Effects of aldosterone and related steroids on LPS-induced increased expression of inducible NOS in rat aortic smooth muscle cells

BACKGROUND AND PURPOSE

Expression of inducible NOS (iNOS) is important in certain inflammatory diseases. We determined if the hormone aldosterone, a mineralocorticoid receptor (MR) agonist, affects LPS activation of iNOS expression in rat aortic smooth muscle cells (RASMC).

EXPERIMENTAL APPROACH

Cultured RASMC were treated with LPS, with or without agonists/antagonists of steroid receptors. iNOS expression was determined by nitrite assays on culture medium removed from treated cells and by immunoblotting of cell protein extracts.

KEY RESULTS

LPS (1 µg·mL⁻¹) increased nitrite and iNOS protein above that in control (untreated) cells. These effects of LPS were reduced by aldosterone (0.1–10 µM). The MR antagonists, eplerenone (10 µM) and spironolactone (10 or 50 µM), did not inhibit these actions of 1 µM aldosterone, but the latter were prevented by 10 µM mifepristone, a glucocorticoid (GR) and progestogen receptor (PR) antagonist. Mifepristone also prevented the reduction of LPS-induced nitrite increase produced by 1 µM dexamethasone (GR agonist) and 10 µM progesterone (PR agonist). Spironolactone (10–50 µM) by itself decreased LPS-induced increases in nitrite and iNOS protein. Mifepristone (10 µM) partially reversed these effects of 10 µM spironolactone, but not those of 50 µM; the effects of 50 µM spironolactone were also unchanged when mifepristone was increased to 50 µM.

CONCLUSIONS AND IMPLICATIONS

This pharmacological profile suggests that aldosterone, and possibly 10 µM spironolactone, use mechanisms that are dependent on PR and/or GR, but not MR, to inhibit iNOS induction in RASMC. With 50 µM spironolactone, other inhibitory mechanisms requiring further investigation may become predominant.     

Quercetin potentiates insulin secretion and protects INS-1 pancreatic ��-cells against oxidative damage via the ERK1/2 pathway

BACKGROUND AND PURPOSE

Quercetin lowers plasma glucose, normalizes glucose tolerance tests and preserves pancreatic β-cell integrity in diabetic rats. However, its mechanism of action has never been explored in insulin-secreting β-cells. Using the INS-1 β-cell line, the effects of quercetin were determined on glucose- or glibenclamide-induced insulin secretion and on β-cell dysfunctions induced by hydrogen peroxide (H₂O₂). These effects were analysed along with the activation of the extracellular signal-regulated kinase (ERK)1/2 pathway. N-acetyl-L-cysteine (NAC) and resveratrol, two antioxidants also known to exhibit some anti-diabetic properties, were used for comparison.

EXPERIMENTAL APPROACH

Insulin release was quantified by the homogeneous time resolved fluorescence method and ERK1/2 activation tested by Western blot experiments. Cell viability was estimated by the [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] (MTT) colorimetric assay.

KEY RESULTS

Quercetin (20 µmol·L⁻¹) potentiated both glucose (8.3 mmol·L⁻¹)- and glibenclamide (0.01 µmol·L⁻¹)-induced insulin secretion and ERK1/2 phosphorylation. The ERK1/2 (but not the protein kinase A) signalling pathway played a crucial role in the potentiation of glucose-induced insulin secretion by quercetin. In addition, quercetin (20 µmol·L⁻¹), protected β-cell function and viability against oxidative damage induced by 50 µmol·L⁻¹ H₂O₂ and induced a major phosphorylation of ERK1/2. In the same conditions, resveratrol or NAC were ineffective.

CONCLUSION AND IMPLICATIONS

Quercetin potentiated glucose and glibenclamide-induced insulin secretion and protected β-cells against oxidative damage. Our study suggested that ERK1/2 played a major role in those effects. The potential of quercetin in preventing β-cell dysfunction associated with diabetes deserves further investigation.     

Differential hepatoprotective mechanisms of rutin and quercetin in CCl4-intoxicated BALB/cN mice

Aim:

To investigate the mechanisms underlying the protective effects of quercetin-rutinoside (rutin) and its aglycone quercetin against CCl₄-induced liver damage in mice.

Methods:

BALB/cN mice were intraperitoneally administered rutin (10, 50, and 150 mg/kg) or quercetin (50 mg/kg) once daily for 5 consecutive days, followed by the intraperitoneal injection of CCl₄ in olive oil (2 mL/kg, 10% v/v). The animals were sacrificed 24 h later. Blood was collected for measuring the activities of ALT and AST, and the liver was excised for assessing Cu/Zn superoxide dismutase (SOD) activity, GSH and protein concentrations and also for immunoblotting. Portions of the livers were used for histology and immunohistochemistry.

