Seong-Su Nah

Effects of advanced glycation end products on the expression of COX-2, PGE2 and NO in human osteoarthritic chondrocytes

OBJECTIVE Advanced glycation end products (AGE) accumulate in articular cartilage with age. We investigated the effects of AGE in primary-cultured human OA chondrocytes. METHODS Chondrocytes were cultured with/or without AGE-bovine serum albumin (AGE-BSA) and the expression levels of inducible nitric oxide (iNOS), cyclooxygenase (COX)-2 microsomal prostaglandin E synthase-1 (mPGES-1) were evaluated using RT-PCR and western blot analysis. Prostaglandin E(2) (PGE(2)) was analysed by ELISA and nitric oxide (NO) was analysed by Griess reaction assay. Pharmacological studies to elucidate the involved pathway were executed using specific inhibitors of MAPK and receptor for AGE (RAGE). RESULTS We found that treatment of OA chondrocytes with AGE-BSA increased COX-2, mPGES-1 and iNOS mRNA and protein, as well as elevating production of PGE(2) and NO. Pre-treatment with the MAPK inhibitors SP600125 (JNK inhibitor), SB202190 (p38 inhibitor) or PD98059 (ERK inhibitor) significantly inhibited AGE-BSA induction of COX-2 expression and production of PGE(2). In contrast, SN50, a nuclear factor-kappaB (NF-kappaB) inhibitor, had no effect on levels of COX-2 and PGE(2). SB202190 and SN50, but not SP600125 and PD98059, decreased AGE-BSA-induced production of NO. Pre-treatment with soluble receptor for AGE (sRAGE) also reduced AGE-stimulated COX-2, iNOS and PGE(2), implicating the involvement of RAGE. CONCLUSIONS These results show that AGE may augment inflammatory responses in OA chondrocytes by increasing PGE(2) and NO levels, possibly via the MAPK pathway for PGE(2) and the NF-kappaB pathway for NO.

Melatonin inhibits human fibroblast‐like synoviocyte proliferation via extracellular signal‐regulated protein kinase/P21CIP1/P27KIP1 pathways

Abstract:  The excessive proliferation and migration of synoviocytes are well‐characterized phenomena that play key roles in the pathophysiology of rheumatoid arthritis (RA). Melatonin has been shown to have potent anti‐proliferative effect in various cancer cells such as breast and prostate cancer cells. In this study, we examined the role of melatonin on synoviocyte proliferation in primary cultured human fibroblast‐like synoviocytes (FLSs) by analyzing protein expression of P21CIP1 (P21) and P27KIP1 (P27), the cyclin‐dependent kinase inhibitors that are important in cell cycle control, and the phosphorylation of mitogen‐activated protein kinases (MAPKs). RA‐FLS proliferation was determined by a [3H]‐thymidine incorporation assay. Western blot analysis was applied to examine the underlying mechanisms of melatonin’s effect. Melatonin inhibited RA‐FLS proliferation in a dose‐dependent manner. It reduced proliferation of passage 2 FLSs by 25% at 10 μm and by nearly 40% at 100 μm concentrations. The inhibitory effect of melatonin on RA‐FLS proliferation was also observed in passages 4 and 6. Melatonin upregulated the expression levels of P21 and P27 dose‐dependently (24 hr), induced the phosphorylation of extracellular signal‐regulated protein kinase (ERK) time‐dependently (10 μm), but did not affect phosphorylation of P38 in RA‐FLSs. In addition, the expression of P21 and P27 triggered by melatonin was inhibited by the pretreatment of the ERK inhibitor, PD98059 (10 μm). The anti‐proliferative action of melatonin in RA‐FLSs was also blocked by PD98059. Taken together, these results suggest that melatonin exerts the inhibitory effect of the proliferation of RA‐FLSs through the activation of P21 and P27 mediated by ERK. Hence we suggest that melatonin could be used as a therapeutic agent for the treatment of RA.

