The Effects And Mechanism Of NOX4-ROS Mediated Blood-retinal Barrier Damage On The Death Of Photoreceptors Cell In Experimental Retinal Detachment

Objective:Blood retinal barrier?BRB?is a selectively permeable tissue structure similar to the blood-brain barrier,located between the retinal tissue and the blood,whichplaysanimportantroleinmaintainingthestabilityofretinal microenvironment.BRB damage can be caused by most retinal diseases,including diabetic retinopathy,retinal vein occlusion,and retinal detachment.There is a large number of studies showing that excessive ROS produced by oxidative stress can lead to the degeneration and necrosis of cells.Retinal vascular endothelial cells are an important part of BRB.The main source of ROS is NADPH oxidase type 4?NOX4?in retinal vascular endothelial cells.Therefore,we investigated whether the BRB damage of experimental retinal detachment rats was reduced and whether the visual function could be effectively restored after NOX4 interference.Which can provide us an effective treatment for the visual function recovery of patients with retinal detachment.Methods:?1?To Construct and filter plasmids of NOX4 recombinant adeno-associated virus type 2?AAV2?,packaged to determine the viral titer and observe the efficiency of AAV2 virus vector transfection into pc-12 cell lines under fluorescence microscope.?2?Subretinal injection of adeno-associated virus vectors transfected the retina of rats.After 3 weeks,the rat retinas were taken to make frozen sections.To observe the virus transfection in rat retinas under fluorescence microscoperight,The right eye of each rat was treated as the experimental group and the left eye as the control.?3?3 weeks after AAV2 transfection retina,RDs were enlarged via subretinal injection of 1%sodium hyaluronate into the subretinal space using a 30-gauge needle.according to the different interference,divided into attached group?uninfected with AAV2 and received no RD surgery?,untreated group?uninfected with AAV2 and received RD surgery?,sh-NOX4 group?infected with AAV2-NOX4 and received RD surgery?,and sh-neg group?infected with sh-neg and received RD surgery?.After 3 days of modeling,retinal tissue was taken for examination.The right eye of each rat was treated as the experimental group and the left eye as the control.?4?The morphological characteristics of retinal vascular endothelial cells and photoreceptor cells were observed by electron microscopy.?5?The expression level of NOX4 protein was detected by western blot.?6?Observation of Evans blue perfusion retinal patch under confocal microscope,observation of EB leakage from the blood vessels,and evaluation of the damage of the blood-retina barrier.?7?The Morris water maze test was used here with modifications.Behavioral data were acquired with a latency to escape to the platform during the training trials.So as to evaluate the visual function of rats.?8?The expression intensity of retinal ROS in rats was determined by fluorescence microscopy.Results:?1?Three AAV2 virus vectors?NOX4-rat-321?NOX4-rat-578?NOX4-rat-1207?were constructed.and according to the transfection efficiency of pc-12 cells and the knockdown of NOX4,the NOX4-rat-1207 with the highest transfection efficiency and the best knockdown was selected.NOX4-rat-1207 virus titer was 1.12x10¹³V.G./ml,and AAV-NC virus titer was 3.99x10¹⁰V.G./ml.?2?3 weeks after AAV2 transfection retina,GFP fluorescence and DAPI staining were observed under fluorescence microscope using blue and red light.It was observed that most retinas could be effectively transfected by virus,and strong GFP fluorescence was detected in photoreceptor cells,while only a small amount of GFP was detected in the remaining layers.?3?Three days after RD surgery,obvious retinal eminence could be observed under the microscope,no vitreous hemorrhage or cataract was observed.?4?3 days after modeling,the results of transmission electron microscopy showed that the destruction of the tight connection between the retinal vascular endothelium in rats in the sh-NOX4 group was lighter than those in the untreated group and the sh-neg group.The necrotic rate of photoreceptor cells in the sh-NOX4 group was?15.33±1.65?%,which was significantly lower than the unterated group?22.60±1.51?%and sh-neg group?22.99±4.09?%,p<0.01)?5?Western blot results showed that the protein expression of cleaved protein NOX4 in the sh-NOX4 group?1.03±0.14?was lower than those in the attached group?1.41±0.23??untreated group?1.94±0.36?and the sh-neg group?1.71±0.18??P<0.01?.?6?The retinal vascular structure of the rats in the attached group was clear with good filling and uniform thickness,and no EB leakage was observed by confocal microscopy.Untreated group?sh-NOX4 group and sh-neg group,EB leakage occurred in retinal vessels.But sh-NOX4 group was lighter than those in the untreated group and the sh-neg group.?7?Recording time rats from the surface of the platform to incubation period?the escape latency?,The test results showed that the time to find the platform in sh-NOX4group?16.82±5.79s?was lower than untreated group?48.54±8.15s?and sh-neg group?45.46±16.00s??P<0.01?.?8?The fluorescence intensity of ROS was measured by a fluorescence enzyme labeling instrument,and the intensity of ROS was represented by fluorescence intensity/protein concentration.Results showed that the intensity of ROS in sh-NOX4?8621.97±1064.57?waslowerthanthoseintheuntreated group?15881.45±1770.43?and the sh-neg group?14442.48±949.78?,?P<0.01?.Conclusion:NOX4 interference can effectively protect the breakdown of blood-retinal barrier in experimental retinal detachment.The mechanism may be that NOX4interference can reduce the production of ROS in retinal vascular endothelial cells in experimental retinal detachment.Thus the blood retina barrier was protected and the death of photoreceptor cells was reduced,which plays a protective role in vision function.