Selective labeling of κ2 opioid receptors in rat brain by [I]IOXY: Interaction of opioid peptides and other drugs with multiple κ2a binding sites

Recent studies from our laboratory resolved two subtypes of the κ₂ binding site, termed κ_2a and κ_2b, using guinea pig, rat, and human brain membranes depleted of μ and δ receptors by pretreatment with the site-directed acylating agents BIT (μ-selective) and FIT (δ-selective). 6β-Iodo-3,14-dihydroxy-17-cyclopropylmethyl-4,5α-epoxymorphinan (IOXY), an opioid antagonist that has high affinity for κ₂ sites, was radioiodinated to maximum specific activity (2200 Ci/mmol) and purified by high pressure liquid chromotography and used to characterize multiple κ₂ binding sites. The results indicated that [¹²⁵I]IOXY, like [³H]bremazocine, selectively labels κ₂ binding sites in rat brain membranes pretreated with BIT and FIT. Using 100 nM [d-Ala²-MePhe⁴,Gly-ol⁵]enkephalin to block [¹²⁵I]IOXY binding to the κ_2b site, two subtypes of the κ_2a binding site were resolved, both in the absence and presence of 50 μM 5′-guanylyimidodiphosphate. Viewed collectively, these results provide further evidence for heterogeneity of the κ opioid receptor, which may provide new targets for drug design, synthesis, and therapeutics.     

Selective κ Opioid Antagonists nor-BNI,GNTI and JDTic Have Low Affinities for Non-Opioid Receptors and Transporters

Background

Nor-BNI, GNTI and JDTic induce selective κ opioid antagonism that is delayed and extremely prolonged, but some other effects are of rapid onset and brief duration. The transient effects of these compounds differ, suggesting that some of them may be mediated by other targets.

Results

In binding assays, the three antagonists showed no detectable affinity (K _i≥10 µM) for most non-opioid receptors and transporters (26 of 43 tested). There was no non-opioid target for which all three compounds shared detectable affinity, or for which any two shared sub-micromolar affinity. All three compounds showed low nanomolar affinity for κ opioid receptors, with moderate selectivity over μ and δ (3 to 44-fold). Nor-BNI bound weakly to the α_2C-adrenoceptor (K _i = 630 nM). GNTI enhanced calcium mobilization by noradrenaline at the α_1A-adrenoceptor (EC₅₀ = 41 nM), but did not activate the receptor, displace radioligands, or enhance PI hydrolysis. This suggests that it is a functionally-selective allosteric enhancer. GNTI was also a weak M₁ receptor antagonist (K _B = 3.7 µM). JDTic bound to the noradrenaline transporter (K _i = 54 nM), but only weakly inhibited transport (IC₅₀ = 1.1 µM). JDTic also bound to the opioid-like receptor NOP (K _i = 12 nM), but gave little antagonism even at 30 µM. All three compounds exhibited rapid permeation and active efflux across Caco-2 cell monolayers.

Conclusions

Across 43 non-opioid CNS targets, only GNTI exhibited a potent functional effect (allosteric enhancement of α_1A-adrenoceptors). This may contribute to GNTI''s severe transient effects. Plasma concentrations of nor-BNI and GNTI may be high enough to affect some peripheral non-opioid targets. Nonetheless, κ opioid antagonism persists for weeks or months after these transient effects dissipate. With an adequate pre-administration interval, our results therefore strengthen the evidence that nor-BNI, GNTI and JDTic are highly selective κ opioid antagonists.     