The difference between lupus nephritis class IV-G and IV-S in Koreans: focus on the response to cyclophosphamide induction treatment

OBJECTIVES To evaluate the response to induction therapy with intravenous (i.v.) cyclophosphamide (CYC) in Korean patients with class IV-G (diffuse global proliferative glomerulonephritis) and class IV-S (diffuse segmental proliferative glomerulonephritis) lupus nephritis (LN) according to the classification system of the International Society of Nephrology/Renal Pathology Society (ISN/RPS). METHODS Of the 52 patients with biopsy-proven diffuse proliferative LN, who had been treated with i.v. CYC over a 10-yr period, 42 had been treated with i.v. CYC (equal to or more than 500 mg) for 6 consecutive months and had biopsy specimens containing more than nine glomeruli. The renal pathology of these 42 patients was reclassified according to the International Society of Nephrology and the Renal Pathology Society 2003 classification, and their renal response rates and laboratory indices after induction therapy were analysed. RESULTS Of the 42 patients assessed, 30 (71%) had IV-G and 12 (29%) had IV-S. Pre-treatment 24 h urinary protein was significantly higher and pre-treatment concentration of anti-dsDNA antibody was significantly lower in IV-G than in IV-S patients. Following induction therapy, complete remission rates were significantly higher in patients with IV-S (67%, 8/12) than in patients with IV-G (33%, 10/30) LN. CONCLUSIONS Class IV-G LN responded more poorly to induction therapy with i.v. CYC pulse than class IV-S LN.

Serum cholesterol in idiopathic and lupus-related protein-losing enteropathy.

Abstract The characteristics of protein-losing enteropathy were evaluated in patients with systemic lupus erythematosus. Among the patients with systemic lupus erythematosus (n = 380) in a tertiary hospital, we reviewed the records of seven patients with generalized edema, hypoalbuminemia without proteinuria and positive results on 99mTc-labelled human serum albumin scintigrams. Patient characteristics and laboratory findings were compared between these seven patients and patients with lupus enteritis (n = 15) or idiopathic protein-losing enteropathy (n = 11). Compared with the lupus enteritis patients, the erythrocyte sedimentation rate and serum total cholesterol levels were significantly increased in patients with systemic lupus erythematosus–related protein-losing enteropathy. Compared with idiopathic protein-losing enteropathy patients, the level of serum total cholesterol was significantly increased, but the level of serum albumin was decreased in patients with systemic lupus erythematosus–related protein-losing enteropathy. Among patients with systemic lupus erythematosus–related protein-losing enteropathy, four patients had high serum total cholesterol levels (≥248 mg/dL) and achieved complete remission after receiving high doses of steroid treatment. However, three patients who had low serum total cholesterol levels (≤219 mg/dL) responded poorly to the steroid-only treatment, and could achieve complete remission only after 3 months of cyclophosphamide pulse treatment with concurrent corticosteroid therapy. The levels of serum total cholesterol are intriguing feature in systemic lupus erythematosus–associated protein-losing enteropathy patients.

Epidermal growth factor increases prostaglandin E2 production via ERK1/2 MAPK and NF-κB pathway in fibroblast like synoviocytes from patients with rheumatoid arthritis

High concentration of epidermal growth factor (EGF) is found in the synovial fluid of rheumatoid arthritis (RA) that might imply the involvement of EGF in the pathogenesis of arthritic diseases. In order to investigate if EGF is involved in the regulation of cyclooxygenase-2 (COX-2) and the prostaglandin E2 (PGE2) production in fibroblast like synoviocytes (FLS) from patients with RA. The levels of COX-2 and microsomal prostaglandin E synthase-1 (mPGES-1) were evaluated using RT-PCR and Western blot analysis. Electrophoretic mobility shift assay (EMSA) was performed to investigate EGF mediated DNA binding activity of nuclear factor-κB (NF-κB). PGE2 levels were analyzed by ELISA. EGF enhanced both COX-2 protein and mRNA expressions. mPGES-1 mRNA level was also increased by EGF treatment. EGF also stimulated ERK1/2 MAPK activity and the inhibition of ERK1/2 by PD098059 (ERK1/2 specific inhibitor) resulted in the suppression of EGF-induced COX-2 expression. The DNA binding activity of NF-κB was remarkably increased by EGF treatment and the pretreatment of PD098059 abolished EGF-stimulated NF-κB activity. We also observed that the level of PGE2 was significantly elevated with the treatment of EGF in FLS, and the pretreatment of PD098059 abolished this stimulating effect. These results suggest that EGF is involved in the inflammatory process of RA by stimulating COX-2 expression and PGE2 production. And EGF enhanced PGE2 production appears to be mediated via ERK1/2 MAPK and NF-κB pathway in FLS.

