The involvement of RGS9 in l-3,4-dihydroxyphenylalanine-induced dyskinesias in unilateral 6-OHDA lesion rat model.

Chronic dopamine (DA) replacement therapy with L-3,4-dihydroxyphenylalanine (L-DOPA) in Parkinson's disease (PD) often leads to abnormal involuntary movements (AIMs) known as L-DOPA-induced dyskinesia (LID), mediated by DA receptors. However, mechanisms underlying LID occurrence are still unclear. Regulator of G-protein signaling RGS9, a member of the RGS family of GTPase accelerating proteins, is expressed specifically in the striatum, has been reported participated in LID. L-DOPA-induced AIMs can be modeled in rats with 6-hydroxydopamine (6-OHDA) lesions by chronic injection of L-DOPA. Herein, we compared the rotational responses and AIMs in 6-OHDA lesioned rats with L-DOPA/benserazide (10/2.5 mg/kg, once per day, i.p.) administration for 14 days whereas control animals received injections of saline. Furthermore, whether sub-chronic L-DOPA treatment impact RGS9 mRNA or protein expression in 6-OHDA lesion rats were also evaluated. As results shown, rotational behavior was not increased significantly, while an obvious AIMs were observed in rats with L-DOPA/benserazide (10/2.5mg/kg, i.p.) administration sub-chronically. In addition, expressions of RGS9 protein or mRNA analyzed by Western blot or real-time PCR with striatal extracts increased significantly after L-DOPA/benserazide. These data demonstrate that RGS9 expression can be modulated by sub-chronic L-DOPA/benserazide administration and increased RGS9 expression in striatum may be one of the reasons for the side effects such as dyskinesia induced by L-DOPA therapy.

Rotenone-induced neurotoxicity of THP-1 cells requires production of reactive oxygen species and activation of phosphatidylinositol 3-kinase.

Parkinson's disease is characterized by slow and progressive degeneration of dopaminergic neurons. Increasing evidence has suggested an important role for exposure to pesticides such as rotenone in the pathogenesis of Parkinson's disease. Although rotenone can elicit immune responses in microglia, the intracellular signaling events mediating these effects are poorly defined. Here we show that cell-free supernatants of rotenone-treated monocytic THP-1 cells induced cytotoxicity in dopaminergic neuroblastoma SH-SY5Y cells. Exposure of THP-1 cells to rotenone led to transient production of reactive oxygen species (ROS) and phosphorylation of Akt. Akt activation was also induced by exogenous hydrogen peroxide. Pretreatment of THP-1 cells with either a phosphatidylinositol 3-kinase (PI3K) inhibitor or ROS scavengers prevented Akt activation and protected SH-SY5Y cells from the cytotoxic effect of conditioned media from rotenone-treated THP-1 cells. Rotenone treatment of THP-1 cells also led to upregulation of cyclooxygenase-2 and secretion of prostaglandin E2. These results suggest that rotenone-induced activation of ROS/PI3K/Akt pathway in THP-1 cells leads to the release of factors that are toxic to SH-SY5Y cells and have implications for the onset of Parkinson's disease.

Behavioral recovery following sub-chronic paeoniflorin administration in the striatal 6-OHDA lesion rodent model of Parkinson's disease.

In the present studies, the effect of paeoniflorin (PF), one of the main compounds extracted from Paeoniae radix, in alleviating the neurological impairment following unilateral striatal 6-hydroxydopamine (6-OHDA) lesion was examined in Sprague-Dawley rats. Sub-chronic PF (2.5, 5 and 10 mg/kg, s.c., twice daily for 11 days) administration dose-dependently reduced apomorphine (APO)-induced rotation, suggesting that PF had an ameliorative effect on the 6-OHDA-induced neurological impairment. Notably, PF had no direct action on dopamine D(1) receptor or dopamine D(2) receptor indicated by the competitive binding experiments. These results suggest that PF, an active component of Paeoniae radix, might provide an opportunity to introduce a non-dopaminergic management of Parkinson's disease.

The enhancement of dopamine D1 receptor desensitization by adenosine A1 receptor activation.

