Effect Of Fractalkine Gene-modified Bone Marrow-derived Mesenchymal Stem Cells On Inflammatory Response In The Retina Of Early Diabetic Rats

Background Diabetic Retinopathy（DR）is a severe blind-causing disease.For the early treatment of DR,there is still no effective method.Retinal microglia are resident immune cells of the retina and can participate in immune defense by releasing neuroprotective and anti-inflammatory factors.Meanwhile,subtle changes in the retinal microenvironment can cause microglia activation.Activated microglia can further aggravate retinal damage by releasing a large number of inflammatory mediators.With the in-depth study of DR,more and more researchers have demonstrated that the activation of retinal microglia and its related inflammatory response is closely related to the pathogenesis of DR.Therefore,intervention of DR in the early stage has become the hotspot.Fractalkine（FKN）is a chemokine expressed and secreted by neurons and endothelial cells.Previous studies claimed that FKN can regulate the activation of retinal microglia by recognizing its unique receptor CX3C chemokine receptor 1（CX3CR1）.Moreover,theupregulated expression of CX3CR1 in the DR model has a protective effect on neurons.Bone marrow-derived mesenchymal stem cells（BMSCs）have neuroprotective,immunomodulatory and anti-inflammatory properties,and have been used in a variety of degenerative diseases including DR.BMSCs can express Fractalkine,suggesting that there is a signal crosstalk between BMSCs and retinal microglia,which may be involved in the regulation of retinal microglial function.Given this,based on the FKN/CX3CR1 signaling pathway,this study tried to investigated the effects of FKN-BMSCs on modulating retinal microgliarelated inflammation ofearlydiabetic rats.Part 1 Activation of retinal microglia and inflammatory response in early diabetic ratsObjective To investigate the activation of microglia and inflammatory response inthe retina of early diabetic rats.Methods Healthy male SD rats were randomly divided into the control group and the diabetes model group.The model group was injected intraperitoneally with STZ to induce hyperglycemia,and the control group was intraperitoneally injected with an equal volume of citric acid-sodium citrate buffer.Blood glucose of rats was detected 72 hours after injection,and those with≥16.7 mmol/L were deemed successful in modeling.4 weeks after modeling,the retinal vascular morphology and leakage of rats was observed by fundus fluorescein angiography（FFA）;Iba1-staining cells by immunofluorescence was used to observe the activation of retinal microglia;TUNEL method was used to detect the apoptosis of retinal cells;Western blot was used to identify the expression of rat retinal inflammatory factors TNF-αand IL-1β;the Griess method was used to detect the release of NO.Results After aone-time intraperitoneal injection of STZ,theblood glucose was maintained above 16.7mmol/L in 10 of 12 rats,and the symptoms ofdiabetes appeared such as polydipsia,polyphagia,and polyuria.The success rate of modeling was 83.3%;At 4 weeks of disease course,no abnormal retinal vascular morphology and leakage was observed by FFA test in the two groups;Compared with the control group,the retinal microglial cells of the model group were activated.The number of retinal microglia in the control group was（11.33±1.21）/field of vision,the model group was（14.83±1.47）/field of vision.The difference was statistically significant（t=-4.498,P0.05）;The number of TUNEL positive cells in the model group was significantly increased compared with the control group（7.89±1.90vs.3.11±1.05,t=-6.596,P<0.05）;Compared with the control group,the protein expressionof TNF-αand IL-1βwas up-regulated inthe model group(t _TNF-α=-6.550,t _IL-1β=-6.789,both P<0.05).Meanwhile,the concentration of NO in the model group was higher than that in the control group（t=-21.296,P<0.05）.Conclusions1.Stable diabetes model of rats can be induced by a One-time intraperitoneal injection of STZ;2.The retinas of diabetic rats with 4 weeks duration appeared activationof retinal microglia,up-regulation of inflammatory factors,and increased apoptosis of retinal cells.Part 2 Effect of Fractalkine gene-modified bone marrow-derived mesenchymal stem cells on inflammatory response in the retina of early diabetic ratsObjective To evaluate the effect of subretinal transplantation of FKN-BMSCs on retinal microglial activation and inflammatory response in the retina of early diabetic rats.Methods1.Isolation,culture,and identification of BMSCs:The whole bone