alpha-Crystallin downregulates the expression of TNF-alpha and iNOS by activated rat retinal microglia in vitro and in vivo.
OBJECTIVE
Inhibition of microglial activation has become an important strategy to attenuate neurotoxic damage to the central nervous system. We evaluated the effects of alpha-crystallin on the production of cytokines in lipopolysaccharides (LPS) and optic nerve injury-activated retinal microglia.


METHODS
Microglia were collected from retinas of newborn rats, cultured and treated with LPS in vitro. Microglia were also activated by an optic nerve crush in vivo. Pretreatments with and without alpha-crystallin were performed in cultured cells, and by intravitreal injection in adult rats. Expression of tumor necrosis factor-alpha (TNF-alpha), nitric oxide (NO) and inducible NOS synthase (iNOS) were measured by RT-PCR, ELISA, Western blot and the nitrate reductase method.


RESULTS
Activated microglia significantly upregulated TNF-alpha and iNOS mRNA expression and protein production in vitro. An optic nerve crush also increased expression of retinal iNOS and TNF-alpha protein. Treatment with alpha-crystallin in vitro and in vivo downregulated their expression.


CONCLUSIONS
The protective effect of alpha-crystallin may be due to its effect on microglia via a downregulation in the expression and release of 2 key immune regulatory and inflammatory molecules: TNF-alpha and iNOS.
Wu
Nan
N
Department of Ophthalmology, Southwest Eye Hospital, Southwest Hospital, Third Military Medical University, Chongqing, China.
Wang
Yan-Hua
YH
Zhao
Hai-Sheng
HS
Liu
Dong-Ning
DN
Ying
Xi
X
Yin
Zheng-Qin
ZQ
Wang
Yi
Y
D000818:Animals; D000831:Animals, Newborn; D015153:Blotting, Western; D002478:Cells, Cultured; D015536:Down-Regulation; D004797:Enzyme-Linked Immunosorbent Assay; D005786:Gene Expression Regulation / Q000187:drug effects*; D008070:Lipopolysaccharides / Q000494:pharmacology; D017628:Microglia / Q000187:drug effects / Q000378:metabolism*; D009409:Nerve Crush; D009569:Nitric Oxide / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000235:genetics* / Q000378:metabolism; D020221:Optic Nerve Injuries / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D020318:Rats, Long-Evans; D055351:Retinal Neurons / Q000187:drug effects / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics* / Q000378:metabolism; D038201:alpha-Crystallins / Q000494:pharmacology*
D008070:Lipopolysaccharides; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D038201:alpha-Crystallins; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat