Minocycline inhibits apoptotic cell death via attenuation of TNF-alpha expression following iNOS/NO induction by lipopolysaccharide in neuron/glia co-cultures.
We attempted to ascertain the neuroprotective effects and mechanisms of minocycline in inflammatory-mediated neurotoxicity using primary neuron/glia co-cultures treated with lipopolysaccharide (LPS). Neuronal cell death was induced by treatment with LPS for 48 h, and the cell damage was assessed using lactate dehydrogenase (LDH) assays and by counting microtubule-associated protein-2 (MAP-2) positive cells. Through terminal transferase deoxyuridine triphosphate-biotin nick end labeling (TUNEL)-staining and by measuring caspase-3 activity, we found that LPS-induced neuronal cell death was mediated by apoptosis. We determined that pre-treatment with minocycline significantly inhibited LPS-induced neuronal cell death. In addition, LPS induced inducible nitric oxide synthase (iNOS) expression significantly, resulting in nitric oxide (NO) production within glial cells, but not in neurons. Both nitric oxide synthase (NOS) inhibitors (N(G)-monomethyl-L-arginine monoacetate (L-NMMA) and S-methylisothiourea sulfate (SMT)) and minocycline inhibited iNOS expression and NO release, and increased neuronal survival in neuron/glia co-cultures. Pre-treatment with minocycline significantly inhibited the rapid and extensive production of tumor necrosis factor-alpha (TNF-alpha) mediated by LPS in glial cells. We also determined that the signaling cascade of LPS-mediated iNOS induction and NO production was mediated by TNF-alpha by using neutralizing antibodies to TNF-alpha. Consequently, our results show that the neuroprotective effect of minocycline is associated with inhibition of iNOS induction and NO production in glial cells, which is mediated by the LPS-induced production of TNF-alpha.
Lee
Sang M
SM
Biomedical Research Center, Korea Institute of Science and Technology, Seoul, Korea.
Yune
Tae Y
TY
Kim
Sun J
SJ
Kim
Young C
YC
Oh
Young J
YJ
Markelonis
George J
GJ
Oh
Tae H
TH
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D018920:Coculture Techniques; D004790:Enzyme Induction / Q000187:drug effects; D008070:Lipopolysaccharides / Q000037:antagonists & inhibitors / Q000633:toxicity; D008911:Minocycline / Q000494:pharmacology*; D009457:Neuroglia / Q000187:drug effects* / Q000378:metabolism; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism; D018696:Neuroprotective Agents / Q000494:pharmacology; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
D008070:Lipopolysaccharides; D018696:Neuroprotective Agents; D014409:Tumor Necrosis Factor-alpha; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat; D008911:Minocycline