The Effect And Mechanism Of Advanced Glycation End Products On Corneal Dendritic Cells In Diabetic Mice

Objective:To investigate the effect and mechanism of advanced glycation end-products(AGEs)on dendritic cells(DCs)of mice with diabetic keratopathy using bone marrow-derived dendritic cells(BMDCs)and streptozotocin(STZ)-induced type 1 diabetic mice model.Methods:1.The effect of AGEs on mice BMDCs and the activation of key signal pathways.The mice BMDCs were induced by treatment with IL-4(10ng/m L)and GM-CSF(20ng/m L).BMDCs were randomly divided into blank control group,BSA group and AGE-BSA group.BSA(200μg/m L)and AGE-BSA(200μg/m L)were used to stimulate the BMDCs for 12 and 24 hours,respectively.The concentrations of IFN-β in the supernatant with different groups were determined by enzyme linked immunosorbent assay(ELISA),and the transcriptional levels of inflammatory factors(such as IFN-β,CXCL10,IFIT1,IL-6,IL-12p35 and IL-1β)in BMDCs were analyzed using Real-time PCR(RT-PCR).The phosphorylation of TBK1,IRF3 and p65 were examined by Western blot.2.The effect of diabetes mellitus on the expression of AGEs and inflammatory factors in the corneas.Male C57BL/6 mice were randomly divided into normal control group(CON)and diabetes group(DM).The type 1 diabetes was induced by intraperitoneal injection of STZ.The eyeballs were harvested on 6 months after the final injection of STZ.The levels of AGEs in the corneas were determined using western blot,and RT-PCR was performed to detect the transcriptional levels of IFN-β and IFIT1 in the corneas of two groups.3.The effect of cGAS/STING signaling pathway in the activation of BMDCs induced by AGEs.The wild-type(WT)BMDC,cGAS knockout(KO)and STING KO BMDC were induced by IL-4 and GM-CSF,respectively.The BMDCs with different groups were then treated with AGE-BSA(200μg/m L)for 12 hours.After treatment,the BMDCs and cell supernatant were collected for subsequent experiments,respectively.The levels of IFN-β in supernatant of different groups were quantified using ELISA.The m RNA levels of inflammatory factors(such as IFN-β,CXCL10,IFIT1,IL-6,IL-12p35 and IL-1β)in different groups were analyzed by RT-PCR.The phosphorylation of TBK1,IRF3 and p65 were determined through Western blot.4.The influence of Toll-like receptor 4(TLR4)signaling on the activation of BMDCs by AGEs.The BMDCs were induced and randomly divided into blank control group,AGE-BSA(200μg/m L)group and TAK-242 group treated with AGE-BSA(200μg/m L)and TAK-242(5μM,a TLR4 inhibitor).After treatment,the concentrations of IFN-β in supernatant and the transcriptional levels of inflammatory factors in BMDCs were evaluated by ELISA and RT-PCR,respectively.Western blot was performed to analyze the phosphorylation of TBK1,IRF3 and p65.5.The effect of TAK-242(an inhibitor of TLR4 signaling)on diabetic corneal wound healing.The C57BL/6 male mice were randomly divided into three groups: CON+ solvent group(solvent composed of 1% DMSO+99% PBS),DM + solvent group,and DM + TAK-242 group(0.375 mg/m L).TAK-242 was subconjunctival injected when corneal epithelium was scraped using 2.5mm trephine.The fluorescein sodium staining was performed to examine diabetic wound healing at 0h,24 h,and 40 h after scraping.Results:1.AGEs promoted the activation of BMDCs.Compared with the blank control and the BSA group,the concentration of IFN-β in the supernatant of the AGE-BSA group was significantly increased.RT-PCR showed a dramatic upregulation of IFN-β,CXCL10,IFIT1,IL-6,IL-12p35 and IL-1β in AGE-BSA group.Furthermore,the phosphorylated TBK1,IRF3 and p65 were also mounted after treatment with AGE-BSA.The findings revealed that AGE-BSA stimulated BMDCs activation by NF-κB and IRF3 signaling pathways.2.Diabetes mellitus caused the AGE accumulation and elevated the expression of cytokines in corneas.We observed the increased accumulation of AGEs in diabetic corneas by western blot when compared with the age-matched controls.Moreover,the RT-PCR also revealed a much higher transcriptional level of IFN-β and IFIT1 than in normal controls.These results indicated the abnormal accumulation of AGEs and the upregulation of inflammation-related factors in diabetic corneas.3.The AGEs activated BMDCs in a cGAS/STING signaling independent manner.Compared with WT BMDCs,the expression of inflammation-related cytokines in cGAS KO and STING KO BMDCs treated with AGE-BSA was not pronouncedly down-regulated.Accordingly,there were no significant alterations of phosphorylated TBK1,IRF3 and p65 between AGE-BSA-stimulated cGAS KO and STING KO BMDCs with the WT BMDCs.The findings indicated that the activation of BMDCs by AGE-BSA did not depend on cGAS/STING signaling pathway.4.The activation of BMDCs induced by AGEs depending on TLR4 signaling.The findings of ELISA and RT-PCR revealed that the TAK-242 treatment pronouncedly lowered the expression of IFN-β,CXCL10,IFIT1,IL-6,IL-12p35 and IL-1β when compared with the AGE-BSA group.Consistently,the increased phosphorylation of TBK1,IRF3 and p65 in AGE-BSA-treated BMDCs was also significantly reversed by TAK-242 treatment.These results demonstrated that the AGE-BSA-ignited BMDCs activation relied on TLR4 signaling pathway.5.Blocking TLR4 signaling pathway promoted diabetic corneal wound healing.Compared with the normal mice,the corneal epithelial wound healing in DM group was significantly postponed at 24 and 40 hours after scraping.However,the diabetic corneal wound closure accelerated after intervention with TAK-242.These results indicated that the TLR4-mediated inflammation delayed the diabetic wound healing.Conclusion:1.Compared with age-matched normal mice,the diabetic corneas showed the abnormal accumulation of AGEs and inflammatory responses.2.AGEs caused chronic inflammation of the diabetic corneas,mechanistically depending on TLR4 signaling pathway.3.Blocking TLR4 signaling pathway through TAK-242 accelerated diabetic corneal wound healing.