Results:

Pretreatment with rutin and, to a lesser extent, with quercetin significantly reduced the activity of plasma transaminases and improved the histological signs of acute liver damage in CCl₄-intoxicated mice. Quercetin prevented the decrease in Cu/Zn SOD activity in CCl₄-intoxicated mice more potently than rutin. However, it was less effective in the suppression of nitrotyrosine formation. Quercetin and, to a lesser extent, rutin attenuated the inflammation in the liver by down-regulating the CCl₄-induced activation of nuclear factor-kappa B (NF-κB), tumor necrosis factor-α (TNF-α) and cyclooxygenase (COX-2). The expression of inducible nitric oxide synthase (iNOS) was more potently suppressed by rutin than by quercetin. Treatment with both flavonoids significantly increased NF-E2-related factor 2 (Nrf2) and heme oxygenase (HO-1) expression in injured livers, although quercetin was less effective than rutin at an equivalent dose. Quercetin more potently suppressed the expression of transforming growth factor-β1 (TGF-β1) than rutin.

Conclusion:

Rutin exerts stronger protection against nitrosative stress and hepatocellular damage but has weaker antioxidant and anti-inflammatory activities and antifibrotic potential than quercetin, which may be attributed to the presence of a rutinoside moiety in position 3 of the C ring.     

The flavonoid scaffold as a template for the design of modulators of the vascular Ca(v) 1.2 channels

BACKGROUND AND PURPOSE

Previous studies have pointed to the plant flavonoids myricetin and quercetin as two structurally related stimulators of vascular Ca_v1.2 channel current (I_Ca1.2). Here we have tested the proposition that the flavonoid structure confers the ability to modulate Ca_v1.2 channels.

EXPERIMENTAL APPROACH

Twenty-four flavonoids were analysed for their effects on I_Ca1.2 in rat tail artery myocytes, using the whole-cell patch-clamp method.

KEY RESULTS

Most of the flavonoids stimulated or inhibited I_Ca1.2 in a concentration- and voltage-dependent manner with EC₅₀ values ranging between 4.4 µM (kaempferol) and 16.0 µM (myricetin) for the stimulators and IC₅₀ values between 13.4 µM (galangin) and 100 µM [(±)-naringenin] for the inhibitors. Key structural requirements for I_Ca1.2 stimulatory activity were the double bond between C2 and C3 and the hydroxylation pattern on the flavonoid scaffold, the latter also determining the molecular charge, as shown by molecular modelling techniques. Absence of OH groups in the B ring was key in I_Ca1.2 inhibition. The functional interaction between quercetin and either the stimulator myricetin or the antagonists resokaempferol, crysin, genistein, and 5,7,2′-trihydroxyflavone revealed that quercetin expressed the highest apparent affinity, in the low µM range, for Ca_v1.2 channels. Neither protein tyrosine kinase nor protein kinase Cα were involved in quercetin-induced stimulation of I_Ca1.2.

CONCLUSIONS AND IMPLICATIONS

Quercetin-like plant flavonoids were active on vascular Ca_v1.2 channels. Thus, the flavonoid scaffold may be a template for the design of novel modulators of vascular smooth muscle Ca_v1.2 channels, valuable for the treatment of hypertension and stroke.     

Z-ligustilide attenuates lipopolysaccharide-induced proinflammatory response via inhibiting NF-KB pathway in primary rat microglia

Aim:

To investigate the anti-inflammatory effect of Z-ligustilide (LIG) on lipopolysaccharide (LPS)-activated primary rat microglia.

Methods:

Microglia were pretreated with LIG 1 h prior to stimulation with LPS (1 μg/mL). After 24 h, cell viability was tested with MTT, nitric oxide (NO) production was assayed with Griess reagent, and the content of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and monocyte chemoattractant protein (MCP-1) was measured with ELISA. Protein expression of the nuclear factor-κB (NF-κB) p65 subunit, cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) was detected with immunocytochemistry 1 h or 24 h after LPS treatment.

