Effect Of LOX-1 Knockdown On Retinopathy And TPL2 In Type 2 Diabetic Mice

Objective: Construction of Adeno-associated virus vector(AAV)mediated Short-hairpin RNA by gene recombination technique shRNA)knocked down Lectin like oxidized lowdensity lipoprotein receptor-1(LOX-1)in db/db mouse retina,To investigate the role of LOX-1 in Diabetic retinopathy(DR)and its effect on Tumor progression locus 2(TPL2).Methods: U6-MCS-CAG-EGFP(shRNA-LOX-1)as well as pAAV-U6-shRNA-CMV b Globin-e GFP-3Flag(shRNA-Con)viruses were constructed.Male db/m mice(7-8 weeks old,10 mice)were normal controls and were recorded as the(NC)group.Male db/db mice(30 mice)of the same age were randomly divided into 3 groups of 10 mice each.Group 1 was treated with saline and was designated as the(NS)group;group 2 was injected with adenoassociated control virus(shRNA-Con)and was designated as the(shRNA-Con)group;group3 was injected with adeno-associated LOX-1 knockdown virus(shRNA-LOX-1)and was designated as the(shRNA-LOX-1)group.During virus transfection,body weight and fasting blood glucose(FBG)of the experimental animals were monitored weekly.After 14 weeks of viral transfection,the animals were executed and sampled,after which the experiments were performed.Immunofluorescence(IF)observation of viral transfection in the retinal tissues of paraffin sections of shRNA-LOX-1 and shRNA-Con mice was performed.Fundus fluorescence angiography(FFA)and hematoxylin-eosin(HE)staining were performed to observe retinal vascular and pathological changes.Quantitative real-time fluorescence polymerase chain reaction(q RT-PCR)and protein blotting(WB)were performed to detect retinal tissue lectin-like oxidized low-density lipoprotein receptor-1(LOX-1),interleukin(IL)-1β,IL-6,nicotinamide adenine dinucleotide phosphate oxidase 4(NOX4),tumor progression locus 2(TPL2)and phosphorylated tumor progression locus 2(p-TPL2)expression.The levels of vascular endothelial growth factor(VEGF),Nε-carboxymethyl lysine(CML)and pigment epithelium-derived factor(PEDF)were measured by enzymelinked immunosorbent assay(ELISA /ELASA)in mouse ocular blood.Results:1.The body weight and fasting blood glucose(FBG)of all 3 groups of db/db mice were significantly higher than those of db/m mice(P<0.05),and the FBG of all 3 groups of db/db mice exceeded 16.7 mmol/L at 8 weeks of age and was maintained at a more stable state.2.Immunofluorescence showed that adeno-associated LOX-1 knockdown virus(shRNALOX-1)and adeno-associated control virus(shRNA-Con)were successfully transfected into the retinal tissues of both groups of mice.3.Compared with the NC group,the retinal LOX-1 mRNA and protein expression levels were significantly increased in both the NS and shRNA-Con groups of mice(P<0.05).After transfection with adeno-associated LOX-1 knockdown virus(shRNA-LOX-1),LOX-1 was significantly reduced in both mRNA and protein expression levels in the retinal tissues of mice in the shRNA-LOX-1 group(P<0.05).4.Fundus fluorescence angiography(FFA)showed that compared with the NC group,the retinal vessels in the NS and shRNA-Con groups showed tortuous microvascular morphology and several obvious areas of strong fluorescence,while the retinal vessels in the shRNALOX-1 group were similar to those in the NC group,with no retinal microvascular morphology tortuous and obvious strong fluorescence.5.Compared with the NC group,the NS group and the shRNA-Con group showed retinal edema and intercellular edema in the inner and outer nuclear layers,and the cell density in each layer was significantly reduced and disorganized,and neovascularization was seen;while the shRNA-LOX-1 group showed similar to the NC group,with the cells in each retinal layer arranged more regularly and closely,and the cells in the outer nuclear layer were slightly loosened,and neovascularization was not seen.6.Retinal IL-1β mRNA and IL-6 mRNA levels were significantly higher in all three groups of db/db mice compared with db/m mice(P<0.05).However,knockdown of LOX-1significantly downregulated the expression of IL-1β mRNA and IL-6 mRNA in the retinas of db/db mice(P<0.05).7.Retinal TPL2 mRNA and protein expression levels were significantly lower in all three groups of db/db mice compared with db/m mice(P<0.05).However,after knockdown of LOX-1,the expression of TPL2 mRNA and protein in the shRNA-LOX-1 group was significantly upregulated in the retinas of db/db mice(P<0.05).8.Compared with the NC group mice,the total retinal TPL2 protein expression levels in the 3 groups of db/db mice were not significantly different(P>0.05),but p-TPL2(Ser400)was significantly increased in the NS and shRNA-Con groups mice compared with the NC group(P<0.05),and the proportion of p-TPL2 in total TPL2 protein was also significantly increased(P<0.05),while in the shRNA-LOX-1 group,the increase of p-TPL2 was significantly suppressed(P<0.05)and the proportion of p-TPL2 in total TPL2 was significantly downregulated(P<0.05).9.Compared with the NC group,serum VEGF and CML levels were significantly increased(P<0.05)and PEDF levels were significantly decreased(P<0.05)in both the NS and shRNA-Con groups of mice.In contrast,in the shRNA-LOX-1 group,the increase of VEGF and CML contents were significantly suppressed(P<0.05)and PEDF contents were significantly upregulated(P<0.05).Conclusions: LOX-1 was involved in regulating retinal damage and TPL2 expression in db/db mice.LOX-1 knockdown inhibited retinal angiogenesis,inflammation and oxidative stress,and TPL2 phosphorylation,thereby delaying the progression of diabetic-associated retinopathy,and had a protective effect on retinal damage in db/db mice.