[Effect of Zishen Huoxue Recipe on Pathomorphology in Coronary Heart Disease Rats with Shen Deficiency Blood Stasis Syndrome].

OBJECTIVE: To observe the effect of Zishen Huoxue Recipe (ZHR) on pathomorphology in coronary heart disease (CHD) rats with Shen deficiency blood stasis syndrome (SDBSS). METHODS: Totally 60 healthy Wistar rats were divided into the blank control group, the model group, high, middle, and low dose ZHR groups according to random digit table, 12 in each group. Myocardial ischemia SDBSS rat model was prepared by ligating the left anterior descending coronary artery and injecting hydrocortisone. ZHR physic liquor was administered to rats in high, middle, and low dose ZHR groups at the daily dose of 21.6, 10.8, 5.4 g/kg by gastrogavage for 7 successive days, equal volume of pure water was administered to rats in the blank control group and the model group by gastrogavage for 7 successive days. Rat heart was collected for pathomorphological observation under light microscope. RESULTS: In the model group the heart muscle fiber was swollen and deformed with widened space, loose and dropsy tissues. Blood vessels in myocardial mesenchymal were dilated, infiltrated with more inflammatory cells. Myocardial cells were markedly swollen, degenerated, or necrotic, with caryolysis or disappearance of partial nuclear. A large amount of collagen fibrous tissue became hyperplasia. Endocardial blood vessels were swollen and degenerated with infiltration of few inflammatory cells. Epicardium tissue and structure were destroyed and got hyperplasia. Swollen, degenerated, or necrotic vessels could be seen, with infiltration of more inflammatory cells and collagen deposition. Pathomorphological injuries were alleviated in each ZHR group. The higher ZHR concentration, the milder the injury degree of myocardial tissue, the more limited range of damage. CONCLUSION: ZHR could attenuate pathomorphological injuries of myocardial ischemia rats with SDBSS and regulate myocardial function, thus improving myocardial ischemia in CHD rats with SDBSS.

The MADS29 transcription factor regulates the degradation of the nucellus and the nucellar projection during rice seed development.

The MADS box transcription factors are critical regulators of rice (Oryza sativa) reproductive development. Here, we here report the functional characterization of a rice MADS box family member, MADS29, which is preferentially expressed in the nucellus and the nucellar projection. Suppressed expression of MADS29 resulted in abnormal seed development; the seeds were shrunken, displayed a low grain-filling rate and suppressed starch biosynthesis, and contained abnormal starch granules. Detailed analysis indicated that the abnormal seed development is due to defective programmed cell death (PCD) of the nucellus and nucellar projection, which was confirmed by a TUNEL assay and transcriptome analysis. Further studies showed that expression of MADS29 is induced by auxin and MADS29 protein binds directly to the putative promoter regions of genes that encode a Cys protease and nucleotide binding site-Leu-rich repeat proteins, thereby stimulating the PCD. This study identifies MADS29 as a key regulator of early rice seed development by regulating the PCD of maternal tissues. It provides informative clues to elucidate the regulatory mechanism of maternal tissue degradation after fertilization and to facilitate the studies of endosperm development and seed filling.

A gene mutation in RNA-binding protein 10 is associated with lung adenocarcinoma progression and poor prognosis.