Results:

LIG showed a concentration-dependent anti-inflammatory effect in LPS-activated microglia, without causing cytotoxicity. Pretreatment with LIG at 2.5, 5, 10, and 20 μmol/L decreased LPS-induced NO production to 75.9%, 54.4%, 43.1%, and 47.6% (P<0.05 or P< 0.01), TNF-α content to 86.2%, 68.3%, 40.1%, and 39.9% (P<0.01, with the exception of 86.2% for 2.5 μmol/L LIG), IL-1β content to 31.5%, 27.7%, 0.6%, and 0% (P<0.01), and MCP-1 content to 84.4%, 50.3%, 45.1%, and 42.2% (P<0.05 or P<0.01), respectively, compared with LPS treatment alone. LIG (10 μmol/L) significantly inhibited LPS-stimulated immunoreactivity of activated NF-κB, COX-2, and iNOS (P<0.01 vs LPS group).

Conclusion:

LIG exerted a potent anti-inflammatory effect on microglia through inhibition of NF-κB pathway. The data provide direct evidence of the neuroprotective effects of LIG and the potential application of LIG for the treatment of the neuroinflammatory diseases characterized by excessive microglial activation.     

Inhibition of the organic anion-transporting polypeptide 1B1 by quercetin: an in vitro and in vivo assessment

AIM

To investigate the effect of quercetin on organic anion transporting polypeptide 1B1 (OATP1B1) activities in vitro and on the pharmacokinetics of pravastatin, a typical substrate for OATP1B1 in healthy Chinese-Han male subjects.

METHODS

Using human embryonic kidney 293 (HEK293) cells stably expressing OATP1B1, we observed the effect of quercetin on OATP1B1-mediated uptake of estrone-3-sulphate (E3S) and pravastatin. The influence of quercetin on the pharmacokinetics of pravastatin was measured in 16 healthy Chinese-Han male volunteers receiving a single dose of pravastatin (40 mg orally) after co-administration of placebo or 500 mg quercetin capsules (once daily orally for 14 days).

RESULTS

Quercetin competitively inhibited OATP1B1-mediated E3S uptake with a K_i value of 17.9 ± 4.6 µm and also inhibited OATP1B1-mediated pravastatin uptake in a concentration dependent manner (IC₅₀, 15.9 ± 1.4 µm). In healthy Chinese-Han male subjects, quercetin increased the pravastatin area under the plasma concentration – time curve (AUC(0,10 h) and the peak plasma drug concentration (C_max) to 24% (95% CI 15, 32%, P < 0.001) and 31% (95% CI 20, 42%, P < 0.001), respectively. After administration of quercetin, the elimination half-life (t_1/2) of pravastatin was prolonged by 14% (95% CI 4, 24%, P = 0.027), with no change in the time to reach C_max (t_max). Moreover, quercetin decreased the apparent clearance (CL/F) of pravastatin by 18% (95% CI 75, 89%, P < 0.001).

CONCLUSIONS

These findings suggest that quercetin inhibits the OATP1B1-mediated transport of E3S and pravastatin in vitro and also has a modest inhibitory influence on the pharmacokinetics of pravastatin in healthy Chinese-Han male volunteers. The effects of quercetin on other OATP1B1 substrate drugs deserve further investigation.     

Differential sensitivity of basal and acetylcholine-induced activity of nitric oxide to blockade by asymmetric dimethylarginine in the rat aorta

Background and purpose:

Previous work has shown that N^G-monomethyl-l-arginine (l-NMMA) paradoxically inhibits basal, but not ACh-stimulated activity of nitric oxide in rat aorta. The aim of this study was to determine if the endogenously produced agent, asymmetric N^G, N^G-dimethyl-l-arginine (ADMA), also exhibits this unusual selective blocking action.

Experimental approach:

The effect of ADMA on basal nitric oxide activity was assessed by examining its ability to enhance phenylephrine (PE)-induced tone in endothelium-containing rings. Its effect on ACh-induced relaxation was assessed both in conditions where ADMA greatly enhanced PE tone and where tone was carefully matched with control tissues at a range of different levels.

Key results:

ADMA (100 µM) potentiated PE-induced contraction, consistent with inhibition of basal nitric oxide activity. Higher concentrations (300–1000 µM) had no greater effect. Although ADMA (100 µM) also appeared to block ACh-induced relaxation when it enhanced PE tone to maximal levels, virtually no block was seen at intermediate levels of tone in the presence of ADMA. Even ADMA at 1000 µM had no effect on the maximal relaxation to ACh, although it produced a small (two- to threefold) reduction in sensitivity. ADMA and l-NMMA, like l-arginine (all at 1000 µM), protected ACh-induced relaxation against blockade by l-NAME (30 µM).

