Role Of TLR4/NF-κB Signal Pathway In Retinal Ganglion Cells Primary Cultured In High Glucose And Its Implications

Part One:Culture and Identification of Primary SD rat Retinal Ganglion Cell,Establishment and Evaluation of a High Glucose ModelPurpose:To culture and identify primary SD rat retinal ganglion cells,as well as establish and evaluate a high glucose model.Methods:Primary retinal ganglion cells were isolated from SD rats(1-3 days old)and purified with 5 fluorouracil and uridine(final concentration is 20μg/ml and 50μg/ml respectively).And then primary cultured retinal ganglion cells were identified with Bm3a.Primary cultured RGCs were divided into control(OmM),HG1(lOmM glucose),HG2(20mM glucose),HG3(30mM glucose),observation time were at 24h and 48h.Result:Cells began to attach immediately,most of them attached at 24 h.a small part of the cells generation aggregated.After purification with 5-Fu and uridine,the axon form of attached cells became clearly as well as the number of them decreased.Immunofluorescence results show that the adherent cells were bm3a-positive and DAPI-positive,which confirmed that the primary cultured cells of SD rat were retinal ganglion cells.Compared with control group,the attached cells in the high glucose groups showed different degrees of apoptosis.Conclusion:Our study followed a protocol with minor modifications to yield primary SD rat retinal ganglion cells with high purity.The high glucose model was efficient and reliable.Part Two:Role of TLR4/NF-κB signal pathway in retinal ganglion cells primary cultured in high glucose and its implicationsPurpose:To investigate the expression profiles of TLR4,the effect of TLR4/NF-κB signal pathway on the apoptosis of RGCs cultured in high glucose and the underlying mechanism.Methods:High glucose model was established in RGCs that were isolated from Sprague-Dawley(SD)rats(2-3 days old)and identified with Brn3a.Primary cultured RGCs were divided into control(OmM),HG1(lOmM glucose),HG2(20mM glucose),HG3(30mM glucose),HG(20mM glucose)+TAK-242(1.0uM),and HG(20mM glucose)+vehicle(1%DMSO)groups.The expressions of TLR4,its downstream signaling molecules and pro-inflammatory cytokines were measured by real time-PCR,Western blot or ELISA at 24h and 48h,respectively.The apoptosis rate of RGCs was measured by flow cytometry.Results:The mRNA and protein expressions of TLR4 were increased in high glucose groups(10mM,20mM,30mM).Consistent with that,four TLR4 downstream signaling molecules(MyD88,NF-κB,TRAF6,NLRP3)and pro-inflammatory cytokines(IL-1β,IL-18)were upregulated in the three high glucose groups.The apoptosis of RGCs was obviously increased in the high glucose group.The Administration of TAK-242,the antagonist of TLR4,inhibited the inflammation and the apoptosis of RGCs in high glucose group.Conclusion:Our results demonstrated that TLR4/NF-κB signal pathway played a critical role in the inflammation and apoptosis of RGCs induced by high glucose.TLR4 may become a novel potential pharmacological target for the prevention of the progression of DR.