RBM10 regulates the expression of various genes, which are often mutated in male lung adenocarcinoma. The present study confirmed the association of the RBM10 mutation at exon 10 with the clinicopathological data and prognosis of lung adenocarcinoma. The effect of mutant RBM10 on regulating lung cancer cell growth and invasion was investigated in vitro. Tissue specimens from 50 patients with lung adenocarcinoma were subjected to Sanger sequencing for RBM10 exon 10 mutations. Lung adenocarcinoma cells were transfected with pcDNA3.1 carrying wild type RBM10 cDNA or exon mutation cDNA for cell viability, apoptosis and invasion assays. RBM10 exon 10 mutations were identified in 11 out of 50 patients, with a high frequency in male patients [c.763 C>T, p.Arg241Cys for 33.3% (10/30)] and were significantly associated with the American Joint Committee on Cancer stage (P=0.005), lymph node metastasis (P=0.019) and shorter 5-year survival rate compared with the wild type RBM10 (36.4% vs. 46.5%; P=0.019). Multivariate analysis revealed that RBM10 exon 10 mutation was an independent prognostic factor (HR=3.787; P=0.033). RBM10 exon 10 mutation at c.763 C>T significantly promoted tumor cell proliferation and invasion capacity, whereas wild type RBM10 inhibited tumor cell invasion in vitro. In conclusion, RBM 10 mutation at exon 10 (c.763 C>T) occurs frequently and is an independent prognostic predictor in lung adenocarcinoma.

Lenalidomide improvement of cisplatin antitumor efficacy on triple-negative breast cancer cells in vitro.

Lenalidomide is an immunomodulatory drug and possesses anti-angiogenic and immunomodulatory activities against multiple myeloma. The present study assessed the in vitro effect of lenalidomide combined with cisplatin on MDA-MB-231, a triple-negative breast cancer (TNBC) cell line and explored the underlying molecular mechanism of this combination. Cell viability, apoptosis and the protein expression of phosphorylated (p) and total extracellular signal-regulated kinase (ERK), B-cell lymphoma-2 (Bcl-2), caspase-3, cleaved poly-adenosine diphosphate-ribose polymerase (cPARP), vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) were measured in MDA-MB-231 cells treated with different concentrations of lenalidomide, cisplatin and their combination using different biochemical assays. Lenalidomide demonstrated no significant effect on the cell viability of MDA-MB-231 cells, even at high concentrations, whereas lenalidomide in combination with cisplatin, significantly reduced cisplatin IC50 from 7.8 to 3.0 µM in MDA-MB-231 cells. In addition, lenalidomide and cisplatin in combination significantly induced cell apoptosis by 1.6- and 1.38-fold, respectively compared with lenalidomide and cisplatin alone (P<0.05). The expression levels of VEGF, bFGF and Bcl-2 proteins were significantly reduced (P<0.01), whereas caspase-3 and cleaved PARP expression were significantly increased in MDA-MB-231 cells treated with the combination compared to those treated with single agents (P<0.01). Lenalidomide treatment alone significantly reduced the p-ERK level compared with the control (P<0.05) and cisplatin treatment alone significantly increased it (P<0.01), however treatment with them in combination significantly reduced the p-ERK level in MDA-MB-231 cells compared with cisplatin treatment alone (P<0.05). In conclusion, the present study provides the basis for using lenalidomide in combination with cisplatin in TNBC therapy.

Profiling microRNA from Brain by Microarray in a Transgenic Mouse Model of Alzheimer's Disease.

MicroRNAs (miRNAs) are small noncoding RNAs, which regulate numerous cell functions by targeting mRNA for cleavage or translational repression, and have been found to play an important role in Alzheimer's disease (AD). Our study aimed to identify differentially expressed miRNAs in AD brain as a reference of potential therapeutic miRNAs or biomarkers for this disease. We used amyloid precursor protein (APP) and presenilin 1 (PS1) double transgenic mice and age-matched wild-type (WT) littermates to determine the expression of miRNAs in the brain. MiRNAs were profiled by microarray, and differentially expressed miRNAs underwent target prediction and enrichment analysis. Microarray analysis revealed 56 differentially expressed miRNAs in AD mouse brain, which involved 39 miRNAs that were significantly upregulated and 19 that were downregulated at different ages. Among those miRNAs, a total of 11 miRNAs, including miR-342-3p, miR-342-5p, miR-376c-3p, and miR-301b-3p, were not only conserved in human but also predicted to have targets and signaling pathways closely related to the pathology of AD. In conclusion, in this study, differentially expressed miRNAs were identified in AD brain and proposed as biomarkers, which may have the potential to indicate AD progression. Despite being preliminary, these results may aid in investigating pathological hallmarks and identify effective therapeutic targets.