Conclusions and implications:

In the rat aorta, ADMA, like l-NMMA, blocks basal activity of nitric oxide, but has little effect on that stimulated by ACh. Further studies are required to explain these seemingly anomalous actions of ADMA and l-NMMA.     

Huperzine A derivative M3 protects PC12 cells against sodium nitroprusside-induced apoptosis

Aim:

To investigate the effects of M3, a derivative of huperzine A, on the apoptosis induced by sodium nitroprusside (SNP) in PC12 cells.

Methods:

Cell viability was detected using MTT method. Apoptosis was examined with annexin V/prodium iodide (PI) stain. The levels of reactive oxygen species (ROS) were measured using fluorophotometric quantitation. The amount of malonaldehyde (MDA) was determined with MDA detection kits. The expression of caspase-3 and Hsp70 were analyzed using Western blotting.

Results:

Exposure of PC12 cells to SNP (200 μmol/L) for 24 h decreased the cell viability to 69.0% of that in the control group. Pretreatment with M3 (10 μmol/L) or huperzine A (10 μmol/L) significantly protected the cells against SNP-induced injury and apoptosis; the ratio of apoptotic bodies in PC12 cells was decreased from 27.3% to 15.0%. Pretreatment with M3 (10 μmol/L) significantly decreased ROS and MDA levels, and increased the expression of Hsp70 in the cells. Quercetin (10 μmol/L) blocked the protective effect of M3, while did not influence on that of huperzine A.

Conclusion:

M3 protects PC12 cells against SNP-induced apoptosis, possible due to ROS scavenging and Hsp70 induction.     

Quercetin and its major metabolites selectively modulate cyclic GMP-dependent relaxations and associated tolerance in pig isolated coronary artery

Background and purpose:

Quercetin is a major flavonoid that contributes to the reduced risk of cardiovascular disease associated with dietary ingestion of fruits and vegetables. We have pharmacologically characterized the effect of quercetin, and its sulphate and glucuronide metabolites, on vasoconstrictor and vasodilator responses in the porcine isolated coronary artery.

Experimental approach:

Segments of the porcine coronary artery were prepared for either isometric tension recording or determination of cyclic GMP content. The effect of quercetin and metabolites on submaximal responses to U46619 was examined in the presence and absence of substance P, bradykinin, forskolin, sodium nitroprusside (SNP) and glyceryl trinitrate (GTN).

Key results:

Quercetin and quercetin 3′-sulphate inhibited endothelin and U46619-induced contractions with greater potency (three- to fivefold) against the former, while quercetin 3-glucoronide was inactive. Quercetin enhanced both the cyclic GMP content of the artery (threefold) and cyclic GMP-dependent relaxations to GTN and SNP (two to threefold), but forskolin-induced relaxations were unaffected. Although the effect of quercetin was qualitatively similar to that noted for UK-114,542, a selective inhibitor of phosphodiesterase 5, it was still evident against SNP-induced relaxations in the presence of 10 nM UK-114,542. Quercetin and quercetin 3′-sulphate significantly reduced the development of GTN-associated ‘tolerance’.

Conclusions and implications:

Quercetin and quercetin 3′-sulphate inhibited receptor-mediated contractions of the porcine isolated coronary artery by an endothelium-independent action. Quercetin selectively enhanced cyclic-GMP-dependent relaxations by a mechanism not involving phosphodiesterase 5 inhibition. In addition, quercetin and quercetin 3′-sulphate opposed GTN-induced tolerance in vitro, which may be beneficial for patients treated for angina pectoris.     

Toll-like receptor 7/8 agonist resiquimod induces late preconditioning in neonatal cardiac myocytes

Aim:

To investigate whether R-848 (resiquimod, toll-like receptor 7/8 agonist) can induce late preconditioning in neonatal cardiac myocytes.

Methods:

The protective effects of R-848 on neonatal myocytes against anoxia-reoxygenation-induced injury were tested, and intracellular reactive oxygen species (ROS) were determined. The protein synthesis inhibitor cyclohexamide (CH) and the ROS scavenger N-acetylcysteine (NAC) were used in this model to test if new protein synthesis and oxidative stress were necessary for their cardioprotective effects. The activation of nuclear factor kappa B (NFκB) and hypoxia inducible factor 1 (HIF1) was investigated by electrophoretic mobility shift assays (EMSA), and inducible nitric oxide synthase (iNOS) was assessed by immunoblotting. After iNOS was down-regulated by small interfering RNA (siRNA) transfection, the cardioprotective effect was reassessed.

Results:

ROS were triggered soon after R-848 (0.01–1.0 μg/L) administration, however, the cardioprotective effect of which was induced 24 h later. This protection was abolished by CH or NAC pretreatment. NFκB and HIF1 activation and iNOS up-regulation were involved in this protective mechanism. The cardioprotective effect was also attenuated after iNOS was knocked down.

Conclusion:

R-848 provided a cardioprotective effect through a late preconditioning mechanism via a ROS/NFκB-HIF1/iNOS-dependent pathway.     

Vascular responses to 8-nitro-cyclic GMP in non-diabetic and diabetic mice

BACKGROUND AND PURPOSE

8-Nitroguanosine 3′,5′-cyclic monophosphate (8-nitro-cGMP), formed nitric oxide (NO)-dependently, is a physiological second messenger, yet little is known about its role in the pathophysiology of vascular diseases. To study the pharmacological activity of 8-nitro-cGMP in diabetic mice, we compared its effects on vascular reactivity of aortas from non-diabetic and diabetic mice.

EXPERIMENTAL APPROACH

Vascular tension recording was performed in thoracic aortic rings from wild-type (C57BL/6), non-diabetic db/+ and obese/diabetic db/db mice. Endothelial NO synthase (eNOS) uncoupling and superoxide were tested by Western blot and dihydroethidium fluorescence respectively.

KEY RESULTS

8-Nitro-cGMP, at concentrations up to 10 µM, enhanced phenylephrine-induced contractions in aortas from C57BL/6 and db/+ mice, but not from db/db mice. This enhancement was not observed with 8-bromo-cGMP. Pretreatment of aortas from C57BL/6 and db/+ mice with l-NAME (100 µM), superoxide dismutase (100 U·mL⁻¹) or tiron (1 mM), abolished 8-nitro-cGMP-induced enhancement of the phenylephrine contraction. In 8-nitro-cGMP (10 µM)-treated C57BL/6 aortas, eNOS dimer/monomer ratio was significantly decreased and vascular superoxide production increased, suggesting that 8-nitro-cGMP-induced superoxide production via eNOS uncoupling may mediate the enhancement of the phenylephrine contraction. At higher concentrations (>10 µM), 8-nitro-cGMP produced relaxation of the phenylephrine-contracted aortas from C57BL/6, db/+ and db/db mice. The 8-nitro-cGMP-induced relaxation in db/db mouse aortas was found to be resistant to a phosphodiesterase 5 inhibitor, zaprinast (1 µM).

CONCLUSIONS AND IMPLICATIONS

The vasodilator effect of 8-nitro-cGMP may contribute to amelioration of the vascular endothelial dysfunction in diabetic mice, representing a novel pharmacological approach to prevent the complications associated with diabetes.     

Flavocoxid,a dual inhibitor of cyclooxygenase and 5-lipoxygenase,blunts pro-inflammatory phenotype activation in endotoxin-stimulated macrophages

Background and purpose:

The flavonoids, baicalin and catechin, from Scutellaria baicalensis and Acacia catechu, respectively, have been used for various clinical applications. Flavocoxid is a mixed extract containing baicalin and catechin, and acts as a dual inhibitor of cyclooxygenase (COX) and 5-lipoxygenase (LOX) enzymes. The anti-inflammatory activity, measured by protein and gene expression of inflammatory markers, of flavocoxid in rat peritoneal macrophages stimulated with Salmonella enteritidis lipopolysaccharide (LPS) was investigated.

Experimental approach:

LPS-stimulated (1 µg·mL⁻¹) peritoneal rat macrophages were co-incubated with different concentrations of flavocoxid (32–128 µg·mL⁻¹) or RPMI medium for different incubation times. Inducible COX-2, 5-LOX, inducible nitric oxide synthase (iNOS) and inhibitory protein κB-α (IκB-α) levels were evaluated by Western blot analysis. Nuclear factor κB (NF-κB) binding activity was investigated by electrophoretic mobility shift assay. Tumour necrosis factor-α (TNF-α) gene and protein expression were measured by real-time polymerase chain reaction and enzyme-linked immunosorbent assay respectively. Finally, malondialdehyde (MDA) and nitrite levels in macrophage supernatants were evaluated.

