Inhibition of Cytokine-Mediated Nitric Oxide Synthase Expression in Rat Insulinoma Cells by Scoparone

Abstract

Autoimmune diabetes is characterized by a local inflammatory reaction in and around the pancreatic islets, followed by selective destruction of insulin-producing b-cells.1) Much evidence supports an effector role for cytokines in mediating b-cell destruction.2,3) Incubation of rat islets with interleukin1b (IL-1b) alone, or in combination with tumor necrosis factor (TNF)-a and interferon-g (IFN-g) results in the expression of the inducible form of nitric oxide synthase (iNOS), increased production of nitric oxide (NO), and potent inhibition of glucose-stimulated insulin secretion. [4][5][6] Heitmeier et al. 7) demonstrated that IL-1b-induced inhibition of insulin secretion and nitrite production by rat islets were completely prevented by N w -nitro-L-arginine methylester (L-NAME) and aminoguanidine.One cellular target activated in response to IL-1b is the transcriptional regulator nuclear factor kB (NF-kB).8,9) NFkB is normally bound to inhibitory kB (IkB) in the cytosol; this binding prevents its movement into the nucleus. Various stimuli induce the phosphorylation of IkB, which releases NF-kB, and translocates to the nucleus, where it interacts with its DNA recognition sites to mediate gene transcription. 10,11) Scoparone (6,7-dimethoxycoumarin), a derivative of coumarin (1,2-benzopyrone) is known to have potent immunosuppressive, vascular relaxant and lipid lowering properties. [12][13][14][15] In this study, we examined the feasibility of scoparone as a means of preventing IL-1b and IFN-g-induced b-cell destruction. Scoparone inhibited IL-1b and IFN-g-induced NF-kB activation, iNOS expression, NO formation, glucose-stimulated insulin secretion (GSIS), and cell death of RIN cells, which may explain the beneficial effects of scoparone as an anti-diabetic agent. MATERIALS AND METHODSCell Culture RIN, clone 5F (RINm5F), is an insulinoma cell line derived from the NEDH rat islet cell tumor.16) Cells were purchased from the American Type Culture Collection and grown at 37°C under a humidified, 5% CO 2 atmosphere in RPMI 1640 (Gibco BRL) supplemented with 10% fetal bovine serum, 2 mM glutamine, 10000 units/ml of penicillin, 10 mg/ml of streptomycin, and 2.5 mg/ml of amphoptericin B.Cell Viability Assay The viability of cultured cells was determined by assaying the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan as described previously. 17) After treatment, cells in 96-well plates were washed twice with PBS. MTT (100 mg/100 ml of PBS) was added to each well. Cells were then incubated at 37°C for 1 h, and DMSO (100 ml) was added to dissolve the formazan crystals. Absorbance was measured at 570 nm with a model Spectra MAX PLUS (Molecular Devices).Cell Proliferation Assay A cell proliferation enzymelinked immunosorbent assay, BrdU kit (Amersham Biosciences, U.K.) was used to measure the incorporation of 5-bromo-2-deoxyuridine (BrdU) during DNA synthesis according to the manufacturer's protocol. Briefly, cells were seeded overnight in black 96-well tissue culture plates with c...

Coptidis rhizoma extract protects against cytokine-induced death of pancreatic β-cells through suppression of NF-κ B activation

“…We have already reported on the protective effects of herbs and dietary supplements against cytokine-or cytotoxin-induced -cell damage via the suppression of iNOS expression (Park and Park, 2001;Kwon et al, 2003aKwon et al, , b, 2005Kwon et al, , 2006Kim et al, 2007). Many other groups have also attempted to save -cells from those insults by repressing either NF-B activation or NF-B-dependent iNOS expression (Scarim et al, 1998;Evans et al, 2002).…”

Development of a rapid and validated method for investigating the metabolism of scoparone in rat using ultra‐performance liquid chromatography/electrospray ionization quadruple time‐of‐flight mass spectrometry

“…In order to demonstrate predominantly the superiority of UPLC with quadruple time‐of‐flight mass spectrometry (QTof‐MS) in metabolite detection and identification, the rat metabolism of scoparone was investigated. Scoparone (6,7‐dimethoxycoumarin), a derivative of coumarin (1,2‐benzopyrone), is known to have potentially immunosuppressive, vascular relaxant, prevention of diabetes mellitus progression, and lipid lowering properties,12–16 and many studies have demonstrated that scoparone,17, 18 a representative compound in Yin Chen Hao Tang preparation, shows hepatoprotectivity, and thus could be used as a biomarker to evaluate the therapeutic effects of the formulae. The pharmacokinetics of scoparone are well understood,19, 20 and the metabolism of scoparone has been preliminarily investigated;21 however, those works only partly understood the metabolic performance; the analytical efficiency and conclusions were not thoroughly expected.…”

Radix asari extract protects pancreatic β cells against cytokine-induced toxicity: Implication of the NF-κB-iNOS signaling cascade

“…We previously reported that herbs and dietary supplements had protective effects against cytokine-or cytotoxin-induced ß-cell damage that occurred through the suppression of iNOS expression (7,21,27,43,44). Numerous other studies have also attempted to demonstrate that ß cells could be protected from cytokine and cytotoxin-induced damage by repressing either NF-κB activation or NF-κB-dependent iNOS expression (45)(46)(47)(48).…”