Pro-inflammatory effects of commercial alpha-lactalbumin on RAW 264.7 macrophages is due to endotoxin contamination |
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Authors: | I-Chun Lin Cheng-Deng Kuo |
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Institution: | Laboratory of Biophysics, Department of Research and Education, Taipei Veterans General Hospital, Taipei 112, Taiwan |
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Abstract: | This study investigated the effects of alpha-lactalbumin (α-LA) on cellular signaling molecules associated with inflammatory responses in RAW 264.7 macrophages. The results indicated that commercial α-LA could increase prostaglandin E2 (PGE2) and the expression of COX-2 via increased phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), p38 mitogen-activated protein kinase (MAPK) and jun N-terminal kinase (JNK), and increase nitric oxide (NO) and the expression of iNOS via the activation of ERK1/2 and JNK. Furthermore, commercial α-LA could increase nuclear translocation of p65 nuclear factor-kappa B (p65 NF-κB) through stimulation on inhibitor kappa B-alpha (IκB-α) degradation. Since endotoxin also has these effects, we assayed the content of endotoxin in the commercial α-LA. We found to our surprise that endotoxin was there and that α-LA-induced NO and PGE2 production could be suppressed by polymyxin B, a specific inhibitor of endotoxin. Thus, the pro-inflammatory effects of commercial α-LA might be caused by endotoxin contamination through activation and expression of iNOS and COX-2 which were upregulated by MAPKs or nuclear translocation of p65 NF-κB in RAW 264.7 cells. It is therefore crucial to assess the possibility of endotoxin contamination within any biological product being studied for immune augmenting activities before a meaning result can be obtained. |
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Keywords: | α-LA alpha-lactalbumin COX-2 cyclooxygenase-2 Cur curcumin DAPI 4&prime 6-diamidino-2-phenylindole Dex dexamethasone ERK1/2 extracellular signal-regulated kinase 1/2 IκB-α inhibitor kappa B-alpha iNOS inducible nitric oxide synthase JNK jun N-terminal kinase l-NAME Nω-nitro-l-arginine methyl ester LPS lipopolysaccharide MAPK mitogen-activated protein kinase MTT 3-(4 5-dimethylthiazol-2-yl)-2 5-diphenyltetrazolium bromide NO nitric oxide NF-κB nuclear factor-kappa B PGE2 prostaglandin E2 NS-398 N-[2-(cyclohexyloxy)-4-nitrophenyl]methanesulfonamide PMB polymyxin B |
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