The present study was designed to examine the effects of adenosine A(1) receptor on dopamine D(1) receptor desensitization in a human embryonic kidney 293 cell line stably cotransfected with human adenosine A(1) receptor and dopamine D(1) receptor cDNAs (A(1)D(1) cells) by means of cAMP accumulation assay. Long-term exposure of A(1)D(1) cells to dopamine D(1) receptor agonist (+/-)-1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol hydrochloride (SKF38393) caused a rapid desensitization of dopamine D(1) receptor. Coadministration of adenosine A(1) receptor agonist N(6)-cyclopentyladenosine (CPA) potentiated the effect of SKF38393. This enhancement effect of CPA was blocked by adenosine A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) but not by pertussis toxin, indicating that this effect of CPA was mediated by adenosine A(1) receptor and was G(i) protein independent. Furthermore, the blockade of endogenous adenosine by adenosine deaminase or DPCPX attenuated dopamine D(1) receptor desensitization. Collectively, these results suggest that adenosine A(1) receptor plays an important role in the regulation of dopamine D(1) receptor by potentiating ligand-induced desensitization.

Inhibitory effects of procyanidin B(2) dimer on lipid-laden macrophage formation.

A proteomic analysis of procyanidin B(2) isolated from cocoa against oxidized low-density lipoprotein-induced lipid-laden macrophage formation was performed. Of approximately 400 detected proteins, 12 were differentially expressed as a result of B(2) treatment. They were subsequently identified by liquid chromatography-electrospray ionization-tandem mass spectrometry and the SWISS-PROT database. Further reverse transcriptase-polymerase chain reaction and Western blot analysis revealed that B(2) strongly inhibited arachidonic acid inflammatory reactions, apoptosis, and their coupled mitogen-activated protein kinase and NF-kappaB pathways. To highlight proteins or genes with similar expressed patterns and similarly biological function induced by B(2) in lipid-laden macrophages, a cluster and Kyoto Encyclopedia of Genes and Genomes pathway analysis were performed. The data were mapped to multiple pathways. Further validation of the bioinformatic results revealed that activation of Wnt signaling may contribute to the cardioprotection of B(2). The differentially expressed genes and proteins mentioned above induced by B(2) are through regulating nuclear transcription factors, activating peroxisome proliferator-activated receptor-gamma and inhibiting AP-1 mRNA expressions. These in vitro data help to interpret the beneficial effects of B(2) in reducing the risk of atherosclerosis after consumption of flavonoid-rich foods. Many differentially expressed genes induced by B(2) help to uncover novel targets and may help to target disease interactions in atherosclerosis in the future.

Activation of adenosine A1 receptor modulates dopamine D1 receptor activity in stably cotransfected human embryonic kidney 293 cells.

The antagonistic interactions between adenosine A1 receptors and dopamine D1 receptors were studied in a human embryonic kidney 293 cell line stably cotransfected with human adenosine A1 receptor and dopamine D1 receptor cDNAs. In the cotransfected cells, but not in control cells only transfected with dopamine D1 receptors, adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA, 10 microM) increased the Kd of dopamine D1 receptor antagonist [N-methyl-3H]R(+)-7-Chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine ([3H]SCH23390) without affecting the Bmax. Moreover, CPA induced a concentration-dependent decrease in the affinity of dopamine D1 receptors for the agonist (+/-)-1-Phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol hydrochloride (SKF38393) and inhibited dopamine D1 receptor-mediated cyclic AMP response element recruitment. Furthermore, pertussis toxin treatment completely counteracted the effects of low concentrations of CPA but only partially counteracted the effects of high concentrations of CPA. These results suggest that adenosine A1 receptors antagonistically modulate dopamine D1 receptors at the level of receptor binding and the second messenger generation. Furthermore, the antagonistic interactions between these two receptors induced by low concentrations of CPA might have a different manner with those induced by high concentrations of CPA.

Cryptotanshinone inhibits cyclooxygenase-2 enzyme activity but not its expression.

