Molecular Mechanism And Protect Function Of Stem Cell Exosomes MiR-141 During Oxidative Stress Injury In Retina

Background:Age related macular degeneration(AMD)is a common blindness eye disease,mainly due to degenerative lesions in macula.At present,the pathogenesis of AMD is not fully understood,but some studies have found that oxidative stress plays an important role in the pathogenesis of AMD,and excessive oxidative stress will cause dysfunction of retinal pigment epithelial cells(RPE cells).Retinal light damage model is one of the most common in vitro models for AMD research.One of the important mechanisms is that photoreceptors and RPE cells absorb excessive photons in visible light,and produce a large number of reactive oxygen radicals ROS),when ROS is continuously gathered in the cell and can not be removed by antioxidant factors,the oxidative stress response mediated by ROS can lead to the damage of RPE cells,protein denaturation,and membrane structure destruction,and eventually lead to apoptosis.RPE cells are one of the main cell components in retina.The change of RPE function is very important to the occurrence and development of retinal related diseases(retinitis,retinitis degeneration and malnutrition,retinal detachment).At present,most scholars use white LED cold light source to induce light damage animal model,and choose white light to simulate the damage of sunlight to retina,so as to build animal model similar to AMD.Oxidative stress is stimulated by various harmful substances,the high active molecules such as ROS free radicals in the body produce too much,which makes the oxidation system and the antioxidant system dynamically unbalanced,and leads to tissue damage.It is found that NF-E2-related factor 2(Nrf2)is one of the important endogenous antioxidant stress pathways by interacting with anti-oxidative response element(ARE).In physiological state,Nrf2 is bound to kelch like ech2 associated protein 1(Keap1),and it is anchored to the cytoskeleton composed of actin through Keap1 protein,so it can not enter the nucleus for transcription.When stimulated by ROS,Keap1 and Nrf2 were decoupled to transfer Nrf2 into the nucleus,and the transcription of downstream protective protein gene was initiated,and the antioxidant stress ability of cells was improved.Micro RNA(miRNA)is a kind of single strand,long 20-25 short nucleotide sequences.Through complete or incomplete pairing with the 3'UTR region of its target messenger RNA(m RNA),the gene expression is regulated horizontally after transcription,and each miRNA can interfere with many m RNA,whereas each messenger RNA can regulate multiple miRNAs.In recent years,there are about 900unique miRNAs in human genome,and 1/3 genes are regulated by miRNAs.miRNAs play an important role in various physiological and pathological processes of human and animals.They can participate in the control of some biological processes,including cell growth,apoptosis,development,metabolism,stress regulation,hormone signaling pathway and differentiation.Mirna-141(miR-141)is a member of mirna-200 family.It plays an important role in physiological state and disease occurrence and development by regulating different signal pathways.Bone marrow mesenchymal cells,which have high self-renewal,multi-directional differentiation potential and immunomodulatory function,have become a hot spot in the research of tissue and organ repair in regenerative medicine.Stem cells have also been reported in the field of Ophthalmology,such as retinitis pigmentosa,age-related macular degeneration,corneal limbal stem cell deficiency and optic nerve injury.Stem cells have also been proved to be able to differentiate into RPE cells,neuroid cells and photoreceptors.At the same time,stem cells can secrete many kinds of cytokines and neurotrophic factors,resist local inflammation,protect retinal cells and improve the microenvironment of retina.The results show that the biological function of MSCs may be achieved by the exosomes secreted by the side.Exosomes is a kind of lipid bilayer structure secreted by living cells,which contains many molecular regulatory substances such as lipids,proteins and nucleic acids.The function of exocrine depends on the cell type,which can be involved in the pathophysiological process of immune response,antigen presentation,cell migration,cell differentiation,tumor invasion and so on.With the development of exosomes extraction and detection technology,the cytokines secreted by stem cells to improve the microenvironment of retina can be detected in the exocrine secreted by stem cells.Recent studies have shown that stem cells secrete exocrine,which not only contain various protective nutrients,but also contain regulatory factors that can promote the transcription and translation of these nutrients.Therefore,the research on stem cell exosomes has become a hot topic.Exosomes,as a nano-level transport carrier,is smaller than stem cells and has lower immunogenicity,which is considered to be a carrier for cell therapy in the future.Objective:This study mainly discusses the protective effect and mechanism of miR-141 on oxidative stress injury of retina in the exosomes of bone marrow mesenchymal stem cells.Method:1.Extraction and identification of bone marrow mesenchymal stem cells exosomes:The supernatant of BMSCs was centrifuged at ultra-high speed to obtain vesicle material.The extracted vesicle materials were observed by electron microscopy and the specific membrane proteins CD9,CD63 and CD81 were detected.2.Protective effect of bone marrow mesenchymal stem cells exosomes on oxidative stress injury of RPE cells:50?g/m L bone marrow mesenchymal stem cells exosomes were added into RPE cell medium,and equal volume of PBS solution was added into the RPE cell medium of control group.The oxidative stress injury model of RPE cells was established by H₂O₂ induction.RPE cell viability value,apoptosis level,caspase-9 expression level,Bcl2 and Bax expression level were compared between the two groups.3.The molecular mechanism of miR-141/Keap1/Nrf2 pathway in oxidative stress response of RPE cells:Through literature review,it was found that the Keap1-Nrf2-ARE pathway is a classic pathway of anti-oxidative stress injury.Target Scan online bioinformatics analysis software was used to predict miRNAs with potential binding sites to Keap1.The predicted binding sites of miR-141 and Keap1were verified by luciferase reporter gene assay.After miR-141 was overexpressed or inhibited,the expression levels of Keap1,Nrf2,HO-1 and NQO1 were detected by q PCR and protein electrophoresis.2)Cell viability was detected by MTS.3)caspase-9activity kit was used to detect apoptosis level.After overexpression of miR-141,Nrf2inhibitor was used to perform a remedial experiment to observe whether miR-141exerted a protective effect of anti-oxidation through Nrf2.The m RNA and protein expression levels of Keap1,Nrf2,HO-1 and NQO1 were detected by q PCR and protein electrophoresis,and the viability and apoptosis of RPE cells were observed.4.The protective effect of miR-141 on the oxidative stress injury of RPE cells:Extract the exosomes of BMSCs expressing miR-141,inhibit the secretion of miR-141 and normal bone marrow mesenchymal stem cells exosomes.After the three groups of exosomes were added to RPE cell medium for 48 hours,and then 200?Mol H₂O₂ induced RPE cells for 6 hours,and then the effects of BMSCs exosomes expressing miR-141 or inhibiting miR-141 on the antioxidant stress of RPE were detected.1)the expression levels of HO-1,NQO1 and caspase-9 were detected by q PCR and protein electrophoresis.2)The cell activity was detected by MTS.3)caspase-9 active kit was used to detect apoptosis.5.In vivo experiment of the protection of the BMSCs exosomes to the acute retinal light injury:the model of acute retinal light injury in mice was constructed by LED lamp.The BMSCs exosomes were injected into the vitreous cavity of mice,and the retinal morphology was observed by HE staining.The results showed that BCL2,Bax,HO-1,NQO1 and caspase-9 were detected by q PCR and protein electrophoresis.Result:1.Extraction and identification of bone marrow mesenchymal stem cells exosomes:The vesicles in the supernatant of bone marrow mesenchymal stem cells extracted by ultra-high speed centrifugation were identified.The vesicles were found to be double-layer membrane with diameter of about 100nm under electron microscope,and they had the morphological characteristics of exocrine.The results showed that the vesicles were exosomes.2.The protective effect of bone marrow mesenchymal stem cell exosomes on oxidative stress injury of RPE cells:The results of 50 ug/ml of bone marrow mesenchymal stem cell exosomes were added to RPE cell culture medium.It was found that the RPE cells added to the exosomes were more active and apoptosis level decreased than that of RPE cells without exosomes,and the expression of Bcl-2 was increased.3.Molecular mechanism of mir-141/keap1/nrf2 pathway in oxidative stress response of RPE cells:bioinformatics analysis(Target Scan online prediction website)speculates that there is potential binding site between Keap1 and miR-141.The luciferase reporter gene experiment confirmed that miR-141 and Keap1 were binding in 293T cells.The results showed that overexpression of miR-141 could inhibit Keap1,reduce the coupling between Nrf2 and Keap1,increase the transfer of Nrf2 into the nucleus,and open the transcription and translation of HO-1 and NQO1 in the nucleus,and play an antioxidant stress role.Inhibition of miR-141 can reduce its inhibition on Keap1,and reduce Nrf2 transfer into the nucleus,thus reducing the transcription and translation of HO-1 and NQO1.4.The protective effect of miR-141 on the oxidative stress injury of RPE cells:Extract the exosomes of bone marrow mesenchymal stem cells expressing miR-141,inhibit the secretion of miR-141 and normal marrow mesenchymal stem cell exosomes.After the three groups of exosomes were added to RPE cell medium for 48hours,the three groups of exosomes were added to RPE cell medium for 48 hours,and then 200?M was used After 6 hours of induction,H₂O₂ induced RPE cells,it was found that the exosomes of bone marrow mesenchymal stem cells overexpression miR-141 increased the activity of RPE cells induced by H₂O₂,decreased caspase-9activity,and increased the expression level of protein and m RNA of HO-1 and NQO1,thus alleviating oxidative stress damage of RPE cells induced by H₂O₂.However,inhibition of miR-141 mesenchymal stem cell exosomes reduced the activity of RPE cells induced by H₂O₂,caspase-9 activity increased,and the expression of HO-1 and NQO1 protein and m RNA decreased.5.In vivo experiments on the protection of acute retinal light injury by bone marrow mesenchymal stem cell exosomes:the number of nuclear layer cells in the model of light injury was significantly reduced in mice injected with BMSCs,and the expression level of HO-1 and NQO1 in retina increased,while caspase-9 protein and m RNA expression level decreased.Conclusion1.Exosomes of bone marrow mesenchymal stem cells can be extracted by ultra-high speed centrifugation.2.Bone marrow mesenchymal stem cell exosomes can protect RPE cells from oxidative stress injury induced by H₂O₂.3.The software prediction and luciferase reporter gene experiments confirmed that miR-141 and Keap1 have binding ability.Mi R-141/Keap1/Nrf2 signaling pathway plays a key role in the regulation of RPE cells during oxidative stress.4.Mi R-141 is the protective effect of bone marrow mesenchymal stem cell exocrine on oxidative stress injury of RPE cells.5.In vivo experiments confirmed that bone marrow mesenchymal stem cells exosomes has protective effect on the acute retinal light injury.