Impact Of Bone Marrow Mesenchymal Stem Cells On Autophagy And Lysosome Function Of Impaired Photoreceptor Cells

Background:The metabolic wastes and damaged organelles are wrapped into autophagosomes under cells' starvation or stress and then get into lysosomes for further degradation.The effective degradation processes are essential for cell homeostasis.Studies have shown that lysosomal dysfunction is closely related to neurodegeneration diseases,and lysosome-related abnormalities occur in the pathological processes of various neurodegenerative diseases,while metabolic abnormalities caused by lysosomal dysfunction or gene mutations can also cause severe neurological dysfunction.Photoreceptor cells are the primary neurons of visual transmission,its damage or death under various pathological conditions will cause visual impairment.At present,the research on its death mechanism is focused on oxidative stress,endoplasmic reticulum stress,mitochondrial damage,cell apoptosis and other fields.The relation of lysosome and photoreceptor cells is increasing with the progress in lysosome researches recently.Although the abnormalities of autophagy and lysosomal metabolism in the process of disease have attracted the attention of scholars,they are mostly limited to the discusses on autophagy,and there is a lack of research on the process of autophagic lysosomal degradation.mTOR is a key signaling factor that regulates cell metabolism,synthesis,proliferation and death.Recent studies have shown that mTOR can locate in lysosomes and plays an important regulatory role in autophagy initiation,lysosome biosynthesis,lysosome regeneration and other processes.In addition,the role of mTOR in promoting photoreceptor cell survival in degenerative diseases of the retina,such as retinitis pigmentosa and age-related macular degeneration,has been confirmed by several studies.Current studies suggest that mTOR promotes photoreceptor cell survival under stress by promoting processes of energy metabolism,biosynthesis,and anti-apoptosis.However,whether this process involves the regulation of autophagy and lysosome function remains unknown.Bone marrow mesenchymal stem cells(BMSCs)have the potential of multidirectional differentiation,and their role in damaged cells and tissue repair has made them a focus of cell therapy.In the field of retinal diseases,the protective effects of stem cells on retinal ganglion cells,photoreceptor cells,and retinal pigment epithelium have been demonstrated.At present,it is believed that stem cells mainly affect the damaged tissue or cells by paracrine and cell differentiation.In studies on stem cells related to diseases of the central nervous system,it has been reported that stem cells can promote lysosomal metabolism in neurons,reduce lysosomal deposition in neurons and improve cell phenotypic abnormalities.Whether this mechanism exists in the protection of damaged photoreceptor cells by stem cells remains to be demonstrated.The purpose of this study was to determine whether there was autophagy-lysosomal metabolism abnormality in stressed photoreceptor cells during the process of photoreceptor cell injury.Whether bone marrow mesenchymal stem cells can protect photoreceptor cells under stress by regulating lysosomal metabolism.Objective:MNU was used to induce 661 w cell injury to establish an in vitro experimental model of photoreceptor cell injury,and to evaluate autophagy process and lysosomal degradation during cell injury.Co-culturing BMSCs with 661 w cells to observe the protective effect of stem cells on 661 w cells,and to clarify the effects of stem cells on autophagy and lysosomal functions.Methods:1.Establishment and evaluation of MNU-induced 661 w cell damage model661w cells were cultured with medium containing 100,300,500,and 1000 ug /ml MNU.The changes of cell viability at different time of culture were detected by MTS method to determine the required concentration for subsequent experiments.661 w cells were cultured in 500ug/ml MNU medium.The changes in cell morphology?the reactive oxygen species(ROS)labeled by DCFH-DA probe,and the expression of apoptotic protein caspase3 detected by western blot(WB)were observed with the prolongation of MNU action time to evaluate the effect of MNU action on cell morphology,oxidative stress and apoptosis.2.Observing the autophagy and lysosomal degradation of 661 w cells in the process of cell injuryTo assess the autophagy procedure of cells in the process of MNU-induced cell injury,MNU 500ug/ml medium was used to culture the cells.The morphology of autophagic vesicles in the cells was observed under electron microscopy at different time.The expressions of autophagy-related proteins LC3 and P62 were detected by WB.To clarify whether autophagic flow was blocked during the process,the lysosomal inhibitor bafilomycin A1 acted together with MNU for 24 hours,and the expressions of LC3 and P62 were detected by WB.To identify the effect of autophagy inhibition on cells,autophagy inhibitor 3MA and MNU were added together into the medium.MTS method was used to detect the effect of autophagy inhibition on the cell viability of damaged cells.WB method was used to detect the effect of autophagy inhibition on the expression of LC3 and casepase3.To observe the lysosome and enzyme distribution in damaged cells,Lysosome tracker and cathepsin were used to mark the lysosome and its protease after 24 h MNU treating.WB was used to detect the phosphorylation level of mTOR to determine whether there was mTOR inhibition during cell damage.3.To evaluate the protective