poorly sensitive to check for IL-1- and/or rhein-induced changes.

Rhein, a diacerhein-derived metabolite, modulates the expression of matrix degrading enzymes and the cell proliferation of articular chondrocytes by inhibiting ERK and JNK-AP-1 dependent pathways.

OBJECTIVE: To determine the effects of rhein on the expression of matrix metalloproteinases (MMP-1, -3, 13) and ADAMTs 4, 5 (a disintegrin and metalloproteinase with thrombospondin type-I repeat)/aggrecanases-1, -2 in interleukin-1-stimulated bovine articular chondrocytes, and to investigate the signalling pathways involved in the effects of the drug on gene expression and cell proliferation. METHODS: Bovine chondrocytes were treated with 10(-4) M rhein for 18 h, followed by 10 ng/ml IL-1Beta for 30 min (cytoplasmic extracts) or 24 h (RNA extraction and EMSA). mRNA was assessed by RT-PCR for the expression of MMPs and aggrecanases, and the phosphorylation of MAP kinases was studied by Western blotting. NF-kappaB and AP-1 DNA binding were determined by gel retardation assay. The effects of inhibitors of these signalling pathways were compared to those of rhein. The proliferation of human chondrocytes and synoviocytes treated with the drug was also investigated. RESULTS: IL-1Beta-induced stimulation of the MMPs and aggrecanase-1 was markedly inhibited by rhein. The drug reduced IL-1Beta-induced NF-kappaB and AP-1 DNA binding, as well as the phosphorylation of ERK and JNK. Similar effects were produced by the specific inhibitors of these signalling pathways. In addition, rhein reduced the proliferation of both human chondrocytes and synoviocytes. CONCLUSION: Our data indicate that rhein may reduce the deleterious effects of IL-1Beta on osteoarthritic cartilage through its effects on the ERK- and JNK-dependent pathways. Both its anti-catabolic and anti-proliferative properties may explain its value in the treatment of joint diseases.

Inhibition of interleukin-1beta-induced activation of MEK/ERK pathway and DNA binding of NF-kappaB and AP-1: potential mechanism for Diacerein effects in osteoarthritis.

In the present report we have shown that bovine articular chondrocytes cultured in low oxygen tension, i.e. in conditions mimicking their hypoxic in vivo environment, respond to IL-1beta (10 ng/ml) by an increased DNA binding activity of NF-kappaB and AP-l transcription factors. Incubation of the cells with 10(-5) M Rhein, the active metabolite of Diacerhein, for 24 h was found to reduce this activity particularly in the case of AP-1. Mitogen activated kinases (ERK-1 and ERK-2) were activated by exposure of the chondrocytes to a 1 h treatment with IL-1beta. This effect was greater in hypoxia (3% O2) than in normoxia (21% O2). Rhein was capable of reducing the IL-1beta-stimulated ERK1/ERK2 pathway whatever the tension of oxygen present in the environment. The mRNA steady-state levels of collagen type II (COL2A1) and aggrecan core protein were found to be significantly increased by a 24-h treatment with 10(-5) M Rhein. This stimulating effect was also observed in the presence of IL-1beta, suggesting that the drug could prevent or reduce the IL-1beta-induced inhibition of extra cellular matrix synthesis. IL-1-induced collagenase (MMP1) expression was significantly decreased by Rhein under the same conditions. In conclusion, Rhein can effectively inhibit the IL-1-activated MAPK pathway and the binding of NF-kappaB and AP-1 transcription factors, two key factors involved in the expression of several pro-inflammatory genes by chondrocytes. In addition, the drug can reduce the procatabolic effect of the cytokine, by reducing the MMP1 synthesis, and enhance the synthesis of matrix components, such as type II collagen and aggrecan. These results may explain the anti-osteoarthritic properties of Rhein and its disease-modifying effects on OA cartilage, in spite of the absence of inhibition at prostaglandin level.

Comparison of the effects of fasting morning, fasting evening and fed bedtime administration of tenatoprazole on intragastric pH in healthy volunteers: a randomized three-way crossover study.

BACKGROUND: The effectiveness of proton pump inhibitors is influenced by meals and administration time. AIM: To compare the effects on intragastric acidity of times of dosing of tenatoprazole, a novel imidazopyridine-based proton pump inhibitor with a prolonged plasma half-life. METHODS: This randomized three-period crossover study included 12 Helicobacter pylori-negative healthy subjects, who received tenatoprazole 40 mg either fasting at 7.00 AM, fasting at 7.00 PM or fed at 9.30 PM for 7 days, with a 2-week washout between periods. Twenty-four hour intragastric pH was monitored on day 7 of each period. RESULTS: On day 7, median 24-h pH was 4.7, 5.1 and 4.7 after breakfast, dinner and bedtime dosing, respectively (P = 0.11), whereas night-time pH was 4.2, 5.0 and 4.4 (P = 0.13). The mean 24-h percentage of time over pH 4 was 62, 72 and 64 after breakfast, dinner and bedtime dosing, respectively (N.S.), and 54, 68 and 56 during night-time (P = 0.06). Nocturnal acid breakthrough incidence decreased from 100% at baseline to 83%, 55% and 75% after 7.00 AM, 7.00 PM and 9.30 PM dosing, respectively (P = 0.18), and its mean duration dropped from 6.2 to 2.8, 1.0 and 2.2 h, respectively (P < 0.05). CONCLUSION: Seven-day administration of tenatoprazole provides a prolonged duration of acid suppression, especially during the night-time, with little effect of food or time of dosing.

