Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
2130056 | Experimental Cell Research | 2015 | 11 Pages |
Abstract
The aim of this paper is to investigate the protective effects of adrenomedullin (ADM) on interleukin-1β (IL-1β)-induced inflammation and apoptosis in rat Leydig cells and its underlying molecular mechanisms. Leydig cells were isolated from adult Sprague-Dawley rats. The cell culture was established by adding ADM 2 h prior to 24 h treatment with IL-1β-induced cytotoxicity. We detected cell viability and concentrations of testosterone, reactive oxygen species (ROS), malondialdehyde (MDA), and reduced glutathione (GSH). Gene expression levels were measured for inducible nitric oxide synthase (iNOS) and cyclo-oxygenase-2 (COX-2). Concentrations were detected for nitric oxide (NO) and prostaglandin E2 (PGE2). Apoptosis was assessed using terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). Levels of gene expression and protein were detected for Bcl-2, Bax, caspase-3, and poly adenosine diphosphate-ribose polymerase (PARP). Protein levels were measured for nuclear factor kappa B (NF-κB) p65 and IκBα. ADM reduced IL-1β-induced cytotoxicity. ADM pretreatment significantly increased testosterone concentrations and decreased ROS, MDA, and GSH concentrations. ADM pretreatment inhibited IL-1β-induced inflammation in Leydig cells by decreasing the gene expression levels of iNOS and COX-2, as well as the concentrations of NO and PGE2. ADM pretreatment further decreased the number of TUNEL-positive stained Leydig cells, as confirmed by the increase in gene expression and protein levels of Bcl-2 and the decrease of Bax, caspase-3, and PARP levels. Moreover, ADM pretreatment inhibited NF-κB p65 phosphorylation and IκBα phosphorylation and degradation. ADM has potential anti-inflammatory and anti-apoptotic properties in IL-1β-induced rat Leydig cells, which might be related to NF-κB signaling pathway.
Keywords
cyclo-oxygenase-2PGE2iNOSTBARSDAPICOX-2GSHIL-1βRIAADMPARPFBSDMEMPBSH2DCF-DAMDANF-κB2′,7′-dichlorodihydrofluorescein diacetateDulbecco's modified Eagle's mediumMTTROSadrenomedullininflammationInterleukin-1βterminal deoxynucleotidyl transferase dUTP nick-end labelingELISAEnzyme-linked immunosorbent assayTUNELApoptosisradioimmunoassayfetal bovine serumLeydig cellinducible nitric oxide synthaseNuclear factor-kappa Bmalondialdehydephosphate buffer solutionthiobarbituric acid reactive substancesNitric oxidepolymerase chain reactionPCRProstaglandin E2GlutathioneReactive oxidative species
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Authors
Wei Hu, Pang-hu Zhou, Ting Rao, Xiao-bin Zhang, Wei Wang, Li-jun Zhang,