Cyclooxygenase-2 (COX-2) is a key enzyme that catalyzes the biosynthesis of prostaglandins from arachidonic acid and plays a critical role in some pathologies including inflammation, neurodegenerative diseases and cancer. Cryptotanshinone is a major constituent of tanshinones, which are extracted from the medicinal herb Salvia miltiorrhiza Bunge, and has well-documented antioxidative and anti-inflammatory effects. This study confirmed the remarkable anti-inflammatory effect of cryptotanshinone in the carrageenan-induced rat paw edema model. Since the action of cryptotanshinone on COX-2 has not been previously described, in the present study, we further examined the effect of cryptotanshinone on cyclooxygenase activity in the exogenous arachidonic acid-stimulated insect sf-9 cells, which highly express human COX-2 or human COX-1, and on cyclooxygenases expression in human U937 promonocytes stimulated by lipopolysaccharide (LPS) plus phorbolmyristate acetate (PMA). Cryptotanshinone reduced prostaglandin E2 synthesis and reactive oxygen species generation catalyzed by COX-2, without influencing COX-1 activity in cloned sf-9 cells. In PMA plus LPS-stimulated U937 cells, cryptotanshinone had negligible effects on the expression of COX-1 and COX-2, at either a mRNA or protein level. These results demonstrate that the anti-inflammatory effect of cryptotanshinone is directed against enzymatic activity of COX-2, not against the transcription or translation of the enzyme.

Potentiation of adenosine A1 receptor agonist CPA-induced antinociception by paeoniflorin in mice.

The effect of paeoniflorin (PF), a major constituent isolated from Paeony radix, on N6-Cyclopentyladenosine (CPA), a selective adenosine A1 receptor (A1 receptor) agonist, induced antinociception was examined in mice. In the tail-pressure test, CPA (0.05, 0.1, 0.2 mg/kg, s.c.) could induce antinociception in a dose-dependent manner. PF (5, 10, 20 mg/kg, s.c.) alone failed to exhibit any antinociceptive effect in mice; however, pretreatment of PF (20 mg/kg, s.c.) could significantly enhance CPA-induced antinociception. Additionally, pretreatment of 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX, 0.25 mg/kg, s.c.), a selective A1 receptor antagonist, could antagonize the antinociceptive effect of combining CPA with PF. Furthermore, in the competitive binding experiments, PF did not displace the binding of [3H]-8-Cyclopentyl-1,3-dipropylxanthine ([3H]-DPCPX) but displaced that of [3H]-2-Chloro-N6-cyclopentyladenosine ([3H]-CCPA, a selective A1 receptor agonist) to the membrane preparation of rat cerebral cortex. These results suggested that PF might selectively increase the binding and antinociceptive effect of CPA by binding with A1 receptor.

Involvement of multitargets in paeoniflorin-induced preconditioning.

Paeoniflorin (PF) is the principal component of Paeoniae radix prescribed in traditional Chinese medicine. The delayed neuroprotection induced by PF preconditioning and its underlying mechanisms were investigated in rat middle cerebral artery occlusion (MCAO) and reperfusion model. At a dosage of 20 or 40 mg/kg, PF preconditioning 48 h before MCAO followed by 24-h reperfusion significantly reduced the mortality and infarct volume and reversed the neurological deficits caused by ischemia. Likewise, the ameliorative effects on mortality, infarct size, and neurological impairment induced by MCAO emerged as well when PF was administered 24 h, 48 h, or 5 days before MCAO at the dose of 20 mg/kg. Furthermore, comparative proteomics analysis was adopted to identify the differentially expressed proteins induced by PF preconditioning itself. The relative levels of 42 proteins were altered after PF preconditioning, among which 20 were elevated and 22 reduced. In summary, A(1) receptor-regulator of G protein signaling-K(ATP) signaling, arachidonic acid cascade, nitric oxide system, markers of neuronal damage, mitochondrial damage-related molecules, and the mitogen-activated protein kinase and nuclear factor-kappaB pathway are associated with the mechanisms of PF preconditioning.

Monocyte-mediated rotenone neurotoxicity towards human neuroblastoma SH-SY5Y: role of mitogen-activated protein kinases.

