The Effect And Mechanism Of Extracellular RNA/TLR3 Signaling Pathway And RNase On Retinal Ischemia Reperfusion Injury

Background and ObjectiveRetinal Ischemia reperfusion injury(IRI)is a common pathophysiological process of ophthalmic diseases,which is mainly caused by acute angle-closure glaucoma,diabetic retinopathy,retinal arteriovenous occlusion,etc.At present,the exact mechanism of retinal IRI is still not clear.Although many new methods and technologies have been applied to the diagnosis and treatment of retinal IRI related diseases,the clinical treatment effect is poor.Therefore,it is of great clinical value to find safe and effective methods to reduce retinal injury.Studies have confirmed that Toll-like receptor(TLR)3 mediated immune regulation is the body's first line of defense against stress response.More and more evidences have shown that TLR3-mediated inflammatory and apoptotic mechanisms play an important role in organ ischemia reperfusion(I/R)injury,such as heart,lung and liver.Extracellular RNAs(exRNAs),including double-stranded RNA(dsRNA),act as endogenous ligands of TLR3 and regulate the development of IRI by activating TLR3 signaling pathway.At the same time,Ribonuclease(RNase),as the hydrolytic enzyme of exRNAs,has a certain protective effect on organ damage caused by I/R.Therefore,the purpose of this study is to explore the role of exRNAs/TLR3-mediated signaling pathway in retinal IRI,and whether RNase treatment can alleviate retinal IRI.MethodsWild type(WT)and TLR3 gene knockout mouse in C57BL/6 J mice were used to build retina I/R model.After I/R,the retinal tissue protein and RNA were extracted.The expression of exRNAs in circulating serum and retinal tissue was detected by Spectrophotometer and RNA select green fluorescent cell stain technology respectively.The levels of TLR3,Glial cells fiber acidic protein(Glial fibrillary acidic protein,GFAP),Caspase 3,and inflammatory cytokines were detected by RT-qPCR and Western blot respectively.Also,immune-fluorescent staining was used to test the expression of dsRNA/TLR3 in retinal tissue sections,TUNEL assay and HE staining were used to observe the levels of apoptosis and changes of retinal tissue structure.After recovery,the original generation Muller cells were subcultured and transfected.Then,MTT method was used to detect the cell vitality,physics gas box method to build the model of Muller cell hypoxia reoxygenation(H/R).After that,collecting Muller cells,the expression of dsRNA/TLR3 and GFAP in retinal tissues were tested by immune-fluorescent staining.In addition,using the RT-PCR method to test the expression of Caspase-3 and inflammatory cytokines,TUNEL method to detect Muller cells apoptosis.ResultsAfter I/R,the expressions of TLR3 and GFAP in retinal tissues of WT mice were significantly up-regulated,the levels of inflammatory factors were increased,the apoptotic factor caspase-3 and TUNEL apoptotic index were significantly added,and the retinal injury was aggravated.After treatment with TLR3 inhibitor,the expression levels of TLR3 and GFAP in retinal tissue were significantly down-regulated,the expression levels of inflammatory factors were decreased,the apoptotic factor caspase-3 and TUNEL apoptotic index were reduced,and the retinal injury was alleviated.Also,TLR3 gene knockout significantly down-regulated the expression of GFAP in retinal tissues,reduced the generation of inflammatory factors,decreased the apoptotic index,and alleviated retinal IRI.After H/R treatment,the immunofluorescence expression of TLR3 and GFAP in Muller cells was significantly up-regulated,the expression levels of inflammatory factors were increased,TUNEL apoptotic index was significantly added,and cell viability was decreased.Inhibition of TLR3 expression by dsRNA/TLR3 inhibiter complex significantly down-regulated the immunofluorescence expression levels of GFAP,decreased the production of inflammatory factors,reduced the TUNEL apoptosis index,and enhanced the viability of Muller cells.The expression levels of exRNAs and dsRNA in retinal tissues of WT mice were increased significantly after I/R.RNase treatment significantly reduced the production of exRNAs and dsRNA,down-regulated the immunofluorescence expression of TLR3 and GFAP,decreased the expression of inflammatory factors,reduced tissue apoptosis,and alleviated the retinal injury caused by I/R.ConclusionsTLR3-mediated inflammatory mechanism and apoptosis mechanism are involved in the pathophysiological changes of retinal IRI.Inhibition of TLR3 expression or knockout of TLR3 gene can significantly reduce the occurrence of retinal IRI.Meanwhile,studies have found that increased exRNAs,including dsRNA,promote retinal I/R-induced damage by activating the TLR3 signaling pathway,and what's more,RNase treatment can significantly inhibit the inflammatory response and cell apoptosis of the body,and has a certain protective effect on retinal IRI.