Key results:

LPS stimulation induced a pro-inflammatory phenotype in rat peritoneal macrophages. Flavocoxid (128 µg·mL⁻¹) significantly inhibited COX-2 (LPS = 18 ± 2.1; flavocoxid = 3.8 ± 0.9 integrated intensity), 5-LOX (LPS = 20 ± 3.8; flavocoxid = 3.1 ± 0.8 integrated intensity) and iNOS expression (LPS = 15 ± 1.1; flavocoxid = 4.1 ± 0.4 integrated intensity), but did not modify COX-1 expression. PGE₂ and LTB₄ levels in culture supernatants were consequently decreased. Flavocoxid also prevented the loss of IκB-α protein (LPS = 1.9 ± 0.2; flavocoxid = 7.2 ± 1.6 integrated intensity), blunted increased NF-κB binding activity (LPS = 9.2 ± 2; flavocoxid = 2.4 ± 0.7 integrated intensity) and the enhanced TNF-α mRNA levels (LPS = 8 ± 0.9; flavocoxid = 1.9 ± 0.8 n-fold/β-actin) induced by LPS. Finally, flavocoxid decreased MDA, TNF and nitrite levels from LPS-stimulated macrophages.

Conclusion and implications:

Flavocoxid might be useful as a potential anti-inflammatory agent, acting at the level of gene and protein expression.     

Role of connexin 43 in the maintenance of spontaneous activity in the guinea pig prostate gland

BACKGROUND AND PURPOSE

To investigate the role of connexin 43 in the maintenance of spontaneous activity in prostate tissue from young and old guinea pigs.

EXPERIMENTAL APPROACH

Conventional intracellular microelectrode and tension recording techniques, coupled with Western blot analysis and immunohistochemistry for connexin 43 (CX43) were used. The effects of three gap junction uncouplers, 18β glycyrrhetinic acid (10 µM, 40 µM), carbenoxolone (10 µM, 50 µM) and octanol (0.5 mM, 1 mM), were studied in cells displaying slow wave activity and on spontaneously contracting tissue from prostate glands of young (2–5 months) and old (9–16 months) guinea pigs.

KEY RESULTS

18β Glycyrrhetinic acid (40 µM), carbenoxolone (50 µM) or octanol (0.5 mM) abolished slow wave activity in prostate tissue from young and old guinea pigs and depolarized membrane potential by approximately 5 mV. These treatments also abolished all contractions in both sets of prostate tissue. These effects were reversed upon washout. Western blot analysis and CX43 immunohistochemistry showed that there was no age-related difference in the expression and distribution of CX43 in prostate tissues.

CONCLUSION AND IMPLICATIONS

When gap junctional communication via CX43 was disrupted, spontaneous activity was abolished at a cellular and whole tissue level; CX43 is therefore essential for the maintenance of spontaneous slow wave activity and subsequent contractile activity in the guinea pig prostate gland.     

Effects of drug interactions on biotransformation and antiplatelet effect of clopidogrel in vitro

BACKGROUND AND PURPOSE

The conversion of clopidogrel to its active metabolite, R-130964, is a two-step cytochrome P450 (CYP)-dependent process. The current investigations were performed to characterize in vitro the effects of different CYP inhibitors on the biotransformation and on the antiplatelet effect of clopidogrel.

EXPERIMENTAL APPROACH

Clopidogrel biotransformation was studied using human liver microsomes (HLM) or specific CYPs and platelet aggregation using human platelets activated with ADP.

KEY RESULTS

Experiments using HLM or specific CYPs (3A4, 2C19) revealed that at clopidogrel concentrations >10 µM, CYP3A4 was primarily responsible for clopidogrel biotransformation. At a clopidogrel concentration of 40 µM, ketoconazole showed the strongest inhibitory effect on clopidogrel biotransformation and clopidogrel-associated inhibition of platelet aggregation with IC₅₀ values of 0.03 ± 0.07 µM and 0.55 ± 0.06 µM respectively. Clarithromycin, another CYP3A4 inhibitor, impaired clopidogrel biotransformation and antiplatelet activity almost as effectively as ketoconazole. The CYP3A4 substrates atorvastatin and simvastatin both inhibited clopidogrel biotransformation and antiplatelet activity, less potently than ketoconazole. In contrast, pravastatin showed no inhibitory effect. As clopidogrel itself inhibited CYP2C19 at concentrations >10 µM, the CYP2C19 inhibitor lansozprazole affected clopidogrel biotransformation only at clopidogrel concentrations ≤10 µM. The carboxylate metabolite of clopidogrel was not a CYP substrate and did not affect platelet aggregation.

CONCLUSIONS AND IMPLICATIONS

At clopidogrel concentrations >10 µM, CYP3A4 is mainly responsible for clopidogrel biotransformation, whereas CYP2C19 contributes only at clopidogrel concentrations ≤10 µM. CYP2C19 inhibition by clopidogrel at concentrations >10 µM may explain the conflicting results between in vitro and in vivo investigations regarding drug interactions with clopidogrel.     

Quercetin sensitizes human glioblastoma cells to temozolomide in vitro via inhibition of Hsp27

Aim： Quercetin is an effective Hsp27 inhibitor and has been reported to facilitate tumor cell apoptosis. The aim of this study was to investigate whether quercetin could sensitize human glioblastoma cells to temozolomide （TMZ） in vitro.
Methods： Both U251 and U87 human glioblastoma cells were treated with quercetin and/or TMZ for 48 h. Cell viability was detected using the MTT assay. Cell apoptosis was analyzed with caspase-3 activity kits and flow cytometry. Hsp27 expression and phosphorylation were examined using Western blot analysis. RNA interference using Hsp27 siRNA oligos was performed to knock down the gene expression of Hsp27.
Results： TMZ （200 or 400 μmol/L） alone effectively inhibited the viability of U251 and U87 cells. When combined with quercetin （30 μmol/L）, TMZ （100 μmol/L） significantly inhibited the cell viability, and the inhibition of TMZ （200 and 400 μmol/L） was enhanced. TMZ or quercetin anole did not affect caspase-3 activity and cell apoptosis, while TMZ combined with quercetin significantly increased caspase-3 activity and induced cell apoptosis. TMZ anole significantly increased Hsp27 phosphorylation in U251 and U87 cells, while quercetin or Hsp27 siRNA oligos combined with TMZ attenuated TMZ-induced Hsp27 phosphorylation and significantly inhibited Hsp27 expression.
Conclusion： Combined treatment with TMZ and quercetin efficiently suppressed human glioblastoma cell survival in vitro.     

5α-reduced glucocorticoids exhibit dissociated anti-inflammatory and metabolic effects

BACKGROUND AND PURPOSE

Dissociating anti-inflammatory efficacy from the metabolic side effects of glucocorticoids is an attractive therapeutic goal. 5α-Tetrahydro-corticosterone (5αTHB), produced from corticosterone by 5α-reductases, activates glucocorticoid receptors. This study compares the effects of 5αTHB on inflammation and metabolism in vitro and in vivo.

METHODS

Suppression of cytokine release by 5αTHB and corticosterone were studied following LPS activation of mouse bone marrow derived macrophages. In vivo the efficacy of these steroids to dysregulate metabolic homeostasis and modulate immune suppression and the responses to thioglycollate-induced peritonitis in C57BL/6 mice were studied following acute injection (1.5–15 mg) and chronic infusion (50 µg·day⁻¹, 14 days).

RESULTS

In macrophages, 5αTHB increased secretion of IL-10 similarly to corticosterone (180%, 340%; data are % vehicle, treated with 5αTHB and corticosterone, respectively) and suppressed LPS-induced secretion of TNF-α (21.9%, 74.2%) and IL-6 (16.4%, 69.4%). In mice with thioglycollate-induced peritonitis, both 5αTHB and corticosterone reduced the numbers of neutrophils (58.6%, 49.9%) and inflammatory monocytes (69.5%, 96.4%), and also suppressed MCP-1 (48.7%, 80.9%) and IL-6 (53.5%, 86.7%) in peritoneal exudate. In mice chronically infused with 5αTHB and corticosterone LPS-induced production of TNF-α from whole blood was suppressed to the same degree (63.2%, 37.2%). However, in contrast to corticosterone, 5αTHB did not induce body weight loss, increase blood pressure or induce hyperinsulinaemia.

CONCLUSIONS

5αTHB has anti-inflammatory effects in vitro and in vivo. At doses with equivalent anti-inflammatory efficacy to corticosterone, 5αTHB did not induce metabolic toxicity and thus may be a prototype for a safer anti-inflammatory drug.     

Riluzole protects against cardiac ischaemia and reperfusion damage via block of the persistent sodium current

Background and purpose:

Current strategies to ameliorate cardiac ischaemic and reperfusion damage, including block of the sodium-hydrogen exchanger, are therapeutically ineffective. Here we propose a different approach, block of the persistent sodium current (INaP).

Experimental approach:

Left ventricular pressure was measured as an index of functional deficit in isolated, Langendorff perfused, hearts from adult rats, subjected to 30 min global ischaemia and reperfusion with vehicle only (control) or riluzole (1–10 µM) in the perfusate. Cell shortening and intracellular Ca2+ concentrations [Ca2+]_i were measured in adult rat isolated myocytes subjected to hypoxia and re-oxygenation. The block of transient and persistent sodium currents by concentrations of riluzole between 0.01 and 100 µM were assessed in rat isolated myocytes using patch clamp techniques.