Increasing evidence has suggested an important role for rotenone in the pathogenesis of Parkinson's disease (PD). In this report, sequential linking of two culture systems, monocytic THP-1 cell line and SH-SY5Y neuroblastoma, was utilized. The supernatant from rotenone-stimulated THP-1 cells was used as the incubating medium for the second culture which adopted cells of the SH-SY5Y neuroblastoma. At 6.25-50 nM, concentrations that were nontoxic to SH-SY5Y directly, rotenone induced dose-dependent cell death on SH-SY5Y through stimulating monocyte THP-1 within a period of 48 h. Cytotoxicity was determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Hoechst 33258 staining revealed that the treatment of SH-SY5Y with rotenone-stimulated THP-1 supernatant resulted in condensed nuclei and a decrease in cell size. Apoptotic rate measured by flow cytometric analysis indicated that at 25 and 50 nM, the percentage of apoptotic SH-SY5Y cells accumulated to 31.5% and 37.0% respectively. We further investigated whether rotenone (50 nM) activated mitogen-activated protein kinase (MAPK) cascades, and found it had effect on p38 MAPK and ERK in THP-1 cells, but not JNK. Pretreatment of THP-1 cells with the MAPK kinase inhibitor, PD98059, inhibited THP-1 cell-mediated rotenone neurotoxicity towards SH-SY5Y, whereas the p38 MEK inhibitor, SB203580, had no effect. These results suggested that activation of microglia intracellular signaling pathway may also involve in microglia-enhanced rotenone neurotoxicity.

Procyanidin dimer B2 [epicatechin-(4beta-8)-epicatechin] suppresses the expression of cyclooxygenase-2 in endotoxin-treated monocytic cells.

The anti-inflammatory activity of the predominant procyanidin dimer in cocoa, dimer B2, was investigated in this study. Pretreatment of the procyanidin dimer B2 reduced COX-2 expression induced by the endotoxin lipopolysaccharide (LPS) in differentiated human monocytic cells (THP-1) in culture. To further elucidate the underlying mechanism of COX-2 inhibition by procyanidin, we examined their effects on the activation of extracellular signal-regulated protein kinase (ERK), Jun-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK), which are upstream enzymes known to regulate COX-2 expression in many cell types. Pretreatment with procyanidin dimer B2 decreased the activation of ERK, JNK, and p38 MAPK. In addition, procyanidin dimer B2 suppressed the NF-kappaB activation through stabilization of IkappaB proteins, suggesting that these signal-transducing enzymes could be potential targets for procyanidin dimer B2. By affecting the expression rather than the activity of COX-2, these in vitro data reported herein give further evidence on the anti-inflammatory protection by procyanidins.

Paeoniflorin attenuates chronic cerebral hypoperfusion-induced learning dysfunction and brain damage in rats.

Chronic cerebral hypoperfusion, a mild ischemic condition, is associated with the cognitive deficits of AD. Paeoniflorin (PF), a major constituent of peony root, was proved to be neuroprotective in middle cerebral artery occlusion model. In this study, we investigated whether PF could attenuate chronic cerebral hypoperfusion-induced learning dysfunction and brain damage in rat. Seven weeks after permanent bilateral occlusion of the common carotid arteries, the rats were tested in the Morris water maze. Subsequently, the animals were sacrificed and neurons, astrocytes and microglias were labeled with immunocytochemistry in hippocampus. PF at the dose of 2.5 mg/kg ameliorated cerebral hypoperfusion-related learning dysfunction and prevented CA1 neuron damage. Chronic cerebral hypoperfusion increased the immunoreactivity of astrocytes and microglias in hippocampus. The increase was prevented by PF at the dose of 2.5 mg/kg. Cerebral hypoperfusion also increased expression of nuclear factor-kappaB (NF-kappaB), mostly in astrocytes, but not in neurons. With the treatment of PF (2.5 mg/kg), NF-kappaB immunostaining was diminished in hippocampus. Our results demonstrated that PF could attenuate cognitive deficit and brain damage induced by chronic cerebral hypoperfusion and that suppression of neuroinflammatory reaction in brain might be involved in PF-induced neuroprotection.

Lignans from Saussurea conica and their NO production suppressing activity.

