α-黑素细胞刺激素对早期糖尿病大鼠视网膜血管渗漏的保护作用

Alleviating effects of α-melanocyte-stimulating hormone on retinal vessel leakage in diabetic rats

背景 糖尿病视网膜病变（DR）的主要病理基础是视网膜的微血管变化和炎性改变.研究认为α-黑素细胞刺激素（α-MSH）玻璃体腔注射可对视网膜脉络膜组织发挥抗氧化应激和抗凋亡作用,从而改善血-视网膜屏障（BRB）功能,但缺乏组织学研究的验证.目的 研究α-MSH对视网膜血管渗漏的改善作用.方法 应用随机数字表法将清洁级SD大鼠90只随机分为正常对照组、糖尿病（DM）模型组和α-MSH组,DM模型组和α-MSH组大鼠采用尾静脉注射链脲佐菌素（STZ）法制备模型,正常对照组大鼠同法注射枸橼酸钠缓冲液.α-MSH组大鼠分别于造模后1周和3周玻璃体腔注射3.3 μg/μl α-MSH 3 μl,正常对照组大鼠同法注射生理盐水3μl,DM模型组不行玻璃体腔注射.造模后第5周,按照45 mg/kg的剂量经鼠尾静脉注射30 mg/ml伊文思蓝溶液,注射后2h每组取2只大鼠制备视网膜铺片,于荧光显微镜下观察视网膜血管形态;收集各组8只大鼠内眦部血液,检测BRB渗漏状况;用37℃枸橼酸钠缓冲液行左心室灌流,处死大鼠,分离大鼠视网膜,透射电子显微镜下观察脉络膜和视网膜血管超微结构的变化;采用实时定量PCR法检测细胞间黏附分子-1（ICAM-1）、肿瘤坏死因子-α（TNF-α）、闭合蛋白（occludin）和血管内皮生长因子（VEGF）mRNA的相对表达水平.结果 DM模型大鼠体质量明显降低,饮水量明显增多;造模后3d和5周,DM模型组大鼠的血糖分别为（29.69±4.77） mmol/L和（24.64±2.72） mmol/L,DM模型成功建立.视网膜铺片显示,DM模型组大鼠视网膜血管走行异常,血管壁大量伊文思蓝渗漏,而α-MSH组大鼠视网膜血管形态接近正常对照组.造模后5周,DM模型组大鼠视网膜伊文思蓝的渗漏量为（10.04±8.18） μl/（g·h）,明显多于α-MSH组的（2.62±3.73） μl/（g·h）和正常对照组的（3.10±1.13） μl/（g·h）,差异均有统计学意义（P=0.035、0.041）.α-MSH组大鼠脉络膜和视网膜血管的超微结构接近正常对照组,而DM模型组大鼠脉络膜血管内皮细胞肿胀,视网膜色素上皮（RPE）空泡样变性.此外,DM模型组大鼠视网膜中ICAM-1 mRNA和TNF-αmRNA水平明显高于α-MSH组和正常对照组,而occludin mRNA的相对表达水平明显低于o-MSH组和正常对照组,差异均有统计学意义（均P＜0.05）.3个组大鼠视网膜中VEGF mRNA相对表达量的整体比较,差异无统计学意义（F=0.791,P=0.466）.结论 在STZ诱导的DM模型大鼠中,玻璃体腔注射α-MSH可通过减少视网膜血管的渗漏,改善脉络膜和视网膜血管的超微结构,下调视网膜中促炎因子的表达和上调细胞间紧密连接成分的表达发挥对视网膜的保护作用.

Background The primary pathogenic basis of diabetic retinopathy（DR） is microangiopathy and inflammatory procedure.Studies showed that α-melanocyte stimulating hormone （ct-MSH） can play anti-oxidative stress and anti-apoptosis effects on retina via intravitreal injection and therefore mend blood-retinal barrier （BRB）.However,experiment has not yet proved its mechanism.Objective This study was to investigate the protective effects of α-MSH on retinal vessel in diabetic rats.Methods Ninety clean SD rats were assigned to the normal control group,diabetes mellitus （DM） model group and α-MSH group.DM models were established by streptozocin （STZ） injection via tail vein in the rats of the DM model group and the α-MSH group,and sodium citrate buffer was injected in the same way in the normal control group,α-MSH of 3 μl （3.3 μg/μl） was intravitreally injected 1 week and 3 weeks after modeling in the rats of the α-MSH group,and normal saline solution was used in the same way in the normal control group;while no operation was performed in the DM model group.In the fifth week after modeling,Evans blue at the dose of 30 mg/ml （45 mg/kg） was injected via tail vein of rats,and retinal mount was prepared 2 hours later to examine the morphology of retinal vessels under the fluorescent microscope.The blood samples were collected from inner canthus vein for the assessment of leakage amount of Evans blue.The rat retinas were isolated after perfusion of sodium citrate buffer via the left ventricle for the observation of choroidal and retinal vessel ultrastructure by transmission electron microscopy （TEM）.The relative expressions of intercellular cell adhesion molecule-1 （ICAM-1） mRNA,tumor necrosis factor-α （TNF-α）,occluding mRNA and vascular endothelial growth factor （VEGF） mRNA were detected by quantitative real-time PCR.Results The body weight was lighter and the water intake was higher in the DM rats than those in the normal control rats.The blood glucose levels in the DM model group were （29.69±4.77） mmol/L at the third day and （24.64±2.72） mmol/L in the fifth week following the injection of STZ,respectively.An amount of Evans blue leaked from vessels in the rat retina of DM model group,but the retinal vessels were intact in the rats of the α-MSH group and the normal control group.The leakage amount was （10.04-±8.18） μl/（g · h） in the D M model group,which was significantly more than that of （2.62±3.73） μl/（g · h）in the α-MSH group and （3.10± 1.13）μl/（g · h） in the normal control group at the fifth week after modeling （P =0.035,0.041）.The ultrastructure of choriodal and retinal vessels was almost normal in the α-MSH group and the normal control group,but the swelling of choriodal vascular endothelial cells and vacuolar-like changes of the retinal pigment epithelium（RPE） were found in the DM model group.In addition,the relative expression levels of ICAM-1 mRNA and TNF-α mRNA were significantly elevated,but those of occludin mRNA were significantly declined in the DM model group compared with the α-MSH group and the normal control group （all at P＜0.05）.No significantdifference was found in the relative expression level of VEGF mRNA among the three groups （F=0.791,P=0.466）.Conclusions α-MSH ameliorates retinal vascular leakage in STZ-induced diabetic rats after intravitreal injection by improving ultrastructure of choroid and retinal vessels,down-regulating the expression of proinflammatory factors and up-regulating tight junction component in retina.