The Protective Mechanism Of α-MSH Against Retinal Damage In Type Ⅱ Diabetes Via LncRNA TSIX/NCOA5 Pathway

ObjectiveTo study the antagonistic effects ofα-melanocyte-stimulating hormone（α-MSH）on vascular leakage and blood-retinal barrier（BRB）damage in mouse retinas of type2 diabetes（T2D）and the molecular mechanisms underpinning theα-MSH’s effects through in vivo and in vitro models.MethodsEight-week-old type 2 diabetic mice（BKS db/db）were randomly divided into diabetic group（db/db）and diabetic+α-MSH group（db/db+α-MSH）;meanwhile,the wild type mice（BKS+/+）were employed as normal controls,10 mice in each group.The metabolic parameters of all mice were monitored for four weeks.At 10 and 12weeks of age,the mice in diabetic+α-MSH group were intravitreally injected withα-MSH（1μl at 3.3μg/μ1）;whereas diabetic and normal control groups received equal amount of sterile saline.In another 3 weeks,electroretinogram（ERG）,staining of hematoxylin and eosin（H&E）,extravascular albumin,glial fibrillary acidic protein（GFAP）,dihydroethidium（DHE）,and terminal deoxynucleotidyl transferase dUTP nick end labeling（TUNEL）were performed to characterize the protective effects ofα-MSH in type 2 diabetic retinas.In vitro,the simian retinal vascular endothelial cells（RF/6A）that had been treated withα-MSH at the doses of 0.1μM,0.5μM,or 1μM and high glucose and palmitate were stained with CM-H₂DCFDA to determine the optimal working concentration ofα-MSH.Subsequently,cells were seeded into a24-well format Transwell plate,establishing a cell culture model of inner BRB.The cells were divided into four groups:normal control group was maintained in conventional cell culture media;the cells in model group were stimulated by sodium palmitate（PAM）and high glucose（HG）,mimicking the hyperlipidemic and hyperglycemic conditions under T2D;and the cells in twoα-MSH-related groups were pretreated withα-MSH orα-MSH with a melanocortin receptor 4（MC4R）-specific antagonist HS024,and then were stimulated by PAM+HG as in the model group.Eight hours later,fluorescein isothiocyanate（FITC）-labeled dextran was used to examine the monolayer endothelial cell leakage,and the monolayer transendothelial electrical resistance（TEER）was also measured.Afterwards,the first three groups of cells were collected for high-throughput RNA sequencing（RNA-seq）,and the selected molecular targets downstream melanocortin receptor,the cognate receptor ofα-MSH,were verified by quantitative real-time PCR（qPCR）.ResultsPhysiological monitoring showed that the db/db mice exhibited typical symptoms of T2D,such as hyperphagia,polydipsia,hyperglycemia,and dyslipidemia（all P<0.001）.ERG tests suggested thatα-MSH restored partial electrophysiological functions in the diabetic mouse retinas（P<0.05）.H&E staining revealed thatα-MSH increased the thickness of retinal layers,except outer nuclear layers,and total retina in the db/db mice（P<0.05）.Moreover,the vascular leakage of albumin was normalized,and the reactive gliosis was profoundly suppressed byα-MSH（both P<0.001）.Additionally,DHE and TUNEL staining demonstrated thatα-MSH significantly alleviated the oxidative stress and apoptosis in the db/db retinas,respectively（both P<0.001）.In vitro,under the stimulation of hyperglycemia and hyperlipidemia,the optimal concentration ofα-MSH against oxidative stress was 0.5μM,which was used for the following experiments.The cell culture results revealed that under the stimulation of PAM and HG,the FITC-dextran leakage from the monolayer endothelial cells was increased as compared to the normal group（P<0.05）,which,however,was reduced byα-MSH to the normal level（P<0.01）.Theα-MSH’s anti-leakage effect was abrogated by the presence of HS024,the MC4R-specific antagonist.Conversely,PAM and HG stimulation in the BRB model decreased the TEER（P<0.05）,which was augmented byα-MSH.Furthermore,theα-MSH’s augmenting effect was also abolished by HS024（P<0.001）.According to the RNA-seq results,α-MSH dramatically and significantly upregulated lncRNA TSIX(P=1.59×10^-6)and a transcriptional co-regulator nuclear receptor coactivator5（NCOA5）（P=0.004）in the retinal vascular endothelial cells stimulated with PAM and HG.The expression patterns of both genes were verified by qPCR.ConclusionsIntravitreal administration ofα-MSH restores morphology and electrophysiological functions,inhibits vascular leakage,oxidative stress and apoptosis,and remarkably suppresses gliosis in T2D retinas.Further,the anti-vascular leakage effects ofα-MSH in the diabetic retinas might be attributed to the MC4R-mediated up-regulation of a novel antisense lncRNA TSIX and the downstream co-regulatory factor NCOA5.