ABSTRACT
It has previously been reported that 16α, 17α-epoxypregnenolone-20-oxime (EPREGO) exerts an inhibitory effect on nitric oxide (NO) production and inducible NO synthase (iNOS) expression in microglia. The present study aimed to investigate the effects of EPREGO on the lipopolysaccharide (LPS)induced inflammatory response in RAW264.7 macrophage cells, and to determine the underlying molecular mechanisms using western blot analysis, enzymelinked immunosorbent assays and fluorescenceactivated cell sorting. The present study demonstrated that LPSinduced production of NO and interleukin (IL)-6, and the protein expression levels of iNOS, were reduced by EPREGO in a dose and timedependent manner, whereas, EPREGO did not affect tumor necrosis factorα production. In addition, EPREGO suppressed LPSinduced cellular reactive oxygen species production and phagocytosis. Furthermore, EPREGO significantly inhibited the LPSinduced activation of mitogenactivated protein kinases and inhibitor of κB α degradation in LPSstimulated RAW264.7 cells, thus resulting in modulation of the production of NO and IL6. Taken together, these results suggest that EPREGO exhibits anti-inflammatory activity in macrophages, thus validating the hypothesis that EPREGO may be useful as a therapeutic agent for the treatment of macrophage-mediated inflammation.
Subject(s)
Interleukin-6/biosynthesis , Macrophages/drug effects , Macrophages/metabolism , Nitric Oxide/biosynthesis , Oximes/pharmacology , Animals , Cell Line , Cytokines/metabolism , Inflammation Mediators/metabolism , Lipopolysaccharides/immunology , MAP Kinase Signaling System/drug effects , Macrophage Activation/immunology , Macrophages/immunology , Mice , Phagocytosis/drug effects , Phagocytosis/immunology , Reactive Oxygen Species/metabolismABSTRACT
The free radical nitric oxide (NO), a main member of neuroinflammatory cytokine and a gaseous molecule produced by activated microglia, has many physiological functions, including neuroinflammation. In the present study, we evaluated the effects of serial 16-dehydropregnenolone-3-acetate derivatives on lipopolysaccharide (LPS)-induced NO production and inducible nitric oxide synthase (iNOS) expression in BV-2 microglial cells. Among the six derivatives tested, the increases in NO production and iNOS expression observed in BV-2 microglial cells after LPS stimulation were significantly inhibited by treatment with 16α, 17α-epoxypregnenolone-20-oxime. Moreover, the inhibitory effect of 16α,17α-epoxypregnenolone-20-oxime on NO production was similar to that of S-methylisothiourea sulfate (SMT), an iNOS inhibitor. Further studies showed that 16α,17α-epoxypregnenolone-20-oxime inhibited c-Jun N-terminal kinase (JNK) phosphorylation but not inhibitor kappa B (IκB)-α degradation. Our data in LPS-stimulated microglia cells suggest that 16α,17α-epoxypregnenolone-20-oxime might be a candidate therapeutic for treatment of NO induced neuroinflammation and could be a novel iNOS inhibitor.
Subject(s)
JNK Mitogen-Activated Protein Kinases/metabolism , Lipopolysaccharides/pharmacology , Microglia/drug effects , Nitric Oxide Synthase Type II/antagonists & inhibitors , Nitric Oxide/antagonists & inhibitors , Oximes/pharmacology , Pregnenolone/analogs & derivatives , Animals , Blotting, Western , Cell Line , Dose-Response Relationship, Drug , Flow Cytometry , MAP Kinase Signaling System/drug effects , Mice , Microglia/enzymology , Microglia/metabolism , Nitric Oxide/biosynthesis , Nitric Oxide Synthase Type II/genetics , Oximes/chemical synthesis , Oximes/chemistry , Phagocytosis/drug effects , Phosphorylation , Pregnenolone/chemical synthesis , Pregnenolone/chemistry , Pregnenolone/pharmacology , Reactive Oxygen Species/metabolismABSTRACT
A steroid-converting fungus isolated from soil samples, was identified as Mucor racemosus according to its morphological characters. The application of M. racemosus for biotransformation of 4-ene-3-one steroids had been investigated to obtain hydroxylated derivatives of 4-ene-3-one steroids. The substrates were incubated with M. racemosus in rotary shaker (220 rpm) culture for a period of four days at 27 degrees C. All of the fermentation media were exhaustively extracted with ethyl acetate and filtered to separate the both from the mycelium. The transformation products were separated on silica gel column chromatography. Each microbial metabolite was characterized by spectroscopic methods such as IR, MS and NMR. Fermentation of progesterone yielded 14alpha-hydroxypregn-4-en-3, 20-dione and 7alpha, 14alpha-dihydroxypregn-4-en-3, 20-dione. Incubation of androstenedione resulted in three transformation products: 14, 17-dihydroxyandrost-4-en-3-one, 14alpha, 17beta-dihydroxyandrost-4-en-3-one and 6alpha, 17beta-dihydroxyandrost-4-en-3-one. This fungus was found to biotransformation steroids. The results showed that the fermentation of 4-ene-3-one steroids with M. racemosus yielded mainly 14alpha-hydroxy steroids.
Subject(s)
Mucor/metabolism , Soil Microbiology , Steroids/metabolism , Biotransformation , Fermentation , Molecular Structure , Mucor/chemistry , Mucor/isolation & purification , Steroids/chemistryABSTRACT
Acetylated 3beta-O-beta-glycosyl steroid derivatives were synthesized by the reaction of a new polyhydroxysteroid 3beta,5alpha,6beta-trihydroxypregn-16-en-20-one (2) with the peracetylated 1-bromo derivatives of d-glucose and d-galactose, respectively. Subsequent protection by excess acetic anhydride in pyridine selectively gave the 6beta-O-acetylated steroid glycosides. Deprotection of the acetylated steroid glycosides separately with moderate catalysis dibutyltin oxide in methanol selectively removed all acetyl groups of sugar moiety, whereas the acetyl group of the steroid part was retained. The structures of the steroid glycosides were confirmed by mass spectrometry, NMR and IR. The complete protocol was shown to be non-destructive at all stages to the sugar moieties and the steroid nucleus. These regioselective reactions open a route to the synthesis of a series of closely related isomers of 2 and other widespread polyhydroxysteroids and steroid glycosides in marine organisms and some terrestrial species.
