ADSC-EVs Regulates PLCD1 Expression Via MiR-23a-5p And Alleviates Retinal Excitotoxicity

PURPOSE: Adipose mesenchymal stem cells(ADSCs)have a protective effect against glutamate-induced excitotoxicity,but the exact mechanisms involved are not well understood.Studies have shown that the extracellular vesicles(EVs)secreted by ADSC(ADSC-EVs)not only have the function of ADSC but also have the unique advantages of being non-immunogenic,having a low probability of abnormal growth and being easily taken up by target cells.Therefore,based on the investigation to verify whether ADSC-EVs can alleviate glutamate-induced excitatory neurotoxicity in the retina,this study hopes to continue to explore whether ADSC-EVs affect intracellular calcium ion concentration and alleviate glutamate-induced excitatory neurotoxicity by regulating the phosphorylation modification levels of PKCA and GluA2.Finally,this study will further explore the key micro RNAs that play a regulatory role in ADSC-EVs and identify key upstream target genes that influence PKCA activation during regulation by ADSC-EVs through bioinformatic analysis.METHODS: We obtained human adipose tissue and isolated ADSCs from it,and we collected cell supernatants and extracted ADSC-EVs from them and used them for subsequent experiments.Then we established a glutamate-induced excitotoxicity model in SD rats and rat retinal neuronal precursor cells(R28).The experiments were divided into Con,Glu,Glu+EVs,and Glu+PBS groups,we used HE staining,TUNEL staining,and electroretinography(ERG)to observe the changes in retinal morphology and function.We also used propidium iodide(PI)staining and Western blot to observe the protective effect of ADSC-EVs in R28 cells and Fluo-4AM staining to observe the intracellular calcium ion concentration in R28 cells.Western blot and immunofluorescence(IF)were used to detect the phosphorylation of GluA2 and its expression on cell membranes in an in vitro and in vivo model.We further set up a Glu+EVs+TPA group in in vitro experiments: R28 cells were pre-treated with the PKCA agonist 12-O-tetradecanoylphorbol 13-acetate(TPA),followed by Western blot to detect the phosphorylation of GluA2 and PKCA to further verify whether the protective effect of EVs was inhibited.Finally,small RNA sequencing was performed on in vitro cell models of the Glugroup and Glu+EVs group to find differentially expressed micro RNAs(p < 0.05,Log2 Fold Change ≥ 1).Target genes were identified by the KEGG database and Miranda database.Dual luciferase was performed to verify whether micro RNAs interacted directly with target genes.To verify the regulatory interaction of micro RNAs with target genes,the study was verified by micro RNA-mimic and micro RNA-inhibitor,and the expression of related molecules was observed by RT-PCR assay,PI staining view,Western blot assay,and IF assay.RESULTS:(1)ADSC-EVs were found to be taken up by retinal ganglion cells(RGCs)and R28 cells,and affected glutamate-induced death of RGCs,thinning of the inner plexiform and inner nuclear layers and decreased inner retinal function in in vivo experiments,while in vitro experiments also confirmed that ADSC-EVs inhibited glutamate-induced R28 cell death.(2)ADSC-EVs can regulate the expression of GluA2 in cell membranes and the calcium permeability of AMPARs by regulating the phosphorylation of PKCA/GluA2.(3)ADSC-EVs target and inhibit PLCD1 expression via miR-23a-5p,which in turn modulates PKCA/GluA2 phosphorylation and GluA2 expression on cell membranes.CONCLUSION: ADSC-EVs are potential therapeutic agents for the treatment of retinal excitotoxicity and deserve further in-depth study.