The Effects Of Fluid-flow-shear-stress-activated-connexin Hemichannel On Protecting Lens Cortical Fiber Cells Against Oxidative Insults

Objective:Oxidative stress is the main factor that induces age-related cataracts.In physiologic condition,metabolic-active lens epithelia cells fight against oxidative stress through multiple mechanisms.However,how metabolic-inactive lens cortical fiber cells fight against oxidative stress still remains unclear.In this project,we aim to explore the role of fluid flow shear stress（FFSS）in regulating the function of homomeric and heteromeric connexin（Cx）hemichannels（HCs）and the glutathione（GSH）transport.And to investigate the mechanism of FFSS-activated Cx HCs in protecting long-lived lens cortical fiber cells against oxidative stress.Thus,providing new therapeutic targets for age-related cataracts.Methods:Site-directed mutagenesis was used to prepare recombinant RCAS（A）DNA constructs containing different Cx50 site mutants.High-titer recombinant RCAS（A）retrovirus was prepared and used to infect chick embryonic fibroblast cells（CEFs）and lens primary cells with exogenous Cxs.Western blot was performed to determine expressions of Cxs.The activity of Cx HCs was determined by lucifer yellow（LY）/rhodamine dextran（RD）uptake assay.Hydrogen peroxide（H₂O₂）and ultraviolet B（UVB）were used to induce oxidative stress.Intracellular GSH and reactive oxygen species（ROS）were determined by using commercial fluorescence-based kits.Cell apoptosis and necrosis were measured by Annexin V and PI staining.Results:1.Compared to non-FFSS-treated groups,the LY uptake ratio and intracellular GSH level were significantly higher in groups with functional Cx HCs after treated with FFSS（Cx50,Cx50E48K,Cx46 and Cx46+Cx50）,whereas there was no significant difference between FFSS-treated groups and non-FFSS-treated groups when Cx HCs were impaired（V,Cx50P88S,Cx50H156N,Cx46+Cx50H156N）.2.FFSS-activated Cx50 HCs showed higher Km than that of Cx46 HCs（Cx50:0.72±0.12mmol/L;Cx46:0.43±0.14 mmol/L）and Vmax（Cx50:44.50±2.00 AU/20min;Cx46:17.18±1.34 AU/20 min）in transporting GSH.3.The intracellular GSH level was significantly elevated in both lens cortical fiber cells and epithelia cells after treated with FFSS when Cx HCs were functional（Cx50）,whereas this effect was abolished when Cx HCs were impaired.4.FFSS+GSH significantly decreased the elevation of intracellular ROS and cell death/apoptosis induced by H₂O₂and UVB in lens cortical fiber cells when Cx HCs were functional（Cx50）,whereas this protection effect was abolished when Cx HCs were impaired（Cx50H156N）.5.In the groups where CEFs express Cx46,Cx50 or Cx46+Cx50（Cx HCs were functional）,FFSS+GSH also significantly decreased the elevation of intracellular ROS and early-stage apoptosis induced by H₂O₂ and UVB,whereas this effect was diminished when CEFs express Cx50H156N or Cx46+Cx50H156N（Cx HCs were impaired）.6.In the groups with functional Cx HCs,FFSS without GSH decreased UVB-induced elevation of intracellular ROS and cell apoptosis,whereas this protection effect was diminished when Cx HCs were impaired（Cx50H156N）.7.After treated with H₂O₂/UVB,the intracellular ROS level was much higher in lens single fiber cells from Cx46^-/-mice than in those from Cx50^-/-mice.Additionally,the intracellular ROS level was much higher in lens single nuclear fiber cells than in lens single cortical fiber cells.Conclusion:1.FFSS can open both homomeric and heteromeric Cx46/Cx50 HCs and mediate the transport of GSH into lens cortical fiber cells.2.FFSS-activated homomeric and heteromeric Cx46/Cx50 HCs and transport of GSH can protect lens fiber cells against H₂O₂/UVB-induced oxidative stress.In addition,FFSS-activated homomeric and heteromeric Cx46/Cx50 HCs can protect lens fiber cells against UVB-induced oxidative stress.3.Cx50 exhibits higher anti-oxidative stress ability than Cx46 in lens single fiber cell,and lens single cortical fiber cell has higher anti-oxidative stress ability than lens single nuclear fiber cell.