Synthesis and pharmacological evaluation of 6-naltrexamine analogs for alcohol cessation

A series of substituted aryl amide derivatives of 6-naltrexamine, 3 designed to be metabolically stable were synthesized and used to characterize the structural requirements for their potency to binding and functional activity of human mu (μ), delta (δ) and kappa (κ) opioid and nociceptin (NOP) receptors. Binding assays showed that 4–10 had subnanomolar K_i values for μ and κ opioid receptors. Functional assays for stimulation of [³⁵S]GTPγS binding showed that several compounds acted as partial or inverse agonists and antagonists of the μ and δ, κ opioid or NOP receptors. The compounds showed considerable stability in the presence of rat, mouse or human liver preparations and NADPH. The inhibitory activity on the functional activity of human cytochrome P450s was examined to determine any potential inhibition by 4–9. Only modest inhibition of CYP3A4, CYP2C9 and CYP2C19 was observed for a few of the analogs. As a representative example, radiolabeled 6 was examined in vivo and showed reasonable brain penetration. The inhibition of ethanol self-administration in rats trained to self-administer a 10% (w/v) ethanol solution, utilizing operant techniques showed 5–8 to have very potent efficacy (ED₅₀ values 19–50 μg/kg).     

The Cytoprotective Effects of E-α-(4-Methoxyphenyl)-2’,3,4,4'-Tetramethoxychalcone (E-α-p-OMe-C6H4-TMC)—A Novel and Non-Cytotoxic HO-1 Inducer

Cell protection against different noxious stimuli like oxidative stress or chemical toxins plays a central role in the treatment of many diseases. The inducible heme oxygenase isoform, heme oxygenase-1 (HO-1), is known to protect cells against a variety of harmful conditions including apoptosis. Because a number of medium strong electrophiles from a series of α-X-substituted 2’,3,4,4’-tetramethoxychalcones (α-X-TMCs, X = H, F, Cl, Br, I, CN, Me, p-NO₂-C₆H₄, Ph, p-OMe-C₆H₄, NO₂, CF₃, COOEt, COOH) had proven to activate Nrf2 resulting in HO-1 induction and inhibit NF-κB downstream target genes, their protective effect against staurosporine induced apoptosis and reactive oxygen species (ROS) production was investigated. RAW264.7 macrophages treated with 19 different chalcones (15 α-X-TMCs, chalcone, 2’-hydroxychalcone, calythropsin and 2’-hydroxy-3,4,4’-trimethoxychalcone) prior to staurosporine treatment were analyzed for apoptosis and ROS production, as well as HO-1 protein expression and enzyme activity. Additionally, Nrf2 and NF-κB activity was assessed. We found that amongst all tested chalcones only E-α-(4-methoxyphenyl)-2’,3,4,4''-tetramethoxychalcone (E-α-p-OMe-C₆H₄-TMC) demonstrated a distinct, statistically significant antiapoptotic effect in a dose dependent manner, showing no toxic effects, while its double bond isomer Z-α-p-OMe-C₆H₄-TMC displayed no significant activity. Also, E-α-p-OMe-C₆H₄-TMC induced HO-1 protein expression and increased HO-1 activity, whilst inhibition of HO-1 by SnPP-IX abolished its antiapoptotic effect. The only weakly electrophilic chalcone E-α-p-OMe-C₆H₄-TMC reduced the staurosporine triggered formation of ROS, while inducing the translocation of Nrf2 into the nucleus. Furthermore, staurosporine induced NF-κB activity was attenuated following E-α-p-OMe-C₆H₄-TMC treatment. Overall, E-α-p-OMe-C₆H₄-TMC demonstrated its effective cytoprotective potential via a non-toxic induction of HO-1 in RAW264.7 macrophages. The observed cytoprotective effect may partly be related to both, the activation of the Nrf2- and inhibition of the NF-κB pathway.     