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.

Arabidopsis PROTEASOME REGULATOR1 is required for auxin-mediated suppression of proteasome activity and regulates auxin signalling.

The plant hormone auxin is perceived by the nuclear F-box protein TIR1 receptor family and regulates gene expression through degradation of Aux/IAA transcriptional repressors. Several studies have revealed the importance of the proteasome in auxin signalling, but details on how the proteolytic machinery is regulated and how this relates to degradation of Aux/IAA proteins remains unclear. Here we show that an Arabidopsis homologue of the proteasome inhibitor PI31, which we name PROTEASOME REGULATOR1 (PTRE1), is a positive regulator of the 26S proteasome. Loss-of-function ptre1 mutants are insensitive to auxin-mediated suppression of proteasome activity, show diminished auxin-induced degradation of Aux/IAA proteins and display auxin-related phenotypes. We found that auxin alters the subcellular localization of PTRE1, suggesting this may be part of the mechanism by which it reduces proteasome activity. Based on these results, we propose that auxin regulates proteasome activity via PTRE1 to fine-tune the homoeostasis of Aux/IAA repressor proteins thus modifying auxin activity.

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.

The involvement of central cholinergic system in (+)-matrine-induced antinociception in mice.

The antinociceptive effect of (+)-matrine was examined in mice by writhing, tail-pressure and hot-plate tests. (+)-Matrine (5, 10 and 20 mg/kg s.c.) produced antinociception in a dose-dependent manner. In hot-plate test, the antinociception produced by (+)-matrine (10 mg/kg s.c.) was attenuated by muscarinic receptor antagonists atropine (5 mg/kg i.p.) and pirenzepine (0.1 mug/mouse i.c.v.) and acetylcholine depletor hemicholinium-3 (HC-3) (1 mug/mouse i.c.v.), but not by opioid receptor antagonist naloxone (2 mg/kg i.p.), dopamine D(2) receptor agonist (-)-quinpirole (0.1 mg/kg i.p.) or catecholamine depletor reserpine (2.5 mg/kg i.p.). Radioligand binding assay demonstrated that (+)-matrine had no affinity for mu-, kappa- or delta-opioid receptors in a wide concentration range (1 x 10(-11)-1 x 10(-3) M). The results suggest that (+)-matrine exerts its antinociceptive effect through multiple mechanism(s) such as increasing cholinergic activation in the CNS rather than acting on opioid receptors directly.

The effect of 7-nitroindazole on the acquisition and expression of D-methamphetamine-induced place preference in rats.

The present study investigated the role of nitric oxide (NO) in the rewarding effects of D-methamphetamine using 7-nitroindazole, a potent inhibitor of neuronal nitric oxide synthase (nNOS), as determined by the conditioned place preference paradigm. Male Sprague-Dawley rats treated with D-methamphetamine (1 mg/kg) or saline every other day for 8 days (four drug and four saline sessions) developed marked place preference for the drug-paired side. The administration of 7-nitroindazole (12.5-50 mg/kg) 30 min prior to the exposure to D-methamphetamine dose-dependently attenuated the acquisition of D-methamphetamine-induced conditioned place preference. In addition, when it was acutely administered 30 min prior to the testing session of an already established D-methamphetamine-induced conditioned place preference, 7-nitroindazole (12.5-50 mg/kg) attenuated the expression of this conditioned response in a dose-dependent manner, while 7-nitroindazole (25 and 50 mg/kg) alone showed no place preference effects. These findings indicate that nitric oxide (NO) is involved in the rewarding properties of methamphetamine and suggest that selective nNOS inhibitors maybe useful in the management of methamphetamine abuse.

Concomitant depletion of PTEN and p27 and overexpression of cyclin D1 may predict a worse prognosis for patients with post-operative stage II and III colorectal cancer.