marrow adherence method was used to isolate and culture BMSCs of SD rats.The cells were identified by osteoblast/adipogenic differentiation induction and flow cytometricdetection of the cell surface markers.2.Construction of FKN-BMSCs:To study the infection of BMSCs with lentivirus overexpressing FKN,and to detect the content of FKN in FKN-BMSCs cell supernatant.3.The first part of the method was used to establish a diabetic rat model.44 male SD rats with successful modeling were randomly divided into 4 groups:control group,DMEM group,BMSCs group,and FKN-BMSCs group.At 4w of disease,the DMEM group was injected with 2μL of DMEM solution in the subretinal space;the BMSCs group was injected with 2μL of BMSCs cell suspension.In the FKN-BMSCs group,2μL of FKN-BMSCs cell suspension was injected into the subretinal space,and the control group was not treated.FFA was used to observe the retinal vascular morphology and leakage of rats;Immunofluorescence Iba1staining was used to find the activation of rat retinal microglia;TUNEL method was used to detect the apoptosis of rat retinal cells;Western blot was used to identify the expression of retinal inflammatory factors TNF-αand IL-1βin rats;the Griess method was used to detect the release of retinal NO.Results1.Isolation,culture,and identification of BMSCs:successfully isolated and cultured SD rat BMSCs,the cells showed adherent growth and uniform morphology.They could be induced to differentiate into osteoblasts and adipocytes.The results of flow cytometry showed that BMCSs highly expressed CD29,CD44,and CD90,and hardly expressed CD11b/c,CD34,and CD45,which met the definition standard of MSCs.2.Construction of FKN-BMSCs:Lentivirus overexpressing FKN can infect BMSCsefficiently,with an optimal multiplicity of infection（MOI）of 40,and an infection efficiency of（82.82±2.45）%;The secretion of FKN in FKN-BMSCs supernatant was significantly higher than that of BMSCs（P<0.05）.3.At 1w or 3w after subretinal transplantation,there was no visible abnormal retinal vascular morphology and leakage by FFA in each group.Subretinal transplantation of BMSCs and FKN-BMSCs can reduce the degree of activation and proliferation of retinal microglia.At 1w after transplantation,the morphology of retinal microgliain the control group and DMEM group showedlargercell bodies and thicker and shorter processes.The majority of cells in the BMSCs group and FKN-BMSCs group were branchy.The number of retinal microglia in the BMSCs group and FKN-BMSCs group was lower than that in the control group and the DMEM group（F=22.331,all P<0.05）,and the FKN-BMSCs group was smaller than the BMSCs group（P<0.05）.At 3 weeks after transplantation,the ratio of retinal microglia coverage area in the BMSCs group and FKN-BMSCs group was higher than that in the control group and DMEM group（F=76.054,all P<0.05）,andin particular,the ratio in FKN-BMSCs group was higher than that in BMSCs group（P<0.05）.The density of retinal microglia in FKN-BMSCs group was lower than that in other groups（F=120.100,all P<0.05）.At 3 weeks after transplantation,the expression of CX3CR1 protein in the BMSCs group and FKN-BMSCs group was up-regulated compared with the control group and DMEM group,while the expression levels of TNF-αand IL-1βprotein were down-regulated compared with the control group and DMEM group(F_CX3CR1=85.280、F_TNF-α=45.762、F_IL-1β=34.727,all P<0.05).Compared with the transplantation of BMSCs,transplantation of FKN-BMSCs had a more significant impact on those three proteins.BMSCs group and FKN-BMSCs group had lower NO concentration than the control group and DMEM group(F_1w=68.229、F_3w=153.146,all P<0.05),FKN-BMSCs group was lower than BMSCs group（P<0.05）.Subretinal transplantation of BMSCs and FKN-BMSCs can inhibit the apoptosis of retinal cells.At 3 weeks after transplantation,the number of TUNEL-positive cells in the BMSCs group and FKN-BMSCs group was lower than that in the control group and DMEM group（F=85.876,all P<0.05）,and the number of TUNEL-positive cells in FKN-BMSCs group was lower than that in BMSCs group（P<0.05）.Conclusions 1.BMSCs can be easily isolated and cultured from the bone marrow of SD rats.Identification of BMSCs can be confirmed by the cell morphology,differentiation potential and surface markers.2.FKN gene can integrate into the BMSCs genome by lentivirus,andmaintain high expressionin BMSCs.3.FKN gene modified BMSCs may inhibit the activation of retinal microglia,reduce the inflammatory response of retina and inhibit the apoptosis of retinal cells in the early stage of diabetes through FKN/CX3CR1 axis.