Effects of morphine-3-glucuronide on the antinociceptive activity of peptide and nonpeptide opioid receptor agonists in mice

The effects of morphine-3-glucuronide (M3G), a metabolite of morphine, were determined on the antinociceptive actions, as measured by the tail flick test, of morphine, a μ-opioid receptor agonist, of U-50,488H, a κ-opioid receptor agonist, of [ -Pen², -Pen⁵]enkephalin (DPDPE), a δ₁-opioid receptor agonist, and of [ -Ala²,Glu⁴]deltorphin II (deltorphin II), a δ₂-opioid receptor agonist in mice. Morphine administered ICV (2.5 μg/mouse) or SC (10 mg/kg), U-50,488H (25 mg/kg, IP), DPDPE (15 μg/mouse; ICV), and deltorphin II (15 μg/mouse, ICV) produced antinociception in mice. Intraperitoneal or ICV injections of M3G did not produce any effect on the tail flick latency nor did it affect the antinociception-induced by morphine, U-50,488H, DPDPE, or deltorphin II. Previously M3G has been shown to antagonize the antinociceptive effects of morphine in the rat. It is concluded that in the mouse, M3G neither produces hyperalgesia nor modifies the actions of μ-, κ-, δ₁-, or δ₂-opioid receptor agonists.     

Distinct involvement of the Jun-N-terminal kinase and NF-kappaB pathways in the repression of the human COL1A2 gene by TNF-alpha

We used a gene knockout approach to elucidate the specific roles played by the Jun-N-terminal kinase (JNK) and NF-κB pathways downstream of TNF-α in the context of α(2) type I collagen gene (COL1A2) expression. In JNK₁^−/−-JNK₂^−/− (JNK^−/−) fibroblasts, TNF-α inhibited basal COL1A2 expression but had no effect on TGF-β-driven gene transactivation unless jnk1 was introduced ectopically. Conversely, in NF-κB essential modulator^−/− (NEMO^−/−) fibroblasts, lack of NF-κB activation did not influence the antagonism exerted by TNF-α against TGF-β but prevented repression of basal COL1A2 gene expression. Similar regulatory mechanisms take place in dermal fibroblasts, as evidenced using transfected dominant-negative forms of MKK4 and IKK-α, critical kinases upstream of the JNK and NF-κB pathways, respectively. These results represent the first demonstration of an alternate usage of distinct signaling pathways by TNF-α to inhibit the expression of a given gene, COL1A2, depending on its activation state.     

Effects of dibuturyl cAMP, LHRH, and aromatase inhibitor on simultaneous outputs of prostaglandin F2α, and 13,14-dihydro-15-keto-prostaglandin F2α by term placental explants

Explants from term human placentas were maintained in culture with daily changes of medium. Daily output of PGF_2α and PGFM¹ decreased during the course of the incubation. Addition of 4 μg/ml DHEAS or 67 μg/ml LDL cholesterol had no effect on output of PGF_2α or PGFM. Addition of 1.6, 3.2, or 6.4 μg/ml of LHRH to the culture plates had no effect on output of PGFM or PGF_2α, but LHRH increased hCG output. Dibutyryl cAMP (1mM, 2mM, and 4mM) increased output of PGF_2α, PGFM, and hCG. Aromatase inhibitor decreased hCG output, but it was without effect on output of PGF_2α, or PGFM. Significant correlations were demonstrated between progesterone, PGFM, PGF_2α, and hCG, suggesting that PGF_2α originates in the syncytiotrophoblast cell. The ability of LHRH to stimulate output of hCG but not PGF_2α while dbcAMP stimulated both suggests that either PGF_2α and hCG arise in different cells or that LHRH does not act through cAMP.     

The Nuclear IκB Family Protein IκBNS Influences the Susceptibility to Experimental Autoimmune Encephalomyelitis in a Murine Model

The nuclear IκB family protein IκB_NS is expressed in T cells and plays an important role in Interferon (IFN)-γ and Interleukin (IL)-2 production. IκB-ζ, the most similar homolog of IκB_NS, plays an important role in the generation of T helper (Th)17 cells in cooperation with RORγt, a master regulator of Th17 cells. Thus, IκB-ζ deficient mice are resistant to Th17-dependent experimental autoimmune encephalomyelitis (EAE). However, IκB-ζ deficient mice develop the autoimmune-like Sjögren syndrome with aging. Here we found that IκB_NS-deficient (Nfkbid^−/−) mice show resistance against developing Th17-dependent EAE. We found that Nfkbid^−/− T cells have decreased expression of IL-17-related genes and RORγt in response to Transforming Growth Factor (TGF)-β1 and IL-6 stimulation. Thus, IκB_NS plays a pivotal role in the generation of Th17 cells and in the control of Th17-dependent EAE.     