Prognostic markers for colorectal cancer (CRC) have not yet been fully investigated. Phosphatase and tensin homolog (PTEN), p27 and Cyclin D1 play significant roles in tumorigenesis and cell cycle regulation, and therefore require evaluation for their prognostic value in this disease. The aim of the present study was to assess the prognostic value of the single and combined expression of PTEN, p27 and Cyclin D1 in CRC patients. Protein expression levels of PTEN, p27 and Cyclin D1 were examined by immunohistochemistry from 61 patients with CRC in either stage II or III. In the CRC tissues, the frequencies of PTEN(-), p27(-) and Cyclin D1(+) expression were 42.62% (26/61), 32.79% (20/61) and 45.90% (28/61), respectively. Depletion of PTEN and p27 was more common with respect to stage III, low grade and lymph node metastasis compared with stage II, moderate grade and no lymph node metastasis (P<0.05). Cyclin D1-positive expression was frequently detected in CRC of stage III, with lymph node metastasis and deeper invasion (P<0.05). The depletion of PTEN was significantly correlated with the loss of p27 (P<0.001) and with the increased expression of Cyclin D1 (P<0.001). PTEN(-) and/or p27(-) expression was significantly correlated with Cyclin D1(+) expression (P<0.05). Combined PTEN(-)/p27(-)/Cyclin D1(+) expression was correlated with a significant decrease in overall survival time (P<0.05). Combined p27(-) and Cyclin D1(+) expression indicated a worse overall survival time than other combined expression patterns. These findings indicate that the single expression of PTEN(-), p27(-) and Cyclin D1(+) and the combined detection of p27(-) and Cyclin D1(+) may be used as prognostic markers for overall survival time in CRC.

Functional genomics based understanding of rice endosperm development.

Seed development, especially the relevant regulatory mechanism and genetic network are of fundamental scientific interest. Seed development consists of the development of embryo and endosperm; and endosperm development of rice (model species of monocots) is closely related to grain yield and quality. Recent genetic studies, together with other approaches, including transcriptome and proteomics analysis, high-throughput sequencing (RNA-seq, ChIP-seq), revealed the crucial roles of genetic and epigenetic controls in rice endosperm development. Here we summarize and update the genetic networks involved in the regulation of endosperm initiation, cell cycle regulation, aleurone layer specification, starch synthesis, storage protein accumulation and endosperm size, and the interactions between embryo and endosperm.

Epimedium flavonoids ameliorate experimental autoimmune encephalomyelitis in rats by modulating neuroinflammatory and neurotrophic responses.

The present study was designed to determine whether epimedium flavonoids (EF) had effect on the development of experimental autoimmune encephalomyelitis (EAE) in rats and to elucidate its underlying mechanisms. EAE was induced by immunization of adult female Lewis rats with partially purified myelin basic protein (MBP) prepared from guinea-pig spinal cord homogenate. EF was administrated intragastrically once a day after immunization until day 14 post immunization (p.i.). Histopathological staining, enzyme-linked immunosorbent assay (ELISA), biochemical methods and western blotting approaches were used to evaluate the disease incidence and severity, neuroinflammatory and neurotrophic response in the central nervous system (CNS). Intragastrical administration of EF (20 and 60 mg/kg) significantly reduced clinical score of neurological deficit in EAE rats; alleviated demyelination and inflammatory infiltration; and inhibited astrocytes activation, production of proinflammatory molecules such as interleukin-1ß (IL-1ß), tumour necrosis factor-α (TNF-α), nitric oxide (NO) and nuclear transcription factor (NF-κB) in the spinal cord of EAE rats. Treatment with EF also enhanced the expression of 2', 3'-cyclic nucleotide 3'-phosphohydrolase (CNPase) and nerve growth factor (NGF), increased the number of oligodendrocytes and protected the ultrastructure of myelin sheaths and axons in the spinal cord of EAE rats. Our results showed that EF inhibited the development of partial MBP-induced EAE in rats. This effect involved reducing neuroinflammation and enhancing myelination and neurotrophins and our findings suggest that EF may be useful for the treatment of multiple sclerosis.

