Effect Of Bone Marrow Mesenchymal Stem Cell Exosomes On Autophagy Of Retinal Pigment Epithelial Cells Under High Glucose Injury

Background:Diabetic retinopathy(DR)is a common microvascular and neurodegenerative complication of diabetes mellitus.It is also one of the leading adult blinding eye diseases worldwide.The development of DR is a complex and continuous process,of which the specific pathogenesis is still not fully understood.It is generally accepted that long-term chronic hyperglycemia is the main pathogenetic basis.Retinal pigment epithelium(RPE)cells,a special type of cell,are located between the retinal nerve cell layer and the choroid,forming the outer barrier of the retina.This cell has several important physiological functions.The development of DR causes different degrees of damage to the functions of RPE cells.Autophagy is a process of self-degradation.Under stress,cells can regulate autophagy to promote the metabolism of waste products such as misfolded proteins and damaged organelles into lysosomes as a way to supply energy to the organism and remove cytotoxic substances.These processes are essential for the maintenance of cellular homeostasis.Dysregulation of autophagy is often closely associated with the development of several diseases.Recent studies have shown that in in-vitro model of DR,reduced levels of autophagy can exacerbate retinal damage,and a certain degree of autophagy activation facilitates cellular response to microenvironmental changes and injury.bone marrow mesenchymal stem cells(BMSCs)have a multi-differentiation potential and their role in repairing damaged cells and tissues has made them a hot topic for cell therapy research.In the field of retinal diseases,BMSCs can exert autophagic regulatory effects on various tissues and organs,and their protective effects on retinal ganglion cells,photoreceptor cells,and RPE cells have been demonstrated.Exosomes are one of the important mechanisms by which stem cells exert their effects on other cells locally or systemically.It has been shown that after extracting exosomes,the effect of BMSCs exosomes was found to be similar to that of BMSCs,i.e.,they reduced apoptosis and activated and prolonged cellular autophagy.This suggests that BMSCs exosomes may have a protective effect on injured RPE cells by regulating autophagy.Objective:In this study,we proposed to establish an in-vitro model of high glucose injury in the retinal pigment epithelium,and further investigate whether BMSCs exosomes can protect RPE cells under high glucose stress state through regulating autophagy by coculturing BMSCs exosomes with RPE cells.By investigating the protective effect of BMSCs exosomes on RPE cells and the function of autophagy in it,the signaling pathway activated by autophagy was finally clarified.To provide experimental evidence for the regulation of resistance to high glucose damage through autophagy in diabetic retinopathy.Methods:1、Isolation and extraction of BMSCs exosomes BMSCs exosomes were isolated and extracted using a kit,and identified by transmission electron microscopy observation and western blot detection of marker proteins.2、Establishment and evaluation of high glucose-induced RPE cell injury model The cells were divided into normal control,hyperosmolar control and high glucose groups.ARPE-19 cells were cultured in vitro for 48 h by applying normal DME/F12 medium,mannitol 50 mmol/L medium and glucose 50 mmol/L medium respectively.The changes of cell viability were detected by MTS method and the expression levels of apoptotic proteins were detected by western blot(WB).The effect of high glucose on cell morphology,viability and apoptosis was evaluated.3、Evaluate the protective effect of stem cells on RPE cells in the co-culture system of BMSCs exosomes and RPE cells A co-culture system was established and the cells were divided into normal,high glucose and co-culture groups.The viability of RPE cells in the three groups was detected,and the expression of apoptotic proteins was detected by WB method to clarify the effect of BMSCs exosomes on damaged cells.4、Observation of cell autophagy during the process of RPE cell injury The cells were cultured by applying high sugar medium,q PCR to detect the expression level of miR-183-5p,WB to detect the expression of HMGB1 and autophagy-related protein LC3,MDC staining to detect the autophagy level,and transfection of HMGB1 and miR-183-5p knockdown and overexpression respectively,WB method to detect the expression level of HMGB1 and LC3-II.5、To investigate the effect of BMSCs exosomes on the autophagy of RPE cells To detect the expression of HMGB1 and LC3 in different groups of cells and to evaluate the effect of BMSCs exosomes on cellular autophagy during cell injury.Results:1,Round and circular-like bilayer membrane-like tiny vesicles could be observed under electron microscope,particle size analysis: 30-150 nm,mean particle size: 78.38 nm.Western blot detected positive expression of TSG101 and CD9,negative expression of calnexin.2,Comparing with normal control and hyperosmotic control,ARPE-19 cell viability decreased,apoptotic protein bcl-2 expression decreased and bax expression increased in high glucose group.The differences were all statistically significant.3,Compared with the high glucose group,the co-culture group showed increased cell viability,increased expression of apoptotic protein bcl-2,and decreased expression of bax.4,miR-183-5p expression decreased,HMGB1 and LC3-II expression increased and autophagy increased in RPE cells under high glucose injury.After overexpression of HMGB1,LC3-II increased and autophagy level was elevated in the overexpression group.Knockdown of miR-183-5p increased expressions of HMGB1 and LC3-II.Autophagy level also increased in the knockdown group.Overexpression of miR-183-5p decreased HMGB1,LC3-II expressions and decreased autophagy level.Simultaneous overexpression of miR-183-5p and HMGB1 reversed the high expression of HMGB1 and LC3-II in the HMGB1-only overexpression group.Knockdown and overexpression of miR-183-5p under high glucose injury further increased HMGB1 expression and autophagy levels in the knockdown group compared to the high glucose group,while reversing them in the overexpression group.5,Increased HMGB1 and LC3-II expression and autophagy in the exosome coculture group under high glucose injury.Overexpression of miR-183-5p in the exosome co-culture group reversed the high expression of HMGB1,LC3-II.Conclusions:1,In vitro high glucose culture of RPE cells can induce their damage.2,BMSCs exosomes have a protective effect against high glucose damage in RPE cells.3.RPE cells can respond to high glucose injury through miR-183-5p/HMGB1/autophagy pathway.4.BMSCs exosomes can help RPE cells resist high glucose damage through the miR-183-5p/HMGB1/autophagy pathway.