effect of stem cells on photoreceptor cells in transwell co-culture systemBMSCs were isolated and extracted from the bone marrow of tibia and femur in rats for primary culture.The surface markers of stem cells,CD105,CD90,CD44,CD34 and CD45 were identified by flow cytometry.BMSCs were planted in transwell inserts to establish the damaged 661 w cells and BMSCs co-culture system.The experiment was divided into normal group,MNU group and co-culture group.The same method was used to detect the cell viability and the activation of reactive oxygen species in 661 w cells after MNU treatment for 24 hours,and the expression of caspase3 protein after MNU treatment for 16 hours and 24 hours were detected by WB to determine the influence of stem cells on the damaged cells.4.To observe the impact of stem cells on autophagy and lysosome functions of injured 661 w cellsAfter MNU treatment for 16 or 24 hours,autophagic vesicles morphology,LC3 and P62 expression,lysosome and lysosomal enzyme intracellular distribution,and mTOR activation were detected by the same method,so as to evaluate the effect of stem cells on autophagy and lysosomal metabolism during cell injury.Results:1.100,300ug/ml MNU has limited effect on the viability of 661 w cells;500,1000ug/ml MNU significantly reduced cell viability,showing a time-dependent trend;500ug/ml was selected as the subsequent experimental concentration.With the extension of MNU-treated time,the dense granules in cells increase,the cell density decreased,the fluorescence of intracellular ROS probes enhanced,and the expression of apoptotic protein caspase3 was increased.2.During the treatment of MNU,the number,diameter and area of autophagic vesicles increased.the levels of autophagy-related proteins LC3 II and LC3II/I increased,accompanied by the accumulation of autophagy substrate marker P62.Along with MNU treatment,lysosomal inhibitor bafilomycin A1 was added to inhibit the fusion of autophagosome and lysosome,and the LC3 II and LC3II/I expression levels increased and P62 accumulated while bafilomycin A1 co-treatment.MNU treatment combined with the addition of autophagy inhibitor 3MA can reduce the expression levels of LC3 II and LC3II/I proteins in cells ? reduce cell activity and promote the expression of the active form of caspase3.After MNU treatment for 24 hours,compared with the scattered fluorescence signals of normal cells,lysosomal probes showed fluorescence aggregation and tended to be distributed around the nucleus.The fluorescent signal of lysosomal cathepsin staining gathered in spots under MNU treating.The expression of phosphorylated mTOR decreased significantly after MNU treatment for 16-24 hours3.The bone marrow mesenchymal stem cells was extracted,cultured and passaged to the third generation with stable morphology.Flow cytometry identification showed that the cells were positive for CD105? CD90? CD44 and negative for CD34 and CD45.MNU was used to coculture system as well to observe the effect of the stem cells on the damaged cells.Compared with the MNU group,the viability of cells in the co-culture group increased significantly,the fluorescence of the ROS marker probe decreased,the expression of the active form caspase 3decreased.4.Compared with the MNU group,the number,diameter and area of autophagic vesicles in the co-culture group decreased.Although no significant difference were detected in LC3 II and LC3II/I levels between the co-culture group and the MNU group,the accumulated P62 decreased in the co-culture group after 16 h MNUtreatment.After MNU treatment for 24 hours,the level of LC3 II in the co-culture group was significantly lower than that in the MNU group,and the cumulative P62 was reduced.Lysosomal fluorescence probes and lysosomal enzyme fluorescence signals tended to be more dispersed in the co-culture group than that in the MNU group.The phosphorylation level of mTOR in the co-culture group increased compared with that in the MNU group after 16 h MNU treatment.Conclusions:1.The pathological processes of MNU induced damage to 661 w cells,which was accompanied by decreased cell activity,increased activation of reactive oxygen species and increased apoptosis,were consistent with the photoreceptor cell death mechanism in the process of retinopathy.Therefore,the model can be used in vitro to study the mechanism of photoreceptor cell damage.2.In the injured 661 w cells,continuous autophagy activation leads to the enlargement of autophagic vesicles,the accumulation of metabolic substrates,and lysosomal metabolic disorders.The cell damage is aggravated while inhibiting autophagy in injured cells.It is the substrates collected by continuous autophagy under the state of cellular stress which cannot be completely degraded by lysosomes lead to cell death finally.This process is accompanied by inhibition on mTOR activity.3.Bone marrow mesenchymal stem cells(BMSCs)can protect the damaged661 w cells in promoting cell viability,weakening ROS activation and reducing apoptosis.4.BMSCs can reduce the diameter and area of autophagic vesicles in cells,decreased the accumulation of metabolic substrates,and promoted the distribution of lysosomes in cells.That is,stem cells promote substrate degradation in damaged cells.This process is accompanied by an up-regulation in mTOR activity.In summary,photoreceptor cell injury is accompanied with lysosomal degradation disorder caused by continuous autophagy activation,and bone marrow mesenchymal stem cells can promote lysosomal degradation and reduce cell death.These processes occur with the change of mTOR activity.