Melatonin inhibits lipopolysaccharide-induced CC chemokine subfamily gene expression in human peripheral blood mononuclear cells in a microarray analysis

Abstract:  Melatonin possesses a number of important biologic activities including oncostatic, anti‐oxidant, and immunostimulatory actions. This study was designed to assess the effects of melatonin on inflammation‐related gene expression in lipopolysaccharide (LPS)‐stimulated human peripheral blood mononuclear cells (PBMCs), using CombiMatrix 2K Human Inflammation chip. After pretreatment with melatonin (100 μm) for 4 hr, cells were incubated with LPS (1 μg/mL) for 24 hr. We compared gene expression profiles between LPS‐treated, melatonin‐treated, LSP/melatonin‐treated, and control groups. LPS induced the upregulation of 95 genes, compared with controls. Melatonin pretreatment in LPS‐stimulated PBMCs suppressed the expression of 23 genes more than twofold. Interestingly, melatonin showed a suppressive effect on the expression of CC chemokine subfamily genes, including CCL2/MCP1, CCL3/MIP1α, CCL4/MIP1β, CCL5/RANTES, CCL8/MCP2, CCL20/MDC, and CCL22/MIP3α, in LPS‐stimulated PBMCs. This result was confirmed by reverse transcriptase polymerase chain reaction. Among the CC chemokine subfamily genes, particularly, the expression of CCL2 and CCL5 was markedly downregulated by melatonin in LPS‐stimulated PBMCs. The secretion levels of CCL2 and CCL5 were measured using enzyme‐linked immunosorbent assay. Stimulation of PBMCs by LPS induced the secretion of CCL2 (2334.3 ± 161.4 pg/mL, mean ± S.E.M.), whereas melatonin pretreatment (153.0 ± 3.8 pg/mL) inhibited the LPS‐induced secretion of CCL2. Melatonin pretreatment (2696.2 ± 385.3 pg/mL) also inhibited the LPS‐induced secretion of CCL5 (4679.6 ± 107.5 pg/mL). Taken together, these results suggest that melatonin may have a suppressive effect on LPS‐induced expression of CC chemokine genes, especially CCL2 and CCL5, which may explain its beneficial effects in the treatment of various inflammatory conditions.

Association of guanosine triphosphate cyclohydrolase 1 gene polymorphisms with fibromyalgia syndrome in a Korean population.

Objective. Guanosine triphosphate cyclohydrolase 1 (GCH1) is the rate-limiting enzyme in the synthesis of tetrahydrobiopterin, which is an essential cofactor in nitric oxide (NO) production. Polymorphisms in the GCH1 gene have been implicated in protection against pain sensitivity. The aim of our study was to determine whether single-nucleotide polymorphisms (SNP) in the GCH1 gene affect susceptibility and/or pain sensitivity in fibromyalgia syndrome (FM). Methods. A total of 409 patients with FM and 422 controls were enrolled. The alleles and genotypes at 4 positions [rs3783641(T>A), rs841(C>T), rs752688(C>T), and rs4411417(T>C)] in the GCH1 gene were analyzed. The associations of the GCH1 SNP with susceptibility and clinical measures in patients with FM were assessed. Results. The frequencies of alleles and genotypes of the 4 SNP did not differ between patients with FM and healthy controls. Among 13 constructed haplotypes, we further examined 4 (CCTT, TTCT, TTCA, and CCTA) with > 1% frequency in both FM and controls. No associations of GCH1 polymorphisms with FM-related activity or severity indexes were found, although the number and total score of tender points in patients with FM differed among the 4 haplotypes (p = 0.03 and p = 0.01, respectively). The CCTA haplotype of GCH1 was associated with significantly lower pain sensitivity and occurred less frequently than the CCTT haplotype in patients with FM (p = 0.04, OR 0.45, 95% CI 0.21–0.96). Conclusion. Our study provides evidence that certain GCH1 haplotypes may be protective against susceptibility and pain sensitivity in FM. Our data suggest that NO is responsible for pain sensitivity in the pathogenesis of FM.