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.

ERK pathway activation is required for amyloid-ß(1-40)(-)induced neurotoxicity of THP-1 human monocytes towards SK-N-SH neuroblastoma.

Alzheimer's disease (AD) is characterized by amyloid-ß peptide deposition, increased activated microglia, and progressive loss of neurons in the brain. Although Aß1₋40 can elicit inflammation in microglia, the intracellular signaling events mediating these effects are poorly defined. Here we show that cell-free supernatant from Aß1₋40-treated THP-1 monocytes induced cytotoxicity towards neuroblastoma SK-N-SH cells. Exposure of THP-1 monocytes to Aß1₋40 leads to increased tyrosine phosphorylation and extracellular signaling-regulated kinase (ERK) and increased levels of inflammatory cytokines (IL-1ß, IL-8, and TNF-α) in the supernatant of THP-1 monocytes. Pretreatment of THP-1 monocytes with either a protein tyrosine kinase (PTK) inhibitor or an ERK inhibitor protects SK-N-SH cells from the cytotoxic effect of conditional supernatant from Aß1₋40-treated THP-1 monocytes. Aß1₋40-treated THP-1 monocytes also lead to upregulation of cyclooxygenase-2 and iNOS expression and increased of nitric oxide production. These results suggest that Aß1₋40-induced activation of PTK/MEK/ERK pathway in THP-1 monocytes leads to the release of inflammatory factors that are toxic to SK-N-SH cells and might contribute to the onset of AD.

Decreased RGS9 protein level in the striatum of rodents undergoing MPTP or 6-OHDA neurotoxicity.

Western blot has been used to study the time-course effect of the two most popular parkinsonian neurotoxins, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP, i.p.) and 6-hydroxydopamine (6-OHDA, intra-substantia nigra), on the expression of several regulators of G-protein signaling (RGS2, 4 and 9) in striatum in rodents. During the few days after MPTP challenge, there was a decline (as expected) in tyrosine hydroxylase expression in the mouse striatum that was accompanied by a decline in RGS9 protein; the latter was specific and did not extend to RGS2 or RGS4 which were resistant to the MPTP challenge. Much the same pattern was observed in rats after 6-OHDA challenge, again, specific to RGS9, although the effect takes a few weeks, rather than a few days, to develop. These results may be helpful for the understanding of molecular mechanism underlying Parkinson's disease (PD) and RGS9 might involve in the striatal function associated with PD.

Tenuigenin protects cultured hippocampal neurons against methylglyoxal-induced neurotoxicity.

Methylglyoxal is a metabolite of glucose. Since serum methylglyoxal level is increased in diabetic patients, methylglyoxal is implicated in diabetic complications such as cognitive impairment. This study aimed to evaluate the effects of tenuigenin, an active component of roots of Polygala tenuifolia Willdenow, on methylglyoxal-induced cell injury in a primary culture of rat hippocampal neurons. MTT and Hoechst 33342 staining, together with flow cytometric analysis using annexin-V and propidium (PI) label, indicated that tenuigenin pretreatment attenuated methylglyoxal -induced apoptotic cell death in primary cultured hippocampal neurons, showing a dose-dependent pattern. Furthermore, 2, 7-dichlorodihydrofluorescein diacetate was used to detect the level of intracellular reactive oxygen species. Tenuigenin decreased the elevated reactive oxygen species induced by methylglyoxal. In addition, tenuigenin inhibited activation of caspase-3 and reversed down-regulation of the ratio of Bcl-2/Bax, both of which were induced by methylglyoxal stimulation. The results suggest that tenuigenin displays antiapoptotic and antioxidative activity in hippocampal neurons due to scavenging of intracellular reactive oxygen species, regulating Bcl-2 family and suppressing caspase-3 activity induced by methylglyoxal, which might explain at least in part the beneficial effects of tenuigenin against degenerative disorders involving diabetic cognitive impairment.

