Inhibitory Effect on LPS-Induced Retinal Microglial Activation of Downregulation of t-PA Expression by siRNA Interference

Abstract

Purpose: 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-1β and TNF-α 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-1β and TNF-α 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.

Keywords:

• activation
• cell culture
• microglia
• retina
• tissue plasminogen activator