Three new lignan derivatives, conicaols A and B and conicaoside, together with seven known compounds were identified from Saussurea conica. Their structures were elucidated by spectral methods including 1D and 2D NMR techniques. Six 2,3-dibenzylbutyrolactone-type lignans were biologically evaluated, and two of them, arctigenin and matairesinol, showed potent activity to suppress NO production in vitro in LPS-activated macrophages of SD rat.

Paeoniflorin attenuates neuroinflammation and dopaminergic neurodegeneration in the MPTP model of Parkinson's disease by activation of adenosine A1 receptor.

1. This study examined whether Paeoniflorin (PF), the major active components of Chinese herb Paeoniae alba Radix, has neuroprotective effect in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of Parkinson's disease (PD). 2. Subcutaneous administration of PF (2.5 and 5 mg kg(-1)) for 11 days could protect tyrosine hydroxylase (TH)-positive substantia nigra neurons and striatal nerve fibers from death and bradykinesia induced by four-dose injection of MPTP (20 mg kg(-1)) on day 8. 3. When given at 1 h after the last dose of MPTP, and then administered once a day for the following 3 days, PF (2.5 and 5 mg kg(-1)) also significantly attenuated the dopaminergic neurodegeneration in a dose-dependent manner. Post-treatment with PF (5 mg kg(-1)) significantly attenuated MPTP-induced proinflammatory gene upregulation and microglial and astrocytic activation. 4. Pretreatment with 0.3 mg kg(-1) 8-cyclopentyl-1,3-dipropylxanthine, an adenosine A1 receptor (A1AR) antagonist, 15 min before each dose of PF, reversed the neuroprotective and antineuroinflammatory effects of PF. 5. In conclusion, this study demonstrated that PF could reduce the MPTP-induced toxicity by inhibition of neuroinflammation by activation of the A1AR, and suggested that PF might be a valuable neuroprotective agent for the treatment of PD.

Triptolide inhibits COX-2 expression via NF-kappa B pathway in astrocytes.

Previous investigations have showed that triptolide possessed potent anti-inflammatory and immunosuppressive properties. In the present study, we examined the protective effects of triptolide on the inflammatory response induced by bacterial lipopolysaccharide (LPS) both in vivo and in vitro. Intrahippocampal injection of LPS (4 microg) in rats significantly increased the immunoreactivity of glial fibrillary acid protein (GFAP) and cyclooxygenase-2 (COX-2) in the injected region, which was reduced by pretreatment with triptolide (10-50 microg/kg) for 5d. In the cultured human differentiated A172 astroglial cells, LPS (1mg/L) increased the expression of COX-2 mRNA and protein, the production of prostaglandin E(2) (PGE(2)) and the DNA binding activity of NF-kappa B, which were markedly attenuated by pretreatment with triptolide (0.2-5 microg/L) for 1h. These results suggested that the protective effect of triptolide on neuroinflammation is mediated by decreasing COX-2 expression, at least partly, via the inhibition of NF-kappa B signaling pathway.

Design and synthesis of a biotin-tagged photoaffinity probe of paeoniflorin.

A trifunctional probe (binding element-photoreactive group-affinity tag) of natural product paeoniflorin was designed and synthesized based on the previous primary structure-activity relationship. This new probe is a potential tool for labeling, purification, and identification of the target proteins.

Design, synthesis, and biological evaluation of isoquinoline-1,3,4-trione derivatives as potent caspase-3 inhibitors.

A series of isoquinoline-1,3,4-trione derivatives were identified as novel and potent inhibitors of caspase-3 through structural modification of the original compound from high-throughput screening. Various analogues (2, 6, 9, 13, and 14) were synthesized and identified as caspase inhibitors, and the introduction of a 6-N-acyl group (compound 13) greatly improved their activity. Some of them showed low nanomolar potency against caspase-3 in vitro (for example, for 6k, IC50 = 40 nM) and significant protection against apoptosis in a model cell system. Additionally, compound 13f demonstrated a dose-dependent decrease in infarct volume in the transient MCA occlusion stroke model. The present small-molecule caspase-3 inhibitor with novel structures different from structures of known caspase inhibitors revealed a new direction for therapeutic strategies directed against diseases involving abnormally up-regulated apoptosis.

