目的：探究利拉鲁肽(LIR)联合神经干细胞(NSCs)减轻大鼠糖尿病视网膜病变(DR)的作用及可能机制。

方法：采用高脂饮食联合一次性腹腔注射链脲佐菌素(STZ)构建DR大鼠模型，对照组大鼠喂养正常饲料并腹腔注射柠檬酸-柠檬酸钠缓冲液。建模后，将大鼠分为对照组、DR组、DR+LIR组、DR+BMSCs组、DR+NSCs组、DR+BMSCs+LIR组和DR+NSCs+LIR组，根据分组说明，分别对大鼠腹腔注射200 μg/(kg·d)利拉鲁肽和玻璃体腔注射骨髓间充质干细胞(BMSCs)或神经干细胞(NSCs)或联合治疗，共治疗8 wk。检测各组大鼠的空腹血糖(FBG)和血清胰岛素水平，以及视网膜氧化应激指标和血清炎症指标水平。通过苏木精-伊红(HE)染色评价视网膜病变，TUNEL染色检测视网膜细胞凋亡，免疫组织化学检测视网膜组织中SIRT1的表达，免疫荧光检测视网膜组织中BDNF的表达。通过qRT-PCR检测视网膜组织中cZNF532、miR-29a-3p、SIRT1、TGF-β1、NRF2、Keap1和BDNF的转录水平，Western blot检测视网膜组织中SIRT1、TGF-β1、SMAD2/3、p-SMAD2/3、NRF2、Keap1、p65和p-p65的蛋白表达水平。

结果：DR+NSCs+LIR组大鼠视网膜形态相较其他组DR大鼠明显改善，FBG、TNF-α、IL-1β、IL-6和MDA水平降低(均P<0.05)，血清胰岛素水平以及SOD、CAT和GSH-Px水平均升高(均P<0.05)，视网膜细胞TUNEL阳性率降低(均P<0.05)，视网膜组织中cZNF532、SIRT1、NRF2和BDNF表达水平升高(均P<0.05)，miR-29a-3p、TGF-β1、p-SMAD2/3、Keap1和p-p65表达水平均降低(均P<0.05)。

结论：利拉鲁肽联合NSCs通过调控cZNF532-miR-29a-3p-SIRT1网络及其下游TGF-β/SMAD、NF-κB和Keap1/NRF2信号通路减轻DR。

METHODS:A DR rat model was established using a high-fat diet combined with a single intraperitoneal injection of streptozotocin(STZ). Rats in the control group were fed a normal diet and received an intraperitoneal injection of citric acid-sodium citrate buffer. After modeling, the rats were divided into the control group, DR group, DR+LIR group, DR+BMSCs group, DR+NSCs group, DR+BMSCs+LIR group, and DR+NSCs+LIR group. According to the grouping instructions, rats were given intraperitoneal injection of LIR at 200 μg/(kg·d)and intravitreal injection of bone marrow mesenchymal stem cells(BMSCs), or NSCs, or the combined therapy accordingly for 8 wk. Fasting blood glucose(FBG)and serum insulin levels, as well as retinal oxidative stress indexes and serum inflammatory indexes, were detected in each group. Retinopathy was evaluated by hematoxylin-eosin(HE)staining, retinal cell apoptosis was detected by TUNEL staining, SIRT1 expression in retinal tissues was detected by immunohistochemistry, and BDNF expression in retinal tissues was detected by immunofluorescence. The transcriptional levels of cZNF532, miR-29a-3p, SIRT1, TGF-β1, NRF2, Keap1, and BDNF in retinal tissues were detected by qRT-PCR, and the protein expression levels of SIRT1, TGF-β1, SMAD2/3, p-SMAD2/3, NRF2, Keap1, p65, and p-p65 in retinal tissues were detected by Western blot.

RESULTS:Compared with the other groups of rats, the retinal morphology of rats in the DR+NSCs+LIR group was significantly improved, the levels of FBG, TNF-α, IL-1β, IL-6 and MDA were decreased(all P<0.05), the serum insulin level and the levels of SOD, CAT, and GSH-Px were increased(all P<0.05), the TUNEL-positive rate of retinal cells was reduced(all P<0.05). The expression levels of cZNF532, SIRT1, NRF2, and BDNF in retinal tissue were up-regulated(all P<0.05), whereas the expression levels of miR-29a-3p, TGF-β1, p-SMAD2/3, Keap1, and p-p65 were down-regulated(all P<0.05).

CONCLUSION:LIR combined with NSCs alleviates DR by regulating the cZNF532-miR-29a-3p-SIRT1 network and its downstream TGF-β/SMAD, NF-κB, and Keap1/NRF2 signaling pathways.