Protective effect of complement receptor 1 on barrier of cultured human retinal epithelial cells under complement-activated oxidative stress

Abstract

Objective To observe the effect of complement receptor 1 (CR1) on barrier of cultured human retinal epithelial cells (hRPE) under complement-activated oxidative stress.Methods The third to fifth passage of hRPE cultured on Transwell insert were used to establish a stable hRPE monolayer barrier.The hRPE monolayer barrier was exposed to 500 μmol/L ten-butyl hydroperoxide and 10％ normal human serum to establish the hRPE monolayer barrier model of complement-activated oxidative stress in vitro.hRPE monolayer barriers under complement-activated oxidative stress were divided into two groups including model group and CR1 treatment (1 μg/ml) group.Model group and CR1 treatment group were treated with 1 μl phosphate buffer solution (PBS) or CR1 for 4 hours.Normal hRPE monolayer barrier were used as control in transepithelial resistance (TER) measurement experiment.TER was measured to evaluate the barrier function of hRPE.The hRPE-secreted vascular endothelial growth factor (VEGF) and chemokine (C-C Motif) Ligand 2 (CCL2),together with complement bioactive fragments (C3a,C5a) and membrane-attack complex (MAC) in the supernatant were detected by enzyme-linked immune sorbent assay.Results Stable hRPE monolayer barrier was established 3 weeks after hRPE seeded on Transwell insert.Complement-activated oxidative stress resulted in a sharp decrease of TER to 54.51％ compared with normal hRPE barrier.CR1 treatment could significantly improve TER of barrier under complement-activated oxidative stress to 63.48％ compared with normal hRPE barrier(t =21.60,P＜0.05).Compared with model group,CR1 treatment could significantly decrease the concentration of VEGF and CCL2 by 11.48％ and 23.47 ％ secreted by hRPE under complement-activated oxidative stress (t =3.26,2.43; P＜0.05).Compared with model group,CR1 treatment could also decreased the concentration of C3a,C5a and MAC by 24.00 ％,27.87 ％,22.44 ％.The difference were statistically significant (t =9.86,2.63,6.94 ; P＜0.05).Conclusions CR1 could protect the barrier function of hRPE cells against complement-activated oxidative stress.The underlying mechanism may involve inhibiting complement activation and downregulating the expression of VEGF and CCL2. Key words: Retinal pigment epithelium; Receptors, complement 3b/antagonists & inhibitors; Complement activation; Animal experimentation