神经胶质成熟因子-β对糖尿病大鼠视网膜Müller细胞活化的影响及可能机制

Effect of glia maturation factor-β on the activation of retinal Müller cells in diabetic rats and its mechanism

目的 探讨神经胶质成熟因子-β(GMFB)对糖尿病大鼠视网膜Müller细胞活化的影响及可能作用机制。方法 采用腹腔注射链脲佐菌素(55 mg·kg-1)的方法制备雄性SD大鼠的糖尿病模型,并按照随机数字表法分成STZ组、STZ+AAV-GMFB组、STZ+AAV-GMFB+colivelin组,每组15只。另取15只正常大鼠作为CON组。STZ+AAV-GMFB组、STZ+AAV-GMFB+colivelin组大鼠于成模8周后玻璃体内单次注射AAV-GMFB腺病毒载体5μL;STZ+AAV-GMFB+colivelin组大鼠在注射腺病毒基础上给予腹腔注射colivelin(2 mg·kg·d-1),共注射4周;CON组和STZ组大鼠腹腔注射2 mL生理盐水。成模12周后,免疫荧光染色检测GMFB在Müller细胞中的表达,免疫组织化学染色检测GFAP的表达,ELISA检测视网膜中TNF-α、IL-1β、IL-6蛋白含量,Western blot检测视网膜中GMFB、p-JAK2和p-STAT3蛋白相对表达。结果 GMFB在STZ组Müller细胞中与GS大量共定位。与CON组相比,STZ组大鼠视网膜中GFAP表达增加,TNF-α、IL-1β、IL-6蛋白含量及GMFB、p-JAK2、p-STAT3蛋白相对表达量均明显增加(均为P<0.05),视网膜神经节细胞排列紊乱,数量明显减少;与STZ组相比,STZ+AAV-GMFB组大鼠视网膜中GFAP表达明显降低,TNF-α、IL-1β、IL-6蛋白含量及GMFB、p-JAK2、p-STAT3蛋白相对表达量均明显降低(均为P<0.05),视网膜神经节细胞排列整齐,数量明显增加。STZ+AAV-GMFB+colivelin组能大部分逆转AAV-GMFB的保护作用。结论 敲减GMFB基因能抑制糖尿病大鼠视网膜Müller细胞的活化,其机制可能与抑制JAK2/STAT3信号通路有关。

Objective To explore the potential mechanism of glia maturation factor-β(GMFB) on activation of retinal Müller cells in diabetic rats. Methods Male diabetic rat model was established by intraperitoneal injection of streptozotocin(STZ)(55 mg·kg-1). According to the random number table method, they were divided into the STZ group, STZ+AAV-GMFB group, and STZ+AAV-GMFB+colivelin group, with 15 rats in each group. Another 15 normal rats were taken as the control(CON) group. Rats in the STZ+AAV-GMFB group and STZ+AAV-GMFB+colivelin group received the single intravitreal injection of 5 μL AAV-GMFB adenovirus vector 8 weeks after modeling. In the STZ+AAV-GMFB+colivelin group, colivelin(2 mg·kg·d-1) was injected intraperitoneally on the basis of adenovirus injection, totally for 4 weeks. The rats in the CON group and STZ group were intraperitoneally injected with 2 mL physiological saline. After 12 weeks, the expression of GMFB in Müller cells was detected by immunofluorescent staining, the expression of the glial fibrillary acidic protein(GFAP) was detected by immunohistochemical staining, the protein levels of tumor necrosis factor α(TNF-α), interleukin-1β(IL-1β) and interleukin-6(IL-6) in the retina were detected by Enzyme-linked immunosorbent assay, and the relative protein expressions of GMFB, porcine-Janus kinase 2(p-JAK2) and porcine-signal transducer and activator of transcription 3(p-STAT3) in the retina were detected by Western blot. Results Both GMFB and glutamine synthetase were expressed in Müller cells in the STZ group. Compared with the CON group, the expression of GFAP increased, the protein levels of TNF-α, IL-1β, and IL-6, as well as relative protein expressions of GMFB, p-JAK2 and p-STAT3 in the STZ group increased significantly(all P<0.05). In addition, the retinal ganglion cells(RGC) were disorganized and decreased significantly. Compared with the STZ group, the expression of GFAP, the protein levels of TNF-α, IL-1β, and IL-6, as well as relative protein expressions of GMFB, p-JAK2 and p-STAT3 in the STZ+AAV-GMFB group decreased significantly(all P<0.05), and the RGC were orderly arranged and increased obviously. The STZ+AAV-GMFB+colivelin group could reverse the protective effect of AAV-GMFB to a great extent. Conclusion GMFB gene silencing can inhibit the activation of retinal Müller cells in diabetic rats, and its mechanism may be related to the inhibition of the JAK2/STAT3 signaling pathway.