The Effects of NF-κB and c-Jun/AP-1 on the Expression of Prothrombotic and Proinflammatory Molecules Induced by Anti-β2GPI in Mouse

Our previous data demonstrated that nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) are involved in the process of anti-β₂GPI/β₂GPI-induced tissue factor (TF) expression in monocytes. However, the role of NF-κB and AP-1 in pathogenic mechanisms of antiphospholipid syndrome (APS) in vivo has been rarely studied. This study aimed to investigate whether NF-κB and c-Jun/AP-1 are involved in anti-β₂GPI-induced expression of prothrombotic and proinflammatory molecules in mouse. IgG-APS or anti-β₂GPI antibodies were injected into BALB/c mice in the presence or absence of PDTC (a specific inhibitor of NF-κB) and Curcumin (a potent inhibitor of AP-1) treatment. Our data showed that both IgG-APS and anti-β₂GPI could induce the activation of NF-κB and c-Jun/AP-1 in mouse peritoneal macrophages. The anti-β₂GPI-induced TF activity in homogenates of carotid arteries and peritoneal macrophages from mice could significantly decrease after PDTC and/or Curcumin treatment, in which PDTC showed the strongest inhibitory effect, but combination of two inhibitors had no synergistic effect. Furthermore, anti-β₂GPI-induced expression of TF, VCAM-1, ICAM-1 and E-selectin in the aorta and expression of TF, IL-1β, IL-6 and TNF-α in peritoneal macrophages of mice were also significantly attenuated by PDTC and/or Curcumin treatment. These results indicate that both NF-κB and c-Jun/AP-1 are involved in regulating anti-β₂GPI-induced expression of prothrombotic and proinflammatory molecules in vivo. Inhibition of NF-κB and c-Jun/AP-1 pathways may be beneficial for the prevention and treatment of thrombosis and inflammation in patients with APS.     

Novel delta opioid receptor agonists exhibit differential stimulation of signaling pathways

A novel family of 1,3,5-trisubstituted 1,2,4-triazoles was discovered as potent and selective ligands for the δ opioid receptor by rational design. Compound 5b exhibited low-nanomolar in vitro binding affinity (IC₅₀ = 5.8 nM), excellent selectivity for the δ opioid receptor over the alternative μ and κ opioid receptors, full agonist efficacy in receptor down-regulation and MAP kinase activation assays, and low-efficacy partial agonist activity in stimulation of GTPγS binding. The apparent discrepancy observed in these functional assays may stem from different signaling pathways involved in each case, as found previously for other G-protein coupled receptors. More biological studies are underway to better understand the differential stimulation of signaling pathways by these novel compounds.     

Mapping of the β2 Subunit Gene (GABRB2) to Microdissected Human Chromosome 5q34-q35 Defines a Gene Cluster for the Most Abundant GABAA Receptor Isoform

The γ-aminobutyric acid receptor (GABA_AR) is a multisubunit C1 channel that mediates most fast inhibitory synaptic transmission in the central nervous system. Molecular evolution has given rise to many genetic variants of GABA_AR subunits, including α_1-6, β_1-4, γ_1-4, δ, and ρ_1-2, suggesting that an enormous number of combinations of subunits are possible. Here we report that the β₂ gene is located on chromosome 5q34-q35, defining a cluster comprising α₁ β₂, and γ₂ genes that together code for the most abundant GABA_AR isoform. The fact that intron position is conserved in the β_1-1 genes, taken together with the observation that chromosomes 4 and 15 also contain distinct α-β-γ gene clusters, strongly suggests that an ancestral α-β-γ cluster was duplicated and translocated to at least two different chromosomes. This organization of GABA_AR gene clusters may have been preserved as linkage provides a mechanism for facilitating coordinate gene expression.     