Role of the second transmembrane domain of rat adenosine A1 receptor in ligand-receptor interaction.

Initial mutagenesis studies exploring the ligand recognition model of A1 adenosine receptor (A1R) mainly focused on the residues in the 5th-7th transmembrane domains (TMs5-7). Little is known about the role of residues in TM2. To explore the importance of reserved hydrophobic region in TM2 of A1R, we mutated the hydrophobic residues at positions 65 and 69 to hydrophilic residues (L65T, Leu-65 to Thr-65; I69T, Ile-69 to Thr-69; I69S, Ile-69 to Ser-69) to change the hydrophobicity at the outer end of TM2. Binding assays showed that the affinities of mutant receptors were significantly decreased for ribose group-containing agonists (2-chloro-N6-cyclopentyladenosine (CCPA) and 5'-N-ethyl-carboxamidoadenosine (NECA)) but not for antagonists, N6-cyclopentyl-9-methyladenine (N-0840), an adenine derivative lacking ribose group, and 8-cyclopentyl-1, 3-dipropylxanthine (DPCPX), a xanthine derivative. This observation suggests that the hydrophobic region at the outer end of TM2 may mediate the recognition of the ribose group of CCPA and NECA.

Comparison of psychic emergence reactions after (+/-)-ketamine and (+)-ketamine in mice.

Ketamine is a racemic mixture containing equal parts of (+)-ketamine and (-)-ketamine. The ketamine enantiomorphs are different in anesthesia and psychic emergence reactions after anesthesia. Therefore, (+)-ketamine was compared with racemic ketamine in a number of randomized studies in volunteers and patients. However, their relations remain controversial. In the present studies, the psychic emergence reactions after injection of (+/-)-ketamine and (+)-ketamine were compared in mice. At equimolar doses, the (+)-isomers elicited episodes of hypnosis nearly 1.4-fold more potent than those of the racemic ketamine. After the administration of equihypnotic doses of (+)-ketamine and (+/-)-ketamine, the posthypnotic stimulation of locomotor activity, stereotype behavior and 5-HT-induced head-twitch response by the (+)-enantiomorph was significantly less intense than that of racemic ketamine. In receptor binding test, (+)-ketamine showed a higher affinity for NMDA receptor than that of (+/-)-ketamine, while (+)-ketamine and (+/-)-ketamine showed no affinity for dopamine D2 and serotonin 5-HT2 receptor. These results suggest that the (+)-ketamine has fewer posthypnotic side effects than (+/-)-ketamine when (+)-ketamine and (+/-)-ketamine were administered at equihypnotic dosages and that dopamine D2 and serotonin 5-HT2 receptor were not involved in the effects of (+)-ketamine and (+/-)-ketamine.

CC 05, a novel anti-inflammatory compound, exerts its effect by inhibition of cyclooxygenase-2 activity.

In the present study, we examined the anti-inflammation of a novel compound, 4-[5-(3-amino-4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide (CC 05) in vitro and in vivo. In an in vitro cell-based assay, CC 05 inhibited cyclooxygenase (COX)-2-derived prostaglandin E(2) (PGE(2)) synthesis with an IC(50) value of 0.328+/-0.04 microM compared with an IC(50) value of 14.34+/-0.05 microM for the inhibition of COX-1-derived PGE(2) synthesis. In two in vivo rodent models, CC 05 (12.5, 25 and 50 mg/kg, i.g.) is a moderate potential and selective inhibitor of COX-2. It can reduce carrageenan-induced paw edema and PGE(2) production in the inflamed pouch dose-dependently without affecting the PGE(2) production in stomachs. Furthermore, CC 05 had no effect on COX-2 mRNA and protein expression in phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 human macrophages stimulated with lipopolysaccharide (LPS). These results demonstrate that CC 05 is a novel COX-2 inhibitor and the anti-inflammatory action is not directed towards the transcription or translation of the COX-2 gene but only to the enzymatic activity of the protein.