Roles of interferon‐gamma and its target genes in schizophrenia: Proteomics‐based reverse genetics from mouse to human

A decreased production of interferon gamma (IFNG) has been observed in acute schizophrenia. In order to explore the possible relationship between IFNG and schizophrenia, we attempted to analyze the differentially expressed proteins in the brains of interferon‐gamma knockout (Ifng‐KO) mice. Five upregulated and five downregulated proteins were identified with 2D gels and MALDI‐TOF/TOF MS analyses in Ifng‐KO mouse brain. Of the identified proteins, we focused on creatine kinase brain (CKB) and triose phosphate isomerase 1 (TPI1). Consistent with the proteomic data, reverse transcriptase‐mediated PCR, immunoblotting, and immunohistochemistry analyses confirmed that the levels of gene expressions of Ckb and Tpi1 were downregulated and upregulated, respectively. When we analyzed the genetic polymorphisms of the single nucleotide polymorphisms (SNPs) of their human orthologous genes in a Korean population, the promoter SNPs of CKB and TPI1 were weakly associated with schizophrenia. In addition, IFNG polymorphisms were associated with schizophrenia. These results suggest that IFNG and proteins affected by IFNG may play a role in the pathogenesis of schizophrenia.

Changes in Dpysl2 expression are associated with prenatally stressed rat offspring and susceptibility to schizophrenia in humans.

Exposure to stress during critical periods of fetal brain development is an environmental risk factor for the development of schizophrenia in adult offspring. In the present study, a repeated-variable stress paradigm was applied to pregnant rats during the last week of gestation, which is analogous to the second trimester of brain development in humans. Behavioral and proteomic analyses were conducted in prenatally-stressed (PNS) adult offspring and non-stressed (NS) adult controls. In the behavioral tests, grooming behavior in the social interaction test, line-crossing behavior in the open field test, and swimming behavior in the forced swimming test were decreased in the PNS group. Western blot analysis and immunohistochemical analysis revealed that the expression of dihydropyrimidinase-like 2 (Dpysl2) or collapsin response mediator protein 2 (Crmp2) was downregulated in the prefrontal cortex and hippocampus of rats in the PNS group. Subsequently, single-nucleotide polymorphisms (SNPs) of the human dihydropyrimidinase-like 2 (DPYSL2) gene were analyzed in a population. Two functional SNPs (rs9886448 in the promoter region and rs2289593 in the exon region) were associated with susceptibility to schizophrenia. The present findings demonstrated that the downregulation of genes such as Dpysl2 and Dypsl3 in a rat model of prenatal stress may affect subsequent behavioral changes and that polymorphisms of the DPYSL2 gene in humans may be associated with the development of schizophrenia. Taken together with previous studies investigating the association between the DPYSL2 gene and schizophrenia, the present findings may contribute additional evidence regarding developmental theories of the pathophysiology of schizophrenia.

Transient complete atrioventricular block associated with curcumin intake.

Curcumin is a polyphenol responsible for the yellow color of turmeric, a curry spice. A large body of evidence showed that curcumin possessed a variety of beneficial activities. We report a case of transient complete atrioventricular block in a 38-year-old man, after intake of curcumin containing pills for on 1 month. Since all other possible causes of conduction disturbance were excluded and causal relation was achieved by re-intake of the same amount of turmeric containing pills, side-effect of the curcumin containing pills was identified as the most likely diagnosis. After cessation of the pills, no further conduction disturbances and associated symptoms were noticed for the ensuing 6 months since discharge.