1α,25-dihydroxy-24-oxo-16-ene vitamin D3, a metabolite of a synthetic vitamin D3 analog, 1α,25-dihydroxy-16-ene vitamin D3, is equipotent to its parent in modulating growth and differentiation of human leukemic cells

1α,25(OH)₂-16-ene-D₃, a synthetic analog of the steroid hormone, 1α,25(OH)₂D₃, has great potential to become a drug in the treatment of leukemia and other proliferative disorders, because of its minimal in vivo calcemic activity associated with a potent inhibitory effect on cell growth. However, at present, the mechanisms through which 1α,25(OH)₂-16-ene-D₃ expresses its biological activities are still not completely understood. Our previous in vitro study in a perfused rat kidney indicated for the first time that 1α,25(OH)₂-16-ene-D₃ and 1α,25(OH)₂D₃ are metabolized differently. 1α,25(OH)₂-24-oxo-16-ene-D₃, an intermediary metabolite of 1α,25(OH)₂-16-ene-D₃ formed through the C-24 oxidation pathway, accumulated significantly in the perfusate when compared to 1α,25(OH)₂-24-oxo-D₃, the corresponding intermediary metabolite of 1α,25(OH)₂D₃. In a subsequent in vivo study, we also reported that 1α,25(OH)₂-24-oxo-16-ene-D₃ exerted immunosuppressive activity equal to its parent, without causing significant hypercalcemia. In order to establish further the critical role of 1α,25(OH)₂-24-oxo-16-ene-D₃, in generating some of the key biological activities ascribed to its parent, we performed the present in vitro study using a human myeloid leukemic cell line (RWLeu-4) as a model. Comparative target tissue metabolism studies indicated that 1α,25(OH)₂-16-ene-D₃ and 1α,25(OH)₂D₃ are metabolized differently in RWLeu-4 cells, and the differences were similar to the ones we previously observed in the rat kidney. The significant finding was the accumulation of 1α,25(OH)₂-24-oxo-16-ene-D₃ in RWLeu-4 cells because of its resistance to further metabolism. Biological activity studies indicated that both 1α,25(OH)₂-16-ene-D₃ and its 24-oxo metabolite produced growth inhibition and promoted differentiation of RWLeu-4 cells to the same extent, and these activities were several fold higher than those exerted by 1α,25(OH)₂D₃. In addition, the genomic action of each vitamin D compound was assessed in a rat osteosarcoma cell line (ROS 17/2.8) by measuring its ability to transactivate a gene construct containing the vitamin D response element of the osteocalcin gene linked to the growth hormone reporter gene. In these studies, both 1α,25(OH)₂-16-ene-D₃ and its 24-oxo metabolite exerted similar but potent transactivation activity which was several fold greater than that exerted by 1α,25(OH)₂D₃ itself. In summary, our results indicate that the production and slow clearance of the bioactive intermediary metabolite, 1α,25(OH)₂-24-oxo-16-ene-D₃, in RWLeu-4 cells contributes significantly to the final expression of the enhanced biological activities ascribed to its parent analog, 1α,25(OH)₂-16-ene-D₃.     