Articular chondrocytes cultured in hypoxia: their response to interleukin-1beta and rhein, the active metabolite of diacerhein.

In the present report, we show that bovine articular chondrocytes cultured in low oxygen tension, i.e. in conditions mimicking their hypoxic in vivo environment, respond to IL-1beta (10 ng/ml) by an increased DNA binding activity of NF-kappaB and AP-1 transcription factors. Incubation of the cells with 10(-5) M rhein for 24 h was found to reduce this activity, particularly in the case of AP-1. Mitogen activated kinases (ERK-1 and ERK-2) were activated by exposure of the chondrocytes to 1-h treatment with IL-1beta. This effect was greater in hypoxia (3% O(2)) than in normoxia (21% O(2)). Rhein was capable of reducing the IL-1beta-stimulated ERK1/ERK2 pathway whatever the tension of oxygen present in the environment. The mRNA steady-state levels of collagen type II (COL2A1) and aggrecan core protein were found to be significantly increased by a 24-h treatment with 10(-5) M rhein. This stimulating effect was also observed in the presence of IL-1beta, suggesting that the drug could prevent or reduce the IL-1beta-induced inhibition of extracellular matrix synthesis. IL-1-induced collagenase (MMP1) expression was significantly decreased by rhein in the same conditions. In conclusion, rhein can effectively inhibit the IL-1-activated MAPK pathway and the binding of NF-kappaB and AP-1 transcription factors, two key factors involved in the expression of several pro-inflammatory genes by chondrocytes. In addition, the drug can reduce the procatabolic effect of the cytokine, by reducing the MMP1 synthesis, and enhance the synthesis of matrix components, such as type II collagen and aggrecan. These results may explain the anti-osteoarthritic properties of rhein and its disease-modifying effects on OA cartilage, in spite of absence of inhibition at prostaglandin level.

[Effect of upright tilt on venous hemodynamics in rat after three-week tail suspension]. / Effet d'une mise en orthostatisme sur l'hemodynamique veineuse chez le rat apres trois semaines de suspension.

The aim of this work was to know if the venous tone measured in vivo in rat was decreased after 3-week tail suspension, a ground-based model to simulate the effects of microgravity. Arterial and venous pressure measurements during upright tilt did not show any cardiovascular deconditioning. A longer period of tail suspension appears to be necessary to induce changes in venous tone.

Roles of NO-synthase and cyclooxygenase in sex- and pregnancy-dependent arterial and venous pressures in the rat.

The roles of NO synthase (NOS) and cyclooxygenase on vascular pressures were studied as a function of sex and pregnancy. After anesthesia, mean arterial pressure (MAP) and mean circulatory filling pressure were lower in pregnant rats compared with male and virgin rats, but N(G)-nitro-L-arginine methyl ester (L-NAME; 30 mg/kg) induced similar increases in MAP. Pithing abolished these pressure differences, suggesting a diminished autonomic reflex in pregnancy, and led in pregnant rats to a lower arterial and venous NO modulation. In separately perfused mesenteries, the lower responses to KCI observed in venous beds of female compared with male rats do not involve any dysfunction of NOS activity in the mesenteries isolated from virgin and pregnant rats. The cyclooxygenase pathway is implicated in the KCl-induced responses of vessels taken from male rats and of venous mesentery from pregnant rats. But prostanoids do not share in the acetylcholine (ACh)-induced relaxations in the arterial and venous K+-contracted mesenteric vasculatures isolated from any of the groups of rats.

Comparison of the nitric oxide and cyclo-oxygenase pathway in mesenteric resistance vessels of normotensive and spontaneously hypertensive rats.

1. The double perfused mesentery was used to compare arterial and venous KCl- and acetylcholine (ACh)-induced responses in tissues taken from normotensive (WKY) and spontaneously hypertensive rats (SHR) in the presence or absence of inhibitors of nitric oxide (NO) synthase (NG-nitro-L-arginine (L-NOARG) and NG-nitro-L-arginine methyl ester (L-NAME)) and cyclo-oxygenase (indomethacin, mefenamic acid). 2. KCl (20 to 120 mM K+) caused concentration-dependent increases in arterial and venous perfusion pressures. The maximal arterial effects were significantly higher in the SHRs than in the WKY, with no differences in the venous pressor responses. 3. L-NAME and L-NOARG (100 microM) had no effect on the basal perfusion pressures in tissues from either WKY or SHRs, and mefenamic acid only induced a significant reduction of the basal perfusion pressures in the venous mesenteric vessels isolated from WKY. 4. L-NAME and L-NOARG (100 microM) potentiated the pressor responses to KCl to the same extent in the venous and arterial beds derived from WKY and SHR, while indomethacin and mefenamic acid (5 microM) only significantly decreased these responses in WKY. 5. Acetylcholine (ACh)-induced relaxations (1 nM to 10 microM) were significantly higher in arterial beds of WKY than in SHR, without differences in the venous relaxant responses. 6. L-NAME (100 microM) inhibited ACh-induced relaxations in arterial and venous beds from both groups of rats. Mefenamic acid was without effect on ACh-induced relaxations in either the arterial or the venous beds from WKY and SHR. 7. In conclusion, the liberation of NO in the perfused mesenteric vasculatures requires an active tone and no dysfunction of NO synthase activity is functionally apparent in the mesenteries isolated from SHRs. The cyclo-oxygenase pathway is only implicated in the KCl-induced responses of tissues derived from WKY, but not in the vasodilatations induced by ACh in either the arterial or the venous vasculatures from WKY and SHR.