Key results:

In perfused hearts, riluzole produced a concentration-dependent cardioprotective action, with minor protection from 1 µM and produced rapid and almost complete recovery upon reperfusion from 3 and 10 µM. In isolated myocytes, riluzole at 3 and 10 µM greatly attenuated or prevented the hypoxia- and reperfusion-induced rise in [Ca2+]_i and the contractile deficit. In patch clamp experiments, riluzole blocked the persistent sodium current with an IC₅₀ of 2.7 µM, whereas the block of the transient sodium current was only apparent at concentrations above 30 µM.

Conclusions and implications:

Riluzole preferentially blocked INaP and was protective in cardiac ischaemia and reperfusion. Thus block of the persistent sodium current would be a viable method of ameliorating cardiac ischaemic and reperfusion damage.     

Signalling pathways involved in sildenafil-induced relaxation of human bladder dome smooth muscle

Background and purpose:

The mechanism(s) of action responsible for the beneficial effects of phosphodiesterase 5 (PDE5) inhibitors including sildenafil on lower urinary tract symptoms suggestive of benign prostate hyperplasia are unclear. In particular, the role of the NO-cGMP signalling pathway in regulating human bladder dome smooth muscle relaxation is questionable. Thus, we assessed the ability of a PDE5 inhibitor, sildenafil, to relax such tissue, and identified the signalling pathways involved in this relaxation.

Experimental approach:

Human bladder samples were obtained from 20 patients with no overactive bladder undergoing cystectomy for bladder cancer. Detrusor strips were mounted isometrically in Krebs–HEPES solution. Concentration–response curves for sildenafil (10 nM–30 µM) were generated in the presence of various inhibitors on carbachol-induced pre-contraction.

Key results:

Sildenafil relaxed carbachol-pre-contracted human detrusor strips, starting at 3 µM. This effect was not modified by NO donors, S-nitroso-N-acetylpenicillamine (10 µM) or sodium nitroprusside (300 nM), but was significantly inhibited by inhibition of guanylate cyclase (with ODQ, 10 µM) or adenylyl cyclase (with MDL-12,330A, 10 µM), by the ATP-sensitive potassium channel inhibitor, glibenclamide (10 µM), or inhibition of the large (with iberiotoxin, 30 nM) or small (with apamin, 100 nM) conductance calcium-activated potassium channels.

Conclusions and implications:

Sildenafil-induced relaxation of human detrusor smooth muscle involved cGMP-, cAMP- and K⁺ channel-dependent signalling pathways, with a minor contribution from NO. The effect of this sildenafil-induced relaxation on the clinical benefit of PDE5 inhibitors on urinary storage symptoms in men deserves further investigation.     

Quercetin protects rat dorsal root ganglion neurons against high glucose-induced injury in vitro through Nrf-2/HO-1 activation and NF-κB inhibition

Aim:

To examine the effects of quercetin, a natural antioxidant, on high glucose (HG)-induced apoptosis of cultured dorsal root ganglion (DRG) neurons of rats.

Methods:

DRG neurons exposed to HG (45 mmol/L) for 24 h were employed as an in vitro model of diabetic neuropathy. Cell viability, reactive oxygen species (ROS) level and apoptosis were determined. The expression of NF-кB, IкBα, phosphorylated IкBα and Nrf2 was examined using RT PCR and Western blot assay. The expression of hemeoxygenase-1 (HO-1), IL-6, TNF-α, iNOS, COX-2, and caspase-3 were also examined.

Results:

HG treatment markedly increased DRG neuron apoptosis via increasing intracellular ROS level and activating the NF-κB signaling pathway. Co-treatment with quercetin (2.5, 5, and 10 mmol/L) dose-dependently decreased HG-induced caspase-3 activation and apoptosis. Quercetin could directly scavenge ROS and significantly increased the expression of Nrf-2 and HO-1 in DRG neurons. Quercetin also dose-dependently inhibited the NF-κB signaling pathway and suppressed the expression of iNOS, COX-2, and proinflammatory cytokines IL-6 and TNF-α.

Conclusion:

Quercetin protects rat DRG neurons against HG-induced injury in vitro through Nrf-2/HO-1 activation and NF-κB inhibition, thus may be beneficial for the treatment of diabetic neuropathy.