磷酸化细胞外信号调节激酶1/2对糖尿病视网膜病变的作用

A pilot study on phosphorylation of extracellular signal-regulated kinase 1/2-mediated diabetic retinopathy

背景 糖尿病视网膜病变（DR）具有复杂的病理表现和发生机制,丝裂原活化蛋白激酶（MAPK）参与高血糖对脑缺血损伤的作用,MAPK信号转导通路在DR发生和发展中的作用有待深入研究.目的 探讨DR与磷酸化细胞外信号调节激酶1/2（ERK1/2）的关系.方法 采用随机数字表法将60只SPF级8周龄SD大鼠随机分为对照组和糖尿病组,将枸橼酸缓冲液配置的链脲佐菌素（STZ）溶液按照60 mg/kg的剂量一次性腹腔内注射制备糖尿病大鼠模型,血糖＞16.7 mmol/L视为建模成功;对照组以同样的方法注射枸橼酸缓冲液.分别于造模后4周和12周处死大鼠并分离视网膜,采用苏木精-伊红染色法进行视网膜的组织形态学观察,采用免疫组织化学法检测各组大鼠视网膜磷酸化ERK1/2和胶质纤维酸性蛋白（GFAP）的相对表达量.结果 造模后4周和12周,糖尿病组大鼠血糖水平均明显高于对照组大鼠,差异均有统计学意义（t=14.174、13.771,P＜0.05）.造模后12周,糖尿病组大鼠平均体质量明显低于对照组;糖尿病组大鼠饮水量明显多于对照组,差异均有统计学意义（t=8.670、18.725,P＜0.05）.与对照组大鼠比较,造模后12周糖尿病组大鼠视网膜变薄,外丛状层水肿,视网膜神经节细胞（RGCs）层、内核层及视细胞层细胞数量减少.免疫组织化学检测可见,造模后4周和12周糖尿病组大鼠视网膜中GFAP阳性细胞数增加,相对表达量（A值）分别为3 197.1±13.1和7 202.0±56.8,均明显高于对照组的2 152.8±16.1和2 337.0±8.6,差异均有统计学意义（t=6.327、16.417,P＜0.05）.造模后12周,糖尿病组视网膜磷酸化ERK1/2相对表达量（A值）为2 850.6±2.4,明显高于对照组的1 274.6±1.3,差异有统计学意义（t=12.771,P＜0.05）.结论 ERKl/2信号通路参与STZ诱导的糖尿病大鼠的早期DR,可能与RGCs和视细胞的凋亡及Müller细胞活化有关.

Background It is well known that diabetic retinopathy（DR） involves in complex pathogenesis.Worse outcome of brain ischemia with hyperglycemia is mediated by mitogen-activated protein kinases （MAPK）.However,the effect of phosphorylation of MAPK signal pathway on DR needs further investigation.Objective This study was to explore the the possible role of extracellular signal-regulated kinase1/2（ERK1/2）in DR.Methods Sixty SPF SD rats aged 8-weeks were grouped into the control group and diabetes group.Diabetic models were established by intraperitoneal injection of streptozotocin （STZ） （60 mg/kg） dissolved in citric acid buffer.Blood glucose level was more than 16.7 mmol/L were used as the diabetic group.The equivalent amount of citric acid buffer was injected in the same way in the rats of the control group.The rats were sacrificed and retinas were isolated in 4 weeks and 12 weeks after modeling.The morphology of rat retinas was examined by hematoxylin and eosin staining.The relative expression levels of phosphorylated-ERK1/2 and glial fibrillary acidic protein （GFAP）（absorption,A value） in rat retinas were detected by immunohistochemistry.Results Blood glucose levels of rats were significantly higher than those in the diabetic group compared with the control group at both 4 and 12 weeks after modeling（t=14.174,13.771,both at P〈0.05）.In addition,the body weight was significantly lower and the drinking was much more in the rats of the diabetic group in comparison with the control group in 12 weeks（t=8.670,18.725,both at P〈0.05）.Twelve weeks after modeling,the decrease of retinal thickness,swelling of outer plexiform layer and decline of number of retinal ganglion cells,rods and cones were seen under the optical microscope.The relative expression levels of GFAP in the retinas were 3 197.1 ±13.1 and 7 202.0±56.8 in the diabetic group at the 4 and 12 weeks,which were significantly higher than those in the control group （2 152.8 ± 16.1 and 2 337.0±8.6） （t =6.327,16.417,both at P〈0.05）.In 12 weeks after modeling,the relative expression level of phosphorylated-ERK 1/2 was significantly higher in the diabetic group compared with the control group （2 850.6±2.4 versus 1 274.6± 1.3）,showing a significant difference between them （t =12.771,P 〈 0.05）.Conclusions Phosphorylation of ERK1/2 signal transduction pathway is involved in the STZ-induced DR through mediating the activation of Müller cells and inducing the apoptosis of photoreceptor and ganglione cells.