Therapeutic Effects Of Mesenchymal Stem Cell-derived Exosomes On Retinal Detachment

ObjectivePhotoreceptor cell death is the ultimate cause of vision loss in various retinal diseases inculding retinal detachment(RD).RD is one of the most common sight-threatening retinal diseases.RD have revealed the occurrence of multiple biological events such as gliosis,inflammatory responses and RPE proliferation.The mechanism of photoreceptor cell death is complicated,including apoptosis,autophagy,necrosis and inflammation.They interact with the other,form a mesh structure.It has been shown that mesenchymal stem cells(MSCs)have various biological functions including multi-lineage differentiation,neuroprotection,immune modulation and suppression of inflammatory responses.There is evidence that MSCs act in a paracrine manner rather than directly to induce differentiation.MSC derived exosomes(MSC-Exos)play important roles in cell-to-cell communication by delivering proteins and RNA.They can mediate the biological functions of MSCs,which has been verified in a variety of animal models.In this study,we observed the therapeutic effect of MSC-exos on retinal detachment animal model,and explored related therapeutic mechanism.In order to lay a certain foundation for the clinical application of MSC-exos in degenerative ocular diseases.Methods1.Culture of MSCs: Sprague—Dawley(SD)bone marrow-derived MSCs were cultured and passaged in vitro.2.Isolation and identification of MSC-exos: to collect the supernatant of cultured MSCs from the 3rd to 5th passage,and then resuspended with phosphate buffer of about 200 ul volume after ultracentrifugation.MSC-exos were identified by western blotting and scanning electron microscope,respectively.Finally,the concentration of BCA protein quantitative kit was measured and stored in-80 °C refrigerator.3.Establishing the retinal detachment model: SD rats were randomly divided into normal group,control group,PNS-treated groupand MSC-exos-treated group.An RD model was developed in rats using a subretinal injection of 1% hyaluronic acid.We subretinally injected 5ul PBS or 5ul MSC-Exos at the time of retinal separation to study their therapeutic function.4.Detecting monocyte chemotactic protein-1(MCP-1),tumor necrosis factor-?(TNF-?),Interleukin 1 beta(1L-1?)and Interleukin 6(IL-6)m RNA levels.To determine the most appropriate treatment concentration of MSC-exos.5.Western blot was used to determine the protein expression of inflammatory factors TNF-?,1L-1? and biochemical markers of autophagy microtubule-associated protein 1 light chain 3 beta(LC3)and Atg5.6.The pathological changes of retina were observed by hematoxylin-eosin(HE)staining: the rats in each group were killed and the eyes were removed at the 7 days.The outer nuclear layer(ONL)thickness on day 7 after experimental RD was determined by Image J software.7.Apoptosis of photoreceptor cells was detected by TUNEL: rats were sacrificed and eyes were enucleated at the third day.The frozen eyeballs were sectioned continuously and stained with TUNEL kit.Finally,apoptosis was counted by Image J software.8.Proteomic analysis of MSC-exos to detect the types and functions of proteins in exosomes.Results1.The supernatant of 3 to 5 generations of MSCs derived from rat bone marrow stem cells was successfully cultured with serum without exosome.The exosome was obtained by classical ultracentrifugation,and the expression of CD9 and CD81 was confirmed by Western blot.Electron microscope showed spheroid shaped vesicles at the diameter of about 40–100 nm.2.The retinal detachment model was successfully established.3.QRT-PCR showed that the m RNA expression levels of MCP-1,TNF-?,1L-1? and IL-6 increased on day 3 post-RD,which indicated that the model was successful.MSC-exos treatment significantly reduced the levels of TNF-? and 1L-1?.The most appropriate treatment concentration of MSC-exos is 0.1mg/ml.4.Western blot showed that compared with PBS treatment group,MSC-exos treatment group significantly decreased the levels of inflammatory factors TNF-? and 1L-1?,and increased the levels of LC3-II/LC3-I and Atg5.5.TUNEL assay showed that there were a large number of apoptotic cells in the detachment areas on day 3 post-RD.The number of apoptotic cells in MSC-exos treatment group was decreased,and the difference was statistically significant(P > 0.05).6.HE staining showed the disordered retina and the thinner retina on day 7 post-RD.MSC-Exo treatment decreased apoptosis of photoreceptor cells and retained retinal structure integrity.7.Proteomic analysis of exosomes showed that MSC-Exos contained proteins with anti-inflammatory,neuroprotective and anti-apoptosis effects.ConclusionMSC-Exos supressed inflammatory cytokine induction,enhanced the level of autophagy,reduced apoptosis and retained retinal structure integrity after retina-RPE separation.