Inhibitory effect on LPS-induced retinal microglial activation of downregulation of t-PA expression by siRNA interference.
OBJECTIVE
This study investigated the expression and release of tissue plasminogen activator (t-PA) in lipopolysaccharide (LPS)-induced retinal microglia activation. We evaluated change in microglia activation following down-regulation of t-PA expression by siRNA interference.


METHODS
The primary cultured microglia cells were isolated from retinas of S-D rats and activated with different concentration of LPS (0, 3, 10, 30, 100, and 300 ng/ml). Double immunofluorescence (OX42 and tPA) and Western blot were used to detect t-PA expression. Next, tPA expression was down-regulated by siRNA interference, the microglia transfected with tPA siRNA lentivirus or blank control lentivirus were activated with 30 ng/ml of LPS, the culture supernatant was collected 1, 3, 6, 12, and 24 hours after LPS treatment for IL-1beta and TNF-alpha ELISA assays, and the cells were collected 24 hours later for immunocytochemistry of microglia markers (OX42 and Iba-1) and quantitative real-time PCR to determine the inhibitory efficiency of t-PA siRNA. Transfection efficiency was evaluated with flow cytometry by EGFP expression.


RESULTS
The microglia expressed t-PA when treated with LPS in a dose-dependent pattern. The expression was down-regulated by siRNA interference markedly; the inhibitory efficiency was 80% as determined by quantitative real-time PCR. Transfection efficiency during siRNA interference was 88%. The expression of Iba-1 and the release of IL-1beta and TNF-alpha were inhibited significantly when the t-PA expression was knocked down.


CONCLUSIONS
Activated microglia express t-PA. Down-regulation of t-PA expression can inhibit the activation of microglial cell.
Wang
Wen-Ying
WY
Department of Ophthalmology, Eye, Ear, Nose and Throat Hospital of Fudan University, Shanghai, People's Republic of China. wenying-wang@hotmail.com
Xu
Ge-Zhi
GZ
Tian
Jie
J
Sprecher
Alicia J
AJ
D000818:Animals; D000831:Animals, Newborn; D015153:Blotting, Western; D002478:Cells, Cultured; D016207:Cytokines / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D019084:Fluorescent Antibody Technique, Indirect; D005786:Gene Expression Regulation / Q000187:drug effects*; D008070:Lipopolysaccharides / Q000494:pharmacology; D017628:Microglia / Q000187:drug effects / Q000378:metabolism*; D034622:RNA Interference / Q000502:physiology; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D055351:Retinal Neurons / Q000187:drug effects / Q000378:metabolism*; D010959:Tissue Plasminogen Activator / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
D016207:Cytokines; D008070:Lipopolysaccharides; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D010959:Tissue Plasminogen Activator