Effects of various prostanoids on th in vitro metabolism of bovine articular chondrocytes

The dose-dependent effects of 9 prostanoids (PGA₁, PGA₂, PGE₁, PGE₂, PGF_1α, PGF_2α, PGD₂, PGI₂, 6 keto- PGF_1α) on metabolism of cultured bovine articular chrondrocytes were investigated. Most prostanoids dose-dependently inhibited ³⁵SO₄⁼ and ³H-glycine incorporation. At 25 μg/ml, the inhibitory sequence was A₂D₂>E₂ = E₁ = A₁>6 keto-F_1α>F₁>F₂, but sensivity (lowest dose eliciting inhibition) followed the sequence E₂ > 6 keto-F_1α = F₁ > A₂ = D₂>E₁>A₁. At 25 μg/ml PGA₂ also inhibited incorporation of ³H-cytidine and ^#H-thymidine, but had no significant effect on ³H-glucose or ¹⁴C-xylose incorporation. The inhibitory effect of PGA₂ was apparent after 30 minutes exposure for ³⁵SO₄= and after 60 minutesd for ³H-cytidine, and was still present up to 72 hours following incubation in fresh non-PG-containing medium. PGI₂ had no significant effect of ³⁵SO₄⁼ incorporation but at concentrations below 10 μg/ml enhanced uptake of ³H-glycine.The PG-induced inhibitory effect was apparently not due to cell damage as indicated by measurement of ³H-glucose metabolism and lactate production.     

Inhibitory Effect of Tanshinone IIA on Rat Hepatic Stellate Cells

Background

Anti-inflammation via inhibition of NF-κB pathways in hepatic stellate cells (HSCs) is one therapeutic approach to hepatic fibrosis. Tanshinone IIA (C₁₉H₁₈O₃, Tan IIA) is a lipophilic diterpene isolated from Salvia miltiorrhiza Bunge, with reported anti-inflammatory activity. We tested whether Tan IIA could inhibit HSC activation.

Materials and Methods

The cell line of rat hepatic stellate cells (HSC-T6) was stimulated with lipopolysaccharide (LPS) (100 ng/ml). Cytotoxicity was assessed by MTT assay. HSC-T6 cells were pretreated with Tan IIA (1, 3 and 10 µM), then induced by LPS (100 ng/ml). NF-κB activity was evaluated by the luciferase reporter gene assay. Western blotting analysis was performed to measure NF-κB-p65, and phosphorylations of MAPKs (ERK, JNK, p38). Cell chemotaxis was assessed by both wound-healing assay and trans-well invasion assay. Quantitative real-time PCR was used to detect gene expression in HSC-T6 cells.

Results

All concentrations of drugs showed no cytotoxicity against HSC-T6 cells. LPS stimulated NF-κB luciferase activities, nuclear translocation of NF-κB-p65, and phosphorylations of ERK, JNK and p38, all of which were suppressed by Tan IIA. In addition, Tan IIA significantly inhibited LPS-induced HSCs chemotaxis, in both wound-healing and trans-well invasion assays. Moreover, Tan IIA attenuated LPS-induced mRNA expressions of CCL2, CCL3, CCL5, IL-1β, TNF-α, IL-6, ICAM-1, iNOS, and α-SMA in HSC-T6 cells.

Conclusion

Our results demonstrated that Tan IIA decreased LPS-induced HSC activation.     

Effects of indomethacin, prostaglandin E2, prostaglandin F2α and 6-keto-prostaglandin F1α on hatching of mouse blastocysts

The present study has been performed to investigate how PGs would participate the hatching process. Effects of indomethacin, an antagonist to PGs biosynthesis, on the hatching of mouse blastocysts were examined in vitro. Furthermore, it was studied that prostaglandin E₂ (PGE₂), prostaglandin F_2α (PGF_2α) or 6-keto-prostaglandin F_1α (6-keto-PGF_1α) were added to the culture media with indomethacin. (1) The hatching was inhibited by indomethacin yet the inhibition was reversible. (2) In the groups with indomethacin and PGE₂, no improvement was seen in the inhibition of hatching and the inhibition was irreversible. (3) In the groups with indomethacin and PGF_2α, inhibition of hatching was improved in comparison with the group with indomethacin. (4) In the groups with indomethacin and 6-keto-PGF_1α, no improvement was seen. The above results indicated that PGF_2α possibly had an accelerating effect on hatching and a high concentration of PGE₂ would exert cytotoxic effect on blastocysts.     

