Effectiveness of conjunctival bleb scarring by knock-down of heat shock protein 47 in rat model.

Abstract

To evaluate the effectiveness of knock-down of heat shock protein 47 (HSP47) on conjunctival bleb scarring in a rat model and its possible mechanism. Male Sprague-Dawley rats were used for glaucoma filtration surgery (GFS) and were treated with either phosphate buffered solution, shControl, mitomycin C, or sh-HSP47 using a microsyringe immediately after GFS. The morphology of filtering blebs was observed postoperatively. The levels of HSP47 were analyzed at 2, 5, 8, and 11d after GFS via real-time quantitative polymerase chain reaction (PCR) and Western blot. The silencing effect of HSP47, the expression of collagen I and III, and the potential signaling pathways of HSP47 during scarification were explored 11d post GFS. The protein levels of transforming growth factor-β1 (TGF-β1), phospho-Smad2 (pSmad2), phospho-Smad3 (p-Smad3), and phospho-p38 (p-p38) were also analyzed using Western blot. Sh-HSP47 treatment significantly prolonged the functional filtration bleb retention. The levels of HSP47 were increased significantly at 5, 8, and 11d postoperatively compared to the control group (P<0.05, P<0.01, and P<0.001). The levels of HSP47 protein at day 11 postoperatively were significantly down-regulated after HSP47 silencing using sh-HSP47 adenovirus transfection (P<0.01). Expression levels of collagen I and III within the blebs were significantly reduced in the absence of HSP47 (P<0.01). Moreover, the protein levels of TGF-β1, p-Smad2/3, and p-p38 were dramatically inhibited after treatment with sh-HSP47 (P<0.01). The inhibitory effects of HSP47 knock-down on scarring after GFS have the potential to be an efficacious therapeutic option for the treatment of conjunctival bleb scarring.