Repression of excessive increase and enlargement of adipocytes that is closely associated with obesity is effective in the prevention and treatment of metabolic syndrome. Generally, apoptosis is induced in cells via a wide variety of intracellular or extracellular substances, and recently, it has been suggested that the FoxO subfamily is involved in the induction of apoptosis. We aimed to elucidate the mechanism of FoxO-mediated apoptosis-induction in the adipocytes under the reactive oxygen species (ROS) stimulus. The treatment of differentiated and undifferentiated 3T3-L1 cells with glucose oxidase (GOD), an enzyme that generates H(2)O(2), induced apoptosis and led to the accumulation of 8-OHdG. Apoptosis analysis revealed that GOD treatment induced apoptosis in differentiated 3T3-L1 cells less efficiently than in undifferentiated preadipocytes. GOD remarkably increased the levels of Bad, Bax, and Bim-the genes that are actively involved in cell apoptosis. GOD treatment also increased the expression of FoxO3a mRNA and protein. The introduction of FoxO3a-siRNA into 3T3-L1 cells suppressed the oxidative stress-induced expression of Bim mRNA, as well as the GOD-induced apoptosis. Furthermore, the expression of MnSOD, Cu/ZnSOD, and catalase, as well as of FoxO, increased significantly along with the progression of adipocyte differentiation. These results indicated that ROS-induced apoptosis in undifferentiated 3T3-L1 cells via the expression of FoxO3a, whereas FoxO expression suppressed the ROS-induced apoptosis in differentiated 3T3-L1 cells via the expression of ROS-scavenging enzymes.
Background Cyclic phosphatidic acid (cPA) has an inhibitory effect on the autotaxin (ATX)/lysophosphatidic acid (LPA) axis, which has been implicated to play an important role in the progression of fibrosis in systemic sclerosis (SSc). The purpose of this study is to assess the antifibrotic activity of cPA for the treatment of SSc using SSc skin fibroblasts and an animal model of bleomycin-induced skin fibrosis. Methods We used a chemically stable derivative of cPA (2ccPA). First, we investigated the effect of 2ccPA on extracellular matrix (ECM) expression in skin fibroblasts. Next, the effect of 2ccPA on the intracellular cAMP levels was determined to investigate the mechanisms of the antifibrotic activity of 2ccPA. Finally, we administered 2ccPA to bleomycin-induced SSc model mice to evaluate whether 2ccPA prevented the progression of skin fibrosis. Results 2ccPA decreased ECM expression in SSc skin fibroblasts and TGF-β1-treated healthy skin fibroblasts without LPA stimulation. 2ccPA increased the intracellular cAMP levels in skin fibroblasts, suggesting that the antifibrotic effect of 2ccPA was the consequence of the increase in the intracellular cAMP levels. Administration of 2ccPA also ameliorated the progression of bleomycin-induced skin fibrosis in mice. Conclusions Our data indicated that 2ccPA had inhibitory effects on the progression of skin fibrosis by abrogating ECM production from activated skin fibroblasts. These cells were repressed, at least in part, by increased intracellular cAMP levels. 2ccPA may be able to be used to treat fibrotic lesions in SSc.
BackgroundSystemic sclerosis (SSc) is a connective tissue disorder with progressive fibrosis in multiple organs including skin, lung and the gastrointestinal tract. Fibrosis is thought to be driven by activated fibroblasts. Therefore, inhibition of the profibrotic activity of activated fibroblasts may be a promising therapeutic approach in skin fibrosis in SSc. The autotaxin (ATX)/lysophosphatidic acid (LPA) axis is reportedly involved in fibrotic pathogenesis in SSc1. 2-carba cyclic phosphatidic acid (2ccPA) is a naturally occurring lipid mediator and one of its pleiotropic properties is to inhibit the ATX/LPA axis.ObjectivesWe investigated the anti-fibrotic effect of 2ccPA on human SSc skin fibroblasts and bleomycin-induced skin fibrosis in mice. Furthermore, we searched signaling pathways related to the anti-fibrotic effects of 2ccPA.MethodsThis study was approved by the ethics committee and the ethical review committee of animal experiments of Tokyo Women’s Medical University. We informed all participants of the content of this study, and written consent was obtained. Skin fibroblasts were taken from SSc patients and adult healthy individuals were purchased. The cells were incubated with 1-10 μM 2ccPA in the presence or absence of 10 ng/ml transforming growth factor-β1 (TGF-β1). Messenger RNA (mRNA) and protein expression for type I collagen, connective tissue growth factor (CTGF), α smooth muscle actin (αSMA), fibronectin (FN) and TGF-β1 were assessed by qRT-PCR or Western blotting. Procollagen type I C-peptide, prostaglandin E2 (PGE2) and hepatocyte growth factor (HGF) levels in the supernatant were assessed by ELISA. Intracellular cyclic adenosine monophosphate (cAMP) levels were calculated using a commercially available EIA kit. Forskolin was used to increase intracellular cAMP levels in cultured SSc skin fibroblasts. An inhibitor of adenylate cyclase (AC), 2’-deoxyadenosine, was used to investigate whether the anti-fibrotic effect of 2ccPA was mediated via the AC/cAMP pathway. Furthermore, we used a mouse model of bleomycin-induced skin fibrosis to investigate the safety and anti-fibrotic effects of 2ccPA.ResultsTen μM 2ccPA significantly reduced mRNA and protein expression for type I collagen, CTGF and αSMA in SSc skin fibroblasts and adult healthy skin fibroblasts treated with TGF-β1. PGE2 and HGF levels in the supernatant of SSc skin fibroblasts were also reduced by 2ccPA treatment. 2ccPA increased intracellular cAMP levels as well as the AC stimulator, forskolin. In addition, forskolin decreased the mRNA expression of profibrotic markers. Reduction of COL1A1 mRNA expression by 2ccPA was blocked by treatment with 2’-deoxyadenosine in cultured SSc skin fibroblasts, suggesting that the anti-fibrotic activity of 2ccPA was partially mediated via AC stimulation. In mouse experiments, intraperitoneal injection of 10 mg/kg 2ccPA significantly reduced the development of skin thickness, collagen content and αSMA-positive cell counts.Conclusion2ccPA suppressed the profibrotic activity of SSc skin fibroblasts and...
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