• Sheng-Hua Wu, Chao Lu, Ling Dong, Guo-Ping Zhou, Zha-Guang He, and Zi-Qing Chen.
• Department of Pediatrics, Central Laboratory, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu, People's Republic of China. kad-yc@163.com
• Kidney Int. 2005 Jul 1;68(1):35-46.

BackgroundStudies have shown that lipoxin A(4) (LXA(4)) and its analogues inhibited proliferation of glomerular mesangial cells induced by leukotriene D(4) (LTD(4)) or platelet-derived growth factor (PDGF), reduced the production of proinflammatory cytokines such as interleukin (IL)-1beta and IL-6 in renal tissue of ischemic injury. In the present studies, we examine whether LXA(4) have inhibitory effects on tumor necrosis factor-alpha (TNF-alpha)-induced productions of IL-1beta and IL-6 and proliferation of glomerular mesangial cells of rat, and explore the molecular mechanisms of signal pathway of LXA(4).MethodsCultured glomerular mesangial cells were treated with TNF-alpha (10 ng/mL), with or without preincubation with LXA(4) at the different concentrations. Cell proliferation was assessed by [(3)H]-thymidine incorporation. Proteins of IL-1beta and IL-6 in supernatant were analyzed by enzyme-linked immunosorbent assay (ELISA). Expressions of mRNA of IL-1beta and IL-6 were determined by real-time polymerase chain reaction (PCR) and cyclin E by reverse transcription (RT)-PCR. Proteins of cyclin E, threonine phosphorylated Akt(1) at 308 site (Thr(308)) and p27(kip1) were analyzed by Western blotting studies. Activities of signal transducers and activators of transcription-3 (STAT(3)), nuclear factor-kappaB (NF-kappaB) were determined by electrophroretic mobility shift assay (EMSA). Expression of Src homology (SH) 2-containing protein-tyrosine phosphatase (SHP-2) was assessed by immunoprecipitation and immunoblotting.ResultsTNF-alpha-stimulated proliferation, release of proteins and expressions of mRNA of IL-1beta and IL-6 in mesangial cells were inhibited by LXA(4) in a dose-dependent manner. The marked increments in mRNA expression and protein synthesis of cyclin E induced by TNF-alpha in parallel with proliferation of mesangial cells were down-regulated by LXA(4). LXA(4) antagonized the phosphorylation of SHP-2 and activity of NF-kappaB induced by TNF-alpha. Pretreatment of the cells with NF-kappaB inhibitor pyrrolidine dithio-carbamate (PDTC) blocked the productions of IL-1beta, IL-6, and activation of NF-kappaB induced by TNF-alpha. Stimulation of mesangial cells with TNF-alpha resulted in enhanced DNA-binding activity of STAT(3). This increment was inhibited by LXA(4) in a dose-dependent manner. Threonine phosphorylated Akt(1) protein at 308 site stimulated by TNF-alpha was reduced by LXA(4.) TNF-alpha-induced decrement in expression of p27(kip1) protein was ameliorated by LXA(4) in a dose-dependent manner.ConclusionTNF-alpha-induced proliferation and increment of cyclin E of rat mesangial cells can be inhibited by LXA(4), and these inhibitory effects might be through the mechanisms of STAT(3) and Akt(1)/p27(kip1) pathway-dependent signal transduction. LXA(4) also antagonized TNF-alpha-stimulated IL-1beta and IL-6 synthesis, and these antagonisms were related to SHP-2 and NF-kappaB pathway-dependent signal transduction.

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