A novel 1,5-diarylpyrazole derivative exerts its anti-inflammatory effect by inhibition of cyclooxygenase-2 activity as a prodrug.

In the present study, we designed and synthesized a novel 1,5-diarylpyrazole derivative, 2-amino-N-(2-methyl-5-(1-(4-sulfamoylphenyl)-3-(trifluoromethyl)-1H-pyrazol-5-yl)phenyl) acetamide hydrochloride (CC06), which was intended to act as a prodrug and would exert potent anti-inflammatory activity after being converted to its parent compound in vivo. In vitro cell-based biological assay, CC06 showed decreased inhibitory effects on cyclooxygenase (COX)-1 and COX-2 compared with its parent compounds, but it exhibited potent anti-inflammatory activity in vivo. The anti-inflammatory effect was evaluated in a carrageenan-induced rat paw edema model and CC06 (15, 30, 60 mg/kg, intragastrically) reduced rat paw edema in a dose-dependent manner. CC06 is also a selective inhibitor of COX-2 since it can reduce prostaglandin E(2) (PGE(2)) production in the inflamed pouch dose-dependently without affecting PGE(2) production in stomach in rat air pouch model. Furthermore, preliminary pharmacokinetics experiments were conducted using high performance liquid chromatography/mass spectrometry (HPLC/MS) to detect whether CC06 can convert to its parent compound or not. Our results supported the hypothesis that CC06 was actually converted to its parent compound. These suggested that CC06 served as an anti-inflammatory prodrug and actually converted to its parent compound to exert its anti-inflammatory effect. This finding will be of great benefit in carrying out structural modifications of prodrug-like selective COX-2 inhibitors.

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.

Design, synthesis, and anti-inflammatory evaluation of a series of novel amino acid-binding 1,5-diarylpyrazole derivatives.

AIM: To design and synthesize a series of novel amino acid-binding 1,5-diarylpyrazole derivatives, which are intended to act as prodrugs with better aqueous solubility than celecoxib, and which will exert potent anti-inflammatory activities after being converted to their parent compounds in vivo. METHODS: To introduce an amino acid, celecoxib analogs containing amino or methylamino group were synthesized first through multi-step chemical reactions. All the synthesized compounds were screened in an intact cell-based assay in vitro and in carrageenan-induced mouse paw edema in vivo. Some active compounds were selected for further evaluation in a carrageenan-induced rat paw edema model. The preliminary pharmacokinetics experiments were conducted using high performance liquid chromatography/mass spectrometry (HPLC/MS). RESULTS: Celecoxib, 6 of the 1,5-diarylpyrazole class of celecoxib analogs, and their amino acid derivatives (hydrochloride salts) were synthesized. In vitro screening, the hydrochloride salts showed decreased inhibitory effects on cyclooxygenase (COX)-1 and COX-2 compared with their parent compounds, but some exhibited potent anti-inflammatory activity in vivo. Compound 4a was selected for further evaluation, and its anti-inflammatory effect was equivalent to that of celecoxib after oral administration in the carrageenan-induced rat paw edema model. At three doses (25 mg/kg, 50 mg/kg, and 100 mg/kg) the percentage inhibition on edema was 20.7%, 52.6%, and 62.6% (for compound 4a) and 27.8%, 38.4%, and 40.1% (for celecoxib), respectively. Preliminary pharmacokinetic evaluations support the hypothesis that compound 4a was actually converted to its parent compound, compound 4. CONCLUSION: The compound bound with amino acid acts like prodrug, which can exert anti-inflammatory effect similar to celecoxib after being converted to its parent compound. This finding will be of great benefit in carrying out structural modifications of prodrug-like selective COX-2 inhibitors.