Abstract
Purpose: :
Ocular inflammation is one of the major cause of blindness due to bacterial infections or autoimmune distresses. We have shown earlier that aldose reductase (AR) mediates the synthesis of inflammatory markers and their autocrine effects induced by various oxidants such as high glucose, cytokines, and bacterial endotoxins. However it is not known how AR mediates ocular inflammatory signals, hence present study was performed.
Methods: :
To examine the effect of AR inhibition on ocular inflammatory signals in vitro, we have incubated human lens epithelial cells (HLEC) with lipopolysaccharide (LPS) without or with AR inhibitors (ARI) or siRNA. Cell viability was assessed by cell counting and MTT assay. NF-ΚB activation was measured by EMSA and reporter gene assays. NF-ΚB upstream signals such as activation of various protein kinases were measured by Western blots. NF-ΚB downstream signals such as expression of inflammatory markers were measured by specific ELISAs. To examine the effect of ARI on ocular inflammation in vivo, rodent models of endotoxin- induced uveitis (EIU) and experimental autoimmune uveitis (EAU) were investigated in the absence and presence of ARI. EIU was developed in Lewis rats by injection of LPS (200 ug/rat, s.c) and EAU by immunizing rats with bovine IRBP (100 ug/rat). The rats were killed at 24 h after LPS injection, and 14 days after IRBP immunization, eyes were enucleated and aqueous humor was collected. The number of infiltrating cells and the levels of inflammatory markers were determined in the aqueous humor. Immunohistochemical analysis was performed in sections of rat eyes to examine the expression of various inflammatory markers.
Results: :
The pharmacological inhibition or siRNA ablation of AR in HLEC prevented LPS-induced generation of reactive oxygen species (ROS), activation of protein kinases such as MAPK, ERK, JNK and PKC, activation of transcription factors such as NF-ΚB and AP1, expression of inflammatory markers such as NO, PGE2, MMP9, MMP2, Cox-2, and TNF-α, and cell growth. Inhibition of AR also significantly prevented the infiltration of inflammatory cells and increased levels of inflammatory markers in the aqueous humor of EIU and EAU rats. AR inhibition also suppressed generation of ROS, activation of NF-kB and expression of inflammatory markers in various ocular tissues of EIU and EAU rat eyes.
Keywords: inflammation • uveitis-clinical/animal model • signal transduction