NF-κB Mediates αvβ3 Integrin-induced Endothelial Cell Survival

The α_vβ₃ integrin plays a fundamental role during the angiogenesis process by inhibiting endothelial cell apoptosis. However, the mechanism of inhibition is unknown. In this report, we show that integrin-mediated cell survival involves regulation of nuclear factor-kappa B (NF-κB) activity. Different extracellular matrix molecules were able to protect rat aorta- derived endothelial cells from apoptosis induced by serum withdrawal. Osteopontin and β₃ integrin ligation rapidly increased NF-κB activity as measured by gel shift and reporter activity. The p65 and p50 subunits were present in the shifted complex. In contrast, collagen type I (a β₁-integrin ligand) did not induce NF-κB activity. The α_vβ₃ integrin was most important for osteopontin-mediated NF-κB induction and survival, since adding a neutralizing anti-β₃ integrin antibody blocked NF-κB activity and induced endothelial cell death when cells were plated on osteopontin. NF-κB was required for osteopontin- and vitronectin-induced survival since inhibition of NF-κB activity with nonphosphorylatable IκB completely blocked the protective effect of osteopontin and vitronectin. In contrast, NF-κB was not required for fibronectin, laminin, and collagen type I–induced survival. Activation of NF-κB by osteopontin depended on the small GTP-binding protein Ras and the tyrosine kinase Src, since NF-κB reporter activity was inhibited by Ras and Src dominant-negative mutants. In contrast, inhibition of MEK and PI3-kinase did not affect osteopontin-induced NF-κB activation. These studies identify NF-κB as an important signaling molecule in α_vβ₃ integrin-mediated endothelial cell survival.     

Long-term Incubation with Proteasome Inhibitors (PIs) Induces IκBα Degradation via the Lysosomal Pathway in an IκB Kinase (IKK)-dependent and IKK-independent Manner

Proteasome inhibitors (PIs) have been reported to induce apoptosis in many types of tumor. Their apoptotic activities have been suggested to be associated with the up-regulation of molecules implicated in pro-apoptotic cascades such as p53, p21^Waf1, and p27^Kip1. Moreover, the blocking of NF-κB nuclear translocation via the stabilization of IκB is an important mechanism of PI-induced apoptosis. However, we found that long-term incubation with PIs (PS-341 or MG132) increased NF-κB-regulated gene expression such as COX-2, cIAP2, XIAP, and IL-8 in a dose- and time-dependent manner, which was mediated by phosphorylation of IκBα and its subsequent degradation via the alternative route, lysosome. Overexpression of the IκBα superrepressor (IκBα-SR) blocked PI-induced NF-κB activation. Treatment with lysosomal inhibitors (ammonium chloride or chloroquine) or inhibitors of cathepsins (Z-FF-FMK or Z-FA-FMK) or knock-down of LC3B expression by siRNAs suppressed PI-induced IκBα degradation. Furthermore, we found that both IKK-dependent and IKK-independent pathways were required for PI-induced IκBα degradation. Pretreatment with IKKβ specific inhibitor, SC-514, partially suppressed IκBα degradation and IL-8 production by PIs. Blockade of IKK activity using insolubilization by heat shock (HS) and knock-down by siRNAs for IKKβ only delayed IκBα degradation up to 8 h after treatment with PIs. In addition, PIs induced Akt-dependent inactivation of GSK-3β. Inactive GSK-3β accelerated PI-induced IκBα degradation. Overexpression of active GSK-3β (S9A) or knock-down of GSK-3β delayed PI-induced IκBα degradation. Collectively, our data demonstrate that long-term incubation with PIs activates NF-κB, which is mediated by IκBα degradation via the lysosome in an IKK-dependent and IKK-independent manner.     

Decreased apoptosis in polyamine depleted IEC-6 cells depends on Akt-mediated NF-κB activation but not GSK3β activity

The PI3-kinase/Akt pathway promotes cell survival in many different cell types including intestinal epithelial cells. Increased AKT activation in polyamine depleted intestinal epithelial cells correlated well with the decrease in TNF-α-induced apoptosis. Increased Akt activation and GSK3β (Ser 9) phosphorylation without significant effect on Bad (Ser136) phosphorylation indicate that Akt-mediated protection is independent of Bad phosphorylation but may depend on GSK3β. Pretreatment of polyamine-depleted cells with LY294002 increased caspase-9 and caspase-3 activation and decreased basal levels of GSK-3β phosphorylation. Inhibition of GSK3β activity using AR-A014418 or lithium chloride or siRNA-mediated downregulation of its expression had no effect on apoptosis. Inhibition of PI3-kinase and over-expression of dominant negative Akt (DN-AKT), significantly increased apoptosis in polyamine depleted cells. DN-Akt expression reversed the protective effect of polyamine depletion on apoptosis. DN-Akt, as well as the PI3-kinase inhibitors, prevented Akt activation and subsequent translocation of NF-κB to the nucleus. Constitutively active Akt (CA-AKT) expression increased resistance to TNF-α-induced apoptosis. Constitutively active-Akt expression increased nuclear staining of NF-κB. Moreover, polyamine depletion of DN-Akt cells prevented basal and TNF-α-induced IκBα phosphorylation. Prevention of NF-κB activation in DN-IκBα-transfected cells increased apoptosis in control cells and restored it in polyamine-depleted cells to control levels. These data indicate that Akt regulates the mitochondrial pathway, preventing activation of caspase-9 and thereby caspase-3 via NF-κB and these effects are independent of GSK-3β activity.     

The Protective Effect of Esculentoside A on Experimental Acute Liver Injury in Mice

Inflammatory response and oxidative stress are considered to play an important role in the development of acute liver injury induced by carbon tetrachloride (CCl₄) and galactosamine (GalN)/lipopolysaccharides (LPS). Esculentoside A (EsA), isolated from the Chinese herb phytolacca esculenta, has the effect of modulating immune response, cell proliferation and apoptosis as well as anti-inflammatory effects. The present study is to evaluate the protective effect of EsA on CCl₄ and GalN/LPS-induced acute liver injury. In vitro, CCK-8 assays showed that EsA had no cytotoxicity, while it significantly reduced levels of TNF-α and cell death rate challenged by CCl₄. Moreover, EsA treatment up-regulated PPAR-γ expression of LO2 cells and reduced levels of reactive oxygen species (ROS) challenged by CCl₄. In vivo, EsA prevented mice from CCl₄-induced liver histopathological damage. In addition, levels of AST and ALT were significantly decreased by EsA treatment. Furthermore, the mice treated with EsA had a lower level of TNF-α, Interleukin (IL)-1β and IL-6 in mRNA expression. EsA prevented MDA release and increased GSH-Px activity in liver tissues. Immunohistochemical staining showed that over-expression of F4/80 and CD11b were markedly inhibited by EsA. The western bolt results showed that EsA significantly inhibited CCl₄-induced phosphonated IkBalpha (P-IκB) and ERK. Furthermore, EsA treatment also alleviated GalN/LPS-induced acute liver injury on liver enzyme and histopathological damage. Unfortunately, our results exhibited that EsA had no effects on CCl₄-induced hepatocyte apoptosis which were showed by TUNEL staining and Bax, Caspase-3 and cleaved Caspase-3 expression. Our results proved that EsA treatment attenuated CCl₄ and GalN/LPS-induced acute liver injury in mice and its protective effects might be involved in inhibiting inflammatory response and oxidative stress, but not apoptosis with its underlying mechanism associated with PPAR-γ, NF-κB and ERK signal pathways.